Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry by Research Signpost

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry Editor

Vinod K. Tiwari Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Co-editor

Bhuwan B. Mishra Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Research Signpost 2011; Rights Reserved Research Signpost T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Vinod K. Tiwari Co-editor Bhuwan B. Mishra Managing Editor S.G. Pandalai Publication Manager A. Gayathri Research Signpost and the Editors assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-308-0448-4

Preface Natural products extracted from tissues of terrestrial plants, marine organisms or microorganism fermentation broths are the evolutionary shaped molecules with a profound impact on human health. Nature's biosynthetic engine produces innumerate metabolites with distinct biological properties that make them valuable as health products or as structural templates for drug discovery. In the early 1900s, before the ‘‘Synthetic Era’’, 80% of all medicines were obtained from roots, barks and leaves with a belief that for every ill there exists a cure in the plants of field and forest. However, with the advent of robotics, bioinformatics, high throughput screening, molecular biology-biotechnology, combinatorial chemistry, in silico (molecular modelling) and other methodologies, the pharmaceutical industry largely moved away from plant derived natural products as a source for leads and prospective drug candidates. There are also several misconceptions that constrained the utilization of plant products for discovery and development of pharmaceuticals. Among some of practical aspects while trying to explain the difficulties associated with natural product research are: low yield, one-sampleone-source problem; high structural complexity and occurrence of multiple stereoisomer; lacking of follow-up studies, since most efforts (e.g. in academic environments) are not the part of focused drug development programs. Pharmaceutical discovery is a numbers game in which thousands of chemicals must be evaluated to find a hit. The interesting chemicals identified as natural products are derived from the phenomenon of biodiversity in which the interaction of organisms among each other and their environment formulate the evolution of diverse complex natural entities in the organisms that enhance their survival by protecting them against a wide variety of microorganisms, arthropods and vertebrates and maintain competitiveness in the ecosystem. Importantly, nature has been “doing” combinational chemistry for eons and supplying almost unimaginable chemical diversity, which yields stereochemically complex structures with diverse functional groups and molecules ideal for interacting specifically with biological targets. As Aristotle said, ‘‘Nature does nothing without purpose or uselessly”, the world of plants, and indeed all natural sources, represents a virtually untapped pool of novel drugs awaiting imaginative and progressive organizations. This book covers almost all natural product drug discoveries that have been made in past few decades. The book editorial (Chapter 1) sumarises the natural products, semi-synthetic natural products and natural product derived compounds that have been registered, undergoing registration or in clinical

development, while chapters 2-12 are focused on natural product drug discoveries by disease area i.e. infectious (bacterial, fungal and parasitic etc.) diseases and Oncology. The chapter 13 is focused on mutasynthesis that couples the power of chemical synthesis with molecular biology to generate derivatives of medicinally important natural products while the last chapter of the book highlights significance of carbohydrate containing natural products in medicinal chemistry. I am thankful to all the authors and reviewers who helped me in compiling this book. Lastly, I want to draw the attention of readers about the increasing loss of much of the world’s forests, particularly in the tropics, where the potentially remarkable properties of plant constituents not yet discovered are threatened. Several plant species are on the brink of extinction and in need of urgent conservation measures, otherwise, many future drugs and other useful plant products would remain undiscovered and the often surprising chemical structures produced by the genetic diversity of plants might not be envisioned by future chemists. Vinod K. Tiwari

Editorial Advisory Board Prof. A. D. Kinghorn, USA Prof. A. W. Lipkowski, Poland Prof. Atta-ur-Rahman, Pakistan Prof. B. Pirotte, Belgium Prof. D. L. Boger, USA Prof. D. S. Bhakuni, India Prof. D. Strack, Germany Prof. De-Yun Wang, Singapore Prof. G. A. Cordell, USA Prof. G. Appendino, Itly Prof. G. Bringmann, Germany Prof. G. H. Veeneman, Netherland Prof. G. P. Bolwell, UK Prof. G. S. Singh, Botswana Prof. G. Vo-Thanh, France Prof. Ganesh Pandey, India Prof. Guisen Zhao, China Prof. H. Ila, India Prof. J. A. R Rodrigues, Brazil Prof. J. C. Stockert, Spain Prof. J. D. Connolly, UK Prof. J. S. Yadav, India Prof. Jamie Simpson, Australia Prof. K. C. Nicolau, USA Prof. K. D. Janda, USA Prof. M. Garson, Austria Prof. M. I. Choudhary, Pakistan Prof. M. J. Chan-Bacab, Mexico Prof. M. P. Kaushik, India Prof. M. Salzet, France

Prof. M. Shibasaki, Japan Prof. N. Tagmatarchis, Greece Prof. Norbert Haider, Austria Prof. P. G. Wang, USA Prof. P. S. Portoghese, USA Prof. Prabhat Arya, Canada Prof. Pradeep Kumar, India Dr. Prabhu P Mohapatra, USA Prof. R. A. Lewis, Switzerland Prof. R. M. Singh, India Prof. R. P. Tripathi, India Prof. R. R. Schmidt, Germany Prof. S. Neidle, UK Prof. R. Robins, France Prof. S. Chandrashekhar, India Prof. S. Komatsu, Japan Prof. Seokjoon Lee, South Korea Prof. Shang-Cheng Hung, Taiwan Prof. Thomas Kurz, Germany Prof. V. K. Singh, India Prof. W. Boland, Germany Prof. Xi Chen, USA Prof. Y. Asakawa, Japan Prof. Y. B. Tripathi, India Prof. Y. H. Wong, Hong Kong Prof. Y. Hashimoto, Japan Prof. Y. Hu, China Prof. Y. Yamamoto, Japan Prof. Yogendra Singh, India Prof. Nity anand, India

Contents

Chapter 1 Natural products in drug discovery: Clinical evaluations and investigations Bhuwan B. Mishra and Vinod K. Tiwari

Chapter 2 Natural products in discovery of potential and safer antibacterial agents Girija S. Singh and Surendra N. Pandeya

Chapter 3 Anti-tubercular activity of natural products: Recent developments L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov

103

Chapter 4 Scope of natural products in fighting against leishmaniasis B. B. Mishra, R. R. Kale, V. Prasad, V. K. Tiwari and R. K. Singh

121

Chapter 5 Naturally occurring antihyperglycemic and antidyslipidemic agents T. Narender, T. Khaliq and G. Madhur

155

Chapter 6 Bio-flavonoids with promising anti-diabetic potentials: A critical survey Goutam Brahmachari

187

Chapter 7 Marine natural alkaloids as anticancer agents Deepak Kumar and Diwan S. Rawat

213

Chapter 8 Microtubule binding natural substances in cancer chemotherapy Ram C. Mishra

269

Chapter 9 Natural products: Anti-fungal agents derived from plants Tasleem Arif, T. K. Mandal and Rajesh Dabur

283

Chapter 10 Sesquiterpene lactones: Structural diversity and their biological activities Devdutt Chaturvedi

313

Chapter 11 A review on natural products with mosquitosidal potentials Navneet Kishore, Bhuwan B. Mishra, Vinod K. Tiwari and Vyasji Tripathi Chapter 12 Soybean constituents and their functional benefits Ajay K. Dixit, J. I. X. Antony, Navin K. Sharma and Rakesh K. Tiwari

335

367

Chapter 13 Mutasynthesis of medicinally important natural products through manipulation of gene governing starter unit Deepak Sharma, Syed Khalid Yousuf and Debaraj Mukherjee

385

Chapter 14 Carbohydrate-containing natural products in medicinal chemistry Hongzhi Cao, Joel Hwang and Xi Chen

411

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 1-62 ISBN: 978-81-308-0448-4

1. Natural products in drug discovery: Clinical evaluations and investigations Bhuwan B. Mishra and Vinod K. Tiwari Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Abstract. Natural products (NPs) have provided the source for the majority of FDA-approved agents and continue to be one of the major sources of inspiration for future drug discovery. The R&D thrust in the pharmaceutical sector today is focused on development of new drugs, innovative/indigenous processes for known drugs, development of NP-based drugs through investigation of leads obtained from the traditional systems of medicine as well as other resources. Present review describes natural products (NPs), semi-synthetic NPs and NP-derived compounds that have been registered, undergoing registration or in clinical development since 1998 till June 2010 by disease area i.e. infectious (bacterial, fungal, parasitic and viral), immunological, cardiovascular, neurological, inflammatory and related diseases and Oncology. This review also highlights the recently launched natural product-derived drugs, new natural product templates and late-stage development candidates.

1. Introduction The interesting chemicals identified as NPs are derived from the phenomenon of biodiversity in which the interactions among organisms and their environment formulate the diverse complex chemical entities within the Correspondence/Reprint request: Dr. Vinod K. Tiwari, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. E-mail: tiwari_chem@yahoo.co.in

Bhuwan B. Mishra & Vinod K. Tiwari

organisms that enhance their survival and competitiveness. Today, R&D thrust in the pharmaceutical sector is focused on development of new drugs, innovative/indigenous processes for known drugs and development of plant-based drugs through investigation of leads obtained from the traditional systems of medicine as well other resources [1,2]. History of medicine dates back practically to the existence of human civilization and use of NPs by human have been traced from ancient records such as the use of Artemisia annua in China, opium poppy (active principle morphine) in Egypt, snakeroot plant (active principle reserpine) in India, willow tree (salicin) & foxglove (active principle digitalis - a mixture of compounds such as digitoxin, digitonin, digitalin) in England and ipecacuanha root (active principle emetine), coca bush (active principle cocaine) and cinchona bark (active principle quinine) in Mesoamerica. The current accepted modern medicine or allopathy has gradually developed over the years by scientific and observational efforts of scientists. However, the basis of its development remains rooted in traditional medicine and therapies. Plants have always been a rich source of NP leads e.g. morphine, cocaine, digitalis, quinine, tubocurarine, nicotine, muscarine, paclitaxel (TaxolTM) and artemisinin. The success of penicillin encouraged the discovery of new antibiotics from microorganisms. Mining of the bacterial genome and identification of crucial targets followed by study of new bacterial or fungal strains have resulted in discovery of significant antibacterial agents such as the cephalosporins, tetracyclines, aminoglycosides, rifamycins and chloramphenicol. Since past five decades, marine sources e.g. coral, sponges, fish and marine microorganisms have attracted scientists from different disciplines leding to the discovery of several marine NPs with promising biological activity such as curacin A, eleutherobin, discodermolide, bryostatins, dolostatins, and cephalostatins. Venoms and toxins (peptides and non-peptides) occurring in snakes, spiders, scorpions, insects, and other microorganisms are also significant in drug discovery due to their specific interactions with macromolecular targets in the body, and have been proved crucial while studying receptors, ion channels, and enzymes. Toxins like α-bungarotoxin (from cobras), tetrodotoxin (from puffer fish) and teprotide (from Brazilian viper) etc. are in clinical trials for drug development. Similarly, the neurotoxins obtained from Clostridium botulinum (responsible for botulism, a serious food poisoning), has been found significant to prevent muscle spasm. The review summarizes the 3 groups of compounds classified as NPs, semi-synthetic NPs and NP-derived compounds that have been registred, undergoing registration or in clinical development since 1998 to June 2010 by disease area i.e. infectious (bacterial, fungal, parasitic and viral),

Natural products in drug discovery

immunological, cardiovascular, neurological, inflammatory and related diseases and oncology. The compounds which have biological activities and are derived from natural sources, e.g., plants, animals and microorganisms have been grouped as NPs. The compounds that are derived from a NP template using semi-synthesis have been grouped in semi-synthetic NPs while the compounds that were synthetically derived or in some cases inspired from a NP template have been classified as NP-derived compounds [3-5]. The review also presents an update of previous reviews published in relevance to present context [6-10]. Table 1. NP-deived drugs launched during 1998-2004; lead compounds and therapeutic area. Year 1998 1998 1999 1999 1999 2000 2001 2001 2001 2001 2002 2002 2002 2002 2002 2003 2003 2003 2003 2003 2004

Trade name orlistat (Xenical®) cefoselis (Wincef®) dalfopristin and quinupristin (70 :30 mixture) (Synercid®) valrubicin (Valstar®) colforsin daropate (Adele, Adehl®) arteether (Artemotil®) ertapenem (InvanzTM) caspofungin (Cancidas®) telithromycin (Ketek®) 55 pimecrolimus (Elidel®) galantamine (Reminyl®) micafungin (Funguard®) amrubicin hydrochloride (Calsed®) biapenem (Omegacin®) nitisinone (Orfadin®) miglustat (Zavesca®)

Lead compound lipstatin cephalosporin streptogramin B 44 & streptogramin A 45

Disease area Antiobesity Antibacterial Antibacterial

doxorubicin 164 forskolin

Oncology Cardiotonic

artemisinin 65 thienamycin 5 pneumocandin B erythromycin 51 ascomycin galantamine FR901379 doxorubicin 164

Antimalarial Antibacterial Antifungal Antibacterial Atopic dermatitis Alzheimer’s disease Antifungal Oncology

thienamycin 5 leptospermone 1-deoxynojirimycin

mycophenolate sodium (Myfortic®) rosuvastatin (Crestor®) pitavastatin (Livalo®) daptomycin (CubicinTM) everolimus (CerticanTM) 24

mycophenolic acid

Antibacterial Antityrosinaemia Type 1 Gaucher disease Immunosuppression

mevastatin mevastatin daptomycin sirolimus 10

Dyslipidemia Dyslipidemia Antibacterial Immunosuppression

Bhuwan B. Mishra & Vinod K. Tiwari

2. Drug approval processes The Investigational New Drug (IND) application is submitted to the FDA or EMA before commencement of clinical trials. Once clinical trials are successfully completed, the applicant files New Drug Application (NDA) in the US or Marketing Authorization Application (MAA) in Europe seeking drug’s approval for marketing, to which the agency replys in the form of “approval letter”, “nonapproval letter” or “approvable letter”. An ‘‘approval letter’’ allows the applicant to begin marketing of product, while a ‘‘non-approval letter’’ rejects the application. An ‘‘approvable letter’’ informs the applicants that the agency have completed their scientific review and determined that the application can be approved pending resolution of minor deficiencies identified in the letter or during an inspection of the manufacturing facilities. Table 2. NP-deived drugs launched during 2005-2010; lead compounds, and therapeutic area. Year 2005

Lead compound Dronabinol 1/cannabidol 2 Fumagillin 3 Thienamycin 5

Disease area Pain

2005 2005

Trade name Dronabinol 1/Cannabidol 2 (Sativex®) Fumagillin 3 (Flisint®) Doripenem 4 (Finibax®/DoribaxTM)

2005 2005 2005

Tigecycline 6 (Tygacil®) Ziconotide 8 (Prialt®) Zotarolimus 9 (EndeavorTM stent)

Tetracycline 7 Ziconotide 8 Sirolimus 10

2006

Anidulafungin 11 (EraxisTM/EcaltaTM) Exenatide 13 (Byetta®) Lisdexamfetamine 14 (VyvanseTM) Retapamulin 16 (AltabaxTM/AltargoTM) Temsirolimus 18 (ToriselTM) Trabectedin 19 (YondelisTM) Ixabepilone 20 (IxempraTM) Methylnaltrexone 22 (Relistor®) Everolimus 24 (Afinitor®) Telavancin 25 (VibativTM) Romidepsin 27 (Istodax®) Capsaicin 28 (Qutenza®) Monobactam aztreonam 29 (CaystonTM)

Echinocandin B 12

Antibacterial Pain Cardiovascul ar surgery Antifungal

Exenatide-4 13 Amphetamine 15 Pleuromutilin 17

Diabetes ADHD Antibacterial

Sirolimus 10 Trabectedin 19 Epothilone B 21 Naltrexone 23 Sirolimus 10 Vancomycin 26 Romidepsin 27 Capsaicin 28 Aztreonam 29

Oncology Oncology Oncology Pain Oncology Antibacterial Oncology Pain Antibacterial

2006 2007 2007 2007 2007 2007 2008 2009 2009 2009 2009 2010

Antiparasitic Antibacterial

Natural products in drug discovery

3. NP based drugs approved during 1998-2004 A total of 21 NP and NP-derived drugs were launched in the United States, Europe or Japan during 1998-2004 that can be classified as 3 NPs, 10 semi-synthetic NPs and 8 NP-derived drugs (Table 1).

3.1. NP based drugs approved during 2005-2010 A total of 19 NP based drugs were approved for marketing worldwide in between the year 2005 to April 2010, among which 7 being classified as NPs, 10 semi-synthetic NPs and 2 NP-derived drugs (Table 2). VeregenTM (Polyphenon® E ointment), a defined mixture of catechins obtained from green tea, is the first ever herbal medicine to receive FDA approval in 2006. VeregenTM was developed by MediGene AG and launched in the US by Bradley Pharmaceuticals in December 2007 for topical use against genital warts. In March 2010, Solvay launched Veregen® (10 % ointment) in Germany. Sativex®, a mixture of dronabinol 1 and cannabidol 2 obtained from the cannabis plant, is the world's first pharmaceutical prescription medicine that was launched in Canada (April 2005) and was later approved by Health Canada (August 2007) as adjunctive analgesic for severe pain in advanced cancer patients [11]. Sativex® has been recommended by FDA to enter directly in Phase III trials and as of November 2009, GW Pharmaceuticals have completed the recruitment for Phase II/III trial against cancer pain. In March 2010, GW Pharmaceuticals provided an update on the progress of regulatory submission for Sativex® oromucosal spray for the treatment of the symptoms of spasticity due to multiple sclerosis. Fumagillin (Flisint®, Sanofi-Aventis) 3, an endothelial cell proliferation inhibitor isolated from Aspergillus fumigatus [12], was approved in France in September 2005 for the treatment of intestinal microsporidiosis. Fumagillin 3 can also block the blood vessel formation through binding to methionine aminopeptidase II and is under clinical investigtions as an angiogenesis inhibitor for the treatment of cancer. Among carbapenem-type β-lactams, doripenem (Finibax®, DoribaxTM) 4 is an ultra-broad spectrum injectable antibiotic launched in Japan (2005) by Shionogi & Co. while ertapenem, a NP derived compound based on structure of thienamycin 5 is being marketed by Merck as InvanzTM. In October 2007, Johnson & Johnson (J&J) obtained formal FDA approval for use of 4 in intraabdominal and urinary tract infections. Tigecycline (Tygacil®) 6, a glycylcycline antibiotic structurally similar to teracycline 7, was approved by FDA in June 2005 against intra-abdominal

Bhuwan B. Mishra & Vinod K. Tiwari

and complicated skin and skin structure infections (SSSIs). Tigecycline 6 was developed by Francis Tally and contains a centralised four-ring carbocyclic skeleton substituted at the D-9 position confering broad spectrum activity. Tigecycline 6 inhibits protein translation by connecting with 30S ribosome and hinders amino-acyl tRNA molecules coming to A site ribosomal subunit [13]. As of May 2006, the 6 has been approved in Europe and later a supplementary NDA for community-acquired pneumonia (CAP) was submitted to the FDA in October 2007. Ziconotide (Prialt®) 8, a synthetic-conotoxin and calcium channel blocker, isolated from Conus magus [14], causes pain relief by inhibiting pro-nociceptive neurochemical releases in the brain and spinal cord [15]. In December 2004, the FDA approved 8 when delivered as infusions into the cerebrospinal fluid using intrathecal pump system. In 2005, Elan launched 8 in US and Europe while rights for marketing 8 in Europe were obtained by Eisai in March 2006. CH3

CH3

H2C H3C O

H3C

CH3 HO

CH3

1 CH3

H CH3

CH3

H3C

S N

OCH3

H N

O O

HO O

N H

NH2 S O

CO2H

3 HO H

4 H3C

CH3

H3C

NH2

CH3 N

H OH

H3C

S N

H N

H3C

O O

H3C

CH3

N H CH3

HO OH

CONH2 O

6 N

CH3

H2N-CKGKGAKCSRLMYDCCTGSCRSGKC-CONH2 CONH2

OH OH

Natural products in drug discovery

Zotarolimus 9, a derivative of sirolimus 10, is an active principle of EndeavorTM stent that is being used as anti-proliferative agent by Medtronic [16,17]. In July 2005 EndeavorTM was approved by European comission for the sale while FDA approved it in February 2008 for the treatment of coronary artery disease. Anidulafungin 11 (EraxisTM in US, EcaltaTM in Europe), a semi-synthetic derivative of echinocandin B 12, was originally developed by Eli Lilly against invasive and oesophageal candidiasis and candidemia. Anidulafungin 11 was later licensed to Vicuron Pharmaceuticals, which was purchased by Pfizer in June 2005. Pfizer gained FDA approval for EraxisTM in the US (February 2006) and EcaltaTM in Europe (July 2007). Exenatide 13 (Byetta®), a 39 amino acid peptide isolated from the oral secretions of Heloderma suspectum (Gila monster) [18], mimics the antidiabetic or glucose-lowering properties of incretins. In April 2005, Eli Lilly obtained FDA approval for 13 while EMEA in November 2006 approved it to Amylin Pharmaceuticals for use in type 2 diabetes mellitus [19]. Amylin Pharmaceuticals, Eli Lilly and Alkermes submitted a NDA in May 2009 for subcutaneous dosing of 13 once weekly that was accepted in July 2009 by the FDA. Attention-Deficit Hyperactivity Disorder (ADHD), a neurodevelopmental disorder in which dopaminergic and noradrenergic neurotransmission are supposed to be dysregulated, is primarily characterized by the co-existence of attentional problems and hyperactivity. Despite abuse potentials methylphenidate and amphetamines were used for Attention-Deficit Hyperactivity Disorder (ADHD) management since many years. 14 Lisdexamfetamine (VyvanseTM, NRP104) consisting of dextroamphetamine coupled with L-lysine was designed by New River Pharmaceuticals produces effects similar to placebo on intravenous administration, however on oral administration it converts into D-amphetamine 15 in the gastrointestinal (GI) tract [20]. In February 2007, FDA approved 14 to treat ADHD. Pleuromutilin 16, a fungal metabolite inhibiting protein synthesis in bacteria [21], is the lead compound of retapamulin (SB-275833) 17 developed by GlaxoSmithKline. In June 2007, EMEA approved an ointment containing 1% retapamulin 17 called AltabaxTM in the US and AltargoTM in Europe for topical treatment of impetigo caused by Staphylococcus aureus or Streptococcus pyogenes. Temsirolimus (Torisel®, CCI-779) 18, a derivative of 10 and mTOR inhibitor [22] developed by Wyeth in various Phase III trials was approved in May 2007 by the FDA and November 2007 by the EMEA for the treatment of renal cell carcinoma (RCC) [23].

Bhuwan B. Mishra & Vinod K. Tiwari

Trabectedin (Yondelis®, ecteinascidin-743, ET-743) 19, an alkaloid obtained from Ecteinascidia turbinate [24], is a DNA minor groove binder that inhibits cell proliferation by disrupting the cell cycle. Trabectedin 19 is sold by Zeltia and J&J against advanced soft tissue sarcoma (STS). In September 2007, the EMEA has approved 19 for use against ovarian cancer and STS. In November 2009, Yondelis® received its second marketing authorisation from the European Commission against relapsed platinumsensitive ovarian cancer when administered in combination with DOXIL®/ Caelyx®. R O CH3

CH3 O

O CH3

O N

CH3

O CH3 CH3

CH3

9R=

N N

O HO

10 R =

O H3C

CH3

O HO

11 R =

NH NH

H3C

N HN

H3C

CH3 H3C

H N

O OH O

12 R = H3C

Natural products in drug discovery

13 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS NH2 H N

NH2 NH2

CH3

14 O

CH2

CH3

CH3 OH

OH O

CH3

O R

CH3

H O CH3 O

H3C H3C O

CH3 CH3

O N

16 R = OH 17 R =

CH3

O CH3

CH3

O CH3

NCH3 O O O CH3 H3C

HO H3C

OCH3

CH3

H3C S

CH3

O O

OH O

H3CO

H3C

OH H3C CH3 H3C

N X

CH3 O

CH3

20 X = NH 21 X = O

Ixabepilone (IxempraTM, BMS-247550) 20, a semi-synthetic derivative of epothilone B 21 produced by Sorangium cellulosum [25], was developed by Bristol-Myers Squibb (BMS) as an anticancer drug that binds to β-tubulin and suppresses the dynamics of microtubule. In October 2007, BMS gained FDA approval for 20 as a monotherapy and in combination with Xeloda® for the treatment of breast cancer, resisting standard therapy [26]. Methylnaltrexone (MOA-728, Relistor® by Wyeth) 22, a derivative of naltrexone 23 that blocks peripheral opioid receptors activated by opioids and

Bhuwan B. Mishra & Vinod K. Tiwari

thus is significant in management of alcohol and opioid dependence [27]. Wyeth and Progenics in May 2007 filed an NDA for subcutaneous doses of 22 against opioid induced constipation (OIC) and other pain indications that was approved in April 2008 by Health Canada and the FDA. As of May 2009 an oral formulation of 22 is under Phase II trials against OIC in chronic pain. HO

CH3 O

H3C

O N HO

O CH3

HO CH3

CH3

O CH3

CH3

O CH3

O HO

N Br

HO O

CH3

H3C

CH3

Everolimus (LuveniqTM or LX211) 24, an mTOR inhibiting derivative of 10 is marketed as immunosuppressant by Novartis under ZortressTM (USA) and CerticanTM (Europe and other countries) in transplantation medicine, and Afinitor® for use in advanced renal cell carcinoma (RCC). CerticanTM was approved in 2004 as immunosuppressant while in March 2009 the FDA has approved 24 against advanced RCC after failure of Sutent® (sunitinib) or Nexavar® (sorafenib). OH R1 HN H3C

O CH3

OH O

O OH

HO Cl O

H N

H N N H

N H

CH3 CH3

O O

CH3

NH2 O OH

H N

CH3

R1 = HO

25 R2 =

N H

26 R1 = H, R2 = H

PO3H2

Natural products in drug discovery

Telavancin (VibativTM, TD-6424) 25, a semisynthetic derivative of vancomycin 26 that inhibits bacterial growth through binding to D-Ala-D-Ala [28], was developed by Theravance in partnership with Astellas for use against Gram-positive cSSSIs and MRSA that was approved in September 2009 by the FDA. Theravance has also submitted telavancin 25 to the FDA in a second indication against nosocomial pneumonia or hospital aquired pneumonia (HAP). In November 2009, the FDA released a complete response letter to Theravance for telavancin 25 NDA against nosocomial pneumonia. Romidepsin (depsipeptide, FK228, FR901228, Istodax®) 27 extracted from the bacteria Chromobacterium violaceum, is a histone deacetylase (HDAC) inhibitor [29] developed by Gloucester Pharmaceuticals under National Cancer Institute (NCI) sponsorship for treatment of cutaneous and peripheral T-cell lymphoma (TCL). In November 2009, the FDA approved 27 to use in the treatment of selective cutaneous TCL patients previously treted with minimum of one prior systemic therapy. In January 2010, Celgene completed the acquisition of Gloucester Pharmaceuticals. Capsaicin (Qutenza®) 28, isolated from chili peppers of genus Capsicum [30], produces burning sensation on contact to tissues though binding to vanilloid receptor subtype 1 (VR 1) [31]. In November 2009, the FDA approved Qutenza® (a transdermal 8% patch of 28) to use in treatment of neuropathic pain combined with postherpetic neuralgia. In April 2010, NeurogesX launched Qutenza® in US. Aztreonam lysine (CaystonTM) 29 is an inhaled lysine salt formulation [32] that was evaluated by Gilead in various Phase III trials against cystic fibrosis (CF) patients infected with the Gramnegative bacteria Pseudomonas aeruginosa. In February 2010, the FDA approved 29 against CF patients. O H3CO

CH3

H N

H3C

CH3

S O

S O O

H3C

CH3 CO2H O

H N

N O

CH3

H2N H3C

CH3

O HN

CH3

N H

CH3

O S

Bhuwan B. Mishra & Vinod K. Tiwari

4. Infectious diseases 4.1. Antibacterial NP-derived drugs have played their crucial role in anti-infective drug discovery and the majorities of antibacterial drugs currently in clinical use are NPs or were designed using NP templates. Despite having complex structure the development of a NP to an antibacterial drug entirely depends on its ability to penetrate bacterial cell membranes. The success of penicillin encouraged the discovery of other compounds from natural sources against bacterial infections and as a result nearly all novel classes of antibiotics belong to NP sourced scaffolds [33]. Ceftobiprole medocaril (BAL-5788) 30, a cephalosporin antibiotic with excellent activity against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, Pseudomonas aeruginosa, and Enterococci [34], was filed for regulatory approval in the US and Europe in July 2007 by Basilea Pharmaceutica and J&J affiliated Cilag GmbH International to use in the treatment of cSSSIs. In November 2008, the approval of 30 was declined by the FDA with recommendation of two new studies to access safety and efficacy in treatment of cSSSIs. Additionly, various Phase III trials are underway for HAP/CAP. Ceftaroline acetate (PPI0903, TAK-599) 31, discovered by Takeda and licensed to Cerexa, shows efficacy against the penicillin-resistant S. pneumoniae and is under Phase II development by Forest Laboratories to use in the treatment of cSSSIs and CAP [35]. Tebipenem pivoxil (ME-1211, L-084) 32, an oral carbapenem antibiotic is under Phase III clinical development by Meiji Seika in Japan for treatment of otolaryngological/respiratory infections. Tomopenem (CS-023, RO4908463, R1558) 33 [36], by Daiichi Sankyo for treatment of common nosocomial infections and PZ601 (SM-216601, Protez) 34 [37], against MRSA and Pseudomonas aeruginosa, are currently in Phase II trials. ME1036 (CP5609) 35, a DHP-1-stable parenteral carbapenem having excellent in vitro activity against multidrug-resistant (MDR) staphylococci and Enterococcus faecalis was licensed by Cerexa and Forest Laboratories from Meiji Seika Kaisha. ME1036 35 is currently under Phase I evaluation. Likewise, sulopenem (CP-70429) 36, is being evaluated by Pfizer in various Phase I trials [38]. Faropenem daloxate (SUN-208, BAY-56-6824) 37 is a penem-type β-lactam licensed to Replidyne by Daiichi Suntory Pharma for marketing in conjunction with Forest Pharmaceuticals [39]. In December 2005, Replidyne submitted an NDA to the FDA for use of 37 in the treatment of bacterial

Natural products in drug discovery

sinusitis (BS), chronic bronchitis (CB), CAP and uncomplicated (SSSIs). In response to Replidyne’s NDA, the FDA in March 2007 agreed for Phase III placebo-controlled trials of 37, one each in BS and CB along with two non-inferiority CAP trials. However, these additional trials have certainly delayed the launch of drug. OH

N H2N

H N

N S N

N O HO

CH3

H3C

N O O

OH H H

CH3

H3C H3C CH3

S N

CH3 CH3 N OH HO

OH H H

O H

H N

OH H H

H3C

H N

N H

N H CH3

O O HN

36 S

Cl H N

H N

O OH OH

OH OH

O H N

38 R = 39 R = OH

Cl HO

OH O

N H

N O

N H

H3C

OH CO2H

CH3

H3C

CH3

NH2

H3C

NH2 O NH

N CH3

O N

CH3

H3C

34 OH H H

CH3

H3C

CH3

N HO

CH3

CH3 N

CH3

N H

CH3

Bhuwan B. Mishra & Vinod K. Tiwari

Dalbavancin (Zeven®, BI-397) 38, a semi-synthetic derivative of the teicoplanin analogue A40926 39, was discovered by Biosearch Italia and being developed by Pfizer for the treatment of cSSSIs [40]. In February 2005, Vicuron Pharamaceutical (now a part of Pfizer) filed an NDA for 38 to use in the treatment of patients suffering from cSSSIs. In response, the FDA released an approval letter in December 2007, however, as of September 2008 Pfizer have withdrawn all the marketing applications of 38 for running another Phase III trial. Oritavancin (NuvocidTM, LY-333328) 40, a chloroeremomycin 41 derivative inhibiting cell-wall synthesis, was discovered and developed by Eli Lilly and acquired by InterMune in 2001 and later tansferred to Targanta Therapeutics in 2005. In February 2008, Targanta submitted an NDA for 40 to the FDA that was not approved due to insufficient data. Aditionally, a MAA was submitted by Targanta for 40 to EMEA that was accepted for review in June 2008. TD-1792 42, a vancomycin-cephalosporin heterodimer successfully evaluated by Theravance in Phase II trials against cSSSIs including MRSA, has been designed to target 2 key targets in bacterial cell wall synthesis. In July 2007, Theravance disclosed to meet primary and secondary endpoints of non-inferiority trial compared to vancomycin 26. Ramoplanin factor A2 (known as ‘‘ramoplanin’’) 43, the major component of the lipopeptide antibiotic drugs obtained from Actinoplanes ATCC 33076 [41], inhibits cell wall synthesis in bacteria by forming U-shaped structures that are able to bind and capture Lipid II (C35-MurNAcpeptide-GlcNAc), a specific intermediate in membrane formation [42]. Oscient Pharmaceuticals hold the North American right and are evaluating orally active doses of 43 in Phase II trials against Clostridium difficile associated GI tract infections [43]. NXL-103 (XRP2868), an orally available mixture (70:30) of flopristin (RPR132552A, streptogramin A-type) 44 and linopristin (RPR202698, streptogramin B-type) 45 that inhibit bacterial protein synthesis through the synergistic binding to different sites on the peptidyltransferase domain of the 50S ribosomal subunit [44], was discovered by Sanofi-Aventis [45]. Novexel in October 2008 announced for positive Phase II trials of NXL-103 against CAP and cSSSIs including MRSA. Friulimicin B 46, a lipopeptide antibiotic produced by Actinoplanes friuliensis HAG 010964 [46], exerts activity through complex formation with bactoprenol-phosphate, resulting in inhibition of peptidoglycan and teichoic acid biosynthesis in bacteria [47], is under Phase I clinical development (July 2007) by MerLion Pharmaceuticals. Structure of friulimicin B 46 was confirmed after the crystal structure of amphomycin tsushimycin (A-1437 B) 47, an aspartic acid analogue of 46 was published in late 2005.

Natural products in drug discovery

15 R HN HO H3C

O CH3

H2N HO H3C

OH O

O CH3

Cl H N

OH OH

O O

N H

O H N

N H

H N

N H

CH3

NH2

CH3 CH3

OH OH

40 R =

41 R = H OH H3C

NH2 O CH3 O

OH OH

O OH

O O

H N

N H

H N

N H

H N

N H

CH3

NH2 NH

H N

N H2N S

OH OH

CH3 CH3

O CO2H

OH H2N

H3C

O H N

H3C

H N

N H

O H N

N H O

N H NH

N H O

NH2 O O

H2N

H3C O

HN H N

O H N

N H

O H N

N H

NH2

N H

CH3

OH OH

O O O OH

CH3

CH3 CH3

HO HO HO

H3C

Bhuwan B. Mishra & Vinod K. Tiwari CH3

H3C N O

N O F

CH3

N O

O O

CH3

H N

H3C

CH3

H N

N H N

N H

45 COOH

CH3 O O

O CH3

H3C

O H3C

NH H3C N O

N H COOH

HOOC CH3 O N H H3C

H N

O O

O NH

NH2

46 R = NH2 47 R = OH H3C

CH3

Moli1901 (duramycin, 2262U90) 48, obtained from Streptomyces cinnamoneum [48], inhances the chloride transport and increases fluid secretion in vitro, thus finds significance for the treatment of CF [49]. Moli1901 48 is currently under clinical development by AOP Orphan in colaboration with Lantibio in Europe. In March 2007, Lantibio announced the positive results of Phase II trial of aerosolized 48 in adolescents and adults suffering from CF. An ophthalmic solution of 48 for treatment of dry eye syndrome is also under Phase II trials by Lantibio. Omiganan 49, originally purified from neutrophils of bovine, is an indolicidin 50 derivative that can interact with the bacterial cytoplasmic membrane and has been found significant against antibiotic-resistant and sensitive bacterial infections [50]. Omiganan 49 was developed by MIGENIX and later licensed to Cadence Pharmaceuticals and Cutanea Life Sciences for catheter-related infections (coded OmigardTM, CPI-226, MBI-226) and dermatological diseases (coded as CLS001, MX-594AN), respectively. Cadence Pharmaceuticals are currently evaluating a gel-based formulation of 49 in Phase III trials while another phase III trials for treatment of rosacea, a chronic inflammatory skin disorder are underway.

Natural products in drug discovery

17 NH

H-Ala-Lys-Gln-Ala-Ala-Ala-Phe-Gly-Pro-Phe-Abu-Phe-Val-Ala-HOAsp-Gly-Asn-Abu-LysOH S S S

48 ILRWPWWPWRRK-NH2

ILPWKWPWWPWRR-NH2

Erythromycin 51, macrolide antibiotic produced by actinomycetes, exerts antibacterial activity through inhibition of protein synthesis by binding to peptidyltransferase site of 50S subunit [51]. Among other derivatives, cethromycin 52, EP-420 53 and BAL-19403 54 are currently in clinical development. Cethromycin (ABT-773) 52 was discovered by Abbott Laboratories and later acquired by Advanced Life Sciences to use in the treatment of CAP and anthrax [52]. Advanced Life Sciences in October 2008 submitted a NDA to use 52 in the treatment of mild-to-moderate CAP which was accepted by FDA in December 2008. The cethromycin 52 (RestanzaTM) has demostrated clinically and statistically significant survival rate in placebo-controlled non-human primate studies with anthrax, plague and tularemia. In September 2009, the FDA has given orphan drug designation to 52 for the treatment of plague and tularemia. Likewise, EP-420 (EP-013420) 53, a bridged bicyclic derivative of 51 is currently under Phase II clinical development by Enanta and Shionogi for treatment of CAP [53]. BAL19403 54, a macrolide antibiotic significant against clinical isolates of Propionibacterium acnes with mutations in the 2057 to 2059 region of 23S rRNA conferring resistance to 51, is under clinical development by Basilea for the treatment of acne [54]. Telithromycin (Ketek®) 55 is the first approved ketolide developed by Sanofi-Aventis that received approval from the European Commission (July 2001) and the FDA (in 2004) for treatment of respiratory infections. Telithromycin 55 displays bactericidal activity by blocking the progression of the growing polypeptide chain through binding with the 50S subunit of ribosome. Tiacumicin B (OPT-80, PAR-101) 56, a macrolactone isolated by Abbott [55] from actinomyces, inhibits RNA synthesis and is under phase III clinical development by Optimer Pharmaceuticals for the treatment of Clostridium difficile-associated diarrhea (CDAD) [56]. PTK-0796 (MK-2764) 57 is an aminomethylcycline inhibiting protein synthesis in bacteria, was discovered and evaluated by Paratek in Phase II trials for the treatment of common hospital infections. PTK-0796 57 was in-licensed

Bhuwan B. Mishra & Vinod K. Tiwari O

H3C

CH3

H3C H3C

OH OH

CH3 HO O

H3C O O

O H3C N O

CH3

H3C N

CH3 HO O

CH3 H3C O

H3C

CH3 OH

CH3

CH3 O

CH3

OCH3

H3C H N

CH3 CH3

CH3

52 N

CH3

H3C

H3C H3C

H3C

CH3 HO O

CH3 OCH3 H3C N CH3 CH3 HO CH3 O O

H3C O

H C S 3

CH3 N

O N

CH3 H3C O

H3C

CH3 CH3

OCH3

O CH3

CH3

53 H3C

H3C

HO O

O H3C

CH3

N H CH3 H3C O

O H3C O

O O

OCH3

H3C H3C

CH3

N CH3

OHO

CH3

H3C OH O

N N

CH3 OH CH3

H3C

CH3 H3C

CH3 OCH3 O

OH O

O O

OH Cl

O H3C

CH3

OH CH3 Cl

CH3

by Novartis form Paratek for collaborative Phase III clinical development. In October 2009, Novartis gained exclusive marketing rights of 57 to use in the treatment of MRSA, MDR Streptococcus pneumoniae and vancomycinresistant enterococci. Eritoran (E5564) 58, a second-generation lipid A antagonist [57] designed by Eisai from Rs-DPLA 59 isolated from Rhodopseudomonas sphaeroides [58], inhibits endotoxin response through antagonism of the Toll-like receptor 4 (TLR4) [59,60].

Natural products in drug discovery

CBR-2092 60, a hybrid antibiotic inhibiting RNA and DNA synthesis is being developed by Cumbre Pharmaceuticals for treatment of gram-positive cocci infections. CBR-2092 60 is supposed to exert antimicrobial activity through combined effects on RNA polymerase, DNA topoisomerase IV and DNA gyrase. Currently, CBR-2092 60 is in Phase IIa trial by Cumbre for treatment of infections caused by gram-positive cocci [61]. H3C CH3

H3C

CH3

H3C H

CH3 OH

H N

H3C

CONH2

OH OH O

57 OCH3 H2O3PO

O O

HO HN

O H2O3PO

O O

HN OPO3H2 O O

H3CO

CH3

O HN

HO O

CH3

O O HN OPO3H2 O O

CH3 H3C H3C

H3C CH3

CH3

58 H3C

59 O

CH3 CH3 CH3

O H3CO

OH CH3

O OH

H3C

CH3 NH

O OH

O CH3

N N CH3

CO2H

N CH3

4.2. Antifungal Invasive fungal infections – infections of the bloodstream and organs within the body (e.g. meningitis, pneumonia, peritonitis) – are important causes of morbidity and mortality in liver, pancreas, heart, kidney and lung (i.e. solid organ) transplant recipients [62]. Fungi are eukaryotes and, despite

Bhuwan B. Mishra & Vinod K. Tiwari

the presence of a cell wall, fungi are more similar to mammalian cells on a cellular level than to bacteria, making the treatment of mycotic infections difficult [63]. Only 2 NP-derived compounds, aminocandin 61 and SPK-843 62 are undergoing clinical evaluation. Due to lack of biological target, 1,3-βD-glucan synthesis in human, echinocandin derivatives have been considered significant against refractory aspergillosis and invasive infections by Candida species [64]. Among other semi-synthetic echinocandins, caspofungin (launch 2001, Cancidas®, Merck), micafungin (launch 2002, Mycamine®/Funguard®, Astellas) and anidulafungin 11 (launch 2006, EraxisTM/EcaltaTM, Pfizer) have been approved. Deoxymulundocandin 63, isolated from Aspergillus sydowii [65], is the lead compound of aminocandin (NXL-201, IP960, HMR-3270) 61 and exhibit excellent activity against Candida albicans and C. tropicalis by destabilizing the fungal cell membrane. SPK-843 62, a semi-synthetic derivative of patrician-A 64, is under clinical development by Dutch company APARTS BV [66] that has acquired world wide rights for the development of 62.

4.3. Antiparasitic The use of medicinal plants against parasitic diseases has been traced to ancient times i.e. bark of Cinchona calisaya and Strychnos pseudoquina, root and leaves of Deianira erubescens, bark of Remijia ferruginea [67]. Artemisinin (Artemotil®) 65, obtained from traditional Chinese medicine Artemisia annua, was approved in the year 2000 for the treatment of chloroquine-resistant Plasmodium falciparum malaria and cerebral malaria. The World Health Organization has strongly discouraged the use of 65 as a monotherapy since malarial parasites are developing resistance to the drug. However, combination therapies that include 65 are the preferred treatment for malaria and are both effective and well tolerated in patients. Artemotil® is currently used only as a second line drug in severe cases of malaria and is also increasingly being used against vivax malaria. As of May 2009, arterolane (RBx11160, OZ-277) 66, a trioxolane modelled on artemisinin 65 pharmacophore, is under Phase III clinical development for the treatment of malaria by Ranbaxy in combination with piperaquine [68]. Paromomycin 67 (HumatinTM, King Pharmaceuticals), an orphan drug extracted from Streptomyces krestomuceticus [69], was approved in September 2006 by Drug-Controller General of India for the treatment of patients suffering from visceral leishmaniasis (VL). Paromomycin 67 was developed by the Institute for OneWorld Health [70] and is an off-patent antibiotic marketed in the US to treat intestinal parasites.

Natural products in drug discovery

H3C

NH2

H N HO

21 OH O

O HO

NH N H

NH H3C

O OH

O HO O

CH3

H N

NH O

CH3 N

H N

H3C OH

H3C

OH H3C HO

H3C

H N OH

OH HO O

H3C

H N

62 R1 =

CH3 R2 =

CH3 N

CH3 OH

H3C

CH3

CH3 OH

64 R1 = OH, R2 = H H H3C

O O

HO HO

CH3

NH O

NH2 O

CH3 CH3 NH2

O H2N HO

NH2

O OH

CH3 O

H2N

NH2 OH

4.4. Antiviral Virus is a small infectious agent that can replicate only inside the living cells of organisms bringing most common (i.e. cold, influenza, chickenpox and cold sores) to greatest human health risk (i.e. ebola, AIDS, avian influenza and SARS). Researches over last 25 years have resulted in the identification of many natural product templates significant to antiviral drug discovery, however fewer are in clinical investigation.

Bhuwan B. Mishra & Vinod K. Tiwari

Betulinic acid 68, a topoisomerase I inhibitor isolated from bark of Betula pubescens [71], is currently in Phase I clinical development. Bevirimat (PA-457) 69, obtained from Syzygium claviflorum, was evaluated by Panacos in Phase IIb trial for development as combination therapy with other standard antiviral drugs. Bevirimat 69 inhibits the final step of the HIV Gag protein processing and thus blocks HIV maturation [72]. In January 2009, Myriad Genetics announced for the acquisition of all rights from Panacos for 69. Ribavarin 70, a NP-derived compound structurally similar to pyrazomycin and showdomycin, was marketed as ‘Rebetol’ until 2005 by Schering Plough with Valeant Pharmaceuticals in the US. Valeant Pharmaceuticals are developing taribavirin (Viramidine®, ribamidine) 71, a liver-targeting prodrug of ribavirin 70 [73], is in various Phase II/III trials for the treatment of chronic hepatitis C virus (HCV). In 2006, 71 failed to meet the non-inferiority efficacy endpoints in Phase III trials by Valeant. In 2007, Valeant initiated another Phase IIb trial for 71 with higher doses and reported the final results in June 2009 against HCV. MBI-3253 (celgosivir, 6-O-butanoylcastanospermine) 72, a glucosidase inhibitor and semi-synthetic derivative of indolizine alkaloid castanospermine 73 isolated from Castanospermum australe seeds [74], is an investigational antiviral drug under clinical development by MIGENIX. As of January 2009, MIGENIX has completed Phase II clinical studies of 72 as a ‘‘triple combination’’ (with peginterferon α-2b and ribavirin 70) and a ‘‘double combination’’ (with peginterferon α-2b) in HCV patients. After discontinuation of exclusive option agreement with United Therapeutics Corporation (UTC) in April 2009, MIGENIX are seeking other strategic options for further development of 72. Cyclosporin 74, a cyclophilin inhibitor obtained from Beauveria nivea, exerts significant antiviral activity. However, due to calcineurin-related and immunosuppressive side effects development of 74 as antiviral drug is not possible [75]. NIM 811 (SDZ NIM 811, cyclosporin 29, MeIle4-cyclosporin) 75, discovered by Sandoz (now Novartis) with 1700 times less immunosuppressive activity than cyclosporin 74 [76], was evaluated in Phase I trial for anti-HIV and HCV activity. Likewise, debio-025 (UNIL025, MeAla3EtVal4-cyclosporin) 76, a cyclophilin inhibitor with 7000 times less immunosuppressive activity than 74, is being evaluated by Debiopharm in various phase IIb trials for the treatment of HCV [77,78]. In February 2010, Novartis in-licensed the exclusive rights to develop and market 76, as potential first-in-class antiviral agent except in Japan. 4-Methylumbelliferone (Heparvit®) 77 is a naturally occurring coumarin that is in Phase II development by MTmedical Institute of Health and BioMonde for the treatment of HBV and HCV. 1,5-DCQA (1,5-di-O-

Natural products in drug discovery

caffeoylquinic acid) 78, a HIV-1 integrase inhibitor extracted from Inula Britannic, is under human clinical trials by Chinese Academy of Military Medical Sciences for treatment of HIV/AIDS and hepatitis B [79]. WAP-8294A2 (JA-002) 79, produced by the Gram-negative Lysobacter species exerts antibacterial activity by interacting selectivly to membrane phospholipids and causes sever damage to bacterial membrane [80]. The aRigen Pharmaceuticals are evaluating injectible, gel and cream of 79 in various Phase I/II trials to treat MRSA and acne. In August 2009, New Energy and Industrial Technology Development Organization (NEDO), Japan has decided for funding two-thirds R&D costs to aRigen Pharmaceuticals until February 2011 for development of 79 as first-line anti-MRSA product candidate. CH2 H3C

R H

N CO2H

CH3 CH3 H H RO H3C

CO2H

CH3 HO CH3 O

H3C

70 R = O

72 R =

71 R = NH

73 R = H

CH3

O H3C O N CH3

CH3

CH3 O H H N

CH3 O CH3 CH3 O H N N H CH3 O

CH3

74 R =

N R N CH3 O O CH3CH3 O CH3 H3C CH3 H3C O CH3 O CH3 H N N N N O N H H O H3C CH3 O CH3 H3C N

HO CH3 CH3 O

O CH3

CH3 O H H N

CH3

H3C

CH3

O CH3

OHOH

69 R =

N HO

H CH3

CH3

68 R = H OH C CH 3 3

H3C

NH2

CH3 CH3 CH3

75 R =

CH3

HO CH3 CH3

CH3 N

O CH3 CH3

CH3

CH3CH3 O H3C CH3 CH3 N

N H

HO O HO

HO2C

HO O

OH OH

Bhuwan B. Mishra & Vinod K. Tiwari NH2

HO H N O H N HN

O O

NH2

H3C

O H N

NH2

H3C

N H

H N H

N H H3C OH H3C

H N

O CH3 HO

CH3

N O

O N H O

CH3 O

NH OH

NH2

5. Neurological diseases Historically, the alkaloids like morphine 80 isolated from Papaver somniferum and physostigmine 81 extracted from Physostigma venenosum, were used to treat sever pain and diseases of central nervous system (CNS). (+)-huperzine A 82, a sesquiterpene alkaloid and acetylcholinesterase (AChE) inhibitor extracted from Huperzia serrata [81], is being evaluated by Chinese scientists against Alzheimer’s disease. The National Institute on Aging (NIA) is evaluating orally administered formulation of 82 in Phase II trials against Alzheimer's disease [82]. Morphine-6-glucuronide (M6G) 83, produced by metabolism of artemisone (BAY 44-9585) 84 (obtained through semi-synthesis from artemisinin 63) in human body, was evaluated successfully by CeNeS Pharmaceuticals as significant analgesic in Phase III trials in Europe. PAION in June 2008 acquired CeNeS Pharmaceuticals and later in November 2008 disclosed for completion of two Phase III trials. A spicamycin derivative KRN-5500 85, obtained from Streptomyces alanosinicus [83], was evaluated by DARA BioSciences in Phase I trials against neuropathic pain. DARA BioSciences are currently running Phase IIa trials of 85 given intravenously (IV) to cancer patients suffering from neuropathic pain [84]. Debio 9902 (ZT-1) 86, synthesized by Shanghai Institute of Material Medica, is a prodrug of 82 licensed to Debiopharm. Debiopharm in June 2007 announced the positive results of a Phase IIa trial of 86 against mild Alzheimer’s disease. As of October 2008, Debiopharm have started tablet

Natural products in drug discovery

formulation bridging study of 86 as Investigational New Drug (IND) to treat Alzheimer’s patients. Lobeline 87, a VMAT2 ligand [85] reducing the methamphetamine induced dopamine release, is a significant tobacco smoking cessation agent occurring in Hippobroma longiflora [86]. Lobeline 87 is being evaluated by Yaupon Therapeutics and NIH as a dopamine modulating agent under Phase II trials against ADHD and methamphetamine addiction. Anabaseine 88, isolated from marine worms of the phylum Rhynchocoela [87], stimulates the neuronal nicotinic receptors thus has been considered significant in the treatment of Alzheimer’s disease as Alzheimer’s brain loses many of its nicotinic receptors by the time of death [88]. The 3(2,4-dimethoxybenzylidene)-anabaseine (DMXBA; also called GTS-21) 89, a synthetic derivative of 88, was evaluated against Alzheimer’s disease in a sponsored research by Taiho Pharmaceutical to Kem’s University of Florida. HO

O H

CH3

H3C

H N

O N O

CH3

N H

CH3

80 R = H

81 HO

83 R =

OH OH CO2H CH3

H3C

H N

H3C

H H

N O O

O O

H3C H2N

CH3

H3C

N H

HO H N

O HO

N H OH

NH N

Bhuwan B. Mishra & Vinod K. Tiwari CH3 O

OCH3

H N H3C

CH3

O N

OCH3

HO N H3CO

The University of Florida licensed 89 to Osprey Pharmaceuticals whose assets were purchased by CoMentis (previously Athenagen) in April 2006. CoMentis are currently assessing 89 in various Phase I/II trials for safety assessment and cognitive improvement in ADHD patients. Tetrodotoxin (TectinTM, Wex Pharmaceuticals) 90, extracted from the puffer fish [89], blocks the action potentials in nerves through binding to sodium channels in cell membrane [90]. Wex are evaluating 90 in colaboratin with Chinese medical institute against cancer pain and management of opiate withdrawal symptoms in Phase III and I trials, respectively. Also a Phase IIa trial of 90 against neuropathic pain caused by cancer chemotherapy is underway by Wex Pharmaceuticals. Capsaicin 28 and related compounds (called as capsaicinoids) are produced by chili peppers as irritants against certain herbivores and fungi. Among capsaicinoids, Xen-2174 91, obtained from venom of Conus marmoreus targeting norepinephrine transporter (NET) was discovered by researchers at the University of Queensland. Xenome are associated with Phase II development of 91 against acute post-operative and chronic pain in cancer patients resistant to morphine and hydromorphone. Anesiva are evaluating capsaicin 28 (coded 4975, ALGRX 4975, AdleaTM) in various clinical trials against pain indications such as severe post-surgical pain, posttraumatic neuropathic pain and musculoskeletal diseases [91]. Anesiva in December 2008, disclosed to meet primary end point in a phase III trial of 28 against acute pain following orthopedic surgery. Winston Laboratories are associated with Phase III trials of civamide (cis-capsaicin, zucapsaicin, WL-1001) to treat episodic cluster headache and knee osteoarthritis. Winston in October 2008 filed a NDS to Canada for Civanex® (civamide 0.075%) to use against osteoarthritis pain. In February 2009, an orphan drug designation to Civanex® was given by FDA with NON release to Winston Pharmaceuticals in October 2009.

Natural products in drug discovery

Phlorizin 92, a flavonoid that belongs to the group of dihydrochalcones obtained from bark of pear (Pyrus communis), apple, cherry and other fruit trees (family-Rosaceae), is a sodium glucose co-transporters (SGLTs) inhibitor that lowers glucose plasma level and improves insulin resistance [92] but has poor intestinal absorption and become inactive by lactasephlorizin hydrolase. Dapagliflozin (BMS-512148) 93, a 92 derivative that selectivly inhibits SGLT2, is under clinical development by Bristol-Myers Squibb (BMS) in collaboration with AstraZeneca for the treatment of 2 diabetes. In October 2009, the BMS announced the positive results of Phase III placebo controlled trial of 93. O HO H2N

O N HN H HO

NGVCCGYKLCHOC OH

Resveratrol 94, a triphenolic stilbene occurring in many plants is significant against clinical indications such as cancer, ischemic injuries and cardiovascular disease [93]. Resveratrol 94 is an agonist of Saccharomyces cerevisiae silent information regulator (Sir2) protein, a class III histone deactylase whose presence causes extention of lifespan in S. cerevisiae, Caenorhabditis elegans and Drosophila melanogaster [94]. Italian scientists in 2006 observed 56% increase in median life span of Nothobranchius furzeri [95], a fish when supplemeted with 94. SRT-501, a formulation of 94 by Sirtris Pharmaceuticals, acts by increasing mitochondrial activity and is under clinical investigations against diabetes and obesity. Sirtris has announced the positive results of Phase IIa trial in which oral doses of 1.25 or 2.5 grams of SRT501 was found safe at twice daily dosing for 28 days in type 2 diabetes. A similar Phase IIa cancer trial with SRT501 is under way. Cannabinoids are a group of secondary metabolites responsible for pharmacological properties of Cannabis sativa (cannabis plant) [96]. CP 7075 (IP 751, ajulemic acid, CT-3) 95, a synthetic cannabinoid, suppressing IL-1β and matrix metalloproteinases (MMPs) through a peroxisome proliferator-activated receptor (PPAR) γ-mediated mechanism [97], was investigated by Indevus Pharmaceuticals in pre-clinical studies. In October 2007, the drug was licensed by Cervelo Pharmaceuticals for Phase I trials in neuropathic pain.

Bhuwan B. Mishra & Vinod K. Tiwari HO HO

HO O

O OH

CH3

92 CO2H

OH HO

H3C OH

H3C

CH3 H3C CH3

6. Cardiovascular and metabolic diseases Natural products have played an important role in development of drugs against cardiovascular and metabolic diseases. Simvastatin (Zocor®, Merck), a lipid-lowering statin obtained from fermentation product of Aspergillus terreus, inhibits 5-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase. Orlistat (Xenical®), a lipstatin derivative isolated from Streptomyces toxytricini [98], inhibits pancreatic lipases and used for the treatment of obesity. Captopril, ramipril and quinapril are the examples of some antihypertensive angiotensin-converting enzyme (ACE) inhibitors derived from the snake venom. An endopeptidase (NEP) inhibitor, ilepatril (AVE-7688) 96, is being developed by Sanofi-Aventis in various Phase IIb/III trials to treat hypertension while phase II trial for diabetic nephropathy. (+)-1-Deoxygalactonojirimycin 97 and (+)-galactonojirimycin 98 obtained from Streptomyces species [99] display strong inhibitory activity toward several β-galactosidases. Miglustat (Zavesca®) was used earlier to treat Type 1 Gaucher disease (GD1) by Actelion. Migalastat (AmigalTM, AT1001, 1-deoxygalactonojirimycin, 1-deoxygalactostatin) 97, a semisynthetic derivative of 98, stabilizes protein structures and restores correct folding through binding with them. (+)-1-Deoxygalactonojirimycin 97 is an orphan designated drug by European Commission in May 2006 to use in treatment of Fabry disease. As of January 2010, the 97 is being evaluated under Phase III trials by Amicus Therapeutics in collaboration with Shire

Natural products in drug discovery

Pharmaceuticals against Fabry disease. Isofagomine (PliceraTM, AT2101) 99, an aza-sugar that mimics the carbocation transition state used by glycosidases [100], is under clinical development by Amicus Pharmaceuticals to treat Gaucher’s disease [101]. Amicus in October 2009 announced the positive results of a Phase II trial for two dose regimens consisting of 225 mg of 99 given three days on/four days off and seven days on/seven days off. Ruboxistaurin (LY333531) 100, inhibiting protein kinase C (PKC), is being developed by Eli Lilly to use in the treatment of microvascular complications in diabetes mellitus [102]. In February 2006, Lilly submitted a NDA for use of 100 in diabetic peripheral retinopathy. In August 2006, the FDA essued an “approvable” letter to Lilly while suggesting another Phase III trial for additional efficacy. SCH 530348 (TRA) 101, a PAR-1 antagonist [103] similar to himbacine 102 obtained from Galbulimima baccata, is in Phase III clinical trials by Schering-Plough for the treatment of cardiovascular diseases such as atherosclerosis, ischemia, myocardial infarction and stroke. H N CO2H

O NH

H3C

H N

OH OH

97 R = H 98 R = OH

SAc

H3C

H N

O H 3C

H N

O O

CH3

O H3C

H3C

N CH3

O N

H3C CH3 F

100

101

102

Bhuwan B. Mishra & Vinod K. Tiwari

Genaera Corporation are associated with clinical development of trodusquemine (MSI-1436) 103 and squalamine 104 extracted from Squalus acanthias [104]. MSI-1436 103 is a protein tyrosine phosphatase 1B inhibitor [105] that is being evaluated by Genaera in a second Phase I trial using an ascending single dose in overweight type 2 diabetics under the obesity IND. Ouabain (g-strophanthin) 105, a cardiac glycoside occurring in ripe seeds of Strophanthus gratus and bark of Acokanthera ouabaio, involves binding to and inhibition of the plasma membrane Na+/K+-ATPase attainable in vitro or with intravenous dosage [106]. Likewise, digoxin 106, isolated from Digitalis lanata (foxglove plant) [107], also exists in the human adrenal gland and is significant in atrial fibrillation and atrial flutter. Rostafuroxin (PST 2238) 107, an ouabain antagonist, is under Phase II development by Sigma-Tau to use in the treatment of chronic arterial hypertension. H3C

OSO3H

CH3 H CH3

H N R

N H

CH3

H3C H OH

103 R = N H

104 R = H

NH2 O

CH3

HO OH

H CH3

H3C

CH3

105

OH O HO

O O

106

CH3

OH CH3

H3C

H3C H3C OH O

CH3 HO O

CH3 O

N O

OCH3

O CH3

CH3

H3C

H3C O

H OH

CH3

CH3 OH

CH3

NH2

CH3 HO

OH H3C

CH3

107

H3C

108

109

Natural products in drug discovery

Mitemcinal (GM-611) 108, an agonist of motilin that lacks the antibiotic properties of 51 and increases the amplitude & frequency of antral contractions and initiates gastric contractions, was discovered by Chugai Pharma. Phase I trials of 108 in Japan has been completed by Chugai while Phase II trials in US are still running against diabetic reflux oesophagitis and idiopathic gastroparesis [108]. Chugai are also conducting Phase II trials of 108 against irritable bowel syndrome (IBS). Pyridoxamine (PyridorinTM) 109, consisting of a pyridine ring bearing hydroxyl, methyl, aminomethyl, and hydroxymethyl substituents, is a vitamin B6 analogue [109] that was evaluated by BioStratum in two phase II trials demonstrating retardation of diabetic nephropathy. In October 2006, BioStratum licensed 109 to NephroGenex, which has initiated a new Phase IIb trial in patients with type 2 diabetes. Taisho Pharmaceutical is evaluating 109 (coded as K-163) in Phase II trials against diabetic nephropathy. In January 2009, the FDA ruled for regulation of 109 as a pharmaceutical drug and awarded a fast track drug designation.

7. Immunological, inflammatory and related diseases Autoimmune and inflammatory disease condition arises through aberrant reactions of the human adaptive or innate immune systems. Aspirin, discovered in the late 1890s, is still a significant analgesic and antiinflammatory drug. Salbutamol, a β2-adrenergic receptor agonist, is marketed by GlaxoSmithKlinen to treat asthma and chronic obstructive pulmonary disease. Cyclosporin 74 (1983), tacrolimus (1993), sirolimus 10 (1999), mycophenolate sodium (2003) and mycophenolate mofetil (1995) are among some important immunosuppressive drugs sourced from natural products. Everolimus (LuveniqTM or LX211) 110, a derivative of 10 inhibiting mTOR, is marketed by Novartis as immunosuppressant Certican® in organ transplantation. Voclosporin (ISA-247, R1524, LX211) 111, a derivative of 74 inhibiting calcineurin [110], is under Phase IIb trial to prevent kidney graft rejection and Phase III trial against psoriasis. Voclosporin 111 was licensed by Lux Biosciences from Isotechnika for ophthalmic indications. As of March 2009, Lux Biosciences have completed Phase III trials of 111 oral capsules against uveitis. In February 2010, Lux Biosciences filed a NDA to the FDA and MAA to the EMA for 111 under LuveniqTM against non-infectious uveitis, which were accepted by respective agencies in March 2010. Eupatilin 112, a flavone isolated from Korean traditional medicine Artemisia argyi possess efficacy against chronic diarrhea [111]. DA-6034 113, a synthetic 112 derivative, is being developed by Dong-A Pharmaceuticals in Phase I and II trials against dry eye and gastritis, respectively.

Bhuwan B. Mishra & Vinod K. Tiwari

H3C

O CH3 O

O CH3

O HO

CH3

O N

CH3

O CH3

CH3

O CH3 O

O HO O H3C

CH3

110 H3C

CH2 CH3

CH3

CH3 H H N

H3C

N O

CH3

CH3 O

CH3

O H N

CH3 CH3

H3C CH3

CH3

N H CH3

CH3

N CH3

CH3

CH3 H3C

CH3 N

H3C

N H

O CH3 CH3

111

OCH3 HO

OCH3

O O

O OCH3

OCH3

H3CO OH

112

OCH3 O

113

8. Oncological diseases 8.1. Small-molecule anticancer agents 8.1.1. Plant-derived compounds Camptothecin 114, a topoisomerase I inhibitor isolated from Camptotheca acuminata [112], exhibits significant anticancer activity. Among other camptothecin class of drugs, belotecan (Camptobell, CDK-602) was developed and launched in 2004 by Chung Kun Dong in Korea [113]. BNP-1350 (Karenitecin®) 115 is an investigational drug under clinical

Natural products in drug discovery

development by BioNumerik Pharmaceuticals for cancer chemotherapy. As of February 2008, BioNumerik are running the Phase III trial of 115 against ovarian cancer [114]. Diflomotecan (BN80915) 116, a 115 analogue [115], is being developed by Ipsen under Phase II trials to treat advance metastatic cancers. Gimatecan (ST-1481) 117, an oral topoisomerase I inhibitor, is currently in Phase II development by Novartis against solid tumors [116]. Elomotecan (BN-80927, LBQ707, R-1559) 118, inhibiting topoisomerase I and II, is a promising Phase I pipeline by Ipsen in oncology (e.g. colon, breast and prostate cancer) [117]. DRF 1042 105 is a 114 derivative evaluated by Dr. Reddy’s Laboratories in Phase I trials to use in the treatment of various cancers [118]. Dr. Reddy’s Laboratories in September 2006 collaborated with ClinTec International for joint Phase II/III development of 119. In September 2006, Sonus Pharmaceuticals initiated a Phase I study of SN2310 120, a prodrug of SN-38 121 to address cancer is presently ongoing. In May 2008, Sonus murged with OncoGenex Technologies and the new company, OncoGenex Pharmaceuticals has included 120 as a strong oncology pipeline. R F

O N

N F

114 R = H

N O

H3C HO

H3C

Si(CH3)3

117 R =

115 R =

116

C(CH3)3

H3C OH N

O O

H3C

O N

N O H3C

CH3

118

H3C

119 CH3 H3C

CH3 O

H3C

O CH3

CH3

O N

120

N O H3C HO

Bhuwan B. Mishra & Vinod K. Tiwari

Combretastatin A-4 phosphate (ZybrestatTM, CA4P) 121, a prodrug of combretastatin A-4 122 obtained from South African Bush Willow Combretum caffrum [119], is a reversible tubulin depolymerizing agent that causes tumour-associated endothelial cells to change from a flat to a round shape, thus by plugging the blood vessels deprives the tumour from oxygen and nutrients. Oxigene are evaluating 121 as a vascular disrupting agent (VDA) [120] and as on September 2008, Phase III trial against anaplastic thyroid cancer (ATC) is underway. Oxigene in November 2009 disclosed the positive results of a Phase II trial of 121 in non-small cell lung cancer (NSCLC). Ombrabulin (AVE8062) 123, another 122 derivative licensed to Sanofi-Aventis from Ajinomoto, is under Phase III trials in advanced STS patients. Combretastatin A-1 diphosphate (OXi4503) 124, a pro-drug of combretastatin A-1 125 that is capable of binding to proteins and nucleic acids [121], is under various Phase I trials by OXGENE to use in the treatment of advanced-stage solid tumors. Noscapine (CB3304, noscapine) 126, a benzylisoquinoline alkaloid occurring in the plants of family Papaveraceae, is a microtubule targeting antitussive currently in Phase I/II trials by Cougar Biotechnology for the treatment of multiple myeloma [122]. Vinblastine (Alkaban-AQ®, Velban®) 127, a microtubule inhibitor isolated from Catharanthus roseus [123], has been found significant when given intravenously to patients suffering from Hodgkin's disease, nonHodgkin's lymphoma, Kaposi's sarcoma, choriocarcinoma, TCL, breast, testicular, lung, neck and head cancers. Vinflunine (Javlor®) 128, a fluorinated vinca alkaloid [124] discovered by Laboratoires Pierre Fabre, was submitted for registration with the EMEA in June 2008, after positive Phase III trial for metastatic treatment of bladder cancer. In June 2009, Pierre Fabre received a positive opinion with recommendation for marketing authorization of 128 in the metastatic treatment of bladder cancer. Paclitaxel (TaxolTM, AbraxaneTM) 129, isolated from Taxus brevifolia [125], is a mitotic inhibitor that stabilizes microtubules and interferes with the normal breakdown of microtubules during cell division. Bristol-Myers Squibb (BMS) are associated with commercial development of 129. Cabazitaxel (XRP6258) 130 and larotaxel (XRP9881) 131 have been designed by Sanofi-Aventis as poor substrates for membrane-associated P-glycoprotein (P-gp), overexpressed in taxane resisting cells [126] and are in Phase III trials against pancreatic and hormone-refractory prostate cancers [127]. Luitpold Pharmaceuticals are developing DHA-paclitaxel (Taxoprexin®) 132, a fatty acid conjugate of 129, in Phase III trials against metastatic melanoma [128]. Spectrum are associated with Phase I/II development of intravenous/oral ortataxel (IDN-5109, BAY-59-8862) 133, a third generation taxane with toxicity/tolerance profile similar to 129. As of

Natural products in drug discovery

June 2009, the 133 is under Phase II trials in taxane-refractory solid tumors [129]. Milataxel (MAC-321, TL-00139) 134, a poor substrate for P-gp, is under Phase II clinical development by Wyeth Pharmaceuticals to use in the treatment of colorectal neoplasms [130]. Tesetaxel (DJ-927) 135, an orally administered semisynthetic taxane, was evlaulated by Genta in various Phase I/II trials against advanced gastric and breast cancer [131]. The Phase II clinical trials of 135 are running for the treatment of patients with advanced melanoma having a normal serum lactate dehydrogenase (LDH) and have progressed after one chemotherapy regimen. Other taxanes that are in Phase II clinical development, i.e. TPI-287 136 by Tapestry Pharmaceuticals and BMS-188797 137 by Bristol-Myers Squibb, to treat patients suffering from pancreatic and advanced malignancies, respectively are currently in Phase II clinical development [132]. R

OCH3 H3CO

NH2

H N O OCH3

OCH3 H3CO

OCH3

OCH3 OCH3

121 R = OPO3Na2 122 R = OH

123

O OCH3 H3CO

CH3 O

H3C

OCH3

OCH3 H3C

124 R = PO3Na2 125 R = H

126

OH CH3

H3CO

CH3 CH3

N OAc H CH3 CO2CH3

128

H3C H3C

O CH3OH

CH3 NH

H3CO H3C

CH3

O HO

O CH3 CH3 O

CH3

OH O O

129

CH3

N OAc H CH3 CO2CH3

127

N H H3CO2C

CH3

H3CO

F CH3

N H H3CO2C

CH3

O O

H3C

O H3C

130

Bhuwan B. Mishra & Vinod K. Tiwari

Acronycine 138, an alkaloid isolated from Acronychia baueri, exhibits activity against various solid tumors such as S-180 and AKR sarcomas, X-5563 myeloma, S-115 carcinoma and S-91 melanoma. S23906-1 139, a benzoacronycine derivative inhibiting DNA synthesis and S-phase cell cycle arrest, is currently in Phase I trials for the treatment of solid tumors [133].

CH3 CH3 O O

CH3 NH

H3C H3C

CH3

CH3 HO

H3C

CH3

O O

H3C

132 CH3 CH3

O H3C

O O CH3 OH

H3C CH3 CH3

H3C

CH3

O O

O OH H3C O CH3 O CH3

O O

CH3

133

H3C

O OH

CH3

131

O CH3 OH

CH3

O O

H3C

CH3

CH3 CH3

H3C H3C O

O H3C

H3C

134

Homoharringtonine (omacetaxine mepesuccinate, Ceflatonin®) 140, a myelosuppressive alkaloid isolated from Cephalotuxus fortuneii, inhibits Mcl-1 protein synthesis and induces apoptosis [134]. The European Commission in October 2004 granted orphan designation to Stragen France SAS for 140 against acute myeloid leukemia (AML) that was later transferred to ChemGenex Europe SAS in January 2009. The FDA in January 2009 designated 140 as orphan drug against myelodysplastic syndromes (MDS). In September 2009, a NDA for 140 under Omapro™ (omacetaxine mepesuccinate) was submitted by ChemGenex to the FDA for use in the treatment of chronic myelogeneous leukemia (CML) patients having T315I

Natural products in drug discovery

mutation or failed in imatinib therapy. ChemGenex are also running Phase II trial of 140 for treatment of refractory or relapseed AML patients failed to intensive chemotherapy. CH3 CH3 O

CH3

CH3 O

CH3 CH3

CH3 N

CH3 CH3 CH3

N O

CH3

H3C

O NH

O O CH3 O

H3C

CH3 CH3

CH2

H3C

CH3

O O

O H3C

136

H3C H3C

H3C

135

O CH3 OH

OCH3

CH3 O

CH3

OH HO

N O

O H3CO

137

CH3

CH3 R

138 R = H 139 R = OAc

3′-O-methyl-nordihydroguaiaretic acid (NDGA) 141, a lignan isolated from Larrea divaricatta exhibits significant anticancer activity by retardation of tumor cell proliferation through inhibition of insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu receptor. Terameprocol 142, a synthetic 141 derivative that induces apoptosis in cancer cells through inactivation of maturation promoting factor, was licensed by Erimos from The Johns Hopkins University. Terameprocol 142 is currently in various Phase I/II trials by Erimos against solid tumors, glioma and leukemia [135]. Epipodophyllotoxin (F11782) 143, a non-intercalating dual inhibitor of both topoisomerases I and II, was originally isolated from root of Podophyllum peltatum [136]. Tafluposide 144, a 143 derivative, is being developed by Pierre Fabre under Phase I/II trials for various tumor types [137]. Ingenol 145, isolated from the sap of Euphorbia peplus, is under clinical development by Peplin Biotech for topical treatment of basal cell carcinomas and squamous cell carcinomas [138]. Afer merger of Peplin with LEO Pharma in November 2009, ingenol mebutate (PEP005) 146, a 145 derivative that

Bhuwan B. Mishra & Vinod K. Tiwari

activates PKC, is currently in Phase III trials against actinic keratosis (AK). In December 2009, LEO Pharma disclosed the positive results of 146 in two Phase III trials against AK lesions on head (including the face and scalp) while announced to meet the primary endpoint in February 2010 with disappearance of AK lesions in non-head locations. Daidzein 147, an isoflavone occurring in Pueraria Mirifica, soybeans and soy products, exhibits clinical indication against tumors [139]. Phenoxodiol 148 is a synthetic 147 derivative that was licensed by Marshall Edwards from Novogen for development as combination therapy against ovarian cancer and as mono-therapeutic agent for the treatment of prostate and cervical cancers, resistant to standard chemotherapy [140]. Phenoxodiol 148 is supposed to inhibit sphingosine-1-phosphate and is under Phase III development by Marshall Edwards to restore chemosensitivity in patients with ovarin cancer resisting platinum drugs. A phase II trial of 148 against castrate and noncastrate prostate cancer is also uderway. Triphendiol (NV-196), an orally-delivered chemosensitizing derivative of 148 that was licensed to Marshall Edwards by Novogen, is under Phase I trials for use in combination therapy against cholangiocarcinoma, advanced prostate cancer and melanoma. An orphan drug status was granted to 148 by the FDA for cholangiocarcinoma, prostate cancer and stage IIb-IV malignant melanoma. In January 2009, FDA granted IND approval to 148. Genistein 149, a soy-derived antineoplastic phytoestrogen, inhibits protein-tyrosine kinase and induces cell differentiation, is under Phase I/II trials by Astellas, Bausch & Lomb for treatment of tumors. Genistein 149 is also supposed to inhibit topoisomerase-II, resulting in DNA fragmentation and apoptosis. (-)-Gossypol (AT-101) 150, a pan-Bcl-2 inhibitor isolated from the cottonseed plant of genus Gossypium [141], is under Phase I/II clinical development by Ascenta Therapeutics to address prostate, brain and lung cancers. In October 2009 Ascenta announced the results of Phase I trial for two combination regeims containing 150 for the treatment of malignant brain tumor. OH H3C

CH3 H3C

CH3 O

OH O

OCH3 H3C

O O

H3CO

OR OR

O H

H3CO

OCH3 OCH3

141 R = H 140 142 R = CH3

143

Natural products in drug discovery

ASA 404 (vadimezan, AS1404 and DMXAA) 151, a tumor-VDA and derivative of flavone-8-acetic acid 152, was discovered at Auckland Cancer Society Research and later in-licensed by Antisoma. Novartis AG in April 2007 signed an agreement with Antisoma for worldwide rights and co-selling of 151 in the US. As of April 2008, the 151 is currently in Phase III clinical trials by Novartis as a second line treatment for NSCLC. The β-Lapachone (ARQ-501) 153, isolated from Tabebuia avellanedae, induces expression of cyclin dependent kinase inhibitor 1A (CDKN1A or p21) and exerts anti-tumor effect by sustained increase of the pro-apoptotic protein E2F-1 [142]. The β-Lapachone 153 is currently in Phase II trials by ArQule as a combination therapy against pancreatic and ovarian cancer. Alvocidib (Flavopiridol, HMR 1275) 154, a CDK inhibitor and synthetic derivative of rohitukine 155 isolated from Dysoxylum binectariferum [143], is being developed by Sanofi-Aventis in collaboration with NCI. As on May 2009, the 154 is under late Phase III clinical development by Sanofi-Aventis against NSCLC while Phase IIb trial for the treatment of chronic lymphocytic leukemia (CLL). F

CH3

CH3 F

H3C

F F

H3C

RO HO

HO HO

O O

145 R = H

CH3

146 R =

CH3

O H

OH R

H3CO

OCH3 OH

O P

144

147

Curcumin 156, isolated from Curcuma longa roots, can interfere with the p53 tumor suppressor pathway and is under various Phase I/II trials world wide while a Phase III trial for the treatment of metastatic colon cancer (MCC) is underway [144]. RTA 402 (CDDO-Me, Bardoxolone methyl) 157,

Bhuwan B. Mishra & Vinod K. Tiwari

an IkB alpha kinase activation inhibitor and synthetic derivative of oleanolic acid 158 [145], is being evaluated by Reata Pharmaceuticals under Phase I/II trials against prostate cancer and Phase II trials for the treatment of type 2 diabetes with chronic kidney disease (CKD). RTA 402 157 is an orphan drug by the FDA against prostate cancer. In January 2010, Kyowa Hakko Kirin gained exclusive rights from Reata Pharmaceuticals to develop and commercialize 157 in Japan and other selected Asian regions to treat type 2 diabetes with CKD. O OH OH

OH HO

HO HO

H3C

CH3 H3C

CH3

148

CH3

149

150 OH

H3C

Cl O HO

O OH

CH3 CH3

HO2C

O N

HO2C

151

H3C

152

CH3

154

153

CH3 O

O HO

O N

HO OH

155

CH3

H3CO

OCH3

156

Betulinic acid (ALS-357) 68, a topoisomerase I inhibitor isolated from Betula pubescens [146], is an orphan drug (by the FDA) in Phase I trial by Advanced Life Sciences for the treatment of malignant melanoma. Silybin 159, a flavonolignan isolated from Silybum marianum, is the active constituent of IdB 1060 (silybin-phosphatidylcholine complex, Siliphos®). Silybin 159 is currently in Phase II trials by American college of gastroenterology for chemoprevention of cancer [147].

Natural products in drug discovery H3C

CH3

H3C

CH3 OH

OCH3

OH CH3 N O H3C

CO2CH3 CH 3

CH3

H CH3

H3C

CH3

CO2H O

CH3

H CH3

157

159

158

8.1.2. Microorganism-derived compounds 8.1.2.1. Actinomycetes Pladienolide D 160, obtained from fermentation broth of Streptomyces platensis Mer-11107, exerts significant antiproliferative activities against variety of cancer cell lines. E7107 161, a synthetic 160 derivative that binds with spliceosome-associated protein 130 (SAP130) and inhibits the splicing of pre-mRNA, is in various Phase I trials by Eisai against solid tumors [148]. Chartreusin (U-7257) 162 isolated from Streptomyces chartreuses and elsamicin A (BMY-28090, elsamitrucin) 163 isolated from actinomycete strain J907-21, are the antibiotics that inhibit RNA synthesis and result in single-strand scission of DNA [149]. Elsamicin A 163 is also a topoisomerase I/II inhibitor being developed by Spectrum Pharmaceuticals in Phase II trials to use in the treatment of advanced solid tumors. OR OH CH3 OH H3C

H3C

O O

CH3

H3C

160 R = Ac O 161 R =

CH3

Doxorubicin 164, an anthracycline antibiotic capable of intercalating with DNA, was isolated from bacteria occurring in soil samples taken from Castel del Monte, an Italian castle. Doxorubicin 164 is an orphan drug by the FDA against acute lymphocytic leukemia (ALL) and AML. Valrubicin (Valstar®), a semi-synthetic 164 derivative was approved in 1999 for the treatment of bladder cancer but was withdrawn in 2002 due to some manufacturing issues and has been relaunched in September 2009. L-annamycin 165, a topoisomerase II inhibitor that was developed at the MD Anderson Cancer Center, is currently in Phase I/IIa trials by Callisto

Bhuwan B. Mishra & Vinod K. Tiwari

Pharmaceuticals for the treatment of younger and adults with refractory or relapsed ALL or AML. Berubicin (RTA744, WP744) 166, a DNA intercalator capable of crossing the BBB, hence is significant for the treatment of primary brain tumor. Reata Pharmaceuticals are associated with Phase II development of 166 against malignant gliomas. Likewise, sabarubicin (MEN-10755) 167 [150], a topoisomerase II inhibitor and disaccharide analogue of 164, is currently in Phase II clinical trials by Menarini Pharmaceuticals against solid tumors [151]. Nemorubicin (MMDX, PNU-152243A) 168, a 3′-deamino-3′[2-(S)-methoxy-4morpholinyl] derivative of 164, is a topoisomerase I inhibitor exhibiting activity against selected tumors resistant to current treatment. Nerviano Medical Sciences are evaluating 168 in Phase I/II trials. Distamycin A 169, a DNA minor grove binder (MGB) and lead compound of brostallicin (PNU-166196) 170, was originally developed by Nerviano [152]. Nerviano had transferred the exclusive world right of 170 to Systems Medicine Inc (SMI) which was taken over by the Cell Therapeutics. Currently, the 170 is in phase II trials by Cell Therapeutics as monotherapy against metastatic or advanced stage STS. H3C

H3C O OH HO

CH3

O CH3

H3C

CH3 O

O NH2

164 R = H 166 R =

163 O

H3C

162

H2N

O OH

OH OH

CH3 O

O O

CH3 O

O O

O OH

OH O

O O

H3C HO

HO O

O HO

CH3

H3C

H3C CH3 O

OH N

O OH

165

H3C

NH2

167

168

Natural products in drug discovery

Geldanamycin 171 is an antineoplastic benzoquinone ansamycin antibiotic and was discovered from broth and mycelium of Streptomyces species [153]. Tanespimycin (17-AAG, KOS-953, NSC-330507) 172, a comparatively less toxic antibiotic derived from 171, can bind to HSP90 and interrupts the MAPK pathway. As on November 2009, Kosan have completed a Phase II/III trial of 172 in combination with Velcade® against relapsed-refractory multiple myeloma. Alvespimycin (17-DMAG, KOS1022, NSC-707545) 173, a second generation HSP90 inhibitor [154] is in clinical development by Kosan to use in the treatment of solid tumors. As on January 2008, 173 is in Phase I trials in combination with trastuzumab & paclitaxel (Taxol®) against solid tumors, Phase II monotherapic trials against HER2-positive metastatic breast cancer and Phase I trials for the treatment of solid tumors. Retaspimycin (IPI-504, 17-AAG hdroquinone salt) 174, a HSP90 inhibitor, is being developed by Infinity Pharmaceuticals in Phase I/II clinical trials to address certain cancers. Currently, the Infinity are evaluating 174 in a Phase II trial against NSCLC while enrolling patients for another Phase II trial in combination with Herceptin® against breast cancer. H NH O H N Br

H2C

CH3

H N

O N

CH3

H N

NH2

169

N CH3

H N

O O

CH3

H N

O N CH3

H N

N CH3

170

H N

N H

N CH3

NH2

Deforolimus (AP23573, MK-8669) 175, is an mTOR inhibitor codeveloped by Merck and ARIAD Pharmaceuticals to address several tumor types including sarcoma. The name of 175 was changed to ‘ridaforolimus’ by ARIAD in May 2009 and as on December 2009, the enrollment for a Phase III study in patients with metastatic STS and bone sarcomas has been completed by ARIAD. Besides, ARIAD are also running several Phase I/II trials of 175 as a single agent and in combination therapies. Salinosporamide A (NPI-0052) 176, a proteasome inhibitor produced by a marine bacterium

Bhuwan B. Mishra & Vinod K. Tiwari

Salinispora tropica [155], exerts activity by modifying the threonine residues of the 20S proteasome. Nereus are associated with Phase I clinical development of 176 to use in the treatment of solid tumors and lymphomas. As on April 2008, Nereus Pharmaceuticals are enrolling patients for a Phase Ib trial of 176 in combination with vorinostat (Zolinza®, Merck & Co.) against selected solid tumor malignancies. O H

O CH3

N H O H3C H3C

171 R = OCH3 172 R =

CH3 OH

O CH3

173 R = NH2

H3C

N H N H

CH3

H2C CH2

CH3

H3C

CH3

CH3 OH O H3C

O CH3

O O

174

NH2

Staurosporine 177, isolated from bacterium Streptomyces staurosporeus [156], a precursor of protein kinase inhibitors like enzastaurin (LY317615) 178 and midostaurin (PKC-412, CGP 41251, 4’-N-Benzoyl-staurosporine) 179, has significant anticancer potenticals. Enzastaurin (LY317615) 178 is a serine/threonine kinase inhibitor [157] that was evaluated in Phase II trials by Eli Lilly to use in the treatment of NSCLC patients. As of April 2010, the 178 is under Phase III trials for the treatment of diffuse large B-cell lymphoma. Midostaurin (PKC-412) 179 inhibits protein kinases including FLT3 [158] and is in Phase II trials by Novartis to treat AML patients carrying FLT3 mutations. K252a 180, an alkaloid isolated from Nocardiopisis species, is the lead compound of lestaurtinib (CEP-701, KT-5555) 181 that inhibits FLT3 and tyrosine phosphorylation of Trk A. As of 2008, the 181 is in Phase II trials against myeloproliferative disorders and Phase III trials for the treatment of AML. Likewise, KRX-0601 (UCN-01, KW-2401) 182, inhibiting a broad spectrum of kinases including CDKs, is being developed by Keryx (Kyowa Hakko) in Phase II clinical trials under sponsorship of NCI against melanoma, TCL and SCLC. Diazepinomicin (ECO-4601, TLN-4601) 183, a dibenzodiazepine alkaloid isolated from the culture of a marine actinomycete of the genus Micromonospora [159], can bind to peripheral benzodiazepine receptor (PBR) and inhibits the Ras/MAP kinase signaling pathway involved in cellular proliferation and migration [160]. ECO-4601 183 was found safe and well-tolerated in Phase I/II trials conducted by the NCI and Thallion. ECO-4601 183 can cross the BBB and as on September 2008, Thallion are enrolling patients for Phase II trial of 183 as a second line treatment for GBM.

Natural products in drug discovery

CH3 OH

CH3 O

O OH

HO P O

O CH3

CH3

O CH3

O N

CH3

O CH3

O H N

176

O HO O H3C

CH3 H N

175

N CH3

N N

N CH3 N

OCH3 NHCH3

178

177

H N

CH3

OCH3

CH3

180 R = CO2CH3

179

181 R = CH2OH

H N

OH O

CH3

N N

CH3

CH3 OCH3

HN CH3

182

CH3

183

Bhuwan B. Mishra & Vinod K. Tiwari

8.1.2.2. Eubacteria Prodigiosin (Streptorubin B) 184, a Bcl-2 inhibitor and lead compound of obatoclax (GX15-070) 185, is produced by strains of the bacterium Serratia marcescens [161]. Gemin X are developing intravenous infusion of 185 in multiple Phase I/II trials as a monotherapy in hematological and solid tumors while in combination with carboplatin & etoposide to treat SCLC and with bortezomib (Velcade®) against mantle cell lymphoma (MCL). In March 2009, Gemin X launched a Phase II study of 185 as first-line treatment for SCLC while disclosed the results of a Phase Ib trial in May 2009 against extensive-stage SCLC. 8.1.2.3. Myxobacteria Patupilone (epothilone B, EPO-906) 21, produced by the myxobacterium Sorangium cellulosum, is a microtubule-stabilizing agent currently in Phase III trials by Novartis against ovarian cancer [162]. Sagopilone (ZK-EPO, ZK-219477) 186, a synthetic 21 derivative, can retain activity in MDR cancer cells overexpressing the P-gp [163]. As of February 2010, Schering AG is evaluating 186 in Phase II trials for the treatment of lung, ovarian and prostate cancers. Epothilone D (desoxyepothilone B) 187, a natural polyketide inhibits the disassembly of microtubules by binding to tubulin. 9,10-Didehydroepothilone D (KOS-1584) 188 [164], a 187 derivative, being evaluated by Kosan Pharmaceuticals to use in the treatment of multiple solid tumors. In Phase I dose escalation trials by Kosan, 188 has demonstrated efficacy and tolerability against patients with ovarian cancer and NSCLC. As of February 2007, Kosan were planning to initiate Phase II clinical development of 188 against multiple solid tumors in collaboration with Roche. OCH3 H C 3

N H

OCH3

N N H

HN HN H3C

184

185

CH3

Natural products in drug discovery

8.1.2.4. Fungi NPI-2350 (halimide, phenylahistin) 189 is a tubulin-depolymerizing agent isolated from a marine fungi Aspergillus ustus [165]. Nereus are developing plinabulin (NPI-2358) 190, a synthetic 189 analog in various clinical trials for the treatment of NSCLC [166]. In November 2009, Nereus announced the positive results of a Phase II trial in NSCLC patients. Irofulven (MGI-114, HMAF) 191 is a DNA synthesis inhibitor and analog of illudin S 192, a sesquiterpene toxin found in mushrooms of the genus Omphalotus [167]. Eisai (MGI Pharma) are currently evaluatig 191 in various Phase II/III trials in patients with advanced-stage prostate cancer and GI solid tumors. 8.1.3. Marine-derived compounds Plitidepsin (Aplidin®) 193, extracted from Aplidium albicans [168], is being evaluated by PharmaMar in Phase II trials to use in the treatment of hematological and solid tumors. Plitidepsin 193 inhibits the vascular endothelial growth factor (VEGF) and is currently in Phase II trials by PharmaMar as a first-line monotherapy treatment and in combination with dacarbazine for advanced unresectable melanoma [169]. Halichondrin B 194, isolated from Halichondria okadai sponge [170], was identified as a significant anticancer agent by NCI. Eribulin mesylate (E7389, ER-086526, NSC-707389) 195, a 194 analog, is being developed by Eisai against advanced breast cancer patients. Eribulin 195 is a microtubule dynamics inhibitor and in March 2010, Eisai has submitted regulatory applications to agencies in Japan, US and EU for approval of 195 to use in the treatment of locally advanced or metastatic breast cancer. Hemiasterlin 196, derived from marine sponges [171], is capable of inhibiting tubulin assembly and disrupts normal microtubule dynamics by depolymerizing the microtubules. E7974 197, a synthetic analogue of 196, can bind to α- and β-tubulin and is under Phase I clinical development by Eisai against a variety of human tumor xenografts. Psammaplin A 198, an inhibitor of key several enzymes that control gene expression, DNA replication and angiogenesis, was originally isolated from the marine sponge Psammaplinaplysilla. Panobinostat (LBH-589) 199, a synthetic 198 analog and pan-deacetylase inhibitor that induces death of tumor cell lines but not the normal cells, is in Phase Ib/II clinical trials by Novartis to use as monotherapy and in combination with chemotherapy and/or targeted therapy against Hodgkins lymphoma, malignant melanoma, AML/MDS and other hematological malignancies [172]. Currently, Novartis are enrolling patients for Phase III trial in relapsed malignant melanoma.

Bhuwan B. Mishra & Vinod K. Tiwari

CH3

H3C

O OH

CH3

H3C CH3 H3C

H3C

CH3

CH2 O

186

S H3C

CH3

187

CH3 O

OH NH H3C

H3C

N NH

CH3

CH2

CH3 O

H3C

CH3

189

188

O OH

CH3 NH

CH3

N NH

HN O

CH3

HO CH3 H3C CH3

H3C

CH3 OH HO H3C

192

191

190

OCH3 H3C

CH3

H3C

CH3 O

O O

H3C H3C

N H NH

O CH3

H3C

CH3

H3C H3C

CH3

193

CH3

Natural products in drug discovery

CH3 H

HO H

H O

O H

CH3 H

O H

CH2 O

CH3

OH O

194 H3C

H2C

H O H

O O

H NH2

H3C

CH3

H3C O

CH3 CH3 CH3

CH2 O O CH3

CO2H

N H O

O H3C

CH3

CH3 CH3

H3C

H2C

195

196 OH

H3C

CH3 H C 3 O N

N H

CH3 CH3 CH3 N O H3C

CO2H CH3 CH3

N H

H N

Br O

198

197

H3C H3C HO

OAc O

O N H

H N

O HO

O OCH3

HO O

OCH3

CH3 O O

H3C

200

OH H3C

H3C

CH3

199

Bhuwan B. Mishra & Vinod K. Tiwari

Bryostatin 1 200, a macrolide lactone isolated from Bugula neritina collected from the Gulf of California and Mexico, inhibits PKC [173] and was granted with orphan drug status by the FDA (2001) and a similar designation by the EU (2002) for use as combination therapy with TaxolTM against esophageal cancer. In 2001, Arizona State University licensed 200 to GPC Biotech, which are associated with current Phase I/II trials under the guidance of the NCI. Jorumycin 201, isolated from Jorunna funebris produces cytotoxic effects through binding to DNA [174] and is the lead compound of Zalypsis® (PM00104/50) 202 being developed by PharmaMar in Phase I trials for the treatment of solid tumors or lymphoma. As on November 2009, the 202 is in Phase II trials for treating cervical and endometrial cancer patients previously treated with standard chemotherapy. Dolastatin 15 203, is an antimitotic agent structurally related to dolastatin 10 205, a five-subunit peptide obtained from Dolabella auricularia [175]. Tasidotin (synthadotin, ILX-651) 204, an analog 203, induces G2/M phase cell cycle arrest by inhibiting tubulin polymerization was evaluated by Genzyme in Phase I/II trials against solid tumors. In May 2009, Genzyme signed an agreement with Ergomed for the co-development of 204 as an antineoplastic agent. Soblidotin (YHI-501, TZT-1027, auristatin PE) 206, a derivative of 205, inhibits tubulin polymerization and is under Phase II trials by Yakult Honsha for treatment of solid tumors. Kahalalide F 207, obtained from the Hawaiian sea slug Elysia rufescens, can alter lysosomal membrane function [176] and is in Phase II trials since October 2008 for the treatment of severe psoriasis. In June 2009, the 207 was licensed by Medimetriks Pharmaceuticals from PharmaMar for uses outside of oncology and neurology. PM02734 (Irvalec®) 208, another 207 derivative, is in Phase II trials against solid tumors by PharmaMar. As on February 2010, PharmaMar are recruiting patients for Phase I trial of 207 in combination with erlotinib against advanced malignant solid tumors.

8.2. NP-antibody anticancer conjugates Anticancer agents conjugated with various supports (antibodies, polymers, liposomes and nanoparticles etc.) have been extensively explored during the last few decades [177]. Zinostatin stimalamer (ZSS), conjugated with a molecule of neocarzinostatin (NCS) chromoprotein and two molecules of polystyrene-co-maleic acid [178], was launched by Yamanouchi (now Astellas) in Japan against hepatocellular carcinoma. Gemtuzumab ozogamicin (Mylotarg®) 209 linked to calicheamicin 210 (obtained from Micromonospora echinospora), was co-developed by Wyeth and

Natural products in drug discovery

OCH3 O H

H3C

H3CO O

CH3

OH OAc

N H

N O

CH3

CF3

CH3

202

CH3H3C

OCH3

O H3C

CH3O

H N

203 H3C H3C

CH3H3C

N H

N CH3

CH3 N

CH3

CH3 CH3 CH3

204 H3C

CH3 H3C

CH3 CH3

H3C

N N H

N CH3

N CH3 OCH3 O

OCH3 O

H N

205 H3C

CH3 H3C

CH3 CH3

H3C

N N H

N CH3

N O

CH3

C CHH 33

CH3

H3C

CH3

201

H3C

CH3 H3C

H3C

OCH3

CH3 OCH3 O

206

OCH3 O

H N

Bhuwan B. Mishra & Vinod K. Tiwari CH3 H2N

CH3

O N H CH3

N H O

H3C O H3C H3C

H3C

NH CH3 H3C H3C

CH3

207 R =

NH O

CH3

CH3 O

O CH3 HN

NH H3C

O H3C

H3C

NH CH3

H N O

208 R = O

CH3

O NH R

UCB Pharma. Likewise, inotuzumab ozogamicin (CMC-544), a calicheamicinantibody conjugated with CalichDMH and hydrazone linker attached to humanized IgG4 anti-CD22 [179], is being developed by Wyeth and UCB Pharma in Phase II/III trials against non-Hodgkin’s lymphoma in combination with rituximab, a chimeric human IgG1 antibody that targets another B-lymphoid lineage-specific molecule, CD20 [180]. Maytansine 211, isolated from plants of the genus Maytenus, is a microtubule inhibitor that failed to show significant activity at non-toxic concentrations in Phase I/II trials. ImmunoGen are associated with clinical development of IMGN-242 (HuC242-DM4) 212, a maytansinoid DM4 and huC242 conjugate, currently in Phase II trials for CanAg-expressing cancers. In June 2009, ImmunoGen discontinued the development of 212 and are seeking for out-licensing. ImmunoGen are also evaluating IMGN-901 (HuN901-DM1) 213, a maytansinoid DM1 and huN901 congugate targeting CD56 expressing tumors, is under Phase I trials against multiple myeloma while Phase II trial for the treatment of SCLC. The FDA in March 2010 awarded orphan drug designation to 213 for use against merkel cell carcinoma (MCC).

9. Conclusion Natural products have been the major sources of chemical diversity for starting materials while driving pharmaceutical discovery over the past century.

Natural products in drug discovery

H N O

hP67.6

O H3C CH3

H3C

N H

O H3C

S O

H3CO

H3C

OCH3

H3C O

N H3CO

209

CH3

O H3C

H3C O

H3C HO H3CO

O O

H3C

OCH3

N H

N H HO

OCH3

O O

HN H3CO

210 CH3

H3C

Cl H3C H3CO

O O CH3

N CH3

HO N H CH3

OCH3

211

S S H3C

S OH

H3C

OCH3

N H HO

OCH3

N H

H3C HO

CH3

O OCH3

Bhuwan B. Mishra & Vinod K. Tiwari

CH3 H3C

H3C

CH3 S S

N H

huC 242

O O

Cl H3C H3CO

O CH3

N CH3

CH3

OCH3

N H

O 3-4

212 CH3 H3C

CH3 S

O O

Cl H3C H3CO

H N

HuN901

O CH3

N CH3

HO N H CH3

OCH3 3-4

213

Today, NPs are finding increasing use as probes to interrogate biological systems as part of chemical genomics and related researches. The modification of natural products in an effort to alter their biochemical capacity is a common technique utilized by synthetic and medicinal chemists. There have been remarkable achievements in the field of ‘natural products drug discovery’ during last three decades and several compounds having profound biological activities have been searched out with the help of modern and sophisticated techniques. The quality of leads arising from NP discovery is better and often more bio-friendly, due to their co-evolution with the target sites in biological systems.

Natural products in drug discovery

The large number of NP-derived compounds in various stages of clinical development indicates that the use of NP templates is still a viable source of new drug candidates. In future, the ‘natural products drug discovery’ will be more holistic, personalized and involve wise use of ancient and modern therapeutic skills in a complementary manner so that maximum benefits can be accrued to the patients and the community.

Acknowledgement Authors are grateful to Prof. Dr. Richard R. Schmidt, Department of Chemistry, Universitat Konstanz, Germany for his useful discussions during the preparation of manuscript. Financial assistance from DST, New Delhi has been greatly acknowledged.

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 63-101 ISBN: 978-81-308-0448-4

2. Natural products in discovery of potential and safer antibacterial agents 1

Girija S. Singh1 and Surendra N. Pandeya2

Chemistry Department, University of Botswana, Gaborone, Botswana; 2Pharmaceutics Department Saroj Institute of Management & Technology, Lucknow-226001, India

Abstract. This article describes the significance of natural products in discovery of potential and safer antibacterial agents. The introductory paragraph is followed by various classes of compounds depending on the structural class. These include β-lactams, macrolides and ketolides, lincosamides, furanomycin, pyrrolidinediones, tetrahydropyrimidinones, biphenomycins, tuberactinomycin and capreomycin, glycopeptides, lysobactins, enopeptin depsipeptides, tetracyclines and aminoglycosides. A brief historical development of each class is described followed by its mechanism of action. Several semi-synthetic compounds are described. Synthetic methods are described in selected cases.

1. Introduction Today, infectious diseases are the second major cause of death worldwide and third leading cause of death in economically advanced countries [1]. Bacterial pathogens are responsible for several serious diseases (Table 1). Strains are getting resistant to antibiotics in clinical use and hence posing threat to mankind (Table 2). The ability of bacteria to deceive any kind of conventional therapy has become apparent and pathogens resistant to one or more antibiotics are emerging and spreading worldwide [2]. Unnecessary use of antibiotics has further fuelled this problem. Correspondence/Reprint request: Prof. Girija S. Singh, Chemistry Department, University of Botswana, Gaborone Botswana. E-mail: singh_gs_57@yahoo.co.in

Girija S. Singh & Surendra N. Pandeya

Table 1. Key bacterial pathogens and related diseases.

Pathogens S. aureus S. pneumonia

Infectious Diseases Skin and wound infections, endocarditis Upper respiratory tract infection, pneumonia, sinusuitis, meningitis Pharyngitis, tonsillitis, skin and soft tissue infection S. pyogenes E. faecalis Endocarditis, urinary tract infection E. faecium Peritonitis, endocarditis, bacteremia E. coli Urinary tract infection, bacteremia, gastrointestinal infection Bacteremia, pneumonia K. neumoniae H. influenza Respiratory tract infection, sinusuitis, meningitis P. aeruginosa Bacteremia, burn infection M. tuberculosis Tuberculosis Table 2. Prevalence of resistance in hospital-acquired infections in USA (2004). Antibiotics Methicillin Vancomycin Cephalosporins (3rd generation)

Imipenem Quinolones (synthetic)

Pathogen S. aureus Enterococci Enterobacter sp. P. aeruginosa E. coli K. pneumoniae P. aeruginosa P. aeruginosa

Resistance (%) 59.5 28.5 31.1 31.9 5.8 20.6 21.4 29.5

The discovery of vancomycin resistant S. aureus (VRSA) and multiresistant S. aureus has generated worldwide concern. It has thus become evident that there is urgent need for novel antibacterial drugs with broader spectrum, lesser side effects, and without cross-resistance to antibiotics in use. More initiative is required to foster the responsible and appropriate use of antibiotic that is another issue. The traditional medicine system based on natural products continues to play an important role in treatment of many diseases especially the infectious diseases. According to the WHO estimation, approximately 80% of the world’s population relies mainly on traditional medicine for their primary health. Indian traditional medicine system relied on plants and their parts to treat various infectious diseases (Table 3). Hundreds of herbs are known to be used for various diseases including many infectious diseases. Acacia, garlic, turmeric, neem, ginger, clove, plum and pomegranate are only few to name.

Antibacterial natural products

Table 3. Some traditionally used herbs and their bioactivity. Plant Triphala i.e. three fruits viz. Haritaki (Terminalia chebula), Bibhitaka (Terminalia belerica) & Amalaki (Emblica officinalis) Turmeric (Curcuma longa)

Plant Parts 90 % ethanolic & aqueous extracts of Triphala

Bioactivity Significant activity on S. aureus, E. coli and P. areoginosa

Leaf extract

Fenugreek (Methika) (Trigonella faenum- graecum) Wildrue (Peganum harmal)

Seed extract

Antibacterial and antifungal activity Antibacterial and antifungal activity Antibacterial and antifungal activity Antibacterial and antifungal activity

Gokarna (Clitoria Ternatea) Sharapunkha (Tephrosia puropurea) Brahmi (Bacopa monnieri) Tulsi (Ocimum sanctum)

Aqueous seed extract Hexane & methanolic root extract Root extract Ethanolic extract of aerial parts Leaf extract

Antibacterial and antifungal activity Antihelminth activity Antibacterial and antifungal activity

Later on extracts from many of such plants and herbs have been screened by investigators in quest for potential and safer antibacterial agents [3-8]. Many comprehensive review articles have been published on the role of natural products in the discovery of antibacterial agents [9-12]. The plethora of literature in the area indicates an urgent need for a coordinated effort for meaningful research and discovery of novel antimicrobial agents. Most of the antibacterial agents in use today are either natural products or their semi-synthetic variations or improved subclasses. The success of natural products as guideposts to new drugs is most obvious in antibacterials (Table 4). Over 75% of new chemical entities submitted between 1984 and 2004 were based on natural product lead structures [13]. This article focuses on significance of selected natural products in discovery of new antibacterial agents with broader spectrum and least side effects. The sections are classified according to established structural characteristics. A brief historical development of each class is described followed by its mode of action. Selected recent examples of synthetic modification of natural antibiotics are discussed. More emphasis is given on β-lactam antibiotics as it consists of several subclasses such as penems, cephems etc. As mentioned in the preceding paragraph there are plethora of

Girija S. Singh & Surendra N. Pandeya

Table 4. Some potential antibiotics and their targets. Class

Representative example H N

β-Lactams

Target

Cell wall

N O

COOH

Penicillin G (1) OH

NMe2 OH

Polyketides

Protein biosynthesis

NH2 OH

OH OH O

Tetracycline (2) O2 N

NHCOCHCl2 OH

Phenylpropanoids

Protein biosynthesis

Chloramphenicol (3) NH2

Aminoglycosides

NH2 O HO

Protein biosynthesis

H2N NH2

OH OH

NH2

Tobramycin (4) O

Macrolides

Protein biosynthesis

HO OH

NMe2

OMeO O

Erythromycin A (5) OH NH

Glycopeptides

H3C

O CH3 O

HO O O NH

N H

Cl H N

OH Cl OH OH

N H

O H N O

O NH2

OH OH HO

Cell wall

OH O

Vancomycin

N H

H N

CH3 CH3

CH3

Antibacterial natural products

Table 4. Continued Streptogramins

Me N

NMe2

O O HN O O N

O O

Protein biosynthesis

N O NH

O NH OH

Pristinamycin IA (7) OH O N O

O O N O

Pristinamycin IIA (8)

literature available in the area and each class has been reviewed by several others from different angles it was considered pertinent to bring a concise account of the material for the convenience of readers.

2. β-Lactam antibiotics The group of antibiotics containing four-membered cyclic amides (azetidin-2-ones) is commonly known as β-lactam antibiotics. It is the first class of antibiotics to be used as a therapeutic treatment for bacterial infections (Figure 1). The first member penicillin was discovered by Fleming from the cultures of Penicillium notatum in 1928. Since then this group has maintained its charm among synthetic and medicinal chemists [14,15]. About half of the antibacterial drugs prescribed today belong to this class. Their broad antibacterial spectrum, clinical efficacy and excellent safety profile make them preeminent in pharmaceutical drug discovery. As a result of extensive research cephalosporins have reached their fourth generation (cefepime). The main approaches in design of the new cephem derivatives involve structural modifications at positions C-3 and C-7, and the development of cephem prodrugs. The compounds with a methoxy [16], carbamoyloxy [17] or heteroaryl ring such as tetrazole [18] or thiazole [19] in the C-3 side chain are known to have potent antibacterial activity.

Girija S. Singh & Surendra N. Pandeya OH H

H N

R O

N O

CO2H

OH H

O N

oxapenem

H N

R O

N oxacephem

H N

R R

CO2H

Cl CO2H

N O

H2N

N CO2-

Cefepime

H N

R N

OMe H N

S CO2H

CO2H sulbactam

carbapenem

CO2H

oxapenam

NH2 OH H

O S

O CO2H

penem

penam

carbacephem

S N

OAc

O CO2H cephem

H N

R O

N SO H 3

monobactam

Figure 1

Natural penicillin G, the first therapeutic antibiotic and lead structure of this class still had a few critical features such as narrow antibacterial spectrum, instability in acidic and alkaline environments, limited solubility and pronounced sensitivity to hydrolyze by bacterial penicillase enzymes that are needed to be improved. For about thirty years, penicillins (penams) and cephalosporins (cephems) remained the only examples of β-lactam antibiotics. Many related subgroups such as the monobactams (aztreonam), oxacephems (moxalactam), carbacephems (loracarbef), oxapenams (clavulanic acid), penems (faropenem), carbapenems (imipenem), and oxapenems (AM-112) were discovered during 1970s and 1980s either from microbes or by synthetic efforts. Numerous structural variations of these βlactam scaffolds provided derivatives with increased potency, low host toxicity, improved physicochemical and pharmacokinetic profiles. The β-lactam antibacterials act on bacteria by inhibiting the final step of the bacterial cell wall biosynthesis. Although several mechanisms might be operating in this inhibition, the most important is probably the inhibition of the terminal peptidoglycan cross-linking. Bacteria have a cytoplasmic membrane similar to that of eukaryotes. This membrane is surrounded by a periplasmic space, which is in turn enclosed by a peptidoglycan layer, and finally the outer membrane. The peptidoglycan layer is a cross-linked polymer that forms a net-like structure, which provides structural rigidity to

Antibacterial natural products

the organism, and allows it to survive in mediums to which it may be strongly hypertonic. As a bacterium grows, a series of covalent cross-links must be formed between adjoining peptidoglycan strands in the cell wall. These cross-links are stitched together by transpeptidase enzymes in the cell membrane through the replacement of a terminal D-alanine unit on one peptidoglycan strand with a glycine residue on a neighboring peptidoglycan strand. The initial cleavage of the D-alanine residue by transpeptidase occurs by a nucleophilic addition of an active site serine onto the amide functionality, as shown in Scheme 1. In a subsequent amidation step, the resulting enzyme-linked peptidoglycan is converted to the cross-linked material, which releases serine for further catalysis. Penicillins, cephalosporins and related β-lactam drugs possess an unusual ability to interrupt this crucial cross-linking process by an irreversible acylation of the hydroxy group of the catalytic serine unit within the enzyme active site resulting in the formation of a catalytically inactive stable enzyme-drug adduct (Scheme 2). The net result is decrease in the number of cross-linked residues within the cell wall making it weak and prone to rupture. Inhibition of the transpeptidase thus inhibits the bacterial growth. The sequences leading to cidal action are still not clearly understood. Nicks may be produced at the growth site of the cell wall. If these nicks are sufficiently severe, the protoplast may protrude into the medium and burst resulting in bacterial death. The major limitation to the potentials of β-lactam antibacterials is the ability of bacteria to produce a family of enzymes called β-lactamases. These enzymes hydrolyze the β-lactam ring which is required for antibacterial activity. There are different types of β -lactamases and their efficacy in

ngtp

H N

CO2-

O N H

ngtp

H N

O O Me enzyme-serine

ngtp

H N

N H

H N ptgn O

enzyme serine

ptgn = Peptidoglycan

enzyme serine

Scheme 1 ROCHN

ROCHN

S N

CO2H OH

enzyme-serine

HN OO

CO2H enzyme-serine

Scheme 2

Bacterial-death

Girija S. Singh & Surendra N. Pandeya

hydrolyzing the ring varies widely. There are four distinct classes of βlactamases, of which class A enzymes are the most common. In order to counter the hydrolysis by β-lactamases, some antibiotics are administered in combination with a β-lactamase inhibitor drug. For example, amoxicillin is administered in combination with clavulanic acid, itself also a β-lactam (oxapenam). However, the discovery of new variants of β-lactamases, which are resistant to known β-lactamase inhibitors, has caused great concern worldwide. The major thrust areas in research on β-lactams have been the development of new stereoselective methodologies to construct the β-lactam ring, and structural modifications in compounds, especially carbapenems and cephems with known activity, to design and develop new molecules with i) a broad spectrum of activity, specially against resistant strains and, ii) least side effects. The succeeding paragraphs describe the synthesis of some new cephems. The biological activity is discussed in selected examples. A series of β-[(Z)-2-(2-aminothiazol-4-yl)-2-hydoxyiminoacetamido]-3[(E) and (Z)-2-substituted vinyl] cephalosporin derivatives 9,10 have been synthesized using palladium-catalyzed coupling reaction of a 3methanesulfonoxy-3-cephem and an E substituted vinyl stannane (Scheme 3) or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an aldehyde (Scheme 4) as key steps [20]. OH N H N

N O H2N

S N

O CO2H

9,10

R = H (Cefdinir), -CH2CH2OMe, -CH2CH2OTMS, -CH2CH2OH, CH2CH2OCONH2, CH2CH2OAc a O

e O N

N N

Antibacterial natural products OHCHN

71 OHCHN

n-Bu3Sn

a, b, c N

OMs CO2CHPh2

H2N

R CO2CHPh2

R CO2CHPh2 c

H2N

vi), vii)

COCl

iii)

S OAc N

H N

O H2N

BocHN

R CO2CHPh2

OAc

N N

S N

ii)

CO2CHPh2

iv), v) CO2CHPh2 iv)

TFA H2N

N 1. iii) H2N

H N

OH N

COCl

OAc

H2N

2. v)

CO2CHPh2

CO2H

9a,c-e

Reagents and conditions: i) Pd(CH3CN)2Cl2, LiBr, DMF; ii) cHCl, MeOH; iii) BSA, CH2Cl2; iv) TFA, anisole, CH2Cl2; v) NaHCO3, NH4Cl, MeOH-H2O; vi) Boc2O, MSA, THF; vii) MeCOCl, Et3N, CH2Cl2

Scheme 3 BocHN O

BocHN

R-CHO a,b,f,m

S PPh3I

BocHN

S N

CO2CHPh2

R CO2CHPh2

CO2CHPh2

OH N

a,e,m

a, e-m b

1. iv) or v) 2. vi) 3. vii)

c H N

N O H2N

j,m j,m

S N

ii) iii)

9j,m = E isomerCO2H 10a,c,d,f-l = Z isomer Reagents and conditions: i) a. 1N NaOH, aq. NaCl, CH2Cl2, b. separation; ii) cHCl, MeOH; iii) a. Cl3CONCO, CH2Cl2, b. SiO2, CHCl3, MeOH; iv) TFA, anisole, CH2Cl2; v) HCO2H, cHCl; vi) BSA or MSA, (Z)-2-(2-aminothiazol-4-yl)-2-acetoxyiminoacetyl chloride hydrochloride, CH2Cl2; vii) NaHCO3, NH4Cl, MeOH, H2O

Scheme 4

The research findings at Lilly in the nineteen seventies that 2,5-dichlorophenylthioacetamido at C-7 as a lipophilic side chain conferred excellent Gram-(+) activity to the cephem class have been exploited further [21]. Through a series of optimization at C-3 and C-7, four cephalosporins 11, 12, 13 and 14 possessing a 2,5-dichlorophenylthioacetamido group at C-7 and a polar thiopyridinium group at C-3 with potent in vitro and in vivo

Girija S. Singh & Surendra N. Pandeya

anti-MRSA activity have been reported [22]. The C-3 thiopyridinium ring was substituted with amino acid and pyruvic acid groups that were designed to provide aqueous solubility as required for IV formulation. These compounds have excellent in vitro activity against a variety of Gram-(+) bacteria including resistant strains such as penicillin-resistant S. pneumoniae, methicillin-resistant S. epidermitis and S. haemolyticus (Table 5). Furthermore, all of them were efficacious in a systemic murine model of infection with PD5os ranging from 4.8-9.6 mg/kg. The aqueous solubility of 13 and 14 was much more (23 and 40 mg/mL, respectively at pH 7) in comparison to 11 (2-3 mg/mL at pH 7 and at room temperature). H N

R O

S N

H N

R S

O CO2

N O

CO2H NH3

11, R = diClPh (double zwitterion) 12, R = diClpyr

O2C

N O

CO2

13, R = diClPh 14, R = diClpyr

Table 5. Antibacterial Activity of Cephalosporin Derivatives. Organism S. aureus/Hetero MR S. aureus/ + 50% calf serum S. aureus/Hetero MR S. aureus/Hetero MR S. aureus /Homo MR S. aureus /Hetero MR S. aureus /Homo MR S. aureus /Homo MR S. aureus /Homo MR S. aureus / MR, PPBP2a IC50 (μg/mL)

A No. A27218

11 0.5

12 1

13 0.5

14 1

M 32

IM 1

A27218

0.5

A27217

0.5

A25795

128

A27223

128

A27223

A27621

A27295

128

A27226

A27225

128

4.5

100

MIC in μg/mL, MR = methicillin-resistant, P- = penicillin negative, M = methicillin, IM = imipenem, NT = not tested.

Antibacterial natural products

The in vitro and in vivo activity of 7-α-methoxy-cephems and 7-αmethoxy-oxacephems 15-18 and their demethoxy congeners 15a-18a on H. felis and H. pylori, human pathogens associated with type B gastritis, peptic ulcer disease and gastric cancer have been studied that showed the significance of 7-α-methoxy substituents in dealing with these bacteria [23]. The in vivo antibacterial activity was studied on a mouse helibacter infection model after oral administration, in which mice were infected with H. felis. All of the compounds except 18 and 18a exhibited very similar MICs for both H. felis (0.25-0.5 mg/L) and H. pylori (0.5-1.0 mg/L). Compounds 18 and 18a had lower MIC for H. felis (0.13 mg/L) than for H. pylori (1.0-2.0 mg/L). Even though the MICs of all four pairs of compounds were within 1to 2-fold dilution for H. felis and H. pylori, the 7-α-methoxy compounds were at least 4-fold more active at bacterial eradication than their demethoxy counterparts (Table 6). Intravenous administration of flomoxef resulted in extremely low eradication activity compared with oral administration. These results, together with the fact that flomoxef is not absorbed orally, indicated that the compound had direct access to the bacteria in the stomach after oral administration. H Y N

R1 O

X N

O 15-18

Compounds 15 Flomoxef

X O

15a Dimethoxyflomoxef O

R2 CO2H

Y OMe

F F

16 1-thiaflomoxef

OMe

16a Dimethoxy-1thiaflomoxef 17 Cefmetazole 17a dimethoxycefmetazole 18 M-1

OMe

18a H-1

N N

Girija S. Singh & Surendra N. Pandeya

The activity of RWJ-54428, a new parenteral cephalosporin originally developed by the R. W. Johnson Pharmaceutical Research Institute, NJ, USA, against recent isolates of Gram-(+) bacteria, including staphylococci with decreased susceptibility to vancomycin 6 has been reported [24]. This compound has been shown to be active against a wide range of multiply resistant Gram-(+) pathogens, including oxacillin-resistant S. aureus (MRSA), E. faecalis (MIC90 = 0.5 mg/L), vancomycin-resistant E. faecalis (MIC90 = 0.25 mg/L), and penicillinresistant pneumococci and streptococci (MIC90 = 1 mg/L). The only group of organisms for which the MIC90 was >2 mg/L was ampicillin-resistant E. faecium. Reinert and coworkers have evaluated cefditoren against penicillin-susceptible strains of S. pneumoniae and penicillin-intermediate strains of S. pneumoniae isolated from patients with respiratory tract infections and suggested it as a promising agent for the treatment of infections caused by pneumococci with reduced penicillin susceptibility [25]. Gerber and coworkers have reported cefepime, considered as fourth generation cephalosporin, having an excellent CSF penetration with level ranging between 10 and 16 mg/L after two intravenous injections (100 mg/kg). The bactericidal activity of cefepime was superior to ceftriaxone and vancomycin in the treatment of rabbits with meningitis caused by an isolate highly resistant to penicillin [26]. Schito and coworkers have studied the activity of many cephalosporins against some common respiratory tract pathogens such as S. pneumoniae, H. influenzae and M. catarrhalis etc., isolated from the patients in Italy, Spain, and Austria [27]. Cefpodoxime has been reported as a suitable choice for use. Table 6. Clearance and Eradication Doses of compound. Compounds 15 Flomoxef 15a Demethoxyflomoxef 16 1-thia-flomoxef 16a Demethoxy-1-thiaflomoxef 17 Cefmetazole 17a Demethoxycefmetazole 18 M-1 18a H-1 Amoxicillin 15 Flomoxef (iv) a

50% Clearance dose (mg/kg/dose)a 1.00 4.00 0.97 3.84 1.00 5.79 0.47 3.59 1.92 >15.0

50% Eradication dose (mg/kg/dose)b 3.67 17.4 3.38 >60 3.67 58.8 Not tested Not tested 9.12 58.8

Compounds were administered orally twice a day for 1 day and mice were killed on the following day. b Compounds were administered orally twice a day for 5 days and mice were killed after 14 days.

Antibacterial natural products

Some amides 19 and imines 20 containing 5-nitrofuryl and 3-methoxy-2nitrophenyl groups from 7-β-aminocephalosporanic acid and 7-βaminodesacetoxycephalosporanic acid have been synthesized and evaluated for antibacterial activity [28]. Many compounds, especially with 5-nitrofuryl moiety, exhibited an activity equal to or better than those of ampicillin or cephalexin against the majority of Gram-(+) organisms tested. None of the compounds showed appreciable activity against E. coli. ArOCHN O

S N

CH2OCOMe CO2Na

ArHC N O

S N

CH2OCOMe CO2Na

Ishikawa and coworkers are involved in the development of new cefozopran (CZOP) 21 derivatives for use against MRSA [29,30]. They observed that the CZOP with lipophilic alkoxyimino groups in the C-7 acyl moiety showed potent anti-MRSA activity. Cyclopentyloxyimino derivatives with amino-based substituent(s) in the C-3’ azole moiety had anti-MRSA activity comparable to vancomycin. In order to further increase the activity they have modified the C-3 linked spacers of cephem derivatives bearing a 1-methylimidazo-[1,2-b]-pyridazinium-6-yl group at the C-3’ position and a 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-cyclopentyloxyiminoacetyl group at the C-7 position [31]. They have found that the optimal spacers are (E)-2vinyl and (E)-2-thiovinyl groups. The anti-MRSA activity of the compounds 22a,b bearing these spacers were 16-32 times higher than CZOP. Taking these two spacers they have modified the alkoxyimino group in the C-7 acyl moiety and the 1-alkylimidazo-[1,2-b]-pyridazinium moiety at C-3’ and discovered compound 22c with anti-MRSA activity comparable to vancomycin both in vitro and in vivo, high affinity (IC50 = 2.7 mg/mL) for PBP2’ of MRSA and potent activity against Gram-(-) bacteria as well. Hakimelahi and coworkers are working on the concept of using antibacterial prodrugs. They have reported the synthesis, antibacterial and β-lactamase inhibitor activity of clavunate derivatives of amoxicillin. The compounds screened by them showed better antibacterial activity than amoxicillin and clavulanic acid combination augmentin [32]. Since the discovery of thienamycin (23a) from the fermentation broth of soil bacteria Streptomyces cattleya in 1976 several carbapenem derivatives have been synthesized and evaluated for their antibacterial activity. Thienamycin itself has excellent antibacterial activity against both Gram-(+) and Gram-(-) bacteria, and is resistant to β -lactamases. Numerous methods

Girija S. Singh & Surendra N. Pandeya

OMe H N

S N

N H2N

OMe H N

S N

H2N

CO2Na

CO222a

OR H N

S N

N Me

H2N O

N N

CO2-

22b, R1 = cyclopentyl; R2 = Me 65b 22c, R1 = CH2F; R2 = Me 65c

are described in literature for the total synthesis of thienamycin [33]. Currently, two 1-H carbapenems, imipenem (23b) and panipenem (23c), and one βmethyl carbapenem, meropenem (24) are available in the market for clinical use [34-36]. Although arbapenems have a broad antimicrobial spectrum and potent bactericidal activity [37] most of them have some limitations as well from the view point of clinical application. For example, imipenem is unstable to the renal dehydropeptidase-I (DHP-I) and has epileptic side effect. Meropenem has good stability to DHP-I due to steric hindrance of β-methyl group at C-1 and an excellent spectrum against Gram-(-)bacteria, but it is relatively less active against Gram-(+) bacteria than imipenem. The studies on carbapenems from a pharmaceutical point of view in the previous decade have been devoted mainly to the synthesis and evaluation of 1-β-methylcarbapenem derivatives as antibacterials. New methodologies for construction of the carbapenem skeleton are under investigation by Mori and

OH H H

X R

N O

CO2H

X = 23a-c: H; 24: Me R = 23a: -SCH2CH2NH2 (Thienamycin) 23b: -SCH2CH2NHCH=NH (Imipenem) 23c:

Me (Panipenem) NH

Me2NOC 24 S

(Meropenem)

Antibacterial natural products

Kozawa [38,39]. A new method involving palladium-catalyzed C-N bond forming reaction (Scheme 5) in azetidinone 25 leading to the synthesis of carbapenem 26 with a carboxylic group on C-3 of the five–membered ring has been reported by them [40]. 10 mol% Pd(OAc)2 15 mol% DP Ephos PhMe, 100 oC, base (2 equiv.)

OSi H H

NH O

OSi H H

CO2Et

CO2Et 26

Scheme 5

3. Macrolide and ketolide antibiotics Macrolide antibiotics belong to the subgroup of polyketide natural products and constitute an important therapeutic class. They act against community-acquired respiratory infections such as community-acquired pneumonia, acute bacterial exacerbations of chronic bronchitis, acute sinusitis and tonsillitis [41,42]. Macrolides account for 20% of all the antibiotics prescribed. The principal representative of macrolides erythromycin A 5 was first isolated from Streptomyces erythreus at the Lilly in 1952 [43]. Its absolute configuration was established by NMR spectroscopic studies and X-ray crystallographic data [44,45]. It helps against the major respiratory pathogens, is considered safe and is widely prescribed for children. However, it has a limited antibacterial spectrum and limited solubility in acidic medium. The second generation macrolide antibiotics, 27-29 have gradually replaced erythromycin A because of their higher potency, broader spectrum of activity, improved physicochemical and pharmacokinetic profiles, and attenuated side effects [46]. However, similar to erythromycin A, the second generation variants also have poor activity against macrolide resistant pathoges. The mode of action of macrolide antibiotics involves blocking bacterial protein biosynthesis by binding to the 23S ribosomal RNA of the 50S subunit and interfering with the elongation of nascent peptide chains during translation [47]. Located in domain V, near the peptyl transferase site, macrolide antibiotics obstruct the peptide exit tunnel without affecting peptidyl transferase activity. Ketolides [48] are derived from 14-membered macrolides by removal of L-cladinone under acidic conditions and selective oxidation of the resulting 3-hydroxy group to the corresponding carbonyl group. The first semisynthetic

Girija S. Singh & Surendra N. Pandeya O N

OMe

HO OH

NMe2

OMeO O

NMe2

OMeO O

O OH

OH O Roxithromycin (28)

Clarithromycin (27) Me N

N OH

HO OH

O O

NMe2 O

OMeO O O

Azithromycin (29)

O O

NMe2 O

Telithromycin (30)

ketolide RU-64004 (HMR 3004) was synthesized at Roussel Uclaf [49]. This ketolide was stable in acidic media, showed good intracellular penetration, and demonstrated potent activity against erythromycin A resistant and penicillin resistant streptococci and H. influenzae. Systematic SAR studies led to the discovery of several ketolide lead structures such as telithromycin, cethromycin, and EP-013420 with potent activity and improved pharmacokinetic profile. Telithromycin 30 was the first ketolide to be approved in Europe (2001), Japan (2003) and in the US (2004) for the once daily oral dose for treatment of respiratory tract infections. It was synthesized from clarithromycin in eight steps [50].

4. Lincosamides Lincomycin 31 and its semi-synthetic congener clindamycin 32 were introduced into clinical use as oral antibiotics in 1960 and 1969, respectively [51]. They exhibit a similar spectrum as macrolides, including activity against most gram-(+) organisms and the anaerobes, but not the Gram-(-) and enterococci [52]. Now a day they are not in much use due to their limited antibacterial spectrum, the emergence of resistance, and severe side effects of this class.

Antibacterial natural products

Clindamycin is a semi-synthetic derivative of the natural product lincomycin, which is produced by fermentation of Streptomyces lincolnensis [53]. This transformation involves selective transformation of only one secalcoholic group of the three present (Scheme 6).

Me N

HO O N H HO

SMe

NCS, PPh3 THF, Δ, 18h

Me N

Cl N H HO

SMe OH

84% 32

Scheme 6

The mode of action of lincosamide involves binding to the ribosome and inhibiting bacterial protein synthesis. Specifically, macrolides, lincosamides, and streptogramin B type antibiotics bind to adjacent sites on the 50S ribosomal subunit. The complexes of bacterial ribosomes with these antibiotics have been studied by X-ray crystallography [54,55]. Several methods for semi-synthetic modification of lincomycin and clindamycin have been published in addition to the methodologies developed during the total synthesis of natural product [56,57]. Substitution of the 7-hydroxy group by a methyl group in conjunction with novel amides resulted into discovery of VIC-105404 33 and VIC-105555 34 (Scheme 7) [58,59]. The latter compound has rapidly been progressed into preclinical development. Hopefully these achievements will translate into clinical benefit.

5. Furanomycin L-(+)-Furanomycin 35 is a low molecular weight (157 g/M) antibacterial natural product. It is a α-amino acid isolated by Katagiri and coworkers in 1967 from the fermentation broth of Streptomyces threomyceticu L-803 (ATCC15795) [60]. It inhibits the growth of bacteria such as M. tuberculosis, E. coli, B. subtilis, and some Shigella- and Salmonella species in the μM range. Its initially assigned absolute configuration was later on revised to (+)(αS,2R,5S) by synthesis starting from D-glucose and by X-ray crystallographic study of the N-acetyl derivative [61-63]. Furanomycin 35 is accepted as a substrate by isoleucyl aminoacyl-t-RNA synthetase and its antibacterial activity results from a substitution for isoleucine during the bacterial protein translation [64]. Therefore, the antibacterial activity of furanomycin is antagonized by isoleucine. Furanomycin hampers the formation

Girija S. Singh & Surendra N. Pandeya Me N

HO O

TMSO O

N H HO

SMe

i), ii), iii)

BocHN

TMSO

SMe

iv), v)

OTMS OTMS

BocHN TMSO

SMe

H2N

vi), vii), viii)

BocHN

OTMS OTMS

Boc N CO2H

SMe

H3C(H2C)4 ix), x), xi)

xii), xiii) NH

Boc N

HN BocHN

SMe

HN BocHN

H3C(H2C)4

OH OH

33 VIC-10555

34 VIC-105404

SMe OH

Reagents and conditions: i) N2H4, H2O; II) (Boc)2O, Et3N, MeOH; iii) BSTFA, Et3N, DMF; iv) DMSO, (COCl)2, Et3N, CH2Cl2, -70 - 40oC; (v) PPh3Me+Br-, t-BuOK; vi) Dowex H+, MeOH; vii) H2 (65psi), Pd/C; viii) TFA/H2O (9:1); ix) HBTU, Et3N; x) TFA/H2O (9:1); xi) oxirane, Et3N; xii) HBTU, Et3N; xiii) TFA/H2O (9:1)

Scheme 7

of isoleucine-tRNA in E. coli, whereas other aminoacyl-tRNA are not affected [65,66]. Aminoacyl-tRNA synthetases are essential in all living organisms and have attracted considerable interest as novel targets in bacterial protein synthesis [67-70].

O H2N

CO2H 35

O H2N

CO2H 36

H2N

CO2H 37

Antibacterial natural products

Several approaches have been developed towards the synthesis of Furanomycin 35. Many syntheses start from carbohydrates such as D-glucose [51], D-ribose [71], D-glucosamine [72], L-xylose [73], and D-mannitol [74]. Glycine [75], serine [76], furans [77], and dimethyl L-tartarate [78], have also been used as substrate for the synthesis of Furanomycin 35. Approaches involving amino acids are appealing as they involve lesser steps. A SAR was studied using many synthetic isomers and derivatives of natural products. Unfortunately, all of them showed either no activity or poor activity against a panel of selected Gram-(+) and Gram-(-) pathogens including S. aureus, and E. coli. Only L-(+)-dihydrofuranomycin 36 showed borderline MIC (32-64 μg/mL) against S. aureus. The chiral carbon analogue 37 exhibited weak antibacterial activity (4 μg/mL) against an efflux-pum-deficient E. coli. Furanomycin, thus, proved an insufficient lead and could not be a valuable compound as a starting point for an antibacterial drug discovery program.

6. Pyrrolidinedione antibacterials Komura and coworkers in 1987 isolated natural peptide antibiotic compound andrimid 38 from cultures of a symbiont of the brown planthopper Nilaparvata lugens [79]. Later on moiramide 39 was discovered in a marine isolate of Pseudomonas fluorescens obtained from a tunicate collected in Moira Sound at Prince of Wales Island, Alaska [80]. The structures of these metabolites contain four characteristic elements: a pyrrolidinedione head group, a valine derived β-ketoamide, a (S)-β-phenylalanine moiety, and an N-terminal polyunsaturated fatty acid. Various diastereoselective and asymmetric total syntheses of andrimid and moiramides are known that allow ready access to these antibiotics [81,82]. Pyrrolidinedione antibiotics act by inhibiting the first committed step in bacterial fatty acid biosynthesis, a reaction catalyzed by the carboxyltransferase subunit of the multimeric bacterial enzyme acetyl-CoA carboxylase [83]. For most living organisms, fatty acid biosynthesis is a vital metabolic process, but the pathway in bacteria and mammals are different. Acetyl-CoA carboxylase is essential for microbial growth and is broadly conserved amongst bacteria [84].

O O

O NH

n N H

N H

n = 2: Andrimid 38; n = 1 moiramide B 39

Girija S. Singh & Surendra N. Pandeya

Using (S)-(-)-methylsuccinic acid 40 as a substrate, the synthesis of a pyrrolidinedione antibacterial 41 is shown in Scheme 8. Through this route, and also by solid-phase synthesis starting with polymer bound (S)-β-phenylalanine, a wide variety of pyrrolidinedione antibacterial are known in literature [85,86]. Broad structural variations were tolerated at the fatty acid side chain without adversely affecting the bioactivity. Inhibitory values (IC50) remained in the nM range for the E. coli and S. aureus acetyl-CoA carboxylase enzymes with polar and lipophilic side chains as well. CO2H

O i), ii)

HO2C

CO2H 40

86%

iii)

iv), v), vi)

NHBoc N OBn

N OBn 40%

BocHN

68%

O O NC

N H

vii)

NH H2N

CO2H

66%

O N H

NH N H

Reagents and conditions: i) MeCOCl, 4h, 60 oC; ii) O-benzylhydroxylamine, CDI, CH2Cl2, 12h, rt; iii) 1. NBoc-(2S)-cyclopentyl glycine, CDI, THF; 2. LiHMDS, THF, 15 min, -65 oC; 3. conc. aq. NH4Cl,- 65 oC - rt; iv) H2, Pd/C (10%), EtOH, 1h, rt; v) 2'-bromoacetophenone, Et3N, cat DMAP, MeCN, 20h, rt; vi) 4N HCl in 1,4-dioxane, 2h, rt; vii) HATU, iPr2EtN, CH2Cl2, DMF, 10h, 0 oC - rt.

Scheme 8

Apparently, the side chain is not involved in key interactions with the enzyme and could be used for tuning the physicochemical profile. On the other hand, the nature of the side chain has a significant effect on antibacterial activity. A comparison of compounds 41a and 41b demonstrated that despite excellent target activity of 41a and the benefit of a polar substituent for other parameters, such as solubility, reasonable lipophilicity was required for penetration into bacterial cells and for good MIC values. Replacement of the (S)-β-phenylalanine by non-aromatic β-amino acids led to a loss in activity. On the other hand significant activity was observed by varying the lead’s β-ketoamide part, for example, by replacing (S)-valine with (2S)-cyclopentyl glycine, whereas aromatic amino acids in this position rendered the molecule inactive.

Antibacterial natural products

O O HO2C

O N H

O O

NH N H

O N H

41a

NH N H

41b

7. Tetrahydropyrimidinone antibiotics The titled class of antibiotics was first isolated by scientist from the Takeda Foundation in Japan from Flexibacter species found in soil samples of the Nachi mountain area of the Wakayama prefecture of Japan [87]. The structures of TAN-1057A-D 42-45 were disclosed in 1993 [88]. The epimeric tetrahydropyrimidinones TAN-1057A/B 42,43 were isolated from Flexibacter species, PK-74, whereas the epimeric dioxo diazepans TAN 1057C/D 44,45 resulted from Flexibacter species PK-176. Total synthesis endeavors and medicinal chemistry optimization focused on TAN-1057A/B 42,43 due to instability problem of TAN 1057C/D 44,45. The antibacterial activity of TAN1057A 42 was studied in detail. Its in vitro antibacterial activity against Gram-(+) organisms such as S. aureus and S. pneumoniae was mediocre under standard conditions (6.25-12.5 μg/mL). However, its in vivo activity was reported superior to vancomycin and imipenem in a murine S. aureus sepsis model. NH O NH H2N

N H

Me N 5 NH2 O

42 TAN-1057A (5S) 43 TAN-1057B (5R)

H2N NH O N H

NH2

N H

N Me 2

NH H N HN 44 TAN-1057C (2R) O 45 TAN-1057D (2S) O

NH2

TAN-1057A/B acts by blocking bacterial protein biosynthesis [89]. The detailed studies found that it inhibited bacterial growth through binding to the 50S subunit of ribosomes [90]. The synthesis of TAN-1057A/B 42,43 was reported by Yuan and Williams which involved a rather linear approach starting from triple-protected β-homoarginine (Scheme 9) [91]. Meijere and coworkers published a more convergent synthesis shortly after [92]. The attractive antibacterial properties and the structure of natural antibiotics TAN-1057A/B attracted several synthetic research groups. Systematic SAR exploration required novel routes to β-lysine and β-homolysine derivatives. The synthetic pyrimidinones (n = 1) 46 and (n = 2) 47 readily accessible on large scale exhibited improved cytotoxicity and tolerability while retaining eminent potency of the natural compound.

Girija S. Singh & Surendra N. Pandeya

8. Biphenomycin An antibiotic with unusual biological properties, LL-AF283a was isolated by fermentation of S. filipinesis at the Lederle Laboratories in 1967 [93,94]. Later on the discovery of peptide antibiotic biphenomycin A 48 (WS-43708A) was reported by scientists from Fujisawa in 1984 [95-97]. In 1991, Border and coworkers found that the two antibiotics were identical [98]. Biphenomycins have unique structure with a cyclic tripeptide containing a biphenyl moiety in a 15-membered ring. The in vitro activity of biphenomycin A was almost limited to Cornbacterium xerosis. It could not affect the growth of other bacteria, such as S. aureus, E. coli, or S. pyogenes up to 200 mg/mL. However, it was NH Z

N H

N Z

i), ii), iii)

+ Me

H N

51%

N H

Me N

N Z

NH O

79%

NHMe

Me N

H N

NHBoc

O O

N H CO2H

Z NPhth CO2tBu NH2 O

iv)

NH Z

N H

Me N

N Z

H N

SMe

NHBoc

, v) NHZ

52%

CO2H

NH O

Z NH Z

vi), vii) TAN-1057A/ TAN-1057B 42,43

N Z

N H

66%

NH Z

MeS

N H

NHZ

Reagents and conditions: i) BOPCl, 16h; ii) MeNH2, MeOH, 5min; iii) TFA/anisole 25:1, 0 oC to rt, 1h; iv) Boc2O, Et3N, H2O/dioxane (1:1), 16h; v) EDC, DMAP, CH2Cl2, 16h; vi) TFA/anisole 10:1, 15 min, evaporation, then Et3N, THF, 10 min; vii) PdCl2, H2, MeOH/CH2Cl2, 2:1, 99%.

Scheme 9 Me N 5

H2N

NH O

NH2 O

N H

O 46 n = 1, 47 n = 2

OH H N

H2N O

R CO2H N H OH

NH2 48 R = OH, Biphenomycin A 49 R = H, Biphenomycin B

NH2

Antibacterial natural products

highly effective in vivo in a murine sepsis model. It protected mice from an otherwise lethal infection against S. aureus Smith (ED50 1.0 mg/kg) and was five times more effective than vancomycin on subcutaneous administration. The reason for this discrepancy between in vitro and in vivo activity is yet not clear. Although both the biphenomycins and vancomycin have a biphenyl group, there is no evidence of binding of to the cell-wall analogues of N-AcD-Ala-D-Ala. Instead, bacterial protein biosynthesis appeared to be the target of these antibacterials [99]. The biphenomycins represented an attractive starting point for an optimization program in medicinal chemistry and need for its synthesis was felt. The first total synthesis of Biphenomycin B 49 was reported by Schmidt and coworkers in 1991 [100]. Its sequence involved 1) synthesis of (S,S)isotyrosine, 2) formation of an ansa-tripeptide, 3) macrocyclization, and 4) removal of protecting groups (Scheme 10). However, besides the total synthesis of natural biphenomycins A and B, neither derivative nor close analogues had been prepared. A first series of simplified amide and ester derivatives, including derivatization at the peptide backbone have been reported recently [101,85]. The novel congeners 50 and 51 of biphenomycin B 49, showed improved in vitro activity (Table 7) [102]. Table 7. In vitro antibacterial activity of 50 and 51 against Gram-(+) pathogens, MIC (μg/mL). Compd No. 50 51

S. aureus 1.5 0.1

E. faecalis 1.0 3.0

B. catarrhalis 1.0 1.0

Thus, the route for the total synthesis of natural compounds and their congeners with improved in vitro efficacy has been established. A further insight into the molecular target of these compounds will definitely pave the way for further development.

OH H N

H2N O

N H O OH

NH2

H N

H2N O

NH2 50

N H O OH NH2

OMe

Girija S. Singh & Surendra N. Pandeya

BnO

OBn i), ii)

CHO

BnO

OBn

94%

BocHN

CO2Me

vii), viii), ix 88%

BocHN CO2TMSE

ZHN

CO2Bn

BocHN

CO2Bn

CO2H O

OBn

OHC

69%

BnO

iii), iv), v), vi) BnO

BnO

x), xi)

N Z

ZHN ETMSO

71%

OBn O BocHN O

N H O

CO2Bn

N Z xii), xiii) HO

OH H N

H2N O

H CO2H N H OH

81%

BnO xiv), xv)

ZHN

60% C6F5O

NH2 Biphenomycin B (49)

OBn O BocHN O

N H OH

CO2Bn

NHZ

Reagents and conditions: i) methyl N-tert-butoxycarbonyl(dimethoxyphosphoryl)glycinate, LiCl, DBU, MeCN, rt, 1h; ii) Et3N, C, EtOH/CHCl3, (1:1), rt, 2days; iii) LiOH, H2O, dioxane, rt, 12h; iv) [Rh(cod)dipamp)]BF4, H2, MeOH, rt, 72h; v) BnOH, DCC, DMAP, EtOAc, - 15 to 20oC, 12h; vi) PPTS, acetone, H2O, Δ, 6h; vii) Nbenzyloxycarbonyl(dimethoxuphosphoryl)glycine tromethyl-silyl ester, LiCl, DBU, MeCN, rt, 2h; viii) Et3N, C, EtOH/CHCl3, (1:1), rt, 2 days; ix) [Rh(cod)dipamp)]BF4, H2, MeOH, rt, 72h; x) HCl, dioxane, 20oC, 2h; xi) EDC, HOBt, CH2Cl2, 15-20oC, 14h; xii) AcOH/H2O (9:1), 50oC; xiii) Bu4NF, DMF, rt, 1h, C6F5OH, EDC, CH2Cl2, - 15 to 20oC, 14h; xiv) HCl, dioxane/CH2Cl2 (1:1) 0oC, evaporation, CHCl3, NaHCO3, 20oC, 5min; xv) trimethylsilyl trifluromethanesulfonate, thioanisole, TFA, rt, 30 min.

Scheme 10

9. Tuberactinomycins and capreomycins Tuberactinomycins 52-55 and capreomycins 56-59 are closely related cyclic homopentapeptides. The first member viomycin (Tuberactiinomycins B) 52 was discovered in 1951 [103] and marketed by Ciba and Pfizer as a tuberculostatic agent in the 1960s. The capreomycins were isolated from the fermentation of Streptomyces capreolus as a four-component mixture, with 56 and 57 as the major and 58 and 59 as the minor components [104]. Both subclasses showed good activity against Mycobacteria including multi-drug resistant strains but had only limited activity against other species [105].

Antibacterial natural products

R1 H2N

OH H N

H N

N H

NH2 O

NH O

O H N

H N

O NH R2

N H

NH2

52 R1 = H, R2 = O, Tuberactinomycin B; 53 R1 = R2 = OH, Tuberactinomycin A (viomycin); 54 R1 = OH, R2 = H, Tuberactinomycin N; 55 R1 = H, R2 = H, Tuberactinomycin O

Tuberactinomycins exert their antibacterial activity as potent inhibitors of the translation step of prokaryotic protein biosynthesis by inhibiting both the initiation and elongation steps. A detailed report on the interaction of tuberactinomycin at the target level illustrated how these compounds interacted with RNA [106]. When tested against a panel of M. tuberculosis strains in vitro, capreomycins compared favorably with streptomycin, cycloserine, and kanamycin [107]. No cross resistance of tuberactinomycins with kanamycin, lividomycin, or paronomycin was observed [108]. The in vivo efficacy of these compounds was low after oral dosing, but good after subcutaneous administration in experimental murine tuberculosis models [109]. Although tuberactinomycins were not devoid of toxicological problems their toxicity profile after i.v. and preoral administration was quite favorable [110]. These biological features warranted further evaluation of this class for their clinical use as antitubercular agents. R1

O H2N

H N

N H NH O

O H N O NH

N H O H N

NH2 O

N NH H 56 R1 = OH, R2 = β-lysyl, Capreomycin IA; 57 R1 = H, R2 = β-lysyl, Capreomycin IB; 58 R1 = OH, R2 = H, Capreomycin IIA; 59 R1 = R2 = H, Capreomycin IIB

Girija S. Singh & Surendra N. Pandeya

Most of the derivatives prepared for biological testing have been obtained through fermentation and semisynthesis. The acetylation of the terminal amino group 60 or both amino groups of β-lysine (Scheme 11) led to a complete loss in activity [111]. Acylation with uncharged or acidic amino acids at the same position also produced inactive compounds whereas introduction of a basic amino acid 61 maintained the original MIC [112]. Similarly, blocking of the serine hydroxyl groups 62 left the activity unaltered [113]. Surprisingly, hydrolysis of the urea functionality produced 63 with comparable in vitro activity as observed for viomycin 52. Oxidation of 52 yielded the inactive bisamide 64 [114]. Finally, reductive opening of the capreomycidine ring of 52 forming 65 led to a complete loss in activity [115]. O P(OiPr)2 O

O H2N NH2

H N

N H

NH2 O

i) N H

iv)

N H

NH2 O

NH2 O O 60 vii)

NH2 N H

HO 65

vi)

OH H N

H N

H2N

O 62

ii), iii)

O P(OiPr)2

N H

O HN

H N

OH O H N

NH2

O NH

52 HN

OH O 64

Reagents and conditions: i) N-acetoxysuccinimide, Et3N, carbonate buffer, dioxane, 1h; ii) Z-D-Orn(Z)OSuc, Et3N, carbonate buffer, THF, 0oC, 12h; iii) H2, Pd, DMF; v) HOH; vi) KMnO4 ; vii) NaBH4

Scheme 11

Antibacterial natural products

Similar modifications of tuberactinomycine N 54, tuberactinomycine O 55, and capreomycins did not result in an improved activity against Micobacteria or an extension of the antibacterial spectrum. The 3,4-dichlorophenylamino analogue 66 of viomycine exhibited good MICs against the animal pathogens P. multocida (MIC = 0.39 μg/mL) but only mediocre activity against E. faecium and E. faecalis (MIC = 25 and 12.5 μg/mL, respectively). Further variation of the substituted ureido analogues of capreomycines IA/IB (mixture of 56 and 57) yielded novel compounds with activities against several multidrug resistant gram-(+) pathogens and gram-(-) E.coli [116]. Despite proven in vitro and in vivo efficacy of the novel analogues no further clinical development in this class has taken place.

H2N

OH H N

H N

N H

NH2 O

NH O

O HN

H N

OH O H N

O NH

Cl Cl

N H

10. Glycopeptide antibiotics Vancomycine 6, the first gylcopeptide introduced into clinical practice in 1959 was isolated from Streptomyces orientalis from soil samples by the Lilly in the mid 1950s [117]. Its structure was unequivocally established in the early 80s [118, 119]. Teicoplanin 67 is the only additional member of this class that is available for human use. Both drugs are unaltered natural antibiotics of the large dalbaheptide group that is produced by various actinomycetes. Their common structural element is a linear heptapeptide backbone (configuration R,R,S,R,R,S,S) in which some aromatic amino acids residues are cross linked (biphenyl and diphenyl ether motives) and build a rigid concave shape. Glycopeptides inhibit bacterial cell walls biosynthesis by recognizing and strongly binding to the L-Lys-D-Ala-D-Ala termini of peptidoglycan precursor strands at the external side of the membrane. In this way, transpeptidases are prevented from executing their cross linking job [120].

Girija S. Singh & Surendra N. Pandeya

Glycopeptides antibiotics are restricted to treating gram-(+) infections as they cannot penetrate the outer membrane of gram-(-) bacteria. With the rise of MRSA infections in hospitals, vancomycines became the antibiotic of last resort but, due to its frequent use, resistant gram-(+) pathogens, in particular vancomycine-resistant enterococci (VRE) has emerged and worryingly spread. By 2003, more than half of the clinical VRE isolates in the US had become resistant to glycopeptides. Three semi-synthetic second generation drugs oritavancin (Ly-333328) [121], dalbavancin (Bi397) [122], and telavancin (TD-6424) [123] have been advanced to clinical developments [124]. O OH O

N H

OH OH

O Cl

OH HO HO

O Cl

O O

NH O

O HN

H N

N H

H N

N H

NH2

N H

CO2H HO HO

O OH

OH OH O

OH OH

11. Lysobactins The lysobactins are good examples of structurally exciting natural products that were isolated from urban soil organisms. Ketanosin B 68 was isolated from the fermentation broth of Lysobacter sp. SC-14076 (ATCC 53042) by scientists from Squibb [125,126]. So far its total synthesis has not

Antibacterial natural products

been published. Recently, katanosin A has been found as a minor metabolite of the Lysobacter sp. ATCC53042 starin [127]. Lysobactin and katanosin A are highly active against Gram-(+) bacteria such as staphylococci and enterococci. Their excellent in vitro antibacterial activity was maintained in vancomycin-resistant enterococci. Promising therapeutic in vivo efficacy has been demonstrated in a systemic murine S. aureus infection model (ED50 = 1.8 mg/kg, i. v., CFU = 105) [128]. The primary target of lysobactin antibiotics appears to be the bacterial biosynthesis. These compounds inhibit consumption of the cell-wall precursor [14C]GlcNAc, a very good indicator for interference with the peptidoglycan biosynthesis. The inhibition of peptidoglycan formation is most likely induced by the binding of lysobactin to lipid intermediates (not through binding to biosynthetic enzymes) that occurs as biosynthetic precursors downstream of the muramyl pentapeptide. The lysobactins are interesting antibacterial lead structures with promising in vitro activity and in vivo efficacy. So far, the knowledge about this class is based on these natural products and some semi-synthetic Edman derivatives. A preliminary SAR has been established for the amino acid position 1 within the liner segment. Further investigations need to be done for a real assessment of the potential of this class.

H2N O

OH H N

H N

O O

H2N

N H

O O

N H NH2

HN NH 68

Girija S. Singh & Surendra N. Pandeya

12. Enopeptin depsipeptide antibiotics The name of the family was derived from two depsipeptides enopeptin A 69 and depsipeptides enopeptin B 70 isolated in 1991, from a culture broth of Streptomyces sp. RK-1051, found in a soil sample collected in Tsuruoka city of Japan [129]. They consist of a 16-membered lactone ring made up of five (S)-amino acids and a lipophilic polyene side chain attached to the serine N-terminus [130]. About a decade ago the Eli Lilly published the isolation of a similar depsipeptide antibiotic A54556 71,72, a complex of eight depsipeptide factors A-H, which was produced by aerobic fermentation of Streptomyces hawaiiensis (NRRL 15010) [131]. Mode of action studies with B. subtilis demonstrated impaired bacterial cell division and induction of filamentation. It has been shown by using RG techniques that lead structures inhibited bacterial growth by binding to caseine lytic protease [132,133]. R O N O O

NH N

O N

O N H

HO O

N H

69 R = Me, Enopeptin A 70 R = H, Enopeptin B 71 R = Me, A54556A 72 R = H, A54556B

The natural enopeptin depsipeptides antibiotics had promising in vitro activity against enterococci and streptococci but only moderate in vitro potency against staphylococci and were inactive against Gram-(-) bacteria. Both lead structures were not effective in vivo in standard lethal bacterial infection models in mice and their ADME profile was critical. Their chemical stability proved to be rather limited. Their solubility was insufficient for parenteral application and they were readily cleared from the body. The in vivo antibacterial activity of some natural and synthetic enopeptins is described in Table 8.

Antibacterial natural products

O R O

O O

N H

C6H11

N H

O 77

73-76

O O NH

O N

O N H

C4H9

O N

O N H

C4H9 O

O 79, 80

Table 8. Antibacterial activity of some natural and synthetic enopeptins. No. 71 72 73 74 75 76 77 78 79 80

R Me H H 3-F 3,5-F2 3,4,5-F3 H COCH2NMe2

S. aureus 8 16 > 64 1 0.5 8 1 ≤ 0.125 0.25 0.5

S. pneumoniae 0.5 1 > 64 0.25 ≤ 0.125 2 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

E. faecium 1 2 > 64 ≤ 0.125 ≤ 0.125 2 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

E. faecalis 1 2 > 64 0.125 0.125 1 ≤ 0.125 ≤ 0.125 ≤ 0.125 ≤ 0.125

13. Tetracycline antibiotics Tetracyclins belong to the group of polyketides. They are old-known class of broad-spectrum antibiotics whose use has been reduced in recent times with the onset of bacterial resistance. They consist of an octahydrotetracene-2-carboxamide skeleton [134]. The first member of the group, chlorotetracycline 81 was discovered in 1940s from a golden-colored fungus-like, soil-dwelling bacterium called Streptomyces aureofaciens. Soon after oxytetracycline 82 was discovered from a similar soil bacterium called Streptomyce srimosus. The structure of oxytetracycline was determined by

Girija S. Singh & Surendra N. Pandeya

Woodword and coworkers [135], which led to its synthesis by Conover and coworkers. Doxycycline 83 is the most commonly known semi-synthetic drug of this class. In 2005, tigecycline [136] 84 belonging to the subclass of glycylcyclines was introduced to treat infections that were resistant to other antimicrobics including conventional tetracyclins [137]. Newer versions of tetracyclins are currently in trials. Tetracyclins inhibit the protein biosynthesis by inhibiting the binding of aminoacyl-tRNA to the mRNAribosome complex. Tetracyclins have also been found to inhibit matrix metalloproteinase. This mechanism does not contribute to their antibiotic activity, but has led to extensive research on chemically modified tetracyclins. Tetracyclins inhibit cell growth by inhibiting translation. It binds to the 16S part of the 30S ribosomal subunit and prevents the aminoacyl tRNA from binding to the A site of the ribosome. The binding is reversible in nature [138].

O OH

OH OH

O OH

OH OH

H2N

H2N O N

OH Cl

OH 82

OH O

OH O OH

O NH2

H OH

N H N

O N H

OH O

OH O 84

OH OH O

NH2

Antibacterial natural products

14. Aminoglycoside antibiotics Aminoglycoside antibiotics are among the oldest known class of antibiotics [139, 140]. Although much has been written on this class it would be worth mentioning the representatives of this class streptomycin 85, neomycin 86 and gentamycin 87 for completeness of the article. The well-known streptomycin was isolated by Waksman and coworkers in 1944 from cultures of Streptomyces griseus. It was the first effective drug for treatment of tuberculosis. Aminoglycosides are often administered into veins or muscle to treat serious bacterial infections. Some aminoglycosides are also used orally to treat intestinal infections or topically to treat eye infections. Among several modes of actions some are protein biosynthesis inhibitors and thus compromising the structure of the bacterial cell-wall. The mechanism of resistance to aminoglycoside antibiotics has been investigated [141]. Recent advances in structure, molecular mechanism, SAR, aminoglycoside mimetics have been reviewed by Silva and Carvalk [142]. The current efforts to develop new aminoglycoside derivatives with modification and reconstruction on each sugar ring and advances in SAR have also been reviewed by Zhou and coworkers [143]. The recent emergence of infections due to Gram-(-) bacterial strains with advanced patterns of antimicrobial resistance has prompted reevaluation of the use of aminoglycoside antibacterial agents [144]. This revived interest has brought back to light the debate on the two major issues related to these compounds, namely the spectrum of antimicrobial susceptibility and toxicity. Current evidences show that aminoglycosides retain activity against the majority of Gram-(-) clinical bacterial isolates in many parts of the world. OH

Me OHC O

HO O HO HO OH

H2N

NH2

N O

O N H

NH2 NH2

NH2

H2N

O OH

NH2

NH2 H2N OH Me HN Me

H2N

O OH O H2N

HO OH 87

O NH2

Me NH2

NH2

Girija S. Singh & Surendra N. Pandeya

However, the relatively frequent occurrence of nephrotoxicity and ototoxicity during aminoglycoside treatment makes physicians reluctant to use these drugs in everyday practice. Recent advances in the understanding of the effect of various dosage schedules of aminoglycosides on toxicity have provided a partial solution to this problem, although more research still needs to be done in order to overcome this problem entirely [145].

15. Concluding remarks Pathogenic bacteria are increasingly evading the standard treatment for antibacterial infections as resistance to multiple antibiotics is spreading worldwide. Resistant pathogens lead to higher expenditure on treatments due to extended stay in hospitals and expensive medicines. There is an urgent need for a sustainable supply of new, potential and safer antibacterial drugs having no cross-resistance to currently used antibiotics. Nature so far has been proved the treasure of potential remedies for diseases. It is more relevant for treatment of infectious diseases. Most of the antibacterials in use today could be discovered on the basis of information obtained from the study of natural products from microbes, marines and plants. Medicinal chemists do systematic studies and provide the tools for optimization of natural products to obtain drug molecules with improved pharmacokinetic, physicochemical and toxicological properties. The diligent selection of natural antibiotics lead structures for medicinal chemistry programs and guideposts for valid targets can reveal pathways for future therapies. Advance studies have been conducted on many classes in order to identify the drug target and mechanism of action. There is still a plenty of scope even within the known antibiotics. Many old classes have not been thoroughly investigated and only partial SAR information is available on their backbone structures. Macrolides are an excellent example that many old classes have not been completely explored. Undoubtedly, more could be done to fully exploit the weapons against bacteria. β-Lactams continue to be one of the most important classes in antibacterial research and development. Their efficacy and compatibility has allowed their broad therapeutic application. Further investigations on second generation lincosamides might be useful for successful treatment of infections by enterococci. Biphenomycins appear promising for the future studies. Further work is anticipated on lysobactins for a real assessment of this class.

References 1. 2.

Nathan, C. Nature, 2004, 431, 899. Clark, N. M., Hershberger, E., Zervosc, M. J., Lynch, J. P. Curr. Opin. Crit. Care., 2003, 9, 403.

Antibacterial natural products

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Girija S. Singh & Surendra N. Pandeya

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 103-120 ISBN: 978-81-308-0448-4

3. Anti-tubercular activity of natural products: Recent developments L. N. Rogoza, N. F. Salakhutdinov and G. A. Tolstikov Department of Chemistry, Russian Acad Sci, Siberian Div, Vorozhtsov Inst Organ Chem Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia

Abstract. An increasing incidence of deaths due to tuberculosis and the known drawbacks of the current existing drugs including the emergence of multi drug-resistant strains have led to a renewed interest in the discovery of new anti-tubercular agents. The recent researches focused on natural products have shown a useful way to obtain a potentially rich source of drug candidates. This review covers the most active naturally occurring compounds with antitubercular properties at minimal inhibitory concentrations (MICs) of 5 mg/mL or less. The literature from January 2001 to 2009 is reviewed. The compounds are presented in order of chemical type, namely alkynes, heterocyclic compounds, phenols and quinones, peptides, alkaloids, terpenoids and steroids.

1. Introduction Tuberculosis is a chronic infectious disease, one of the major enemies of the humanity from times immemorial. Today it still remains one of the most serious medical and social problems. It is responsible for 3 million deaths per year and around 8 million cases of first-recorded disease. The advances in the chemotherapy of tuberculosis in the mid-20th century have recently given way to anxiety over the evolution of drug resistance based on the genetically Correspondence/Reprint request: Prof. L. N. Rogoza, Department of Chemistry, Russian Acad Sci, Siberian Div, Vorozhtsov Inst Organ Chem, Pr Akad Lavrenteva 9, Novosibirsk 630090, Russia E-mail: rogoza@nioch.nsc.ru

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fixed mutations of M. tuberculosis. Moreover, nearly all drugs used for the treatment of tuberculosis and possessing different mechanisms of activity are able to cause adverse side effects on the human organism. Therefore, it is extremely important to search for new, low-toxic substances superior to the available drugs in their activity and efficiency. This primarily concerns the agents possessing activity against M. tuberculosis strains with multidrug resistance. Modern tuberculosis is generally associated with M. tuberculosis and M. bovis, mycobacteria that are pathogenic to the human organism. Because of slow growth and pathogenicity of M. tuberculosis H37Rv, many research groups used fast-growing and/or nonpathogenic mycobacteria including M. tuberculosis H37Ra, M. smegmatis, M. aurum, and others as organisms to be tested. The antimycobacterial activity was also investigated on M. avium and M. intracellulare, which cause bird tuberculosis and are associated with human diseases in advanced countries (AIDS patients and immunocompromised individuals), to find compounds with a wide range of activity. A special group of research works includes investigations on M. tuberculosis clinical isolates and strains possessing multidrug resistance. Multidrug-resistant tuberculosis (MDRTB) is strictly defined as M. tuberculosis strains showing resistance simultaneously against isoniazid and rifampicin [1, 2]. Tuberculosis with a different drug resistance (DDRTB) involves M. tuberculosis strains displaying mono- or polyresistance not including associated resistance against isoniazid and rifampicin [3]. The M. tuberculosis strains may be sensitive (inhibited by first series drugs such as isoniazid) or resistant (not inhibited by isoniazid). Since researchers use different analytical procedures and/or organisms under test, care should be taken in comparing the biological activities obtained by different authors. The review covers publications from 2001 to first half 2009; the selected structures have minimum inhibiting concentrations (MIC) of 5 μg/mL or less. Due to this limitation, the most effective compounds were analyzed within one review. For better insight into the "structure-property" relationship, we occasionally gave structures with higher MIC values. The review includes the introduction section, two chapters on synthetic and natural compounds with an antimycobacterial activity, and the conclusions section. To reveal possible "structure-activity" relationships, we grouped the data according to chemical structures.

2. Alkynes and heterocyclic compounds The metabolite of several strains of the endophytic fungus of the genus Phomopsis, 3-nitropropionic acid (1), actively inhibited growth of

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M. tuberculosis H37Ra (MIC 0.4 μg/mL). Although the high neurotoxicity of this compound was a hindrance to its use as a pharmaceutical, it could be used as a model for the synthesis of new inhibitors of isocitratelyase, an enzyme essential to the catabolism of fatty acids and virulence of M. tuberculosis [4]. Linoleic acid (2) that inhibits growth of M. phlei (MIC 2 μg/mL) is extracted from the plant Humulus lupulus. An example of polyacetylene compounds is 3S,8R stereoisomer (3) isolated from Anethum graveolens and having MIC 2-4 μg/mL when tested on a group of fast growing mycobacteria (M. fortuitum ATCC 6841, M smegmatis ATCC 14468, M. phlei ATCC 11758, M. aurum Pasteur Institute 104482, and M. abscessus ATCC 19977; for ethambutol, MIC 0.5-4 μg/mL) [5]. However, cytotoxicity of this class of polyacetylene compounds can lower the interest in their biological activity [6]. Compounds (4a) and (4b), the synthetic analogs of the natural antibiotic thiolactomycin, inhibit growth of M. tuberculosis with MIC 1-16 μg/mL, including drug-resistant strains [7]. OH HOOC

NO2

CO2H 3

(2)

(1)

C7H15 (3)

O O

C8H17

R (4a), R = O(CH2)10Br (4b), R = O(CH2)8SCH2COOMe

(5)

(6)

Investigation of the components of the plant Cinnamomum kotoense led to the isolation of a number of compounds, of which lincomolide В (5) with MIC 2.8 μg/mL had the highest antituberculosis activity [8]. Micromolide (6), which is a γ-lactone derivative of oleic acid, was isolated from the stem bark of Micromelum hirsutum and has MIC 1.5 μg/mL against M. tuberculosis (H37Rv). Further evaluation of activity on J774 mice cells infected with a more virulent strain of M. tuberculosis Erdman gave MIC 5.6 μg/mL [9]. 2-Substituted furans (7a,b) and (8a,b) isolated from the roots of Polyalthia evecta possess activity against M. tuberculosis (MIC 3.1 and 6.25 μg/mL, respectively) [10]. The synthesized natural compound pamamycin-607 (9) inhibits growth of M. bovis BCG, M. smegmatis and M. tuberculosis (MIC 0.54.7 μg/mL). It does not show cross resistance to isoniazid and rifampicin [11].

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(CH2)13 O

n OR

(7a) R = H (7b) R = Me

(8a) n = 5 R = C15H31 (8b) n = 3 R = (CH2)13OH

NMe2 O O

O O

(9)

3. Phenols and quinones Phenylpropanoids (10) and (11), metabolites of Pimpinella sp., inhibit growth of a number of mycobacteria, including M. intracellulare, M. smegmatis, M. aurum, and M. phlei (MIC 1.25-10 μg/mL) [12]. The tricyclic diphenol ether engelhardion (12) is very active against M. tuberculosis H37Rv (MIC 0.2 μg/mL) [13]. (-)-4-Hydroxy-1-tetralone (13, MIC 4.0 μg/mL) & 3-methoxycarboxy-1,5-dihydroxyantraquinone (15, MIC 3.125 μg/mL) were isolated as an antituberculosis component of Engelhardia roxburghiana [13]. As is known, the level of the intra- and extracellular inhibition of M. tuberculosis by 7-methyljuglon (14) (MIC 0.5 μg/mL) extracted from the plant Е. natalenis, is comparable to that of streptomycin and ethambutol (MIC 1 and 2 μg/mL, respectively). Its derivatives, namely, 5-hydroxy-, 5-alkoxy-, and 5-acetoxy-8-substituted naphthoquinones, are less active (MIC 2.5- >20 μg/mL) and possess low antituberculosis selectivity, probably because of their nonspecific activity with various disulfide reductases found in mammal cells. Optimization of the specificity of these compounds for mycothiol disulfide reductase, which is one of the several biological targets for the antituberculosis activity of naphthoquinones of this structure, is required [14].

Anti-tubercular natural products

107 HO

OH O

O O

H O

(10)

(11)

(12) O

OCH3

H OH

(13)

O (15)

(14)

Marine metabolites pseudopyronines A and B (16a,b, MIC 0.78-3.125 μg/mL) inhibit the growth M. tuberculosis H37Rv [15]. Pyrone (17, MIC 4 μg/mL) is a component of Piper sanctum that is active against M. tuberculosis H37Rv [16]. Ferulenol (18a) isolated from the Sardinian giant fennel Ferula communis is effective against M. smegmatis (MIC 0.5 μg/mL), as well as M. fortuitum, M. phlei and M. aurum (MIC 2 μg/mL). The analogs of this compound, (18b-d), were isolated from the same plant; compound (18b) with a benzyloxy group retained its activity against M. smegmatis and M. phlei, and, to a lesser extent, against M. fortuitum and M. aurum, while the activity of (18c) and (18d) with the hydroxy and acetoxy groups is considerably lower [17]. Ostruthin (19), the metabolite of Peucedanum ostruthin Koch, inhibit the growth M. aurum (МIC 3.4 μg/mL) [18]. OH

OMe

O (16a), R = C5H11 (16b), R = C7H15

(17)

OH R O

O (18a), R = H (18b), R = OBz (18c), R = OH (18d), R = OAc

HO (19)

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OH R

O O

O OH

O O (21)

R1 (20g) R = O (20h) R = OH

(20a) R1 = OH, R2 = H (20b) R1 = OH, R2 = H, Δ saturated (20c) R1 = O, R2 = H (20d) R1 = O, R2 = H, Δ saturated (20e) R1 = O, R2 = OMe, Δ saturated (20f) R1 = O, R2 = OH, Δ saturated

O HO

OAc

(22c)

O AcO

OAc

(22a) R = Ac (22b) R = H

Compounds (20a-h), isolated from the lichen fungus Microsphaeropsis sp., show different activities against M. tuberculosis H37Ra (MIC 25, 3.12, 3.12–6.25, 6.25, 12.5, 25, 1.56-3.12, 50 μg/mL, respectively), but are also characterized by cytotoxicity [19]. The dibenzofuran derivative, usnic acid (21), which is a secondary metabolite of lichen, inhibits growth of M. tuberculosis (MIC 2.5–5 μg/mL) [20]. One of the xanthone dimers isolated from the endophytic fungus of the genus Phomopsis, phomoxanthone A (22a), is very active against M. tuberculosis H37Ra (0.5 μg/mL), while its deacetylated derivative (22b) is inactive. Phomoxanthone B (22c) is less active (MIC 6.25 μg/mL). Both active compounds are cytotoxic [21]. The anthraquinone celastramycin B (23), isolated from the unknown species Streptomyces, is active against M. Vaccae (MIC 3.1 μg/mL) [22]. The anti-HIV agent (+)-calanolide A (24) was tested for the antituberculosis activity; a combination of the anti-HIV and antituberculosis activities in one agent is very attractive in view of the concurrence of these diseases. This compound, isolated from the tropical tree Calophyllum lanigerum, also has an antituberculosis activity against M. tuberculosis (MIC 3.13 μg/mL) and a number of drug resistant strains (MIC 8–16 μg/mL) [23].

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Cl O O (23)

OH (24)

4. Peptides Four cyclic peptides, namely, enniatins H (25a), I (25b), B (25c), and B4 (25d), which are the components of the pathogenic fungus Verticillium hemipterigenum, inhibit growth of M. tuberculosis H37Ra (MIC 3.12–6.25 μg/mL) [24]. Syringomycin E (26), isolated from Pseudomonas syringae pv. Syringae, is active against M. smegmatis (MIC 1.5 μg/mL) [25]. The metabolite of Nocardia sp. (ATCC 202099), namely, the thiazole peptide nocathiacin (27) shows activity against M. tuberculosis ATCC 35828, M. avium A26778, and M. avium A26640 (MIC ≤ 0.008, 0.06, and 0.25 μg/mL, respectively). Unfortunately, compounds from this class typically show poor pharmacokinetics and solubility (the latter problem can be solved by synthesizing analogs with higher solubility in water) [26].

5. Alkaloids Two compounds, namely, the known antibiotic pyrrolnitrin (28) and banegasine (29), isolated from the zoobacterium Aristabacter necator, act synergetically against M. smegmatis (MIC (29) >0.5 μg/mL, (28) 0.3 μg/mL, (28) + (29) 0.075 μg/mL) [27]. Their analog celastramycin A (30), which is a dichloropyrrole metabolite of the Streptomyces strain, has a broad spectrum of antimycobacterial activity (MIC 0.05-3.1 μg/mL against M. smegmatis, M. aurum, M. vaccae, and M. fortuitum) [22]. The bis-1-oxaquinolizidine alkaloid (−)-araguspongine С (32), isolated from the sea sponge Xestospongia exigua, inhibits growth of M. tuberculosis H37Rv (MIC 1.9 μg/mL) [28]. In the series of quinolone alkaloids (33a-d), isolated from the fruits of Evodia rutaecarpa, compounds with usaturated aliphatic chains at 2-position exhibited better antimycobacterial activity as compared with saturated chain compounds [18]. Agelasine E (33a) and agelasine D (33b) were previously isolated from the sea sponge Agelas nakamurai. While agelasine Е is inactive, its methoxy analogs (33c-g), having different terpenoid side chains,

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O N

O O

N O

NH2

H N

NH2

N OH

O O

(CH2)2NH2

(26):R = NHCOCH2CH(OH)(CH2)8CH3 OH

N H

S H N

H N O

H N

CH2OH

N H

Me2N

(25a) R1 = R2 = R3 = R5 = R6 = i-Pr; R4 = s-Bu; (25b) R1 = R2 = R3 = R6 = i-Pr; R4 = R5 = s-Bu; (25c) R1 = R2 = R3 = R4 = R5 = R6 = i-Pr; (25d) R1 = i-Bu; R2 = R3 = R4 = R5 = R6 = i-Pr

MeO

H2N(H2C)2

CH(OH)CH2Cl R O CH(OH)CO2H

H N

O N

O (27)

NH2

NO2

NH Cl

N H

N H (28)

COOH

(30)

(29)

(31a) N

(31b) (31c)

(31d) HO H

O N

N O

H OH (32)

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demonstrate high activity against M. tuberculosis H37Rv (MIC 3.13, 1.56, and 3.13 μg/mL respectively). Possibly, the presence of an alkoxy group at the terminal nitrogen atom is a very important factor for the antimycobacterial activity of these compounds. However, there is only slight difference between the activities of agelasine D (33b) and its alkoxy derivatives (33f) and (33g) [29]. It is interesting that the simpler analog of the compounds, 9-methyladenine (33h), has MIC of 6.25 μg/mL [30]. The tetracyclic alkaloid cryptolepine (34a), isolated from Cryptolepis sanguinolenta, is active against a number of fast-growing mycobacteria, including M. aurum (MIC 2 μg/mL), M. phlei (MIC 4 μg/mL), and M. fortuitum (MIC 16 μg/mL) [31]. Metabolite of Allium neapolitanium (34b) R1O NH2

R N

N N

NH2

N N

Me (33a) R = c (33b) R = d

(33c) R = a, R1 = Me (33d) R = b, R1 = Me (33e) R = c, R1 = Me (33f) R = d, R1 = Me (33g) R = d, R1 = t-Bu

(33h)

a n

Me N R N H

(34a)

(34b), R = H (34c), R = OH

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displayed enhanced activity against the M. smegmatis (mc22700), when compared with that for (34c) (MIC 2-8 μg/mL). Furthermore, the activity of (34b) was greater against M. smegmatis (mc2 2700) than M. smegmatis (ATCC 14468) (MIC 16 μg/mL for (34c) and 8 μg/mL for (34b) [32]. The metabolites of the Thailand pathogenic fungus Hirsutella nivea BCC 2594 hirsutellones A-D (35a-d) inhibit growth of M. tuberculosis H37Ra (MIC 0.78, 3.125, 0.78, 0.78 μg/mL, respectively). Compound (35d) exhibits moderate in vitro cytotoxicity, while other compounds are less cytotoxic [33]. Hirsutellone F (35e), which is a new dimer alkaloid isolated, together with known hirsutellones A, B, and C, from the seeds of the fungus Trichoderma sp. BCC 7579 shows a weaker antituberculosis activity against M. tuberculosis H37Ra (MIC 3.12 μg/mL) than hirsutellones А, В, and С [34]. The known alkaloid ecteinascidin 770 (36a) and the new one, ecteinascidin 786 (36b), both isolated from Ecteinascidia thurstoni, inhibit growth of M. tuberculosis H37Ra (MIC 0.1 and 1.6 μg/mL, respectively) [35]. Manzamine alkaloids isolated from sea sponges are promising from the viewpoint of their antituberculosis activity. Manzamines А (37a), Е (37c), and F (37d) and their hydroxyl derivatives 6-hydroxymanzamine Е (37e) and

O NH

(35a) R = H (35b) R = Me

NH N H

(35d) H

(35c)

O O

(35e)

NH O

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(+)-8-hydroxymanzamine А (37b) show activity against M. tuberculosis H37Rv (MIC 1.5, 3.8, 2.6, 0.4, and 0.9 μg/mL, respectively) [36]. Manadomanzamines A (38a) and B (38b) inhibit growth of M. tuberculosis H37Rv (MIC 1.9 and 1.5 μg/mL, respectively) [37].

OMe R

HO OAc

N N

N H

N O O MeO

R1 N

O NH

HO (37a), R = R1 = H (37b), R = H, R1 = OH

(36a) R1 = CN, R2 = none (36b) R1 = CN, R2 = O

R N

N H

H R1

22 O

N H N

N H H H OH

N HN

O (37c), R = R1 = H (37d), R = H, R1 = OH (37e), R = OH, R1 = H

(38a), 22βH (38b), 22αH

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6. Terpenes Compound (39), isolated from Indigofera longeracemosa, is active against M. tuberculosis (MIC 0.38 μg/mL) [39]. Diterpenes (40) and (41) from Calceolaria pinnifolia [40], the structurally related lecheronol A (42), isolated from Sapium haematospermum (MIC 4 μg/mL) [40], and metabolite of Melica volkensii 6-hydroxyculactone (43) [18] have the same value of MIC against M. tuberculosis H37Rv. Ugandensidial (44, from Warbugia ugandensis) inhibit growth of M. aurum and M. phlei at this value of MIC [18].

OMe O

O OAc CH2OCOCH2COOH

MeOOCH2COCOH2C

(40)

(39)

(41)

OHC

OH CHO

O H OCOCH3

O H

(42)

(43)

(44)

The diterpenes diaportheines A (45a) and B (45b) were isolated from the fungus Diaporthe sp. Compound (45b) has antituberculosis activity against M. tuberculosis H37Ra (MIC 3.1 μg/mL) and cytotoxicity, while compound (45a) is much less active and cytotoxic (MIC 200 μg/mL) [41]. These data indicate that the presence of a carbonyl group is important for the antituberculosis activity. A metabolite of the African tree Combretum imberbe, traditionally used in folk medicine is imberbic acid (46), which shows activity against M. fortuitum (MIC 1.56 μg/mL) [42].

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HOOC HO OH O OH

OH R2 R1

HO Me

(46)

(45a) R1 = OH, R2 = H (45b) R1 + R2 = O

The chemical modifications of the parent structure of ursolic acid (at the C-3 position to cinnamate-based esters) resulted in an 4-fold increase in antimycobacterial activity ((47а), MIC 3.13 μg/mL for M. tuberculosis H37Ra, for ursolic acid MIC 12.5 μg/mL) [43].

COOH O CO R MeO

(47a) R = OAc (47b) R = OH

O R1

COOH

MeO2C MeO2C

(48)

(49a) R1 + R2 = O (49b) R1 = H, R2 = OH

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24 OH H H

(51) saturated (52) unsaturated

(50a) 24R (50b) 24S

O O

R1O

(54a) R1 = CO(CH2)16Me, R2 = Me (54b) R1 = H, R2 = Me (54c) R1 = H, R2 = Et

(53)

H H O

H CHO

(54d)

(55a) R = Me (55b) R = Et

Triterpene (48), isolated from Elateriospermum tapos, is active against M. tuberculosis H37Ra (MIC 3.13 μg/mL, for isoniazide and kanamicin sulfate MIC 0.05 & 1.25 μg/mL, respectively) [44]. Aegicerin (49a) and protoprimulagenin A (49b) were isolated from Aegiceras sp., Embelia Schimperi, and the Peruvian plant Clavija procera. Aegicerin (49a) was tested on 37 different strains of tuberculosis (MIC 1.6-3.1 μg/mL against one strain of H37Rv, 21 sensitive clinical strains, two clinical isolates resistant to isoniazid, and 13 MDR clinical strains). The inactivity of protoprimulagenin

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A (49b) (MIC 200 μg/mL) demonstrates that as in the case of (45a) and (45b), the presence of a carbonyl group is critical for the antituberculosis activity. For the first time, an oleane type triterpene shows uniformly high activity against a wide range of both sensitive and resistant strains. Regretfully, for many MDR strains, its excellent antituberculosis activity (for comparison, MIC is 4-32 μg/mL for isoniazid and 2-16 μg/mL for rifampicin) has not yet been effected [45].

7. Steroids Saringosterol, isolated from brown seaweeds Sargassum ringgoldianum and Lessonia nigrescens in the form of a 1:1 mixture of the 24R isomer (50a) and 24S isomer (50b), inhibits growth of M. tuberculosis H37Rv (MIC 0.25 μg/mL) and has low cytotoxicity. In pure form these isomers possess different levels of activity (MIC is 0.125 μg/mL for the 24R isomer and 1 μg/mL for the 24S isomer) [46]. Lipids that inhibit growth of M. tuberculosis H37Rv were isolated from an extract from Morinda citrifolia (Rubiaceae), traditionally used in folk medicine in the Philippines for the treatment of tuberculosis and respiratory diseases. The highest activity was found for a mixture of (51) and (52) (MIC <2.0 μg/mL for the 2:1 mixture) and endoperoxide (53) (MIC 2.5 μg/mL) [47]. Sterines (54a-d), isolated from an extract from the Argentinian plant Ruprechtia triflora, are active against M. tuberculosis (MIC 2-4 μg/mL) [39]. Synthetic analogues (55a,b) of 5(6→7) abeo-sterol from the Caribbean Sea sponge Svenzea zeai inhibit growth of M. tuberculosis H37Rv ATCC 27294 (MIC 3.8 & 3.9 μg/mL, respectively) but possess moderate cytotoxicity [48].

8. Conclusions More than 50 % of the medicines introduced in world medical practice are connected with natural compounds to some extent. It can be as native metabolites and synthetically the modified derivatives. Enthusiastic examples of discovery of natural compounds with remarkable pharmacological properties such as antitumoral metabolite taxol or antimalarial metabolite artemisinin and also tens other compounds of a plant and animal origin with various high biological activity allow to hope for prompt discovery of highly effective low-toxic natural compound which will be leader in struggle against a tuberculosis.

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References Bastian, I., Portaels, F. In Multidrug-Resistant Tuberculosis, Bastian, I., Portaels, F., Eds., Medicine and life, Moscow, 2003, p. 17. (Multidrug-Resistant Tuberculosis, Bastian, I., Portaels, F., Eds., Kluwer: Dortrecht, Neth., 2000). 2. Bastian, I., Portaels, F. In Multidrug-Resistant Tuberculosis, Bastian, I., Portaels, F., Eds., Medicine and life, Moscow, 2003, p. 21, 22. 3. Bastian, I., Portaels, F. In Multidrug-Resistant Tuberculosis, Bastian, I., Portaels, F., Eds., Medicine and life, Moscow, 2003, p. 23. 4. Chomcheon, P., Wiyakrutta, S., Sriubolmas, N., Ngamrojanavanich, N., Isarangkul, D., Kittakoop, P. J. Nat. Prod., 2005, 68, 1103-1105. 5. Stavri, M., Gibbons, S. Phytother. Res., 2005, 19, 938-941. 6. Bernart, M.W., Cardellina, J.H., Balaschak, M.S., Alexander, M.R., Shoemaker R.H., Boyd, M.R.. J. Nat. Prod., 1996, 59, 748-753. 7. Kamal, A., Ali Shaik, A., Sinha, R., Yadav, J.S., Arora, S.K. Bioorg. Med. Chem. Lett., 2005, 15, 1927-1929. 8. Chen, F.-C., Peng, C.-F., Tsai, I.-L., Chen, I.-S. J. Nat. Prod., 2005, 68, 1318-1323. 9. Ma, C., Case, R. J., Wang, Y., Zhang, H.-J., Tan, G.T., Hung, N.V., Cuong, N. M., Franzblau, S.G., Soejarto, D.D., Fong, H.H.S., Pauli, G.F. Planta Med., 2005, 71, 261-267. 10. Kanokmedhakul, S., Kanokmedhakul, K., Kantikeaw, I., Phonkerd, N. J. Nat. Prod., 2006, 69, 68-72. 11. Lefevre, P., Peirs, P., Braibant, M., Fauville-Dufaux, M., Vanhoof, R., Huygen, K., Wang, X.-M., Pogell, B., Wang, Y., Fischer, P., Metz, P., Content, J. J. Antimicrob. Chemother., 2004, 54, 824-827. 12. Tabanca, N., Bedir, E., Ferreira, D., Slade, D., Wedge, D.E., Jacob, M.R., Khan, S.I., Kirimer, N., Baser, K.H.C., Khan, I.A., Chem. Biodiversity, 2005, 2, 221-232. 13. Lin, W.-Y., Peng, C.-F., Tsai, I.-L., Chen, J.-J., Cheng, M.-J., Chen, I.-S. Planta Med., 2005, 71, 171-175. 14. Mahapatra, A., Mativandlela, S.P.N., Binneman, B., Fourie, P.B., Hamilton, C. J., M. Meyer, J.J., van der Kooy, F., Houghtond, P., Lalla, N. Bioorg. Med. Chem., 2007, 15, 7638-7646. 15. Demaray, J.A., Thuener, J.E., Dawson, M.N., Sucheck, S.J. Bioorg. Med. Chem. Lett., 2008, 18, 4868. 16. Mata, R., Morales, I., Perez, O., Rivero-Cruz, I., Acevedo, L., EnriquezMendoza, I., Bye, R., Franzblau, S., Timmermann, B. J. Nat.Prod., 2004, 67, 1961-1968. 17. Schinkovitz, A., Gibbons, S., Stavri, M., Cocksedge, M.J., Bucar, F. Planta Med., 2003, 69, 369-370. 18. Negi, A.S., Kumar, J.K., Luqman, S., Saikia, D., Khanuja, S.P.S. Med. Res. Rev., DOI 10.1002/med.20170. 19. Seephonkai, P., Isaka, M., Kittakoop, P., Palittapongarnpim, P., Kamchonwongpaisan, S., Tanticharoen, M., Thebtaranonth, Y. Planta Med., 2002, 68, 45-48. 1.

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 121-154 ISBN: 978-81-308-0448-4

4. Scope of natural products in fighting against leishmaniasis B. B. Mishra, R. R. Kale, V. Prasad, V. K. Tiwari and R. K. Singh Department of Chemistry & Biochemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Abstract. Leishmaniasis, a group of tropical diseases resulting from infection of macrophages by obligate intracellular parasites of genus Leishmania, is a major health problem worldwide. Growing incidence of resistance for the generic pentavalent antimony complex for treatment in endemic and non-endemic regions has seriously hampered their use. The second line drugs such as amphotericin B, paromomycin and miltefosine are the other alternatives, but they merely fulfill the requirements of a safe drug. The recent researches focused on natural products have shown a wise way to get a true and potentially rich source of drug candidates against leishmaniasis. The present review initially highlights the current status of leishmaniasis, synergy of the disease with HIV, therapeutic options available and in later sections summarizes natural products that have shown significant antileishmanial activities. In order to highlight any possible mechanism based action, the review has been organized according to chemical structural classes.

1. Introduction The Leishmania are Kinetoplastid protozoans that cause four main clinical syndromes: Cutaneous Leishmaniasis; Muco-cutaneous Leishmaniasis (also known as espundia); Visceral Leishmaniasis (VL; also known as kala-azar); and Difuse Leishmaniasis. Leishmaniasis continues to be one of the six entities Correspondence/Reprint request: Dr. Rakesh K Singh, Department of Chemistry & Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. E-mail: rakeshbhu@yahoo.com

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on the World Health Organization tropical disease list [1]. Leishmania species are transmitted by 30 species of sand fly and essentially requires two different hosts: an invertebrate insect vector, Phlebotomus (in the OldWorld) or Luztomiya (in the NewWorld) sandfly-mosquito and a vertebrate host (human, dog or even a wild vertebrate) [2]. Leishmaniasis is prevalent in tropical and temperate regions of world, ranging from rainforests in Central and South America to deserts in West Asia and the Middle East. Current epidemiological reports estimate about 350 million populations at risk with 12 million people affected worldwide, while 1.5-2 million new cases being recorded each year. The visceral leishmaniasis has an estimated incidence of 500,000 new cases and 60,000 deaths each year with more than 90 % of cases are centralized to India, Bangladesh, Nepal, Sudan, and Brazil [3]. There are a growing number of reports of Leishmania/human immunodeficiency virus (HIV) co-infections across the world. LeishmaniaHIV co-infection has been globally controlled in Southern Europe since 1997 by highly active anti retroviral therapy (HAART), but it appears to be an increasing problem in other countries such as Ethopia, Sudan, Brazil or India where both infections are becoming more and more prevalent [4]. The situation is particularly alarming in southern Europe, where 50-75% of adult VL cases are HIV positive and among the 45 million people infected by HIV worldwide, an estimated one-third lives in the zones of endemic Leishmania infections [5]. To date, the greatest prevalence of Leishmania/HIV co-infection has been in the Mediterranean basin. Among more than 2,000 cases notified to the WHO, 90 % of them belong to Spain, Italy, France and Portugal [6]. The symptoms of leishmaniasis include fever, weight loss, enlarged spleen, swollen glands, skin sores (changing in size and appearance over time), splenomegaly, lymphadenopathy, hepatomegaly, pancytopenia, progressive anemia, and hypergammaglobulinemia with hypoalbunemia. Leishmaniasis is always fatal when left untreated and some times patients (50% in Sudan and 1-3% in India) develop post kala-azar dermal leishmaniasis (PKDL) [7]. The present review briefly illustrates the current status of Leishmaniasis, occurrence and treatment around the world, and also critically discusses the key points in natural products based drug discovery protocols. Finally, a comprehensive coverage of natural products with significant activity against Leishmania species has been given in detail. In order to highlight any possible structure-activity relationships, the review has been organized according to chemical structural class.

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2. Taxonomy of Leishmania spp. The Leishmania are protozoa belonging to the order Kinetoplastida and family Trypanosomatidae. Earlier various classifications have been successively applied to the genus Leishmania; however the simplest one can be summarized from Figure 1. ORDER Kinetoplastida

FAMILY Trypanosomatidae

GENERA Leishmania

Viannia

SUB-GENERA Leishmania

L. tropica L. mexicana L. aethiopica L. lainsoni L. donovani L. major L. tropica L. mexicana L. major L. donovani SPECIES L. killicki L. amazonensis L. chagasi L. garhani L. infantum

L. archibaldi

L. braziliensis L. guyanensis L. braziliensis L. guyanensis L. peruviana L. panamiensis

L. pifanoi L. venezuelensis

Figure 1. Classification of Leishmania parasite.

3. Morphology and life cycle Leishmania are the obligate intracellular parasites existing in two morphologic forms: promastigotes and amastigotes. Promastigotes are found in digestive tract of sandfly and are long spindle-shaped with a single delicate flagellum (15-28 μM long) attached to cytoplasmic organelle called, kinetoplast containing intertwined circular DNA (kDNA) molecules known as maxicircles and minicircles, which make up 5-10% of total DNA [8]. A fully developed promastigote measures about 114.3 to 20 μM in length and 1.5 to 1.8 μM at their widest part [9]. The small, round to oval bodies called amastigotes (2 to 3 μM in length) are the non-infective Leishmania parasites occurring in monocytes, polymorphonuclear lecucocytes or endothelial cells of vertebrates (hosts) while promastigotes represent the infective stage in sandfly (vector).

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Figure 2. Life cycle of Leishmania parasite.

The Leishmania promastigotes are transmitted by sandfly to vertebrate hosts e.g. canines, marsupials, edentates and rodents. Once inside the bloodstream of reservoirs for the disease, promastigotes are phagocytosed by the mononuclear phagocytic cells and are transformed to amastigotes that multiply by means of binary fission. On lyse of host cell, the free parasites spread to new cells and tissues of different organs including the spleen, liver and bone marrow. Amastigotes in the blood as well as in the monocytes are ingested during a blood meal by female sandfly. Once ingested, the amastigotes migrate to the midgut of the sand fly and transform into the promastigotes. After a period of four to five days, promastigotes move forward to the oesophagus reach to salivary glands of the sandfly. Infected sandfly during the second blood meal regurgitates the infectious promastigotes from its pharynx into the bloodstream of the host vertebrates and the life cycle is repeated [10].

4. Chemotherapy of leishmaniasis The leishmanicidal agents with the most favorable therapeutic index are the antimony compounds known as antimonials. Pentostam® (sodium stibogluconate) and Glucantime® (meglumine antimoniate), able to interfere with the bioenergetics of the Leishmania amastigotes [11], are the mainstay therapy for VL. They bind to and inhibit enzymes involved in the glycolysis and oxidation of fatty acids. Since ADP phosphorylates to ATP using NADH generated by glycolysis and citric acid cycle, the intracellular ATP levels

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essential for the survival of Leishmania are depleted. However, due to high cost (approx 200 USD per patients) of branded sodium stibogluconate, a generic sodium antimony gluconate (SAG, Albert David Ltd, India, 13USD per patients) was used to treat patients satisfactorily without any significant difference in final cure. However, due to serious side effects (pain at the site of injection, stiff joints, gastrointestinal problems, cardiotoxicity, hepatic and renal insufficiency) and declining efficacy, the SAG is no longer used in VL hyper endemic regions of India. COOH

COOH

.3 Na .9 H2O

O Sb HO

H OH

Sb O

OH H OH

Sodium stibugluconate Pentamidine (1) that hampers replication and transcription at the mitochondrial level in pathogen was the first drug used for the treatment of patient refractory to Sbv [12]. Biophysical analysis, foot-printing studies and the crystal structure has proved that the charged amidinium groups of pentamidine establish hydrogen bonding with O2 of thymine or N3 of adenine and form complexes with the minor groove of DNA. However, the efficacy of 1 has gradually declined over the years and now it cures only 70% of patients producing serious adverse events like shock, hypoglycemia and death in significant proportion. HN O H2N H2N O HN 1

Amphotericin B (2) is a pollen antibiotic that was recommended as first line drug in India by National Expert Committee for Sbv refractory regions of VL. At doses of 0.75-1.0 mg/kg for 15 infusions on alternate days its cures more than 97% of patients. The drug can perturb both parasitic and mammalian cells, but the selective lethality of 2 for parasitic cells is the result of its great affinity towards 24-substituted sterols, called ergosterol, the major cell membrane sterols [13].

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Miltefosine (3) originally developed as anti tumor agent, was approved in India at 50–100 mg (~2.5 mg/kg) doses for four weeks against VL patients including children. The drug 3 blocks Leishmania proliferation, alters phospholipid and sterol composition and activates cellular immunity. However, due to high cost and serious side effects, medical advisors generally avoid 3 in their prescriptions [14]. OH OH H3C

HO CH3

OH O

COOH

H3C O

CH3

OH NH2

Paromomycin (4), an amino glycoside antibiotic originally identified as an antileishmanial drug in the 1960s, acts synergistically with antimonials in vitro, and was demonstrated significant (93% cure rate) at a dose of 16 mg/kg when given intramuscularly for 21 days to VL patients in India. Like other amino glycosides, the drug 4 acts by impairing the macromolecular synthesis and alters the membrane properties of Leishmania [15]. O

CH3

P H3C

H3C

O O

H3N

OH O O NH3

CH3

NH3

CH3

NH3

N O

H3N

OH O

N HN

H3C

CH3

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Sitamaquine (5), an orally active analog of 8-aminoquinoline, is in clinical development by the Walter Reed Army Institute in collaboration with GlaxoSmithKline (formerly SmithKline Beecham) to use for the treatment of VL. In a randomized, open label and multicenter Phase II trial in India and Kenya, the drug 5 was found efficacious and well tolerated at various dose levels [16]. As on March 2002, the drug 5 is currently in Phase III trials for the treatment of VL.

5. Natural products as folk medicines for treatment of leishmaniasis Utility of natural products in drug discovery and development is not surprising as many of medicinal plants i.e. Cinchona calisaya (bark), Strychnos pseudoquina (bark), Deianira erubescens (roots and leaves) and Remijia ferruginea (bark) were historically used against different parasitic diseases. Ancient records as well as recent literature reports have established the effectiveness of natural products as potentially rich sources of new and selective agents for the treatment of important tropical diseases caused by protozoans and other parasites. In 1970s, artemisinin, an important antimalarial drug was identified from traditional Chinese medicine Artemisia annua and since then many artemisinin derivatives were prepared and evaluated in various pre-clinical and clinical trials to use for the treatment of malaria. Likewise, paromomycin 4 (HumatinTM, King Pharmaceuticals), obtained from Streptomyces krestomuceticus, is an orphan drug that was approved by Drug-Controller General of India in September 2006 against VL. Paromomycin 4 was originally developed by the Institute for OneWorld Health and is an off-patent antibiotic marketed in the US to treat intestinal parasites also. Natural products literature provides a growing research on plant derived antileishmanial agents and several natural products so far have been discovered with excellent activity against leishmania parasites, however, none of them have been clinically evaluated in studies or projected to reach the clinical applications in near future. This review is focused to cover the entire formal and constant research on leishmanicidal natural products from the mid-1980 to June 2010 with special attention on structure-activity relationship (SAR) based activity and mechanism of action.

6. Alkaloids The alkaloids constitute an important class of natural products exhibiting significant anti-leishmanial activities. The quinoline alkaloids,

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2-n-propylquinoline (6), chimanine-D (7) and chimanine-B (8), isolated from Galipea longiflora (Rutaceae), exhibit antileishmanial activity against L. braziliensis promastigotes with an IC90 values of 50, 25 and 25 μg/mL, respectively. Oral in vivo studies using 6 in BALB/c mice demonstrates 99.9% suppression of liver parasites while subcutaneous treatment with 7 causes 86.6% parasite suppression when given for 10 days at 0.54 mmol/kg [17]. However, oral treatment with 7 for 5 days results in 72.9% parasite suppression only. Likewise, dictylomide-A (9) and B (10), isolated from the bark of Dictyoloma peruviana (Rutaceae), causes total lyses of L. amazonensis promastigotes at 100 μg/mL concentration [18].

6.1. Indole alkaloids Dihydrocorynantheine (11), corynantheine (12) and corynantheidine (13), isolated from the bark of Corynanthe pachyceras (Rubiaceae), are the respiratory chain inhibitors exhibiting IC50 of 3 μM against L. major. Pleiocarpine (14) isolated from stem bark of Kopsia griffithii (Apocynaceae), shows in vitro antileishmanial activity with an IC50 < 25 μg/mL against L. donovani promastigotes. Gabunine (15), a bis-indole alkaloid obtained from stem bark of Peschiera van heurkii (Apocynaceae), exhibits in vitro activity with an IC50 25 μg/mL against L. amazonensis amastigotes [19].

6.2. Isoquinoline alkaloids O-methylmoschatoline (16) and liriodenine (17), isolated from Annona foetida (Annonaceae), display in vitro activity against promastigote forms of

O N

CH3

CH3 HO CH3 9

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L. braziliensis with an IC50 < 60 μM [20]. The SAR study among these oxoaporphine alkaloids reveals that 17 bearing methylenedioxy moiety is eight times more active against L. braziliensis and L. guyanensis than the 16. Berberine (18), occurring in many plant species of Annonaceae, Menispermaceae and Berberifaceae, exhibits in vivo leishmanicidal activity with an IC50 value of 10 μg/mL against L. major. Isoguattouregidine (19) isolated from Guatteria foliosa (Annonaceae), shows activity at 100 μg/mL

N N H

N H

H C2H3

C2H5 H

OCH3

OCH3 H3COOC

H3COOC

H 12

N N N H

C2H5

H N

H H OCH3

H3COOC

H H

N H3CO

COOCH3

N H H3COOC

CH3

H3COOC

H N N H

15 CH3

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H3CO CH3

H OCH3

OCH3 17

OCH3

H3CO

O N

H3CO

CH3

O O 21

19 O

O N

H3CO

N H

O 22

concentrations against L. donovani and L. amazonensis. Anonaine (20) isolated from Annona spinescens (Annonaceae), exhibits activity against promastigotes of L. braziliensis and L. donovani [21]. The alkaloids, (+)-neolitsine (21) and cryptodorine (22), isolated from Guatteria dumetorum (Annonaceae), display significant activity against promastigotes of L. maxicana at 15 and 3 μM concentrations, respectively. Xylopine (23), an aporphine alkaloid isolated from Guatteria amplifolia (Annonaceae) shows activity against promastigotes of L. mexicana (IC50 value 3 μM) and L. panamensis (IC50 value 6 μM) [22]. Unonopsine (24), a dimeric aporphine alkaloid isolated from the Unonopsis buchtienii (Annonaceae), displays antileishmanial activity (IC100 value 25 μg/mL) against L. donovani promastigotes [23].

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6.3. Naphthylisoquinoline alkaloids Among the naphthylisoquinoline alkaloids, ancistroealaine-A (25) isolated from Ancistrocladus ealaensis (Ancistrocladaceae), exhibits activity against L. donovani promastigotes with an IC50 value 4.10 μg/mL. Ancistrocladinium A (26) and B (27) isolated from yet un-described Congolese Ancistrocladaceae species, require 2.61 and 1.52 μg/mL concentrations, respectively to reach the IC50 towards L. major promastigotes. An apoptosis-like death pathway is the possible mode of action for compounds 26 & 27. Ancistrocladidine (28), isolated from Ancistrocladus tanzaniensis (Ancistrocladaceae) shows relatively weak activity by a factor of 2 against L. donovani when compared to ancistrotanzanine-B (29) (IC50 = 1.6 μg/mL), while by a factor of 10 in comparison to miltefosin (positive control). Likewise, ancistrotanazanine-A (30), exhibits activity against promastigotes of L. donovani. SAR based studies among the alkaloids suggest that the compound bearing C,C-biaryl axis connecting the naphthyl and isoquinoline moiety shows weak or no leishmanicidal activity [24].

6.4. Bisbenzylisoquinolinic alkaloids Daphanandrine (31) isolated from Albertisia papuana (Menispermaceae), obaberine (32) obtained from Pseudoxandra sclerocarpa (Annonaceae), gyrocarpine (33) produced by Gyrocarpus americanus (Hernandiaceae) and limacine (34) isolated from Caryomene olivasans (Menispermaceae), display OCH3 OCH3 CH3

H3CO CH3 H3CO

CH3

H3CO

TFA OH

TFA

OCH3

OCH3 CH3

CH3

OCH3

H3C

OCH3 CH3

OCH3 OCH3 H3CO H3CO

CH3

OCH3 OH N

CH3

H3C

CH3 H3CO

H3CO

OCH3 CH3

CH3

OCH3 CH3

CH3

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activity against L. donovani, L. braziliensis and L. amazonensis with an IC100 of ~50 μg/mL. SAR studies among these alkaloids demonstrate that alkaloids with methylated nitrogen are more active than those with non-substituted or aromatic nitrogens while quaternization of one or more nitrogen atoms results in the loss of antileishmanial activity [25]. OCH3 H3CO HN

OCH3 H3CO N

H3C

CH3

O OCH3

OCH3

OCH3 H3CO

OCH3 HO

H 3C

H3CO O

CH3 H3C

CH3 O

OCH3

6.5. Steroidal alkaloids Among the alkaloids, holamine (35), 15-α-hydroxyholamine (36), holacurtine (37) and N-desmethylholacurtine (38), obtained from Holarrhena curtisii (Apocynaceae), the metabolite 35 exhibits strongest activity against L. donovani (1.56>IC50>0.39 μg/mL) in compared to 36, 37 and 38 (6.25>IC50>1.56 μg/mL) [26]. O

O CH3

CH3 CH3

CH3

CH3 H

H2N

H2N 35

O CH3

CH3

H CH3 H

CH3 H

H NH H3C

OH CH3 O O

H NH2 OCH3

H OCH3

CH3

O O

CH3

H 38

H OH

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6.6. Benzoquinolizidine alkaloids Klugine (39), cephaeline (40), isocephaeline (41) and emetine (42), demonstrating significant leishmanicidal activity against L. donovani have been isolated from Psychotria klugii (Rubiaceae). Among these metabolites, the compound 39 (IC50 of 0.40 μg/mL) and 41 (IC50 0.45 μg/mL) exhibit <13- and <15-fold less potent activity in compared to 40, while compound 40 with IC50 of 0.03 μg/mL demonstrates >20- and >5-fold more in vitro activity against L. donovani when compared to pentamidine and amphotericin-B, respectively. The alkaloid 42, exhibits activity against L. donovani with an IC50 value 0.03 μg/mL, however produces toxicity in treatment of cutaneous leishmaniasis caused by L. major [27]. H3CO

N H3CO

H3CO

CH3

CH3 R2

OCH3

H OCH3

OH 41 R = OCH3 42 R= OH

39 R1 = OH; R2 = OH 40 R1 = OCH3; R2 = H

6.7. Diterpene alkaloids The alkaloids, 15,22-O-Diacetyl-19-oxo-dihydroatisine (43), azitine (44) and isoazitine (45), isolated from Aconitum, Delphinium and Consolida species, show significant leishmanicidal activities. The metabolite 45 exhibits strongest activity against promastigotes of L. infantum with IC50 values 44.6, 32.3 and 24.6 μM at 24, 48 and 72 h of culture, respectively. The compound 44 and 43 with IC50 values of 33.7 and 27.9 μM at 72 h of culture, respectively, exhibit activity against promastigotes of L. infantum [28]. CH2

CH2

OAc

CH3 CH3 O

CH3

CH3 44

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6.8. Pyrrolidinium alkaloids The pyrrolidinium alkaloid (2S,4R)-2-carboxy-4-(E)-p-coumaroyloxy1,1-dimethylpyrrolidinium inner salt (46), isolated from Phlomis brunneogaleata (Lamiaceae), display activity with an IC50 of 9.1 μg/mL against axenic amastigotes of L. donovani [29].

6.9. Acridone alkaloids The rhodesiacridone (47) and gravacridonediol (48) isolated from Thamnosma rhodesica (Rutaceae), exhibit 69% and 46% inhibition at 10 μM concentration, respectively against promastigotes of L. major. The compounds also display activity against L. major amastigotes and cause over 90% and 50% inhibition at 10 and 1 μM concentration, respectively [26]. O

OH O

OOC

N H3C

N CH3

O CH3 46

R 47 R = C(OH)(CH2OH)COCH3 48 R = C(OH)(CH3)CH2OH

6.10. β-Carboline alkaloids The harmaline (49), isolated from Peganum harmala (Nitrariaceae), exhibits amastigotespecific activity (IC50 of 1.16 μM). Harmine (50) isolated from same plant species reduces spleen parasite load by approximately 40, 60, 70 and 80% in free, liposomal, niosomal and nanoparticular forms, respectively in mice model. Canthin-6-one (51) and 5-methoxycanthin-6-one (52) occurring in plant species of Rutaceae and Simaroubaceae, demonstrate in vivo activity against L. amazonensis in BALB/c mice model. Nhydroxyannomontine (53) and annomontine (54) isolated from Annona foetida (Annonaceae), show efficient leishmanicidal potentials. The SAR studies suggest that the metabolite 54 (IC50 = 34.8 μM) displays 6 times more activity compared to 53 against L. braziliensis promastigotes. The compound 53 also exhibits activity against promastigotes of L. guyanensis while 54 remain inactive [25].

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N N

H3CO

N H

R N

O R

R H2N

49 R = H 50 R = CH3

51 R = H 52 R = OCH3

53 R = OH 54 R = H

6.11. Alkaloids from marine sources Many marine sponges e.g. Amphimedon viridis, Acanthostrongylophora species, Neopetrosia species, Plakortis angulospiculatus and Pachymatisma johnstonii serve as rich sources of alkaloids with significant antileishmanial potentials. Renieramycin A (55) isolated from Neopetrosia species, is a La/egfp (expressing enhanced green fluorescent protein) inhibitor that shows efficient antileishmanial activity against L. amazonensis with IC50 0.2 μg/mL. Araguspongin C (56), isolated from a marine sponge Haliclona exigua, displays leishmanicidal activity against promastigotes as well as amastigotes at 100 μg/mL concentrations [30]. N

OCH3

H CH3

O H

H3C

CH3

H3CO O

CH3

O O

H N

CH3 55

Among the ciliatamides A-C (57-59) isolated from Aaptos ciliate, the peptide 57 and 58 at 10.0 μg/mL concentrations inhibit 50% growth L. major promastigotes [31]. The lipopeptides, almiramides A-C (60-62) isolated from cyanobacterium Lyngbya majuscule, exhibit significant in vitro antileishmanial activity against L. donovani. The SAR studies among these peptides suggest that 61 and 62 exhibit strong activity against L. donovani

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with EC50 values of 2.4 and 1.9 μM, respectively. The metabolites 61 and 62 also display weak cytotoxicity to mammalian Vero cells at 52.3 and 33.1 μM concentrations, respectively [32]. Dragonamide A (63), E (64) and herbamide B (65), isolated from same cyanobacterium strain, exhibit in vitro activity against L. donovani with EC50 values of 6.5, 5.1 and 5.9 μM, respectively [33]. Viridamide A (66) isolated from Oscillatoria nigro-viridis, shows activity against L. mexicana with EC50 of 1.5 μM [34]. Venturamides A (67) and B (68) obtained from cyanobacterium Oscillatoria species, exhibit activity against L. donovani with EC50 >19.0 μM. Valinomycin (69), a dodecadepsipeptide isolated from Streptomyces strains, exhibits activity against promastigotes of L. major with EC50 < 0.11 μM, but at the same time shows cytotoxicity to 293T kidney epithelial cells and J774.1 macrophages [35].

7. Quinones Primin (2-methoxy-6-pentylcyclohexa-2,5-diene-1,4-dione), occurring in Primula obconica and other species (Primulaceae), shows significant leishmanicidal activity against L. donovani with an IC50 of 0.711 μM. Diospyrin (70), a bis-naphthoquinone inhibiting topoisomerase I, isolated from the bark of Diospyros Montana (Ebenaceae), demonstrates antileishmanial activity against L. donovani promastigotes with an MIC of 1.0 μg/mL [36]. The hydroxylated derivative of 70 at 3 μM concentration eliminates 73.8% of amastigotes in infected macrophages [37]. Plumbagin (72), originally isolated from Plumbago zylenica, shows leishmanicidal activity against amastigotes of L. donovani (IC50 = 0.42 μg/mL) and L. amazonensis (IC50 = 1.1 μg/mL). At a concentration of 10 μg/mL, the

O H N

H N

N O

CH3

60 R = 61 R = 62 R =

H3C CH3

CH3 59

57 R = (CH2)7CHCH2 58 R = (CH2)6CH3 O

CH2

CH3 N

O H3C

O N CH3 CH3

CH3 H N O H3C

CH3

N CH3 CH3

CH3

O NH2

Scope of natural products against leishmaniasis CH3

H3C O

CH3

H3C O

CH3

N O

CH3

H3C

CH3

H N

Cl3C NH2

N CH3

CH3

137

CH3

O N

63 R = 65 64 R = CH3

CH3 OCH3

H N

H3C

N H3C

N O

H3C

N CH3 S

S O

H3C

CH3

H3C

O NH

N H

CH3 CH3

HN O

CH3

H3C

N H

CH3

N H

CH3 H3C

H3C

COOCH3

CH3

O H3C

CH3

N H

O H3C

CH3

CH3 CH3

H3C O

CH3

H3C

CH3

CH3 CH3

H3C

NH CH3

CH3

N H CH3

H3C

CH3

compound 72 presents an amastigote survival index (SI) of 16.5% against L. amazonensis with the absence of toxic effects against the macrophages. The metabolite 72 also shows in vivo activity against L. amazonensis and L. venezuelensis at concentrations 2.5 and 5 mg/kg/day, respectively. The mechanism of the action of compounds 72 and 71 involves generation of oxygen free radicals from which the parasites remain unable to defend. The dimeric products 3,3-biplumbagin (73) and 8,8′-biplumbagin (74), isolated from the bark of Pera benensis (Euphorbiaceae), display significant

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antileishmanial activity. Among these, the metabolite 73 shows lower activity (IC90 = 50 μg/mL) compared to 72 and 75 (IC90 = 50 μg/mL) against L. braziliensis, L. amazonensis, and L. donovani promastigotes [38,39]. Lapachol (75), a prenylated hydroxynaphthoquinone isolated from Tecoma species (Bignoniaceae), displays activity with mechanism of action similar to 71 and 72 against L. donovani amastigotes in peritoneal mice macrophages. The metabolite 3,4-dihydronaphthalen-1(2H)-one (76), isolated from the bark of Ampelocera edentula (Ulmaceae), exhibits leishmanicidal activity (IC90 of 10 μg/mL) against L. braziliensis, L. amazonensis and L. donovani promastigotes. The metabolite 76 demonstrates strong in vivo activity on subcutaneous treatment in BALB/c mice infected with L. amazonensis or L. venezuelensis when compared to Glucantime® (25 mg/kg/day vs 56 mg SbV/kg/day). However, the use of tetralones is limited due to cytotoxic, carcinogenic and mutagenic properties in animals [40]. Jacaranone (77), a quinone isolated from the leaves of Jacaranda copaia (Bignoniaceae), exhibits a strong activity with an ED50 of 0.02 mM against L. amazonensis promastigotes but at the same concentration shows toxicity to peritoneal mice macrophages. The prenylated dihydroquinone hydropiperone (78), isolated from Peperomia galioides (Piperaceae), shows activity at a concentration of 25 μg/mL against promastigote forms of L. braziliensis, L. donovani and L. amazonensis. At 100 μg/mL concentration the metabolite 78 causes total lysis of the parasites [41]. OH

OCH3 OH OH

H3C

OCH3 OH

CH3

H3C O H3C

OH H3C

OH 72

70 O

O CH3 O

OH O

H3C

CH3

O OH

O H3C

CH3

O OH 73

O 74

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The anthraquinone-2-carbaldehydes, 79 and 80, isolated from the roots of Morinda lucida (Rubiaceae), shows leishmanicidal potential selective to L. major promastigotes. SAR studies suggest that presence of an aldehyde group at C-2 and a phenolic hydroxy group at C-3 in both structures, are essential for their antiprotozoal activity [42]. O

O H3C

CH2COCH3

OH 76

77 O

CH3

CH3 CH3

OH CH3 O

78 OH

CHO

OH O 79 R1 = OCH3 80 R1 = H

CH2OH O 81

The aloe-emodin (81) isolated from Stephania dinklagei (Menispermaceae), shows leishmanicidal activity at IC50 values of 185.1 and 90 μM against L. donovani promastigotes and amastigotes, respectively [43]. Vismione D isolated from Vismia orientalis (Clusiaceae) exhibits activity against axenic amastigotes of L. donovani with an IC50 value of 0.37 μg/mL but shows cytotoxicity when tested on human L6 cells (IC50 of 4.1 μg/mL) [29].

8. Terpenes 8.1. Iridoids Iridoids, a class of monoterpenoid glycosides often serve as intermediates in the biosynthesis of indole alkaloids are well known for significant leishmanicidal activity. The arbortristosides-A (82), B (83), C (84) and 6-βhydroxyloganin (85), isolated from Nyctanthes arbortristis (Oleaceae) exhibit in vitro activity against L. donovani amastigotes. The in vivo studies using intraperitoneal and oral treatment (10 and 100 mg/kg concentrations for 5 days) of hamsters infected with L. donovani, the metabolite 82 displays significant leishmanicidal activities [44]. Picroside I (86) and kutkoside (87),

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obtained from Picrorhiza kurroa, exhibits a high degree of protection against the infection of promastigotes of L. donovani in hamsters [45]. Picroliv, a standardized fraction of iridoid glycosides 86 and 87, increases the nonspecific immune response and induces a high degree of protection against the infection of promastigotes of L. donovani in hamsters. Picroliv is an adjuvant proposed to increase the efficacy of leishmanicidal drugs and has demonstrated excellent therapeutic index in Phase I and II clinical trials [46]. HO

OH H CO2CH3 O R1O

O O

R1O

H OR2 O O OH OH OH 86 R1 = Vanilloyl, R2 = H 87 R1 = H, R2 = Cinnamoyl

OH CH3H O O HO OH OH 82 R1 = p-Methoxycinnamoyl 83 R1= Caffeoyl 84 R1 = Coumaroyl 85 R1 = H

Amarogentin (88), a secoiridoid glycoside isolated from Swertia chirata (Gentiaceae), produces leishmaincidal effect at a concentration > 60 μM against L. donovani through inhibition of catalytic activity of topoisomerase I [47]. The metabolite 88 exerts inhibitory effect with a mechanism of action similar to Pentostam® i.e. by binding to the enzyme and preventing the formation of a binary complex with DNA. The evaluation of 88 in the form of liposomes and niosomes shows an enhanced leishmanicidal activity (without toxic effects) than those observed for free 88 when tested in hamsters [48]. O

H CH2

O O

HOH2C

O O

C HO

HO OH 88

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8.2. Monoterpenes Espintanol (89), isolated from the bark of Oxandra espintana (Annonaceae), shows antileishmanial activity against promastigotes of twelve Leishmania species. However, the metabolite 89 exhibits only a weak activity in vivo in mice infected with L. amazonensis. Grifolin (90) and piperogalin (91) obtained from Peperomia galoides, causes total lysis of L. braziliensis, L. donovani and L. amazonensis promastigotes at 100 μg/mL concentrations. At 10 μg/mL concentration, metabolite 91 causes more than 90% lysis of the promastigotes [49]. CH3

CH3

CH3 OH

H3C

CH3

OH OH

H3C H3CO H3C

CH3

OCH3 CH3 89

H3C

CH3 91

8.3. Sesquiterpenes A sesquiterpene lactone, dehydrozaluzanin C (92), isolated from the leaves of Munnozia maronii (Asteraceae), shows activity at concentrations between 2.5-10 μg/mL against promastigotes of eleven Leishmania species. The in vivo test using the metabolite 92 in BALB/c mice results in reduction of the lesions caused by L. amazonensis [50]. Sesquiterpene dilactone, 16,17-dihydrobrachycalyoxide (93), isolated from Vernonia brachycalyx (Asteraceae), exhibits activity (IC50 = 17 μg/mL) against L. major promastigote but also inhibits the proliferation of human lymphocytes [51]. Kudtriol (94), a sesquiterpene alcohol isolated from the aerial parts of Jasonia glutinosa (Asteraceae), shows toxic activity against promastigotes of L. donovani at 250 μg/mL concentration. SAR study with metabolite 94 indicates that the presence of a C-5 hydroxy group in the α-orientation is essential for the expression of the leishmanicidal activity [52]. The (+)-curcuphenol (95), isolated from sponge Myrmekioderma styx, exhibits in vitro anti-leishmanial activities against L. donovani with an EC50 of 11.0 μM [53].

142

B. B. Mishra et al. H2C H

O CH

O H2C

H2C

O 3

C2H5 HO H CH2

H O O

H3C O 93 OH

CH3

OH H3C OH H3C

OH CH2 H3C

CH3

8.4. Diterpenes A phorbol diester, 12-O-tetradecanoyl phorbol-13-acetate (TPA) 96, also known as phorbol 12-myristate 13-acetate (PMA), was originally identified from the croton plant, which at a concentration of 20 ng/mL displays ability to cause a variety of structural changes in the parasites of L. amazonensis by activation of protein kinase C, an important enzyme in the development of several cellular functions [54]. Among the other diterpenoids isolated from Euphorbiaceae species with leishmanicidal potentials are jatrogrossidione (97) and jatrophone (98). These metabolites possess toxic activity against the promastigote forms of L. braziliensis, L. amazonensis and L. chagasi. SAR studies with these metabolites revealed that 97 with IC100 value of 0.75 μg/mL displays activity higher than 98 (IC100 = 5 μg/mL), but remains inactive in vivo [55]. The 15-monomethyl ester of dehydropinifolic acid (99), obtained from the stem bark of Polyalthia macropoda (Annonaceae), and ribenol (100), an ent-manoyl oxide derivative isolated from Sideritis varoi (Lamiaceae), show in vitro activity against promastigotes of L. donovani [56]. Also the different derivatives of this metabolite, obtained through chemical or biological transformations, exhibit strong leishmanicidal activity. Additionally, 6-βhydroxyrosenonolactone (101), a diterpene isolated from the bark of Holarrhena floribunda (Apocynaceae), has a moderate and weak activity against promastigotes and amastigotes of L. donovani, respectively [57].

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H3C H3C(H2C)12OCO H3C H

H3C

HO O HO

OCOCH3 CH3

CH3

CH3 CH3

CH2 H3C

H O

H3C CH2OH

CH3

H2C

CO2CH3

CH3

H3C CH3 CH3 CH3

CH3 O

CH2

CH2 CH3

CH2 O

CH3

HO H H3C CO2H 99

H O

H3C CH3

H CH3 100

OH 101

8.5. Triterpenes The ursolic acid (102) and betulinaldehyde (103), obtained from the bark of Jacaranda copaia and the stem of Doliocarpus dentatus (Dilleniaceae), respectively show activity against the amastigotes of L. amazonensis. However, the metabolite 103 exhibits toxicity to peritoneal macrophages in mice while 102 displays limited activity in vivo. The triterpenes, (24Z)-3-oxotirucalla-7,24-dien-26-oic acid (104) and epi-oleanolic acid (105), isolated from the leaves of Celaenododendron mexicanum (Euphorbiaceae), display leishmanicidal activity against L. donovani with IC50 values of 13.7 and 18.8 μM, respectively. The quassinoids, simalikalactone D (106) and 15-β-heptylchaparrinone (107), obtained from species of Simaroubaceae family show activity against promastigotes of L. donovani but at the same time exhibit toxicity to macrophages [58]. Triterpene glycosides obtained from marine sources e.g. holothurins A (108), isolated from the sea cucumber Actinopyga lecanora, causes 73.2 ± 6.8% and 65.8 ± 6% inhibition of L. donovani promastigotes and amastigotes, respectively at 100 μg/mL concentration. The other isomer B (109) obtained from same source shows 82.5 ± 11.6% and 47.3 ± 6.5% inhibition against promastigotes of L. donovani at 100 and 50 μg/mL concentrations, respectively [59].

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CH2

CH3 H3C

H3C H

CH3 H

CH3

CO2H

CH3

CHO

CH3 CH3

HO H CH3

H3C

H CH3

H3C 102

103 H

CH3

CO2H CH3

CH3

CH3 H

CH3

H3C

CH3

H3C

CO2H

CH3

O CH3

H3C

H CH3

CH3

105

104

OH OH HO OH CH3

HO OH CH3

CH3 O

(CH2)6CH3

OCOCH(CH3)C2H5

O H

CH3 O

CH3

CH3 107

106

CH3 HO

CH3

O OH

CH3 CH3 O O

H3C

CH3

O NaO3SO HO H3C

O OH OH

OH HO O

HO OR 108 R1 = HO MeO 109 R2 = H

CH3

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9. Saponins The α-hederin (110), β-hederin (111) and hederagenin (112), obtained from the leaves of Hedera helix (Araliaceae), show lishmanicidal activity against L. infantum and L. tropica. Among these, the metabolite 112 also shows significant activity against the amastigote forms while both 110 and 111 exhibit strong anti-proliferative activity on human monocytes [60]. The saponins 110-112 appear to inhibit the growth of Leishmania promastigotes by acting on the membrane of the parasite with induction of a drop in membrane potential [61]. The hederecolchiside-A1 (113), isolated from Hedera colchica, shows strong activity against the promastigotes and amastigotes of L. infantum, but also displays a notable activity on human monocytes. The saponin, mimengoside-A (114), isolated from the leaves of Buddleja madagascariensis (Loganiaceae) [62], exhibits activity against promastigotes of L. infantum. Muzanzagenin (115), obtained from the roots of Asparagus africanus (Liliaceae), displays activity with an IC50 value 31 μg/mL against the L. major promastigotes. However, the metabolite 115 also inhibits the proliferation of human lymphocytes [63].

10. Phenolic derivatives 10.1. Chalcones The chalcone, (E)-1-[2,4-hydroxy-3-(3-methylbut-2-enyl)phenyl]-3-[4hydroxy-3-(3-methylbut-2-enyl)phenyl]-prop-2-en-1-one (116) shows toxicity to promastigotes of L. donovani, while 2′,6′-dihydroxy-4′methoxychalcone (117), isolated from inflorescences of Piper aduncum (Piperaceae), exhibits significant in vitro activity against promastigotes and amastigotes of L. amazonensis by affecting the ultrastructure of the parasite mitochondria without causing damage or inducing NO production in the macrophages [64,65]. The metabolite 117 with an IC50 value of 0.5 μg/mL shows strong antileishmanial activity against the promastigotes of L. amazonensis, while exhibit lower activity (IC50 = 24 μg/mL) against amastigote forms. Encapsulated formulation of 117 when administered at 1.0 μg/mL causes the reduction in the level of L. amazonensis infected macrophages by 53% [66]. Ultrastructural studies suggest that 117 produces selective toxicity to the intracellular amastigotes without affecting macrophage organelles even when exposed to 80 μg/mL concentration. The licochalcone-A (118), isolated from roots of the Chinese licorice plant Glycyrrhiza species (Fabaceae), shows in vitro

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H3C

CH3

CO2H

CH3 R1O CH3

R2H2C

110 R1 = Ara 2-1 Rha, R2 = OH 111 R1 = Ara 2-1 Rha, R2 = H 112 R1 = H, R2 = OH 113 R1 = Ara [Glc 4-1] 2 Rha, R2 = H Ara: α -L-arabinopyranose Glc: β -D-glucopyranose Rha:α -L-rhamnopyranose Fuc: β -D-fucopyranose H3C

CH3

O CH3

CH3 CH3

RO H3C OH 114 3-0-α -L-rhamnopyranosyl-(1-4)- β -D-glucopyranosyl(1-3)-[ β -D-glucopyranosyl-(1-2)]-β -D-fucopyranoside of 16-dehydroxysaikogenin G H

CH3

H OH CH3

CH3

H H H

O 115

O CH3

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OH CH3 H3CO

CH3

H3C CH3

117

116 CH2

H3C H3C

OCH3 118

119

OH HO

R1O

HO OH

OH 120

OR2

121 R1 = H, R2 = H 122 R1 = H, R2 = OCH3 123 R1 = OCH3, R2 = OCH3

activity against L. major and L. donovani promastigotes. The intraperitoneal administration of 118 prevents the development of lesions in BALB/c mice infected with L. major [67,68]. The intraperitoneal and oral administration of 118 significantly reduces the parasite load in the spleen and liver of hamsters infected with L. donovani. The compound 118 appears to affect the parasite respiratory chain without damaging the organelles of macrophages or phagocytic function by altering the ultrastructure and function of mitochondria only. However, at lower concentrations 118 inhibits the proliferation of human lymphocytes. Subsituents that hinder free rotation in chalcones have been demonstrated to be inactive. The introduction of polar chemical moieties (like hydroxyl and glycosyl groups) led to a reduction of the antileishmanial activity. The modification at the α,β-double bond in chalcones results in marginal reduction of the leishmanicidal activity compared to parent compounds, thus this part is just a chemical spacer

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necessary only. The sulfuretin (2-[(3,4-dihydroxyphenyl)methylene]-6hydroxybenzofuran-3(2H)-one) (119), is an aurone, a group of metabolites related biosynthetically to the chalcones, exhibit activity with EC50 values of 0.09-0.11 μg/mL against promastigotes of Leishmania species. The metabolite 119 with an EC50 value of 1.24 μg/mL displays activity against L. donovani amastigotes, but remains non-toxic to bone marrow-derived macrophages [69].

10.2. Flavonoids The compound 5,7,4′-trihydroxyflavan (120) shows activity against the amastigotes of L. amazonensis [70], while the biflavonoids amentoflavone (121), podocarpusflavone A (122) and B (123), isolated from the leaves of Celanodendron mexicanum, exhibit weak activity against L. donovani promastigotes. The flavones fisetin (124) (isolated from Acacia greggii and A. berlandieri), 3-hydroxyflavone (125), luteolin (126) (isolated from Salvia tomentosa), and quercetin (127) (isolated from plants of family Alliaceae) exhibit potent antileishmanial activity against the intracellular forms of the L. donovani with IC50 values 0.6, 0.7, 0.8 and 1.0 μg/mL, respectively. Biochanin A (128), an O-methylated isoflavone occurring in legumes, shows activity against L. donovani with an IC50 value of 2.5 μg/mL [3]. O

124

125

O 126

OH HO

O OH OH

O 127

OH OH

OCH3

128

10.3. Lignans The lignans (+)-medioresinol (129), (-)-lirioresinol B (130) and (+)nyasol (131), show activity against the amastigotes of L. amazonensis, whereas 131 also exhibits high selectivity in its activity against the promastigotes of L. major. Dyphillin, isolated from Haplophyllum bucharicum (Rutaceae), modulates phagocytosis of macrophages and selectively inhibits the amastigotes of L. infantum with an IC50 value 0.2 μg/mL [71].

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R2 HO HO

H3CO H

H OCH3

H2C

OH OH

R1 129 R1 = R2 = OCH3 130 R1 = R2 = CH3

131

10.4. Coumarins The coumarin isomers 2-epicycloisobrachycoumarinone (132) and cycloisobrachycoumarinone (133), isolated from Vernonia brachycalyx (Asteraceae), display selective activity against promastigotes of L. major. R1 CH3

R2 CH3

CH3 O

132 R1 =CH3, R2 = H 133 R1 =H, R2 = CH3

CH3

134 R1 = R2 = OCH3 135 R1 = H, R2 = OCH3 136 R1 = H, R2 = H

10.5. Curcumins The curcumins, curcumin (134), desmethoxycurcumin (135) and bis-desmethoxycurcumin (136), isolated from the rhizomes of Curcuma longa, show significant anti-leishmanial activity against promastigotes of L. major. However, these metabolites also inhibit the proliferation of human lymphocytes [72].

11. Other metabolites Acetogenins like senegalene (137), squamocine (138), asimicine (139) and molvizarine (140), isolated from the seeds of Annona senegalensis (Annonaceae), show activity against promastigotes of L. major and L. donovani at concentrations that vary between 25 and 100 μg/mL. However, these metabolites also show cytotoxicity greater than that of

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vinblastine against KB and VERO cell lines [73]. Other acetogenins such as rolliniastatin-1 (141), isolated from Rollinia emarginata (Annonaceae), annonacin A (142) and goniothalamicin (143), obtained from Annona glauca (Annonaceae), display promicing activity against the promastigote of L. braziliensis, L. donovani, L. amazonensis, however a clear SAR has not been established [74]. O O

OH O

H3C

(CH2)3CH3

7 OH O O

137 R1

OH O

H3C

OH O

(CH2)5CH3

threo-trans-threo-trans-* 138 R1 = H, R2 = OH, n = 10, *= erythro 139 R1 = OH, R2 = H, n = 10, *= threo 140 R1 = OH, R2 = H, n = 8, *= erythro

CH3

OH O

H3C

CH3

6 141 R2

H3C

O OH n

O CH3

142 R1 = OH, R2 = H, n = 5, m = 8, *= erythro 143 R1 = H, R2 = OH, n = 3, m = 10, *= threo

Future prospectives Despite the advances in the parasitological and biochemical researches using various species of Leishmania, the treatment options available against leishmaniasis are far from satisfactory. In current situation, development of new drugs to combat leishmaniasis require increased input from the disciplines of chemistry, pharmacology, toxicology and pharmaceutics to complement the advances in molecular biology that have been made in past 21 years. Natural products are potential sources of new and selective agents for the treatment of important tropical diseases caused by protozoans and other parasites. The tremendous chemical diversity present in natural products and the promising leads that have already been demonstrated significant against parasitic diseases are needed to be addressed also against leishmania

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parasites. The development of antileishmanial natural products or their analogs in accordance to the considerations outlined above would have a dramatic positive impact on the treatment of leishmaniasis. A safe, non-toxic and cost-effective drug is urgently required to eliminate this problem from every corner of world. A safer, shorter & cheaper treatment, identification of the most cost effective surveillance system and control strategies, suitable vector control approach are among some important aspect for the control and complete eradication of this deadly disease.

Acknowledgement Financial assistance from DST, New Delhi is greatly acknowledged.

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65. Torres-Santos, E.C., Moreira, D.L., Kaplan, M.A.C., Meirelles, M.N., RossiBergmann, B. Antimicrob. Agents Chemother., 1999, 43, 1234. 66. Torres-Santos, E.C., Rodrigues, J.M., Moreira, D.L., Kaplan, M.A.C., RossiBergmann, B. Antimicrob. Agents Chemother., 1999, 43, 1776. 67. Chen, M., Christensen, S.B., Blom, J., Lemmich, E., Nadelmann, L., Fich, K., Theander, T.G., Kharazmi, A. Antimicrob. Agents Chemother., 1993, 37, 2550. 68. Chen, M., Christensen, S.B., Theander, T.G., Kharazmi, A. Antimicrob. Agents Chemother., 1994, 38, 1339. 69. Kayser, O., Kiderlen, A.F., Folkens, U., Kolodziej, H. Planta Med., 1999, 65, 316. 70. Sauvain, M., Dedet, J., Kunesch, N., Poisson, J. J. Nat. Prod., 1994, 57, 403. 71. Chan-Bacab, M.J., Pena-Rodriguez, L.M. Nat. Prod. Rep., 2001, 18, 674. 72. Oketch-Rabah, H.A., Lemmich, C.E., Dossaji, S.F., Theander, T.G., Olsen, E., Cornett. C., Kharazmi, A., Christensen, S. B. J. Nat. Prod., 1997, 60, 458. 73. Shapaz, S., Bories, C., Loiseau, P.M., Cortes, D., Hocquemiller, R., Laurens, A., Cave, A. Planta Med., 1994, 60, 538. 74. Waechter, A., Yaluff, G., Inchausti, A., Rojas de Arias, A., Hocquemiller, R., Cave, A., Fournet, A. Phytother. Res., 1998, 12, 541.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 155-185 ISBN: 978-81-308-0448-4

5. Naturally occurring antihyperglycemic and antidyslipidemic agents T. Narender, T. Khaliq and G. Madhur Medicinal and Process Chemistry Division, Central Drug Research Institute Lucknow-226 001, U.P., India

Abstract. Diabetes mellitus is an independent risk factor for the development of coronary artery diseases, myocardial infarction, hypertension, and dyslipidemia. Clinically diabetic patients are characterized by marked increase in blood glucose level followed by mild hyperlipidemia. Non-insulin dependent diabetes mellitus (NIDDM) accounts for approximately 80–90% of all cases and it is the fastest growing global threat to public health. If the current trend continues, it is likely to result in an estimated 215 million sufferers from NIDDM worldwide by the year 2010. When carbohydrates are in low supply or their breakdown is incomplete, fats become the preferred source of energy. Fatty acids are mobilized into the general circulation leading to secondary triglyceridemia in which total serum lipids in particular triglycerides as well as the levels of cholesterol and phospholipids increases.This rise is proportional to the severity of the diabetes. Uncontrolled diabetes is manifested by a very high rise in triglycerides and fatty acid levels. These conditions are responsible for one third of deaths in industrialized nations Plants have always been a rich source of drugs and many of the currently available drugs have been derived either from natural products or its templates. We here in present a precise description of naturally occurring compounds possessing potential antihyperglycemic action or antidyslipidemic action against specific drug targets. Correspondence/Reprint request: Dr. T. Narender, Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow-226 001, U.P., India. E-mail: t_narendra@cdri.res.in

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1. Diabetes mellitus Diabetes is a disease in which the body does not produce or properly use insulin. Insulin is a hormone that converts sugar, starch and other food into energy needed for daily life. The causes of diabetes are not known clearly, although both genetics and environmental factors such as obesity and lack of exercise appear to play roles. Diabetes mellitus and glucose intolerance are common in adolescent and adult patients with cystic fibrosis. Diabetes is invariably associated with pancreatic exocrine dysfunction (malabsorption). The prevalence in patients over 20 years of age may be as high as 53% [1]. The major types of diabetes include type-I and type-II diabetes. The former results from the body's failure to produce insulin, the hormone that "unlocks" the cells of the body, allowing glucose to enter and fuel them while the latter results from insulin resistance, a condition in which the body fails to properly use insulin combined with relative insulin deficiency. Type-II insulin-resistant diabetes mellitus accounts for 90-95% of all diabetes. This heterogeneous disorder afflicts an estimated 6% of the adult population in western society; its worldwide frequency is expected to continue to grow by 6% per annum, potentially reaching a total of 200-300 million cases in 2010 [2].

2. Drug targets At present, therapy for type-II diabetes relies mainly on several approaches intended to reduce the hyperglycemia itself. Table 1. Current therapeutic agents for type-II diabetes. Drug class

Molecular target

Site(s) of action

Insulins

Insulin receptor

Liver, muscle, fat Hypoglycemia, weight gain Pancreatic β-cell Hypoglycemia, weight gain

Sulphonylureas SU receptor/ K+ (e.g. ATP channel glibenclamide) plus nateglinide & repaglinide Biguanides Unknown Metformin Acarbose

α-glucosidase

Thiazolidinediones PPARγ Rosiglitazone, Pioglitazone

Liver (muscle) Intestine Fat, muscle, liver

Adverse events

Gastrointestinal disturbances, lactic acidosis Gastrointestinal disturbances Weight gain, anemia, oedema,

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3. Antihyperglycemic isolates from nature Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them. The ethnobotanical information reports that about 800 plants may possess anti-diabetic potential [3]. Several such herbs have depicted antidiabetic activity while assessed using currently available experimental techniques [4]. A wide array of plant derived active principles representing numerous chemical compounds has demonstrated activity consistent with their possible use in the treatment of non-insulin dependent diabetes mellitus (NIDDM) [5]. Amongst these are flavonoids, alkaloids, glycosides, polysaccharides, peptidoglycans, hypoglycans, guanidine, steroids, carbohydrates, glycopeptides, terpenoids and amino acids. Even the discovery of widely used hypoglycemic drug, metformin was developed on the basis of the natural products lead isolated from Galega officinalis [6]. Thus, plants are a potential source of anti-diabetic drugs. Herein, is presented a precise description of naturally occurring compounds possessing potential antihyperglycemic action.

3.1. Flavonoids The flavonoids are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear three carbon chain. Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognized as flower pigments in most angiosperm families (flowering plants). However, their occurrence is not restricted to flowers but include all parts of the plant. They show wide variety of activities including antihyperglycemic activity. Bio-flavonoids with promising anti-diabetic potential: A critical survey by Goutam Brahmachari will give comprehensive information on the flavonoids and their antihyperglycemic activity.

3.2. Triterpenoids and steroids There are at least 4000 known triterpenes, which are derived from mevalonic acid pathway. Triterpenes are precursors to steroids in both plants and animals. Steroids are hormonal substances in animals, but they are components of membranes in most organisms. Many triterpenes and sterols occur free, but others occur as glycosides or in special combined forms. Momordica charantia belongs to the family of Cucurbitaceae the fruits of the plant is also known as bitter melon or bitter guard. Cucurbitane class of

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triterpenoids isolated from M. charanta such as 5-β,19-epoxy-3-β,25dihydroxycucurbita-6,23-(E)-diene (1) and 3-β,7-β,25-trihydroxycucurbita5,23-(E)-dien-19-al (2) have blood hypoglycemic effects in the diabetesinduced male ddY mice strain at 400 mg/kg [7]. Hypoglycemic activity guided fractionation together with chemical analysis on the stem of Agarista mexicana led to the isolation of 12-ursene (3) and 23,24-dimethyl-24-ethyl-stigmast-25-ene (4) from the chloroform fraction. The isolated triterpenes showed hypoglycemic activity in normal and alloxan-diabetic CD1 mice at a dose of 50 mg/kg body weight. Comparison was made between the action of the triterpenes and a known hypoglycemic drug, tolbutamide (50 mg/kg). The 12-ursene (3) was found to be less potent than tolbutamide where as 23,24-dimethyl-24-ethyl-stigmast25-ene (4) was shown to be more effective than tolbutamide [8]. 20 17

OHC

H O

From the roots of Salacia oblonga a friedelane-type triterpene, kotalagenin 16-acetate (5), maytenfolic acid (6), 3β,22α-Dihydroxyolean-12en-29-oic acid (7) and a unique thiosugar sulfonium sulfate named Salacinol (60) was isolated. They were screened for inhibitory activity on aldose reductase and were found to be responsible components for the inhibitory activity [9]. Bioassay-guided isolation work on Cabernet Sauvignon’s grape skin yielded antihyperglycemic active compounds which were identified as, oleanolic acid (8) and oleanolic aldehyde (9). These compounds were assayed for insulin production using an INS-1 cell assay. In a dose-response study,

Naturally occurring antihyperglycemic and antidyslipidemic agents

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COOH

H OAc OH

oleanolic acid stimulated insulin production of INS-1 cells by 20.23, 87.97, 1.13 and 6.38 ng of insulin/ mg of protein at a dose of 6.25, 12.5, 25 and 50 μg/mL respectively. The activity was similar to the dose-dependent insulin production of INS-1 cells by glucose. Oleanolic aldehyde also showed a dose-dependent insulin production in the same assay [10]. Our activity guided fractional and isolation work on the plant Ficus racemosa yielded moderately active antihyperglycemic principle, α-amyrin acetate (10). Several ester derivatives of α-amyrin were prepared to study their structure activity relationship [11].

CHO

COOH

O HO

Triterpenoid and steroidal glycosides referred to collectively as saponins are bioactive compounds present naturally in many plants and known to possess potent hypoglycemic activity [12]. Glucuronide saponin named betavulgaroside (11) was isolated from the roots and leaves of Beta vulgaris L. (sugar beet) exhibited hypoglycemic effect in rats [13].

H CO2H HO2C

O HO HO2C HO2CH2CO

O O

OH 11

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The root cortex of Aralia elata provided another triterpnoid glycoside, Elatoside E (12), which was shown to affect the elevation of plasma glucose level by oral sugar tolerance test in rats [14]. Hypoglycemic activity-guided fractionation on the rhizomes of Anemarrhena asphodeloides yielded steroidal glycosides, pseudoprotoimosaponin AIII, (13) and prototimosaponin AIII, (14). These compounds exhibited hypoglycemic effects in a dose-dependent manner in streptozotocin-diabetic mice but showed no effects on glucose uptake and insulin release, suggesting that the hypoglycemic mechanism may be due to inhibition of hepatic gluconeogenesis and/or glycogenolysis [15]. HO

HO O

O HO

COO

O O

O OH

OH OH

O OH OH OH

HO OGlu

OGlu

Glu GalO 2

Glu 13

GalO 2 14

Charantin (15) a steroidal saponin, obtained from Momordica charantia is known to have an insulin-like activity [16]. Charantin stimulates the release of insulin and blocks the formation of glucose in the bloodstream. Similar steroidal saponin (16) was isolated from the fruiting bodies of Ganoderma applanatum, which exhibits Rat lens aldose reductase (RLAR) inhibiting activity. The same plant also produced few other class of compounds (65-67) with RLAR inhibiting property [17]. A steroidal saponin, chloragin (17) was isolated from the aerial part of Chlorophytum nimonii (Grah) Dalz. The saponin characterized as tigogenin3-O-α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranosyl-(1 → 3)-β-Dxylopyranosyl-(1 →4)-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside showed potent antihyperglycemic activity in streptozotocin induced diabetic rats [18].

Naturally occurring antihyperglycemic and antidyslipidemic agents

OH OH OH

161

OH O OH OH

O OH

O O OH

O 16

O H

OH OH OH

O OH

OH O

OH O OH

O OH

Yoshikawa and co-workers isolated elatoside G (18), H (19) and I (20) from a garnish foodstuff "Taranome," the young shoot of Japanese Aralia elata were found to exhibit potent hypoglycemic activity in rats [19]. From Gynostemma pentaphyllum Makino (Cucurbitaceae) a gypenoside saponin, named phanoside (21,23-epoxy-3-β-20,21-trihydroxydammar-24ene-3-O-([α-D-rhamnopyranosyl-(1→2)]-[β-D-glucopyranosyl-(1→3)]-β-Dlyxopyranoside) (21), has been isolated. Phanoside is a dammarane-type saponin and found to stimulate insulin release from isolated rat pancreatic islets. Phanoside (40 and 80 mg/mL) improved glucose tolerance and enhanced plasma insulin levels at hyperglycemia, when given orally to rats [20]. Coagulin C (22), 17β-hydroxywithanolide K (23), withanolide F (24), coagulanolide (25) and coagulin L (26), isolated from the fruits of Withania somnifera, showed significant inhibition on postprandial rise in hyperglycemia post sucrose load in normoglycemic rats and in streptozotocin-induced diabetic rats. Coagulin L (26) showed significant fall in peripheral blood glucose profile and also improved the glucose tolerance of db/db mice [21]. Methanolic extract of the leaves of Boussingaultia baselloides yielded four nor-saponins and a saponin with hypoglycemic activity (27-31). Amongst these, boussingoside A1 (31) exhibited very strong hypoglycemic activity in rats [22].

3.3. Diterpenoids Diterpenoids are composed of four isoprene units and have the molecular formula C20H32, which are derived from geranylgeranylpyrophosphate pathway. Andrographolide (32), a diterpenoid lactone, obtained from

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COOH

COOH O O OH

COOH CH2OH

CH2OH

O CH2OH

OH OH

OH 19

O HO

COOH OH COOH O O O

CH2OH O

O CH2OH OH OH

OH OH

CH2OH

OH OH

OH HO O

H O O

H O

24 OH

OH H O O

H H

OH 25

H O O

OH HO HO

O OH

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H CO2R1 R3 HO HO

O HO HO

O R2

CO2 H

HOOC O HO O

27- R1 = H, R2 = Me, R3 = CO2H 28- R1 =β-D-glucosyl, R2 = Me, R3 = CO2H 29- R1 =β-D-glucosyl, R2 = R3 = CH2OH 30- R1 = H, R2 = CH2OH, R3 =CO2H

Andrographis paniculata was found to possess significant hypoglycemic activity [23]. A modified diterpene, saudin (33) was isolated from the leaves of Cluytia richardiana (Euphorbiaceae) growing in Saudi Arabia. It is related to the labdane-type of diterpenes with a novel rearrangement of lactone groups, was found to possess hypoglycemic activity when tested in alloxan induced diabetic rats [24]. Bioassay-guided fractionation of the EtOH extract of Maprounea africana, on noninsulin-dependent diabetes mellitus db/db mouse model, resulted in the isolation of a daphnane-type diterpenoid, maprouneacin (34) which showed potent glucose-lowering properties by the oral route [25]. O O

O O CH2

O O

CH2OH 32

O H Me

O O H

O O

O 33

OH OH

OH 34

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3.4. Sesquiterpenoids Sesquiterpeniods consist of three isoprene units and have the molecular formula C15H24. A sesquiterpene lactone, lactucain C (35) and furofuran lignan, lactucaside (77), were isolated from Lactuca indica which showed in vivo antihyperglycemic activity profile Δ -22.74 ± 12.53% and Δ -17.95 ± 5.63% using STZ-diabetic rats at a dose of 1 μM/kg [26]. O 14

13'

1 3

H 11'

12' O 6'

H R

15'

14'

35: R =

16 O C

H2C

3.5. Alkaloids An alkaloid is a naturally occurring nitrogenous organic molecule that has a pharmacological effect on humans and other animals. Berberine (36) is known to have potent hypoglycemic activity. It was obtained from the traditional medicinal plant Tinospora cordifolia [27]. The mode of its antihyperglycemic activity was investigated in the Caco-2 cell line. Berberine effectively inhibited the activity of disaccharidases in Caco-2 cells, decreased sucrase activity after pre-incubation with Caco-2 cells for 72 h but failed to produce any significant effect on gluconeogenesis and glucose consumption of Caco-2 cells, suggesting that the antihyperglycemic activity of berberine is at least partly due to its ability to inhibit α-glucosidase and decrease glucose transport through the intestinal epithelium [28]. Other alkaloids such as catharanthine (37), vindoline (38) and vindolinine (39) obtained from Catharanthus roseus also lower blood sugar level [29]. Arecoline (40), an alkaloid isolated from Areca catechu was investigated and reported to have hypoglycemic activity in an animal model of diabetes upon subcutaneous administration [30].

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Cryptolepine (41) is a rare example of a natural product whose synthesis was reported prior to its isolation from Cryptolepis sanguinolenta. Cryptolepine and its salts form lower blood glucose in rodent models of type II diabetes. To optimize this natural product lead, a series of substituted and hetero substituted cryptolepine analogs was synthesized [31]. Aegeline (42), an alkaloidal-amide from the leaves of Aegle marmelos, was isolated by our group and was found to have antihyperglycemic activity as depicted from the lowering of the blood glucose levels by 12.9% and 16.9% at 5 and 24 h, respectively, in sucrose challenged streptozotocin induced diabetic rats (STZ-S) model at the dose of 100 mg/kg body weight. The reasonable mapping of compound to a validated pharmacophoric hypothesis and 3D QSAR model with an estimated activity (283 nM) suggested that aegeline might be a β3-adregenic receptor (AR) agonist [32]. Hypoglycemic activity of trigonelline (43) and 4-hydroxyisoleucine (57) isolated from seeds of Trigonella foenum graecum viz was evaluated in alloxan induced diabetic mice. The combination of 4-hydroxyisoleucine and trigonelline [4-HIT, 40: 30, 120 mg/kg] was administered orally in alloxan induced diabetic mice. After 28 days treatment with 4-HIT, there was significant decrease in blood glucose level. 4-HIT increased the glucose threshold as compared to only alloxan treated group. Histology of pancreas showed formation of new islets near the vicinity of the pancreatic duct. Glyburide was used as a standard antidiabetic drug and its effect on pancreatic cell was also studied. The pancreatic β cells of glyburide treated mice did not show any islets in the vicinity of pancreatic duct. LD50 was found to be more than 5000 mg/kg. These results suggested that 4-HIT showed hypoglycemic effect in alloxan induced diabetic mice. The presence of the pancreatic islets in the vicinity of duct suggested that 4-HIT might act by regeneration of new islets [33]. The therapeutic potential of Galega officinalis for the management of diabetes was defined in the first half of the twentieth century. G. officinalis is a rich source of guanidine and related molecules, which account for its biological effects. The toxicity of guanidine precludes its use clinically, and experiments by Georges Tanret in the years immediately before the Great War identified a less toxic guanidine-like alkaloid, galegine (44) [34]. The synthetic biguanides such as metformin (45) and its analogues were synthesized on the basis galegine chemical structure.

3.6. β-Carbolines The β-carboline alkaloids Harmane (46), norharmane (47) and pinoline (48), were found to increase insulin secretion two to three-fold from isolated

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+ N

OCOCH3

OCH3 N H

OCH3

COOC H3

COOCH3 OH

N H

H3CO

37 O

N O N

N COOCH3

N H

O OH

H N + N

MeO

H2N

N H

NH2

human islets of langerhans. Harmane and norharmane obtained from Tribulus terrestris may account for the hypoglycemic property of the plant [35]. Harmane stimulates insulin secretion in a glucose-dependent manner. The results strongly substantiated the claim of β-carbolines as potent insulin secretagogues [36]. Harmine (49) is found in Syrian rue (Peganum harmala) and other plants. Recently Waki and co-workers through a small-molecule library screen has identified it as a proadipogenic that acts by inducing PPARγ expression. Obese (db/db) mice treated with harmine show a delay in the onset of diabetes, coincident with increased oxygen consumption and thermogenesis. A 2-fold increase in PPARγ levels was selectively seen in white adipose tissue, while there was a 50% decrease in PPARγ levels in the liver and no change in muscle, brown adipose tissue, or kidney. The effect of harmine on PPARγ expression in the brain and pancreas is currently unknown [37].

H3CO

N H

MeO

Naturally occurring antihyperglycemic and antidyslipidemic agents

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3.7. Carbohydrates Two hypoglycemic principles, ganoderan B (50) and C (51), isolated from the fruit bodies of Ganoderma lucidum were shown to be peptidoglycans with mol wts of 7400 and 5800, respectively. Physicochemical and chemical studies demonstrated that the backbone and side chains of ganoderan B contain D-glucopyranosyl β-1→3 and β-1→6-linkages while those of ganoderan C contain D-glucopyranosyl β-1→3 and β-1→6-linkages and a D-galactopyranosyl α-1→6-linkage [38]. β -D-Glcp1

3)- β -D-Glcp-(1

6β -D-Glcp1 1

3)-β -D-Glcp-(1

6 β -D-Glcp

[

3)-β -D-Glcp-(1

]5 [

3)- β -D-Glcp

6)-α -D-Galp-(1

3.8. Amino acids FR225659 (52) and four related compounds (53-56) are gluconeogenesis inhibitors that consisted of an acyl-group and three unusual amino acids. They were isolated from the culture broth of Helicomyces sp. and purified by absorptive resin and reverse-phase column chromatography. They were found to be potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents [39]. T. foenum-graecum (Leguminosae family) is an annual herbaceous plant commonly known as fenugreek and is widely distributed across Asia, Africa, and Europe. Fowden [40] was the first to isolate and identify the unusual amino acid, 4-hydroxyisoleucine (57). Christophe et al. [41] discovered that the major isomer 2S,3R,4S of 4-hydroxyisoleucine induces insulin secretion through a direct effect on pancreatic β cells in rats and humans. Recent studies by our group also confirm the antihyperglycemic activity [42]. The plant Blighia sapida belongs to sapindacae family, which is known for its poisonous properties. Two unusual amino acids such as hypoglycin A (58) and hypoglycin B (59) isolated from this plant possess antihyperglycemic activity [43].

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T. Narender et al. NH2 HO N

NH2 COOH

NH O R1

NH2

-OH

-CH3

53 54

-OH

-OH -OH -H

-OCH3 -OCH3

COOH

-OH

COOH

O NH2

-CH2CH3 COOH

-OH

-CH3 -CH2CH3

-OH

-CH3

COOH

3.9. Miscellaneous Salacinol (60) has been isolated from an antidiabetic ayurvedic traditional medicine, Salacia reticulata, through bioassay-guided separation and was found to be most potent natural α-glucosidase inhibitor [44]. Allicin (thio-2-propene-1-sulfinic acid S-allyl ester) (61), a sulphur compound isolated from garlic (Allium sativum) has resulted in pronounced hypoglycemia in mildly diabetic rabbits upon oral administration (0.25 mg/kg) [45]. S-allyl cysteine sulphoxide (62), a sulphur containing amino acid which is the precursor of allicin and garlic oil, has been found to show significant antidiabetic effects in alloxan diabetic rats at a dose of 200 mg/kg body weight [46]. Leporin B (63), a demethylated analog of leporin A (64) was isolated from a taxonomically unidentified fungal strain to discover compounds with the ability to increase expression levels of the enzyme hexokinase II [47].

O O HO HO

-O3SO S+

CH2OH

H H CH2OH OH 60

S S

H2N OH

N O

O 61

63 R=Me 64 R=H

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Rat lens aldose reductase (RLAR) inhibitors (65-67 and 16) from the fruiting bodies of Ganoderma applanatum were isolated, protocatechualdehyde (67) was the most potent RLAR inhibitor (IC50 = 0.7 μg/mL) equivalent to that of the positive control TMG (IC50 = 0.6 μg/ml) [17]. O CHO OH

OMe HOOC

HO HO HO

n 65

NH O

n = 12-15

n = 7-9

OH OH 67

2-Arylbenzofuran, puerariafuran (68) was isolated from MeOH extract of the roots of Pueraria lobata as active constituent, using an in vitro bioassay based on the inhibition of advanced glycation end products (AGE). The compound (68) and coumestrol (69) exhibited a superior inhibitory activity against AGEs formation with IC50 values of 0.53 and 0.19 μM, respectively, compared to a well known positive control, aminoguanidine (IC50 value of 473 μM) [48]. Two compounds viz, kodaistatin A (70) and kodaistatin C (71) were isolated from cultures of Aspergillus terreus. The kodaistatins are effective inhibitors of the glucose-6-phosphate translocase component of the glucose6-phosphatase system (EC 3.1-3.9), an enzyme system which is important for the control of blood glucose levels. The IC50 was 80 nM for kodaistatin A and 130 nM for kodaistatin C [49]. H HO

OH HO

O H3CO

69 HO O

HO OH HO O

O R OH

70 R = H 71 R = OH

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The glucose lowering effect of mangiferin (72), a xanthone glucoside, isolated from the leaves of Mangifera indica was studied in streptozotocininduced diabetic rats. Hypoglycemic activity of mangiferin (10 and 20 mg/kg, i.p. once daily for 28 days) at different time intervals in STZ induced diabetic rats and improvement in oral glucose tolerance in glucose-loaded normal rats upon chronic administration (10 and 20 mg/ kg, i. p.) for 28 days was observed [50]. O

HO O

HO HO

O OH

A xanthone, which is close analogue of mangiferin was isolated from the hexane fraction of the plant, Swertia chirayita, identified as 1,8-dihydroxy3,5-dimethoxyxanthone (swerchirin: 73). It has a very significant blood sugar lowering effect in fasted, fed, glucose loaded, and tolbutamide pretreated albino rat models. The ED50 for 40% blood sugar lowering in CF male albino rats (body weight 140-165 g) is 23.1 mg/kg/oral [51]. Various active components like (−)-epicatechin (74), the benzofuranone, marsupsin (75) and the stilbene, pterostilbene (76) isolated from the bark and heartwood of Pterocarpus marsupium were evaluated for their putative antihyperglycemic activity against streptozotocin-induced hyperglycemic rats and were found to possess blood sugar lowering activity. The phenolic constituents viz, marsupsin (75) and pterostilbene (76) significantly decreased the plasma glucose level of STZ-induced diabetic rats by -33% and -42% respectively. The antidiabetic activity of pterostilbene (-42%) was comparable to that of the reference compound, metformin (-48%) [52]. OH HO

O OH

OH OH

OH 74

OCH3 O 75

MeO

OMe 76

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A furofuran lignan, lactucaside (77) along lactucain (35) was isolated from Lactuca indica which showed in vivo antihyperglycemic activity profile Δ -17.95 ± 5.63% using STZ-diabetic rats at a dose of 1 μM/kg [26]. OCH3 OH O 9' 7

8' 1'

O 4' HO

R = glucose

3' OCH3 77

HO OH

MeO 78

H 79

Ferulic acid (78) is polyphenolic compound found in many medicinal plants such as Curcuma longa. Ohnishi and co-workers from Japan demonstrated its antihyperglycemic activity in insulin dependent (IDD) and non-insulin dependent diabetes milletus models (NIDDM) [53]. Similar class of compound that is cinnamaldehyde (79) was isolated from Cinnamonum zeylanicum (cinnamon) exhibits potent antihyperglycemic activity in streptozotocin (STZ)-induced male diabetic wistar rats [54]. Both the compounds also possesses hypolipidemic properties [51,52].

4. Dyslipidemia Dyslipidemia is elevation of plasma cholesterol, triglycerides (TGs), or both, or a low high density lipoprotein level that contributes to the development of atherosclerosis. Causes may be primary (genetic) or secondary. Diagnosis is by measuring plasma levels of total cholesterol, TGs, and individual lipoproteins. When carbohydrates are in low supply or their breakdown is incomplete, fats become the preferred source of energy in diabetic patients. As a result, the fatty acids are mobilized into the general circulation leading to secondary triglyceridemia in which total serum lipids in particular triglycerides as well as the levels of cholesterol and phospholipids

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increase. This rise is proportional to the severity of the diabetes. Uncontrolled diabetes is manifested by a very high rise in triglycerides and fatty acid levels. An increase in plasma lipids, particularly cholesterol, is a common feature of atherosclerosis, a condition involving arterial damage, which may lead to ischemic heart disease, myocardial infarction, and cerebrovascular accidents. These conditions are responsible for one-third of deaths in industrialized nations [55].

5. Current therapeutics Current antidyslipidemia drugs include statins, fibrates, niacin, ezetimibe, and bile acid binding resins (Table-2). These drugs target one component of the lipid profile, with smaller additional effects on other parameters. For instance, statins and fibrates produce sizable reductions primarily in plasma LDL-C and TG, respectively. Meanwhile, niacin has the greatest HDL-C raising capacity. However, many high CHD risk patients fail to reach strict guideline target levels with currently Table 2. Currently available pharmaceuticals for dyslipidemia. Medication

Effects on lipid parameters ↓ ↓ LDL-C, ↓ TG Minimal effects on HDL-C (rosuvastatin can increase HDL-C levels)

Adverse effects

Fibrates (PPAR- α agonists)

↓ LDL-C, ↓ ↓ TG, ↑ HDL-C (mild)

Myalgias, Rhabdomyolysis Cholelithiasis, Elevations in serum creatinine

Ezetimibe (intestinal cholesterol absorption inhibitor) Niacin

↓ LDL-C, ↓ TG

Myalgias (very rare) Rhabdomyolysis (very rare)

Statins (HMG-CoA reductase inhibitors)

Myalgias, Myositis/rhabdomyolysis Transaminitis

↓ ↓ TG, ↑ ↑ HDL-C, ↓ Flushing/vasodilation Impair insulin sensitivity ↓ LDL-C, ↓ ↓ LP (a) Gout, gastric

Bile acid ↓ LDL-C resins (inhibitors of enterohepatic circulation)

↑ TG Bloating, constipation Interference with absorption of other, medications such as levothyroxine, warfarin, digoxin, statins

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available drugs. A small but clinically relevant proportion of patients experience adverse effects. Thus, additional pharmaceutical strategies are required to fill these gaps in efficacy and tolerability. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them.

5.1. Sterols and triterpenoids A number of studies, both in animal models and human clinical trials, have shown that guggulipid (80,81) isolated from the Resin of the gum of the guggul tree, Commiphora mukul, has beneficial effects on serum lipoprotein profiles [56]. A pregnane glycoside roylenine (82) was isolated from Marsdenia roylei. The glycoside (82) and its acetylated derivative showed singnificant antioxidant and antidyslipidemic activities [57]. H3C

OH O

H3C

OH 82

H3C AcO AcO

O O

A steroidal saponin, chloragin (17) [tigogenin-3-O-α-Lrhamnopyranosyl-(1 → 4)- -D-glucopyranosyl-(1 → 3)-β-D-xylopyranosyl(1 → 4 )-β-D-glucopyranosyl-(1 → 4)-β-D-xylopyranoside] was isolated from the aerial part of Chlorophytum nimonii (Grah) which showed potent antidyslipidemic activities in albino rats [18]. Coagulin L (26) isolated from Withania somnifera showed significant fall in peripheral blood glucose profile and also improved the glucose tolerance of db/db mice. It also showed antidyslipidemic activity in db/db mice that is comparable to median effective dose of fenofibrate i.e., 50 mg/kg body weight [21]. Sudhahar and co-workers reported hypercholesterolemia in lupeol (83) and linoleate ester of lupeol (84) [58]. We have also prepared several ester derivatives of lupeol and studied their structure activity relationship. Some of the derivative showed potent activity than the lupeol. Lupeol nicotenate (85) was found to be the most potent triglyceride lowering agent in addition to antihyperglycemic activity [59].

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O O

HO N 84

Wiedendiol-A (86) and B (87), sesquiterpene-hydroquinones which inhibit cholesteryl ester transfer protein (CETP), have been isolated from the marine sponge Xestoepongia wiedenmayeri [60] HO

HO OCH3 CH3

OCH3 CH3

H 87

Statins are currently marketed drugs used to lower the plasma cholesterol levels in humans. Natural statins obtained from different genera and species of filamentous fungi. Lovastatin (88) is mainly produced by Aspergillus terreus strains and mevastatin (89) by Penicillium citrinum. Pravastatin (90) was obtained by the biotransformation of mevastatin by Streptomyces carbophilus and simvastatin (91) by a semi-synthetic process, involving the chemical modification of the lovastatin side chain. The hypocholesterolemic effect of statins lies in the reduction of the very low-density lipoproteins (VLDL) and LDL involved in the translocation of cholesterol, and in the increase in the high-density lipoproteins (HDL), with a subsequent reduction of the LDL- to HDL-cholesterol ratio, the best predictor of atherogenic risk [61]. HO O

O O

COOH OH

HO O

O O

HO 88

Naturally occurring antihyperglycemic and antidyslipidemic agents

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Several synthetic statins such as atorvastatin (92), cerivastatin (93), pitavastatin (94) and rosuvastatin (95) were developed on the basis of structures of natural statins. HO

COOH OH

F F

COOH OH

O 92

COOH OH

S O

N N O 95

A diterpene, (96) which has close structural features of statins was isolated from the leaves of Polyalthia longifolia [62]. This compound showed significant antidyslipidemic activity in high diet (HFD) fed dyslipidemic hamsters at different doses. O

5.2. Polyphenolic compounds Few naturally occurring flavanones and their glycosides such as hesperetin (97), hesperidin (98), naringenin (99), and naringin (100) have been reported as potential agents for improving the cholesterol metabolism in diet-induced hypercholesterolemic animals [63]. We also isolated three modified furano-flavonoids (101-103) and a rare flavonol glycoside (104) as an antidyslipidemic agents from the aerial parts of Indigofera tinctoria [64]. Flavonoid mixture (101 and 102) showed potent triglyceride lowering activity in high fat fed hamster model.

176

T. Narender et al. O O H

O H H

R2 RO

O 101

OH O

H O

O H H

O 102 OR

O 97: R= H; R1=OH; R2=OCH3 98: R= β -D-Rutinoside; R1=OH; R2= OCH3 O 99: R = H; R1=H; R2=OH 100: R=Neohespiridoside; R1=H; R2=OH

O O

O OH

O OH O

O 103

104: R= Rhamnose

Eriocitrin (105) (eriodictyol 7-O-β-rutinoside) is the main flavonoid in lemon fruit (Citrus). Eriocitrin was effective in lowering effect on serum and hepatic lipids in high-fat and high-cholesterol fed rats [65]. OH HO

OH OH

H3C

OH O

OH OH

OH O

105

Pterosupin (106) and liquiritigenin (107) were isolated from the heartwood of Pterocarpus marsupium showed hypolipidemic activity in Triton model. Both the compounds lowered the serum cholesterol and LDL-cholesterol. Pterosupin also lowered the triglycerides [66]. OH HO

Glc OH O 106

O 107

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Rutin (108) is flavonoid glycoside found in many plants and is also an important dietary constituent of food and plant-based beverages. Several studies demonstrated lipid lowering effect of rutin. Recently Amir and co-workers reported its anti-hyperchloesterolaemic effect (plasma cholesterol and LDL-C) in rat model [67]. Odbayar and co-workers from Japan studied the effect of quercetin (109) and its glycoside (rutin) and their studies indicated that quercetin better than the rutin in reduction of hepatic lipogenesis (hypolipidemic effect) [68]. OH OH HO

OH HO O

OH OH OH O

H3C O HO HO

O OH

O 109

108

Tso-Hsiao Chen and co-workers studied about 40 flavonoids for their HMG-Co-Enzyme reductase activity. Astilbin (110) was the only effective HMG-Co-Enzyme reducates inhibitor in their studies, which demonstrates its hypochelestereamic activity [69]. OH O

O HO

OH OH O

CH3 OH

110

Kurarinol (111) is a prenylated flavanone, which is known for its alpha glucosidase, beta amylase and diacylglyceral transferase activity. Kuraridinol (112) is a prenylated chalcone. Both were isolated from the Sophora flavescens showed significant hyperlipidemic and hypercholesterolemic effect. Kuraridinol was more potent than kurarinol in their studies [70].

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T. Narender et al. OH

OH HO

OH OH

OH O

O 112

111

Resveratrol (113), a naturally occurring stilbenoid commonly available in red wine act as a free-radical trap to halt the progression of LDL oxidation. It is very strong antioxidant and mild lipid lowering agent, which certain extent prevents the development of atherosclerosis [71]. Resveratrol derivatives such as, pterostelbene (76) and trimethylated resveratrol (114) and its analogue Piceatannol (115) have been studied for their PPAR alpha activity and in-vivo hyperlipidemic activity. Pterostelbene showed good PPAR alpha agonist activity and hypolipidemic activity than other compounds [72]. Polydatin (116) is glycoside of resveratrol isolated from Polygonum cuspidtum also has been reported for its lipid lowering effect in high fat diet fed hamster [73]. OH HO

MeO

OH OH

OMe 113

OMe

114

115

Mangiferin (72) a xanthone glucoside, isolated from the leaves of Mangifera indica showed significant antihyperlipidemic activity at a dose of 10 and 20 mg/kg, i.p. Further, in streptozotocin-induced diabetic rats it showed antiatherogenic activities as evidenced by significant decrease in plasma total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C) levels coupled together with elevation of high density lipoprotein cholesterol (HDL-C) level and diminution of atherogenic index in diabetic rats [50].

Naturally occurring antihyperglycemic and antidyslipidemic agents

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HO H OH

H OH H O

O H H OH

HO H

HO H 116

Bergenin (117) is commonly available in many plants of Euphorbiaceae, Saxifragaceae and Myrsinaceae. It is a C-glucoside of 4-O-methylgallic acid. Oral administration of bergenin isolated from the leaves of Flueggea microcarpa reduced the serum cholesterol, triglycerides, low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL)-cholesterol levels were significantly [74]. OH OH

OH O MeO

OH O

HO O 117

5.3. Alkaloids Berberine (36), a natural plant alkaloid isolated from the root of Berberis oblonga. In vitro and in vivo studies have showed its effects on hyperglycemia and dyslipidemia [75]. Our activity guided fraction and isolation work o the leaves of A. marmelos led to isolate an alkaloidal-amide, Aegeline (42) and found to have antihyperglycemic activity as well as hypolipidemic activity [32]. Aegeline has strong triglyceride lowering activity in our studies and the activity was comparable with the marketed drug i.e. fenofirbrate. Hsu and coworkers showed that arecoline (40) inhibited adipogenesis as determined

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by oil droplet formation and adipogenic marker gene expression. There further studies indicated that arecoline induced lipolysis in an adenylyl cyclase-dependent manner [30].

5.4. Amino acid We isolated an unusual amino acid 4-hydroxyisoleucine (57) from the seeds of T. foenumgraecum, which significantly decreased the plasma triglyceride levels by 33% (P < 0.002), total cholesterol (TC) by 22% (P < 0.02), and free fatty acids by 14%, accompanied by an increase in HDL–C/ TC ratio by 39% in the dyslipidemic hamster model [11]. 4-Hydroxyisoleucine is also very good insulin releasing agent.

5.5. Miscellaneous C60-polyprenol (118) was isolated from the chloroform fraction of the ethanol extract of Coccinia grandis. It significantly decreased serum TG by 42%, total cholesterol (TC) 25% and glycerol (Gly) 12% and increased HDL-C/TC ratio by 26% in high fat diet (HFD)-fed dyslipidemic hamsters at the dose of 50 mg/kg body weight as compared to the standard drug fenofibrate at the dose of 108 mg/kg [76]. O S 7

OH H2N OH O

118

119

S-methyl cysteine sulfoxide –SMCS (119) isolated from Allium cepa was investigated for its lipid lowering action in SD rats. SMCS at a dose of 200 mg/kg body weight for 45 days enhanced the hyperlipidemic condition. Concentrations of cholesterol, triglyceride and phospholipids were significantly reduced with respect to control [77]. Itokawa and co-workers also reported the lipid lowering activity in S-methyl cysteine sulfoxide (SMCS) and S-allylcysteine sulfoxide (62) [78]. Ferulic acid (78) and cinnamaldehydes (79) which are commonly available in many medicinal plants have been reported for their lipid lowering activity as well as anthyperglycemic activity [51,52].

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6. Conclusion Type-II diabetes poses a lethal threat to mankind in the present health scenario. The more alarming situation has raised owing to the secondary complications such as atherosclerosis, (ischemic heart disease, myocardial infarction, and cerebrovascular accidents) associated with this silent killer. So, there is an urgent need for broad based drugs which can ameliorate this complex menace. Natural products have always been the inexhaustible source of new drugs from the time immemorial. Notwithstanding the significant headways in synthetic chemistry in the management of hyperglycemia and hyperlipidemia, chemical entities emanating from the natural source still hold promise in alleviating the blood glucose levels and lipids and its concurrent ailments. More has been done but much has remained unexplored in the drug discovery paradigm of natural products attributed with therapeutic virtues. Some targets have been identified for the active principles but unless, their mechanism of action is not determined and clinical studies not performed, their potential as antihyperglycemics and antidyslipidemics will remain unearthed. Moreover, the combination of plant based drugs and synthetic pharmaceuticals for correcting this metabolic error could pave way for costeffective therapies. The scope of plant drugs lies in the rectifying the problem of adverse side effects generated by synthetic drugs, cost-effectiveness and minimal side-effects. The resurgence of natural products in the drug discovery and development may hold the key in the proper utilization of biodiversity for the management of hyperglycemia and hyperlipidemia.

Acknowledgements The authors are grateful to the Director, CDRI, Lucknow for constant encouragement for the program on Indian medicinal plants, CSIR, New Delhi for financial support.

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64. Narender, T., Puri, A., Shweta, Khaliq, T., Saxena, R., Bhatia, G., Chandra, R. Bioorg. Med. Chem. Lett., 2006, 16, 293-296. 65. Miyake, Y., Suzuki, Ohya, S., Fukumoto, S., Hiramitsu, M., Sakaida, K., Osawa, T., Furuichi, Y. J. Food Sci., 2006, 71, S633. 66. Farbooniay, M.A., Ray, A.B. J. Nat. Prod., 1993, 56, 989. 67. Ziaee, A., Zamansoltani, F., Nassiri-Asl, M., Abbasi E. Basic & Clinical Pharmacology & Toxicology, 2009, 104, 253-258. 68. Odbayar, T.O., Badamhand, E., Kimura, T., Takahashi, Y., Tsushida, T., Ide, T. J. Agric. Food Chem., 2006, 54, 8261-8265. 69. Chen, T.H., Liu, J.C., Chang, J.J., Tsai, M.F., Hsieh, M.H., Chan, P. Chinese Medical Journal, 2001, 64, 382-387. 70. (a) Kim, H.Y., Jeong, D.M., Jung, H.J., Jung, Y.J., Yokozawa, Choi, J.S. Biol. Pharm. Bull., 2008, 31,73-78. (b) Woo, E.R., Kwak, J.H., Kim, H.J., Park, H. J. Nat. Prod., 1998, 61, 1552-1554. (c) Chung, M.Y., Rho, M.C., Ko, J.S., Ryu, S.Y., Jeune, K.H., Kim, K., Lee, H.S., Kim, Y.K. Planta Med., 2004, 70, 258260. (d) Kim, J.H., Ryu, Y.B., Kang, N.S., Lee, B.W., Heo, J.S., Jeong, I.Y., Park, K.H. Biol. Pharm. Bull., 2006, 29, 302-305. (e) Zhang, L., Xu, L., Xiao, S.S., Liao, Q.F., Li, Q., Liang, J., Chen, X.H., Bi, K.S. J. Pharm. Biomed. Anal., 2007, 44, 1019-1028. (f) Santos, L., Curi Pedrosa, R., Correa, R., Cechinel Filho, V., Nunes, R.J., Yunes, R.A. Arch. Pharm., 2006, 339, 541-546. 71. Arichi, H., Kimura, Y., Okuda, H., Baba, K., Kozawa, M., Arichi, S. Chem. Pharm. Bull., 1982, 30, 1766-1770. 72. Rimando, A.M., Nagmani, R., Feller, D.R., Yokoyama, W. J. Agric. Food. Chem., 2005, 53, 3403-3407. 73. Du, J., Sun, L.N., Xing, W.W., Huang, B.K., Jia, M., Wu, J.Z., Zhang, H., Qin, L.P. Phytomedicine, 2009, 16, 652-658. 74. Jahromi, M.A.F., Chansouria, J.P.N., Ray, A.B. Phytotherapy Research, 1992, 6, 180. 75. (a) Leng, S.H., Lu, F., Xu, L.J. Acta Pharmacol Sinica, 2004, 25, 496-502. (b) Punitha, I.S.R., Shirwaikar, A., Shirwaikar, A. Diabetologia Croatica, 2005, 117, 34-4. 76. Singh, G., Gupta, P., Rawat, P., Puri, A., Bhatia, G., Maurya, R. Phytomedicine, 2007, 14, 792-798. 77. Kumar, K., Augusti, K.T. J. Ethnopharmacology, 2007, 109, 367. 78. Itokawa, Y., Indue, K., Sasagawa, S., Fujiwara, M. J Nutr., 1973, 103, 88.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 187-212 ISBN: 978-81-308-0448-4

6. Bio-flavonoids with promising antidiabetic potentials: A critical survey Goutam Brahmachari Department of Chemistry, Visva-Bharati University, Santiniketan-731 235 West Bengal, India

Abstract. Bio-flavonoids comprise a group of phenolic secondary plant metabolites that are widespread in nature. Major flavonoids that have well categorized structures and well defined structure function-relationships are: flavans, flavanones, flavones, flavonols, flavanols, flavanonols, cetechins, anthocyanidins and isoflavones. Bio-flavonoids are well-known for their multi-directional biological activities including anti-diabetic efficacy. Numerous studies have been carried out to explore their potential role in the treatment of diabetes. A good number of studies have already demonstrated the hypoglycemic effects of flavonoids using different experimental models and treatments - the drug candidates have been shown to exert such beneficial effects against the disease manifestation, either through their capacity to avoid glucose absorption or to improve glucose tolerance. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications; besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues, and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. As a result, bio-flavonoids are now-a-days regarded as promising and significantly attractive natural substances to enrich the current therapy options against diabetes. Correspondence/Reprint request: Dr. Goutam Brahmachari, Department of Chemistry, Visva-Bharati University, Santiniketan-731 235, West Bengal, India. E-mail: brahmg2001@yahoo.co.in

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The purpose of this resume is to represent promising anti-diabetic flavonoid candidates highlighting their absorption and metabolism along with their mode of action in regulating diabetic symptoms.

1. Introduction Diabetes mellitus is the most prevalent metabolic syndrome world-wide with an incidence varying between 1 to 8% [1,2]. The disease arises when insufficient insulin is produced, or when the available insulin does not function properly. Thus diabetes is characterized by hyperglycaemia (elevation in blood sugar levels) resulting in various short-term metabolic changes in lipid and protein metabolism and long-term irreversible vascular changes. The long-term manifestation of diabetes can result in the development of some complications, broadly classified as microvascular or macrovascular disease. Microvascular complications include neuropathy (nerve damage), nephropathy (renal disease) and vision disorders (retinopathy, glaucoma, cataract and corneal diseases), while macrovascular complications include heart disease, stroke and peripheral vascular disease, which can lead to ulcers, gangrene and amputation [3]. These complications are also found in non-diabetic population, but have a two to five-fold increase in diabetic subjects [4]. The last century has seen a rapid increase in the global prevalence of coronary artery disease (CAD) [5,6]. Current estimates from different countries in Europe and the United States have shown that diabetes and its complications account for 8-16% of the total health costs for society and this will increase dramatically unless major efforts are made to prevent the ongoing epidemic. There are two major categories of diabetes - insulin dependent diabetes mellitus (IDDM, Type 1 diabetes mellitus) and non-insulin dependent diabetes mellitus (NIDDM, Type-2 diabetes mellitus). Type 1 diabetes occurs due to almost 95% destructions of β-cells of islets of Langerhans in the endocrine pancreas caused by an autoimmune process, usually leading to absolute insulin deficiency, this type has an early onset, most often between the ages of 10 and 16 yrs. Insulin resistance in peripheral tissue and an insulin secretive defect of the β-cells characterizes Type-2 diabetes mellitus (NIDDM). It is the most common form of diabetes mellitus constituting above 90% of the diabetic population and highly associated with a family history of diabetes, older age, obesity and lack of exercise [3]. The global prevalence of diabetes is estimated to increase, from 4% in 1995 to 5.4% by the year 2025 [7]. The World Health Organization (WHO) has predicted that the major burden will occur in the developing countries, there will be a 42% increase from 51 to 72 million in the developed countries while 170% increase from 84 to

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228 million, in the developing countries [8]. Prevalence of the complications is greater among the lower socio-economic people due to lack of good control of glycaemia and hypertension and also due to behavioral factors. The direct and indirect costs involved in the treatment of the chronic disease especially when associated with the vascular complications are enormous. The overall global scenario urges to implement cost-effective and at the same time efficacious preventive measures against diabetes to reduce the high morbidity and mortality [4].

2. Currently available therapies Currently available therapies for diabetes include insulin and various oral anti-diabetic agents such as sulfonylureas, biguanides, α-glucosidase inhibitors, and glinides, which are used as monotherapy or in combination to achieve better glycemic regulation. Many of these oral anti-diabetic agents suffer from various adverse effects, thus, managing diabetes without any side effects is still a challenge to the workers [9], and hence the search for more effective and safer therapeutic agents in eradiating diabetic syndromes has continued to be an important area of investigation. Both fasting and postprandial impaired glucose tolerance are associated with an increased risk of developing Type-2 diabetes mellitus and therefore form an important target group for interventions aimed at preventing diabetes [10]. The pharmacological agents with the greatest effect on postprandial hyperglycemia include insulin lispro, amylin analogues, and α-glucosidase inhibitors. In hyperglycemia associated with diabetes, the use of aldose reductase inhibitors has been reported for the treatment of diabetic complications [11]. Aldose reductase as a key enzyme in the polyol pathway has been reported to catalyze the reduction of glucose to sorbitol. Sorbitol does not readily diffuse across cell membranes, and the intracellular accumulation of sorbitol has been implicated in the chronic complications of diabetes such as peripheral neuropathy, retinopathy, and cataracts [12]. A recent study reported that aldose reductase may also be involved with another signal transduction pathway in the pathogenesis of diabetic nephropathy [13].

3. Back to the plant kingdom The use of ethnobotanicals has long folkloric history for the treatment of blood sugar abnormalities. In the India, indigenous remedies have been used in the treatment of diabetes since the time of Charaka and Sushruta (6th century B.C.) [14]. Plants have always been exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly

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from them. The ethnobotanical information reports about 800 plants that may possess anti-diabetic potential [15]. Many of such plants have exhibited anti-diabetic activity when assessed using presently available experimental techniques [17-20]. It may be mentioned in this connection that the discovery of widely used hypoglycaemic drug, metformin came from the traditional approach of using Galega officinalis. In spite of all these, the indigenous system has not yet gained enough momentum in the scientific community. The reasons may be many including lack of belief among the practitioners of conventional medicine over alternative medicine, alternative form of medicine are not very well-defined and natural drug may vary tremendously in content, quality and safety. To cope with severe problems associated with using of synthetic anti-diabetic drugs, there is a need to look for more efficacious drugs with lesser side effects and also of low cost. It is the high time to turn our attention to the plant kingdom in search of natural drugs for diabetes following an integrated approach and using correct procedures. The hypoglycemic effect of several plants used as anti-diabetic remedies has already been confirmed, and the mechanisms of hypoglycemic activity of these plants are being studied; if even a single plant material stands the acidtest of efficacy comparable to commonly used synthetic oral drugs already marketed, it will herald the discovery of cheap and relatively nontoxic drug.

4. Purpose of the present review A number of review articles on the uses of various plants (different parts of plant materials, crude extracts, herbal formulations, etc.) as anti-diabetic agents have been published time to time [22-26]. Naturally occurring chemotypes of varying structural skeletons have also been reported to possess anti-diabetic properties [27,28], and the purpose of this resume is to represent promising anti-diabetic bio-flavonoids highlighting their absorption and metabolism along with mode of action in regulating diabetic symptoms.

5. Anti-diabetic bio-flavonoids of promise Bio-flavonoids comprise a group of phenolic secondary plant metabolites that are widespread in nature. Major flavonoids that have well categorized structures and well defined structure function-relationships are: flavans, flavanones, flavones, flavonols, flavanols, flavanonols, cetechins, anthocyanidins and isoflavones. Bio-flavonoids are well-known for their multi-directional biological activities including anti-diabetic efficacy [29-32]. Numerous studies have been carried out to explore their potential role in the treatment of diabetes [27,28,33]. A good number of studies have already demonstrated the

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hypoglycemic effects of flavonoids using different experimental models and treatments - the drug candidates have been shown to exert such beneficial effects against the disease manifestation, either through their capacity to avoid glucose absorption or to improve glucose tolerance. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications; besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues, and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. As a result, bio-flavonoids are now-a-days regarded as promising and significantly attractive natural substances to enrich the current therapy options against diabetes. This present section embodies the information on promising anti-diabetic efficacies of certain bio-flavonoids. Choi et al. [34] demonstrated that intraperitoneal administration of prunin (naringenin 7-O-β-D-glucoside) produces a significant hypoglycemic effect in diabetic rats. Anti-hyperglycemic effects have also been demonstrated for various flavonoids including chrysin and its derivatives, silymarin, isoquercetrin and rutin [35-37]. Long-term studies carried out with rutin orally administered to diabetic rats showed that it decreased the plasma glucose levels by up to 60% when compared to the control group. However, oral administration of rutin to normal rats did not show any significant effect on fasting plasma glucose levels [38]. Chronic treatment with hesperidin and naringin was found to lower the blood glucose level of db/db mice compared with the control group [39]. Myrciacitrins I, II, III, IV and V (1-5) isolated from the dried leaves of Myrcia multiflora DC. (family: Myrtaceae) were reported to possess significant rat lens aldose reductase inhibitory activity [40], the IC50 values for the flavonoids 1-5 were determined as 3.2 x 10−6, 1.5 x 10−5, 4.6 x 10−5, 7.9 x 10−7, 1.6 x 10−5 and 1.3 x 10−5 M, respectively [40,41]. Hence, myrciacitrin IV (4) exhibited the most potent activity, although it had less activity than epalrestat, a commercially available synthetic aldose reductase inhibitor (IC50 = 7.2 x 10−8 M) [40]. Kawabata et al. [42] isolated five 6-hydroxy-flavonoids (6-10) from the methanol extract of Origanum majorana L. (family: Lamiaceae) leaves and studied their α-glucosidase enzyme inhibitory activity, three of these flavonoids: 6-hydroxyapigenin (scutellarein) (6), 6-hydroxyapigenin-7-O-βD-glucopyranoside (7), 6-hydroxyluteolin-7-O-β-D-glucopyranoside (8) are previously known [43-47], and the other two feruloylglucosides namely, 6-hydroxyapigenin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (9) and 6hydroxyluteolin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (10) are novel compounds. All the isolates showed rat intestinal α-glucosidase inhibitory activity, at an equal concentration of 500 μM, the flavonoid candidates 6-10

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inhibited the enzyme activity by 81%, 44%, 55%, 25% and 26%, respectively. The respective IC50 values for 6-10 were determined as 12, >500, 300, >500 and >500 μM. Another flavonoid, 6-hydroxyluteolin (11) [48], was also found to exhibit potent α-glucosidase inhibitory activity (92% inhibition at a concentration of 500 μM) with an IC50 value of 10 μM [42]. The same group [49] also evaluated 5,6,7-trihydroxyflavone (baicalein, 12), the flanonoid constituent of Scutellaria baicalensis, as an important inhibitor against rat intestinal α-glucosidase (IC50 = 32 μM). The investigators also observed that apigenin (5,7,4′-trihydroxyflavone, 13) and luteolin (5,7,3′,4′-tetrahydroxyflavone, 14), both lacking the 6-hydroxyl substituent, showed negligible activity (12% and 22% inhibition at 500 μM, respectively) in the α-glucosidase inhibitory assay. From their study, the present investigators suggested that 5,6,7-trihydroxyflavone skeleton is crucial for high α-glucosidase inhibitory activity regardless of B-ring hydroxylation, in addition, glycosation of 7-hydroxyl substituent as well as acylation of the sugar reduces the enzyme inhibitory activity [49]. Haraguchi et al. [50] isolated C-glucosidic flavone derivative named as isoaffineyin (5,7,4,3′,5′-pentahydroxyflavone-6-C-glucoside, 15) from Manikara indica (family: Sapotaceae), the flavonoid candidate exerted promising inhibition against porcine lens aldose reductase activity with an IC50 value of 4.6 μM (epalrestat was used as positive control, IC50 = 0.87 μM).

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The genistein derivatives (16-19) isolated from an EtOAc-soluble partition of the MeOH extract of a branch of Tetracera scandens (family: Dilleniaceae) were evaluated to possess promising activities on Type-2 diabetes mellitus treatment since the test compounds significantly stimulated the uptake of glucose, adenosine monophosphate-activated kinase (AMPK), glucose transport protein-4 (GLUT4) and GLUT1 mRNA expressions and protein tyrosine phosphatase 1B (PTP1B) inhibition in L6 myotubes [51]. The IC50 values for isofavonoids 16-19 in inhibiting PTP1B activities were

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determined as 31.75 ± 0.27, 28.13 ± 0.19, 20.63 ± 0.17 and 37.52 ± 0.31 μM, respectively (ursolic acid was used as positive control with IC50 value of 5.13 ± 0.45μM). No muscle cell toxicity was reported with compounds 17-19, while compound 16 reduced muscle cell viability with IC50 value of 18.69 ± 0.19 μM. The investigators, thus, demonstrated that the isoflavonoids constituents (16-19) of T. scandens stimulate glucose-uptake in basal and insulin-stimulated L6 myotubes in a dose-dependent manner - AMPK activation, GLUT4 and GLUT1 expressions and PTP1B inhibition by these bioactive constituents appeared to be involved in the mechanism of the stimulation of basal and insulin-responsive glucose-uptake. Hence, compounds 16-19 may be possible candidates of a novel therapeutic strategy for Type-2 diabetes mellitus treatment, although further studies will be required to clarify the molecular mechanism of these bioactive constituents [51].

Isoorientin (20), isolated from the water and butanolic extracts of Cecropia obtusifolia (family: Ceropiaceae), exhibited potent hypoglycemic activity comparable to that of glibenclamide at a dose of 3 mg/kg body weight in diabetic rats [52]. Kim et al. [53] isolated a new flavonol glycoside, quercetin 3-O-α-L-arabinopyranosyl-(1Æ2)-β-D-glucopyranoside (21) along with the known flavonoid glycosides such as kaempferol 3-O-β-D-glucopyranoside

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(astragalin) (22a) and quercetin 3-O-β-D-glucopyranoside (isoquercetin) (22b) from the leaves of Eucommia ulmoides (family: Eucommiaceae), these flavonoid constituents were found to be glycation inhibitors having comparable activity to that of aminoguanidine, a known glycation inhibitor. The IC50 values for the test compounds 21, 22a and 22b were determined as: 2.95 x 10−7, 4.86 x 10−7, and 3.20 x 10−7 M, respectively (aminoguanidine was used as positive control, IC50 = 4.45 x 10−7 M) [53].

Tabopda et al. [54] reported that six unusual C-4′-prenylated flavonols, dorsilurins F-K (23-28), isolated from the roots of Dorstenia psilurus (family: Moraceae), were found to exhibit glycosidase enzyme inhibitory activity against α-glucosidase, β-glucosidase, and α-mannosidase. Compound 23, with three unmodified prenyl groups, showed the best α-glucosidase inhibitory activity (IC50 4.13 μM), while compound 28, with only one unmodified prenyl group, showed the least α-glucosidase inhibitory activity (IC50 43.95 μM). Thus, it was suggested that α-glucosidase inhibitory activity of the compounds increased with the number of unmodified prenylated groups present. These compounds (23-28) showed very weak enzyme inhibitory activities against β-glucosidase and α-mannosidase [54].

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Two dihydroflavonol glycosides such as engeletin (29) and astilbin (30), isolated from the leaves of Stelechocarpus cauliflorus (family: Annonaceae), exhibited inhibitory activity against a recombinant human aldose reductase, the inhibitory activity of 29 (IC50 = 1.16 μM) was found to be twice that of quercetin (positive control, IC50 = 2.48 μM), and 23 times greater than that of 30 (IC50 = 26.7 μM) [55].

Flavonoid glycosides (FG 1 and FG 2), isolated from Phyllanthus fracternus (family: Euphorbiaceae), at a dose of 100 mg/kg p.o. were found to be hypoglycaemic in alloxanised rats (20 and 25%) at 3 hrs, however, no

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blood sugar lowering was observed in normal rats [56]. A neoflavonoid, coutareagenin [5-hydroxy-7-methoxy-4-(3,4-dihdroxyphenyl)-2H-benzo-1pyran-2-one] isolated from the bark of Hintonia latiflora (family: Rubiaceae), exhibited promising anti-diabetic efficacy in streptozotocin-induced Wistar rats as well as in menopausal diabetic women [57,58]. Kaempferol-3,7-O-(α)-dirhamnopyranoside (kaempferitrin, 31), isolated from the n-butanol fraction of the leaves of Bauhinia forficata (family: Leguminosae), exhibited significant hypoglycemic effect in normal and alloxan-induced diabetic rats on oral administration. In normal rats, reduction in blood glucose level was noticed only with the higher dose of 31 (200 mg/kg) at 1 h after treatment, whenever such efficacy of the test compound in diabetic rats was evident at all doses administered (50, 100, and 200 mg/kg), and this profile was found to be maintained throughout the period studied for both higher doses. However, in glucose-fed hyperglycemic normal rats, kaempferitrin could not down-regulate blood glucose levels [59]. Kaempferol3-neohesperidoside, a glycosylated flavonoid structurally very similar to kaempferitrin, was also shown to demonstrate promising hypoglycemic effect in both oral and intraperitoneal treatments in diabetic rats, in addition, kaempferol-3-neohesperidoside-VO(IV) complex showed potent hypoglycemic efficacy throughout the post-treatment period studied when compared with zero time [60]. When complexed with vanadium, quercetin also demonstrated much promising insulin-enhancing activity in STZ-diabetic mice with no effect on the blood glucose level of normal mice, which is in agreement with the results for kaempferitrin and kaempferol-3-neohesperidoside- VO(IV) complexes [60,61]. Quercetin itself was evaluated to possess anti-diabetic effect by reducing the blood glucose level of diabetic rats in 8-10 days of treatment [62], in the same study by Vessal and his group, the test compound exerted no effect on the glucose tolerance curve either in normoglycemic or in STZ-diabetic rats [62]. These results support the views of Shetty et al. [63] for hypoglycemic effects of quercetin in diabetic rats. Three prenylated flavanones (33-35) isolated from stem barks of Erythrina abyssinica (family: Liguminosae) exhibited inhibitory activity against protein tyrosine phosphatase 1B (PTP1B) in dose-dependent manner with IC50 values >60, 18.9±1.9 and 15.7±0.4 μM, respectively [64], hence, the flavanone (32) bearing a 2,2-dimethylpyran moiety on B ring is less potent than the other two (33 & 34) in the series. The investigators, thus, suggested that substitution of prenyl groups on flavonoids may be important for in vitro PTP1B inhibitory activity and cyclization between a hydroxy group and the prenyl group in B ring without prenyl or methoxy groups may reduce the activity [64]. One more isoprenyl flavonoid (35) isolated from the root barks of Erythrina mildbraedii were also found to exhibit inhibitory

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activity against PTP1B enzyme in dose-dependent manner with IC50 values 21.2±1.6 μM. The present investigators argued that substitution of isoprenyl groups on ring-B might be important for PTP1B inhibitory activity in vitro, and introduction of one more hydroxyl group to C-5 of ring-A or one of the isoprenyl groups in ring-B might be responsible for a loss of such activity [65]. Isorhamnetin 3-O-β-D-glucoside (36) isolated from the ethylacetate fraction of Salicornia herbacea (family: Chenopodiaceae) was evaluated to possess significant inhibitory activity against rat lens aldose reductase (RLAR) in vitro with an IC50 value of 1.4 mM, which is similar to that of tetramethylene glutaric acid (IC50 = 1.7 mM) [66]. The flavonol glycoside (36), when administered orally at 25 mg/kg in streptozotocin (STZ)-induced diabetic rats, caused not only a significant inhibition of serum glucose concentration but also sorbitol accumulation in the lenses, red blood cells (RBC), and sciatic nerves, thereby, advocating the test compound from S. herbacea as a leading compound for further study as a new drug for the prevention and/or treatment of diabetes and its complications [66]. Luteolin 6-C-(6′′-O-trans-caffeoylglucoside) (37) isolated from Phyllostachys nigra (family: Gramineae) showed inhibitory efficacy against advanced glycation end products (AGEs), hence, this compound could be offered as a leading compound for its further study towards development of new natural products drug for diabetic complications [67]. Jang et al. [68] reported two flavan-3-ol derivatives (38 and 39) from the roots of Actinidia arguta (family: Actinidiaceae) that were found to exhibit inhibitory activity in vitro on the formation of advanced glycation end products with IC50 values of 13.5 and 17.9 μg/mL, respectively. Few more advanced glycation end products (AGEs) inhibitors such as the dihydroflavonol glycosides (40 and 41) [55], isoflavone C-glucosides (42 and 43) [69] and the 2,3-dioxygenated flavanone erigeroflavanone (44) have also been reported [70]. The isoflavone C-glucosides (42 and 43) isolated from the roots of Pueraria iobata (family: Pueraria) showed more potent in vitro inhibitory activity against AGEs formation with IC50 values 8.7 and 24.9 μg/mL, respectively [69]. The present investigators [69] suggested that the compound (42) is worthy of consideration as a therapeutic agent for diabetic complications or related diseases. Yoo et al. isolated the 2,3dioxygenated flavanone, erigeroflavanone (44) from the flowers of Erigeron annuus (family: Asteraceae/Compositae), and evaluated its inhibitory activity against AGEs formation with an IC50 value 22.7 μM [70]. A flavone xylopyranoside, 4',5-dihyroxy-6,7-dimethoxyflavone-3-O-βD-xylopyranoside (45), isolated from the roots of Euphorbia leucophylla (family: Euphorbiaceae) by Satyanarayana et al., was found to reduce the

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blood glucose levels (BGLs) and increase the serum insulin levels in normal and diabetic rats [71]. One flavone [1′′(R)-5,4′,1′′-trihydroxy-6,7-(3′′,3′′dimethylchromano)flavone, 46] and one flavanone [(2S)-4′-O-methyl-6methyl-8-prenylnaringenin, 47) both isolated Eysenhardtia platycarpa (family: Leguminosae) were evaluated to possess promising anti-

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hyperglycemic activity by decreasing glucose level of streptozotocin (STZ)induced diabetic rats (31 mg/kg of body weight, P < 0.05) [72]. Matsuda et al. [12] examined a variety of flavonoids for their rat lens aldose reductase inhibitory activity to study structure-activity relationships. Among the flavone constituents, 3′,4′-dihydroxyflavone (48), 3′,4′,7-trihydroxyflavone (49), luteolin (50), and luteolin 7-O-β-D-glucopyranoside (51) were found to possess potent inhibitory activity with IC50 values of 0.37, 0.30, 0.45 and 0.99 μM, the flavonoid glycosides, quercitrin (52), guaijaverin (53) and desmanthin-1 (54) also showed the most potent activity against the enzyme with respective IC50 values of 0.18, 0.18 and 0.082 μM [12]. The activity of desmanthin-1 (54) was equivalent to that of a commercially available synthetic aldose reductase inhibitor, epalrestat (IC50 = 0.072 μM). From their detailed studies, Matsuda et al. suggested the following structural requirements of flavonoids for aldose reductase inhibitory activity - (i) the 5-hydroxyl moiety has no effect, (ii) the 3-hydroxyl and 7-Oglucosyl moieties reduce the activity, (iii) the 2-3 double bond enhances the activity, and (iv) the flavones and flavonols having the catechol type moiety at the B ring (the 3′,4′-dihydroxyl groups) exhibit stronger activities than those of pyrogallol-type moiety (the 3′,4′,5′-trihydroxyl groups) [12].

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6. Absorption and metabolism of flavonoids 6.1. Absorption of flavonoids As far as reports are available, the absorption of dietary flavonoids may be influenced by the matrix in which they are consumed, with enhanced excretion in urine of easily recognized mammalian conjugates observed when presented in foods with a higher fat content [73-78] - although certain reports are there in contrast to [79-83]. However, an important factor in the absorption efficiency of flavonoid glycosides in the intestine is the sugar moiety, as demonstrated for quercetin glycosides, its aglycone and rutin supplements in healthy ileostomy volunteers [84]. Flavonoid aglycones, being hydrophobic in nature, can be transported across membranes by passive diffusion, whereas in flavonoid glycosides the sugar moiety enhances the hydrophilicity of the flavonoid molecules as a whole, thereby, reducing the possibility of passive transport. Hence, it may be argued that flavonoids are absorbed by active transport [85]. A good number of studies in human and animals are in agreement with the fact that some dietary flavonoids such as flavanols [86], quercetin-3-glucoside and quercetin-4′-glucoside [87-89] can be absorbed in the small intestine — however, quercetin, quercetin-3galactoside, quercetin-3-rutinoside (rutin), naringenin-7-glucoside, genistein7-glucoside and cyanidine-3,5-diglucoside have been found not to be [89,90]. It has been suggested that before absorption flavonoids are cleaved by specific enzymes either in the lumen or inside the cells of the gut. Lactasephlorizin hydrolase (LPH) is anchored in the brush-border membrane in the small intestine and catalyzes extracellular hydrolysis of some glucosides [91,92]. Another enzyme, located intracellularly and with broad specificity, is the cytosolic β-glucosidase (CBG). It is found in abundance in the small intestine, liver and kidney of mammals and requires active transport of hydrophilic glucosides into the cells [93]. Concerning LPH activity, it has been shown that the enzyme cleaves some flavonol and isoflavone glycosides such as quercetin-4′-glucoside, quercetin-3-glucoside, quercetin-3,4′glucoside, 3′-methylquercetin-3-glucoside, genistein-7-glucoside, and

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daidzein-7-glucoside. However, quercetin-3-rhamnoglucoside and naringenin-7-rhamnoglucoside (naringin) are not substrates for this enzyme [91,93]. In addition, β-glucosidase activity is reported to act on flavonoid and isoflavone glycosides according to the position and the structure of the sugar moiety attached to the flavonoid aglycone [94]. Mechanism of absorption have still not been completely elucidated but is believed to involve inter alia interaction of certain glucosides with the active sugar transporter-1 (SGLT-1) and luminal lactase-phlorizin hydrolysate (LHP), passive diffusion of the more hydrophobic aglycones, or absorption of the glycoside and interaction with cytosolic β-glucosidase (CBG).

6.2. Metabolism of flavonoids After being absorbed in body, flavonoids undergo three main types of conjugations such as methylation, sulfation and glucuronidation [95-97]. The most important enzymes involved in flavonoids metabolism are catechol-Omethyltransferase (COMT, EC 2.1.1.6), phenol sulfotransferase (P-PST, SULT, EC 2.8.2.1) and UDP glucuronosyl transferase (UDPGT, UGT, EC 2.4.1.17). Catechol-O-methyltransferase methylates polyphenols and has the highest activity in the liver and kidneys [98]. Phenol sulfotransferases are cytosolic enzymes that transfer sulfate moieties to hydroxyl groups from substrates such as iodothyronines, phenols and hydroxyarylamines mainly in the liver [96,97,99]. UDP glucuronosyl transferase catalyzes the conjugation of polyphenols to glucuronic acid in endoplasmic reticulum in the intestine, liver and kidney. In humans, the liver has the greatest capacity for glucuronidation while in rats, the highest level of glucuronyl transferase activity was observed in the intestine [99-101]. Conjugation reactions with glucuronic acid and/or sulfate appear to be the most common type of metabolic pathways for the flavonoids first occurring in the gut barrier [85] and these conjugates then reach the liver, where they are further metabolized [81,99,102]. Otake et al. [103] showed that hepatic UDP-glucuronosyl transferase isoforms were the main factors responsible for galangin metabolism into two major glucuronides conjugated at the 7- and 3- positions by using human liver microsomes. Also, Vaidyanathan and Walle [104] demonstrated no glucuronidation of (−)-epicatechin by human liver and small intestinal microsomes. However, in rats, (−)-epicatechin was efficiently metabolized by liver microsomes with formation of two glucuronides. In the same study, the authors concluded that sulfation also occurred in both the liver and intestine in human and rats. Three (−)-epicatechin metabolites such as (−)-epicatechin-3′-Oglucuronide, 4′-O-methyl-(−)-epicatechin-3′-O-glucuronide, and 4′-Omethyl(−)-epicatechin-5 or 7-O-glucuronide have been isolated from human

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urine [105], whereas the exact fate of (+)-catechin is not known although there is evidence for the formation of (+)-catechin sulfates, sulfoglucuronides, and 4′-methylated conjugates in plasma and urine [76,106]. In contrast, (−)-epicatechin gallate and (−)-epigallocatechin gallate appear to be excreted in bile [79,86,107,108]. The (−)-epicatechin gallate is extensively methylated by human liver catechol O-methyl transferase at the 4′-position and to a lesser extent at the 3′-position [109,110], while (−)-epigallocatechin gallate is metabolized first to the 4′′-methyl ether and then to the 4′,4′′dimethyl ether [110]. Flavonoid glycosides that are not absorbed in the small intestine along with the conjugated metabolites that are excreted in bile can be metabolized by microflora when they reach the colon. Glycoside flavonoid-hydrolyzing enzymes have been identified in fecal flora cultures. Bokkenheuser et al. [111] recovered three enzyme-producing strains that, using β-glucosidases, α-rhamnosidases, and/or β-galactosidases, were capable of converting rutin to quercetin. Also, it was shown that at least some of the bacterial glycosidases are able to cleave glycosidic bonds and flavonoid-saccharide bonds in the gut [91]. Genistein-7-glucoside and daidzein-7-glucoside have not been found in human plasma [112] but the aglycones have been observed [113]. Human metabolism of isoflavone glycosides produces genistein and daidzein 7-glucuronides/7-sulfates and 4′,7-diconjugates (including diglucuronides and mixed conjugates), with monoglucuronides predominant [114,115]. The profile of metabolites has been demonstrated in studies with quercetin, rutin and naringin. The flavonoid metabolism produces aromatic acids such as phenylvaleric, phenylpropionic, phenylacetic and benzoic acids with easy absorption through the colonic barrier [116-118]. Flavonol glycosides and quercetin aglycone have not been convincingly demonstrated in plasma [119-121], although kaempferol aglycone has been detected [122]. The main kaempferol metabolite in human plasma is the 3-glucuronide [122]. The three major metabolites of quercetin are: quercetin-3-glucuronide, quercetin-3′-sulfate, and isorhamnetin-3-glucuronide. Apigenin glucuronides have been detected in urine after volunteers consumed parsley [123], luteolin aglycone administered to volunteers has been detected in plasma as a monoglucuronide accompanied by a trace of unconjugated luteonin [124,125]. Chrysin is transformed primarily to the 7-glucuronide with much smaller yields of the 7-sulfate [126]. Metabolites of flavonoids in general (and also microflora metabolites), aglycones, glycosides and conjugated metabolites which are not absorbed, may follow two pathways of excretion: via the biliary or the urinary route. Large conjugated metabolites are more likely to be eliminated in the bile whereas small conjugates such as monosulfates are preferentially excreted in

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urine [100]. When excreted in bile, the flavonoids are passed to the duodenum and metabolized by intestinal bacteria, which results in the production of fragmentation products and/or the hydrolysis of glucurono- or sulfoconjugates [127]. The resulting metabolites which are released may be reabsorbed and enter an enterohepatic cycle or being excreted in feces [128,129]. For each flavonoid, the beneficial effect will be dependent upon their absorption and availability in the body. Thus, these factors should be considered in any interpretation of the potential health effects of flavonoids.

7. Mode of action of flavonoids Very recently, Cazarolli et al. [130] reviewed on the mode of action of flavonoids including cellular and molecular mechanism. In their review, the authors thoroughly discussed about the various effects of the drug candidates in regulating diabetic syndromes. It has been demonstrated that flavonoid compounds act against diabetes mellitus either through their capacity to avoid glucose absorption or to improve glucose tolerance. In vitro studies have shown that a soybean extract containing the isoflavones genistein and daidzein inhibits glucose absorption into the intestinal brush border membrane vesicles of rabbits [131]. Naringenin was also found to reduce glucose uptake in the intestinal brush border membrane vesicles of diabetic rats to a level similar to that of normal rats [132]. The (−)-epicatechin gallate, myricetin, quercetin, apigenin, (−)-epigallocatechin gallate, and (−)epigallocatechin demonstrated a marked reduction in glucose absorption, when compared with the control, by competitive inhibition of sodiumdependent glucose transporter-1 [133]. The non-glycosylated flavonoids were shown to reduce glucose absorption under sodium-dependent conditions in vivo and in vitro in animal tissues [134,135]. Besides reducing glucose absorption, another possible mechanism followed by flavonoid compounds to control blood glucose levels is the inhibition of α-glucosidase activity in the intestine. Such inhibitory effects against α-glucosidase activity were observed when luteolin, kaempferol, chrysin and galangin were used both in vitro and in vivo to study the potential role in the absorption and metabolism of carbohydrates [136]. Kim et al. [137] also demonstrated the α-glucosidase inhibitory activity of flavonoids in a study, where it was shown that luteolin, amentoflavone, luteolin 7-O-glucoside and daidzein are the strongest inhibitors of the compounds tested. It has also been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleiotropic mechanisms, to attenuate the diabetic complications, besides, the drug candidates have been found to stimulate glucose uptake in peripheral tissues,

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and regulate the activity and/or expression of the rate-limiting enzymes involved in carbohydrate metabolism pathway. In an experimental study by Liu et al. [138], genistein was found to act directly on pancreatic β-cells, leading to activation of the cAMP/PKA signaling cascade to exert an insulinotropic effect. Interestingly, it has found that epigallocatechin 3-gallate mimics the effects of insulin on the gene expression reduction of phosphoenolpyruvate carboxykinase and G-6-Pase in the mouse liver [139], like insulin, the drug candidate enhances tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), mitogen-activated protein kinase, p70s6k, and PI3K activity, and reduces phosphoenolpyruvate carboxykinase gene expression mediated by PI3K [140]. Furthermore, epigallocatechin 3gallate upregulates glucokinase mRNA expression in the liver of db/db mice [141]. In another study, oral administration of rutin to diabetic rats resulted in a decrease in plasma glucose and increase in insulin levels, and restored the glycogen content and hexokinase activity. The activity of enzymes such as G-6-Pase and fructose-1,6-bisphosphatase significantly decreased in the liver and muscles of rutin-treated diabetic rats [142]. Kaempferol-3neohesperidoside has been shown to have the efficacy for prompt stimulating of glycogen synthesis in rat soleus muscle by approximately 2.38-fold, it has also been demonstrated that the phosphatidylinositol-3-kinase (PI3K) glycogen synthase kinase-3 (GSK-3) pathway and mitogen-activated protein kinase (MEK) - protein phosphatase-1 (PP-1) pathway are involved in the stimulatory kaempferol-3-neohesperidoside effect on the glycogen synthesis [143]. Very recently, Cazarolli et al. [144,145] have reported on the mechanism of action of the anti-diabetic effects of apigenin-6-C-β-L-fucopyranoside and apigenin-6-C-(2′′-O-α-L-rhamnopyranosyl)-β-L-fucopyranoside – the former drug candidate was evaluated to stimulate insulin secretion and potentiated glucose-induced insulin secretion in hyperglycemic rats, in addition, this flavonoid stimulated glycogen synthesis in rat soleus muscle through mechanisms well known to insulin signal transduction, thereby, establishing the dual effects of apigenin-6-C-β-L-fucopyranoside as an anti-hyperglycemic (insulin secretion) as well as an insulino-mimetic (glycogen synthesis) agent [144]. In another study, the same group of investigators has characterized apigenin-6-C-(2′′-O-α-L-rhamnopyranosyl)-β-L-fucopyranoside as both an insulin secretagoge and an insulin-mimetic agent [145].

8. Conclusions Diabetes mellitus has already emerged as an alarming disease worldwide affecting the public health much. Though presently available therapies

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against the disease reduce the sufferings to some extent, still it remains inadequate and at the same time is costly, and also associated with a lot of side effects. Hence, there is an urgent need for search of more efficacious drugs with no or minimum side effects. There has been a growing interest in anti-diabetic agents from natural products, particularly those derived from plants. Flavonoids are naturally occurring phenolic compounds with a broad range of biological activities and the beneficial effects of flavonoids have been studied in relation to diabetes mellitus, either through the inhibition of intestinal α-glucosidase enzyme or through their capacity to avoid glucose absorption and/or to improve glucose tolerance. A good number of bio-flavonoids reported over the past 15-20 years discussed in this review clearly demonstrate that these exogenous substances represent an unparalleled source of molecular diversity in relation to the drug discovery process in the treatment of Type-2 diabetes. Although there has been considerable scientific progress over the past few years in unraveling of the effect and mechanism of action of flavonoids, we still need to define the missing steps in the flavonoid-signaling network and elucidate the mechanism of cross-talk based on the complex mechanism of insulin action, in order to provide new insights into the potential role of flavonoids in diabetes treatment. Further study is required concerning safety (assessment of toxic effect) and human trial to develop potential anti-diabetic remedies of choice.

Acknowledgement The author greatly appreciates financial support under Major Research Grant from the University Grants Commission (UGC), New Delhi, India [Project No. F.34-357/2008(SR) dt 02.01.2009].

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93. Day, A.J., Gee, J.M., DuPont, M.S., Johnson, I.T., Williamson, G. Biochem. Pharmacol., 2003, 65, 1199-1206. 94. Lambert, N., Kroon, P.A., Faulds, C.B., Plumb, G.W., McLauchlan, W.R., Day, A.J., Williamson, G. Biochim. Biophys. Acta, 1999, 1435, 110-116. 95. Cazarolli, L.H., Zanatta, L., Alberton, E.H., Figueiredo, M.S.R.B., Folador, P., Damazio, R.G., Pizzolatti, M.G., Silva, F.R.M.B Mini-Rev. Med. Chem., 2008, 8, 1429-1440. 96. Manach, C., Scalbert, A., Morand, C., Rémésy, C., Jiménez, L. Am. J. Clin. Nutr., 2004, 79, 727- 747. 97. Scalbert, A., Williamson, G. J. Nutr., 2000, 130 (8S suppl), 2073S-85S. 98. Nielsen, S.E., Breinholt, V., Justesen, U., Cornett, C., Dragsted, L.O. Xenobiotica, 1998, 28, 389-401. 99. Piskula, M.K., Terao. J. J. Nutr., 1998, 128, 1172-1178. 100. Mojarrabi, B., Mackenzie, P.I. Biochem. Biophys. Res. Commun., 1998, 247, 704-709. 101. Strassburg, C.P., Nguyen, N., Manns, M.P., Tukey, R.H. Gastroenterology, 1999, 116, 149-160. 102. Donovan, J.L., Crespy, V., Manach, C., Morand, C., Besson, C., Scalbert, A., Rémésy, C. J. Nutr., 2001, 131, 1753-1757. 103. Otake, Y., Hsieh, F., Walle, T. Drug Metab. Dispos., 2002, 30, 576-581. 104. Vaidyanathan, J.B., Walle, T. Drug Metab. Dispos., 2002, 30, 897-903. 105. Natsume, M., Osakabe, N., Oyama, M., Sasaki, M., Baba, S., Nakumura, Y., Osawa, T., Terao, J. Free Radical Biol. Med., 2003, 34, 840-849. 106. Donovan, J.L., Kasim-Karakas, S., German, J.B., Waterhouse, A.L. Br. J. Nutr., 2002, 87, 31-37. 107. Yang, C.S., Chen, L., Lee, M.J., Balentine, D.A., Kuo, M.C., Schantz, S.P. Cancer Epidemiol. Biomarkers Prev., 1998, 7, 351-354. 108. van Amelsvoort, J.M., van Hof, K.H., Mathot, J.N., Mulder, T.P., Wiersma, A., Tijburg, L.B. Xenobiotica, 2001, 31, 891-901. 109. Meng, X., Lee, M.J., Li, C., Sheng, S., Zhu, N., Sang, S., Ho, C.T., Yang, C.S. Drug Metab. Dispos., 2001, 29, 789-793. 110. Lu, H., Meng, X., Yang, C.S. Drug Metab. Dispos., 2003, 31, 572-579. 111. Bokkenheuser, V.D., Shackleton, C.H.L., Winter, J. Biochem. J., 1987, 248, 953-956. 112. Setchell, K.D., Brown, N.M., Zimmer-Nechemias, L., Brashear, W.T., Wolfe, B.E., Kirschner, A.S., Heubi, J.E. Am. J. Clin. Nutr., 2002, 76, 447-453. 113. Setchell, K.D., Brown, N.M., Desai, P., Zimmer-Nechemias, L., Wolfe, B.E., Brashear, W.T., Kirschner, A.S., Cassidy, A., Heubi, J.E. J. Nutr., 2001, 131, 1362S-1375S. 114. Adlercreutz, H., van der, W.J., Kinzel, J., Attalla, H., Wahala, K., Makela, T., Hase, T., Fotsis, T. J. Steroid Biochem. Mol. Biol., 1995, 52, 97-103. 115. Doerge, D.R., Chang, H.C., Churchwell, M.L., Holder, C.L. Drug Metab. Dispos., 2000, 28, 298-307. 116. Rechner, A.R., Smith, M.A., Kuhnle, G., Gibson, G.R., Debnam, E.S., Srai, S.K.S., Moore, K.P., Rice-Evans, C.A. Free Radic. Biol. Med., 2004, 36, 212-225.

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117. Jenner, A.M., Rafter, J., Halliwell, B. Free Radic. Biol. Med., 2005, 38, 763-772. 118. Aura, A.M., O´Leary, K.A., Williamson, G., Ojala, M., Bailey, M., PuupponenPimiä, R., Nuutila, A.M., Oksman-Caldentey, K.M., Poutanen, K. J. Agric. Food Chem., 2002, 50, 1725-1730. 119. Walle, T., Otake, Y., Walle, U.K., Wilson, F.A. J. Nutr., 2000, 130, 2658-2661. 120. Day, A.J., Mellon, F., Barron, D., Sarrazin, G., Morgan, M.R.A., Williamson, G. Free Radical Res., 2001, 35, 941-952. 121. Graefe, E.U., Wittig, J., Mueller, S., Riethling, A.K., Uehleke, B., Drewelow, B., Pforte, H., Jacobasch, G., Derendorf, H., Veit, M. J. Clin. Pharmacol., 2001, 41, 492-499. 122. DuPont, M.S., Day, A.J., Bennett, R.N., Mellon, F.A., Kroon, P.A. Eur. J. Clin. Nutr., 2004, 58, 947-954. 123. Nielsen, S.E., Young, J.F., Daneshvar, B., Lauridsen, S.T., Knuthsen, P., Sandstrom, B., Dragsted, L.O. In: Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease, Kumpulainen, J.T. and Salonen, J.T, Eds., Royal Society of Chemistry: Cambrige, 1999. 124. Shimoi, K., Okada, H., Furugori, M., Goda, T., Takase, S., Suzuki, M., Hara, Y., Yamamoto, H., Kinase, N. FEBS Lett., 1998, 438, 220-224. 125. Shimoi, K., Saka, N., Kaji, K., Nozawa, R., Kinase, N. Biofactors, 2000, 12, 181-186. 126. Walle, T., Otake, Y., Brubaker, J.A., Walle, U.K., Halushaka, P.V. Br. J. Clin. Pharmacol., 2001, 51, 143-146. 127. Formica, J.V., Regelson, W. Food Chem. Toxicol., 1995, 33, 1061-1080. 128. Crespy, V., Morand, C., Besson, C., Cotelle, N., Vezin, H., Demigné, C., Rémésy, C. Am. J. Physiol. Gastrointest. Liver Physiol., 2003, 284, G980-G988. 129. Silberberg, M., Morand, C., Mathevon, T., Besson, C., Manach, C., Scalbert, A., Rémésy, C. Eur. J. Nutr., 2006, 45, 88-96. 130. Cazarolli, L.H., Zanatta, L., Alberton, E.H., Figueiredo, M.S.R.B., Folador, P., Damazio, R.G., Pizzolatti, M.G., Silva, F.R.M.B. Mini-Rev. Med. Chem., 2008, 8, 1032-1038. 131. Bhathena, S.J., Velásquez, M.T. Am. J. Clin. Nutr., 2002, 76, 1191-1201. 132. Li, J.M., Che, C.T., Lau, C.B.S., Leung, P.S., Cheng, C.H.K. Int. J. Biochem. Cell Biol., 2006, 38, 985-995. 133. Shimizu, M., Kobayashi, Y., Suzuki, M., Satsu, H., Miyamoto, Y. Bio Factors, 2000, 13, 61-65. 134. Johnston, K., Sharp, P., Clifford, M., Morgan, L. FEBS Lett., 2005, 579, 1653-1657. 135. Zhao, H., Yakar, S., Gavrilova, O., Sun, H., Zhang, Y., Kim, H., Setser, J., Jou, W., Leroith, D. Diabetes, 2004, 53, 2901-2909. 136. Matsui, T., Kobayashi, M., Hayashida, S., Matsumoto, K. Biosci. Biotechnol. Biochem., 2002, 66, 689-692. 137. Kim, J.S., Kwon, C.S., Son, K.H. Biosci. Biotechnol. Biochem., 2000, 64, 2458-2461. 138. Liu, D., Zhen, W., Yang, Z., Carter, J.D., Si, H., Reynolds, K.A. Diabetes, 2006, 55, 1043-1050. 139. Koyama, Y., Abe, K., Sano, Y., Ishizaki, Y., Njelekela, M., Shoji, Y., Hara, Y., Isemura, M. Planta Med., 2004, 70, 1100-1102.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 213-268 ISBN: 978-81-308-0448-4

7. Marine natural alkaloids as anticancer agents+ Deepak Kumar and Diwan S. Rawat Department of Chemistry, University of Delhi, New Delhi-110007, India

Abstract. Alkaloids are naturally occurring nitrogen containing biologically active heterocyclic compounds. Over the last few years, a large number of biologically important alkaloids with antiviral, antibacterial, anti-inflammatory, antimalarial, antioxidant and anticancer activities have been isolated from marine source. Present article summarizes the isolation and anticancer activity evaluation of natural marine alkaloids and their synthetic analogues that includes pyridoacridine, indole, pyrrole, pyridine, Isoquinoline, guanidine and steroidal alkaloids.

Introduction Since ancient times nature has been a source of medicines to cure many deadly diseases. Majority of drugs in use today are either natural products (NP), their derivatives (ND), natural products mimics (NPD) or semisynthetic derivatives (SSD) [1-4]. In natural sources, plants, animals and microorganisms have been the main source of biologically important molecules. Ocean has been considered as the main source of medicines and during the past two decades thousands of compounds and their metabolites with several different type of biological activity such as antimicrobial, anti-inflammatory, antimalarial, antioxidant, anti HIV and anticancer activity have been isolated from marine microorganisms [5-12]. But till date only few anticancer drugs such as citarabine, +

Dedicated to Dr. DS Bhakuni and Prof. Deepak Pental

Correspondence/Reprint request: Prof. Diwan S. Rawat, Department of Chemistry, University of Delhi New Delhi-110007, India. E-mail: dsrawat@chemistry.du.ac.in

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vidarabine etc have been commercially developed from marine compounds while several others are currently in different stages of clinical trials [13]. Over 18000 compounds have been isolated from marine source and approximately 150 compounds are cytotoxic against the different tumor cells [14,15]. Some of the prominent anticancer compounds which are in different stages of clinical trials include aplidine, ecteinascidin-734 (Yondelis), bryostatin-1, squalamine, dolastatin10, ILX651, and KRN7000 (α-galactosylceramide) [16]. The present article summarises the recent development in the area of marine alkaloids that includes pyridoacridine, indole, pyrrole, pyridine, isoquinoline, guanidine and steroidal alkaloids.

1. Pyridoacridine alkaloids Pyridoacridines are highly coloured marine natural products having polycyclic planar heteroaromatic 11H-pyrido[4,3,2,mn]acridine system (1) [17]. They are probably the largest class among marine alkaloids and are almost universally isolated from sponges, ascidians as well as from a mollusc and a coelenterate [18]. Pyridoacridine alkaloids show significant biological activity primarily cytotoxicity and certain specific biological properties viz. fungicidal and bactericidal properties, inhibition of topoisomerase II, anti HIV, intercalation of DNA property, Ca+2 releasing activity, production of reactive oxygen species [19-22]. These activities depends upon the substitution pattern of the basic structure of pyridoacridine, therefore many synthetic analogues have also been synthesized keeping the basic skeleton of pyridoacridine in mind. The synthesis of these analogues and their biological activity evaluation revealed that in most of the cases cytotoxicity of the analogues has improved compared to the parent molecule [23, 24]. During the last few years, numerous additional compounds of this family were isolated; most of them are polycyclic with different substituents such as shermilamine, kuanoniamine, neoamphimedine, arnoamines and styelsamines. It has been observed that almost all the pyridoacridines shows promising cytotoxicity against different type of tumors. Therefore a great interest was developed to modify the pyridoacridine moiety for developing a new generation of therapeutic agents. The first review article on marine pyridoacridines alkaloids was published by Molinski in 1993 [25] followed by Ding et al. in 1999 [26]. The cytotoxicity of the compounds of this family is a manifestation of their DNA binding properties, topoisomerase II inhibition and the production of reactive oxygen species. Pyridoacridines vary structurally by attachment of different side chains or fusion of different rings to ring C of the basic structure (1) and less often to the acridine nitrogen. Halogen substitution in pyridoacridines is quite rare; even if it is present, then it is always bromine at C2 in ring A. Oxidation states of the rings are variable and in some cases ring D is partially saturated. Additional rings are

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often attached to ring C. Pyridoacridines can be divided into tetracyclic, pentacyclic, hexacyclic, heptacyclic and octacyclic alkaloids. 2 1

3 A

11a 11 HN

4 B

10a

4a 5

10 C

7a N 7

N O

1.1. Tetracyclic alkaloids In 1988, Kobayashi et al. reported the isolation of three tetracyclic alkaloids, cystodytins A-C (3-5) from tunicate Cystodytes dellechiajei collected from Okinawa [27]. Then in 1991, the same group reported six other novel tetracyclic alkaloids of cystodytin family, cystodytins D-I (6-11) along with cystodytins A (3) and B (4) [28]. Thus cystodytins A-C (3-5) are the first pyridoacridine alkaloids isolated from a marine tunicate and therefore the first tetracyclic member of this class. The common heterocyclic nucleus of cystodytins A-C (3-5) is an iminoquinone substituted at C10 with a 2-amidoethyl side chain. The N-acyl groups are derived from β,β´-dimethylacrylic, tiglic and 3-hydroxy-3methylbutanoic acids, respectively. Cystodytins D-I (6-11) are chiral, levorotatory compounds. Cystodytins F-I (7-10) are substituted with an O-methyl ether or O-9-octadecenoate ester. The isomeric pairs of cystodytin β,β´-dimethylacrylate and tiglate amides could not be separated and were characterized as 7:2 mixtures. Hydration of cystodytin A (3) in presence of 6% aq. HC1 at 100 οC gives cystodytin C (5). When treated with diazomethane, it afforded monomethyl ether (12) with 23% yield. This transformation is unusual as it constitutes a formal reductive methylation. Cystodytin A (3) is readily reduced in the ionization stage of a mass spectrometer as observed for quinones. Cystodytin A (3), when hydrogenated over Adams catalyst in acetic acid yielded (13) by reduction of the side chain and disubstituted benzene ring, but the iminoquinone part remains intact. Compounds 3, 4 and 5 showed potent cytotoxicity against L-1210 with IC50 values of 0.22, 0.22 and 0.24 µg/mL, respectively. Cystodytins D-I (6-11) were also found to be cytotoxic against murine lymphoma L-1210 cells with IC50 values of 1.1 (6 and 7), 0.068 (8 and 9) and 0.080 (10 and 11) µg/mL and values of 1.4 (6 and 7), 0.078 (8 and 9) and 0.092 (10 and 11) µg/mL against human epidermoid carcinoma KB cells in vitro.

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H N

O O N

N Y=

O 3, R2 = X, R1 = H O 4, R2 = Y, R1 = H 5, R2 = Z, R1 = H 6, R2 = X, R1 = OH Z= 7, R2 = Y, R1 = OH 8, R2 = X, R1 = OMe 9, R2 = Y, R1 = OMe 10, R2 = X, R1 = OCO(CH2)7CH=CH(CH2)7CH3 11, R2 = Y, R1 = OCO(CH2)7CH=CH(CH2)7CH3

12 H N OH

OMe

O N 13

Cystodytin J (14) was isolated from a ascidian Cystodytes sp. [29]. Cystodytin J (14) showed cytotoxic activity against HCT and xrs-6 with IC50 values of 1.6 and 135.6 µM, respectively. It also inhibited the topoisomerase (TOPO) II-mediated decatenation with IC90 value of 8.4 µM. Recently, Appleton et al. reported isolation of cystodytins K (15), a new member of cystodytins, from the extract of an ascidian Lissoclinum notti collected near Leigh Harbour, Northland, New Zealand [30]. Structure of compound was determined by spectroscopic techniques, including 2D 1H-15N NMR experiments and was found to be 12-methoxy derivative of cystodytin J (14). Cystodytins K (15) exhibited cytotoxic activity against P-388 murine leukaemia cell line with IC50 value of 1.3 µM. Two bright crimson pigments, Varamine A (16) and B (17) were isolated from the Fijian ascidian Lissoclinum vareau [31]. Varamines A (16) and B (17) have parent tetracyclic aromatic ring system at the same oxidation level as the methylation product of cystodytin A (12). Varamines also contain a methyl thioether substituent at C9. Varamine A (16) was readily oxidised by aq. cerric ammonium nitrate to imonoquinone (18) with 90% yield. Varamine A (16) and B (17) exhibited cytotoxicity towards L-1210 murine leukemia cells with IC50 values of 0.03 and 0.05 µg/mL, respectively. In 1989, Ireland et al. isolated a new tetracyclic alkaloid, diplamine (19) from the tunicate Diplosoma sp. collected from the Fiji Island [32]. The structure was established by interpretation of spectral data and chemical analysis. Diplamine (19) was found to be cytotoxic towards L-1210 murine leukemia cells with IC50 value of 0.02 µg/mL. Recently, two novel alkaloids, isodiplamine (20) and lissoclinidine (21) along with known diplamine (19) were isolated from an ascidian Lissoclinum notti collected near Leigh Harbour, Northland, New Zealand. All the compounds (19-21) were tested for their cytotoxicity against murine leukaemia (P-388), human colon tumour (HCT-116) and non-malignant African Green Monkey kidney (BSC-1) cell lines. Diplamine (19) was found to be the

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most active compound among the three and it was observed that movement of the thiomethyl group from C-9 (diplamine) to C-5 (isodiplamine) decreases cytotoxicity against all the cell lines and the same pattern also observed, when the thiomethyl group is cyclised into a benzoxathiole ring (lissoclinidine). These results were also found to be consistent with the proposed mechanism of cytotoxicity of diplamine, which includes DNA intercalation, inhibition of topoisomerase II and other DNA processing enzymes and bioreductive activation. Lissoclinidine (21) was also evaluated against the NCI 60 cell line panel and demonstrated moderate activity and selectivity with panel average values of GI50 = 1.0 mM, TGI = 6.9 mM and LC50 = 29 mM.

H N

N O

O MeO

H N

OMe

16, R = CH2CH3 17, R = CH3

O H N

O HN

R3 S

R2 N

S O

20, R1 = H, R2 = SCH3, R3 = H 19, R1 = SCH3, R2 = H, R3 = H

N H CF3COO

In 1998, Copp et al. reported the isolation of four new tetracyclic pyridoacridine alkaloids, styelsamines A-D (22-25) from an extract of the ascidian Eusynstyela latericius [33]. The structures of all the compounds were determined on the basis of 1D and 2D NMR spectroscopy. Styelsamines A-D (22-25) exhibited mild cytotoxicity toward the human colon tumor cell line (HCT-116) with IC50 values of 33, 89, 2.6 and 1.6 µM, respectively. OH H

N H

CF3COO

22, R =

NH3

23, R =

NHCOMe

24, R = 25, R =

CHO NH3

CF3COO

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1.2. Pentacyclic alkaloids Amphimedine (26) was the first example of pyridoacridine alkaloids to be fully characterized [34]. In 1983, Schmitz et al. isolated amphimedine as a sparingly soluble yellow pigment from Amphimedon sp. The structure of amphimedine was established on the basis of spectroscopic data analysis. High resolution mass spectral analysis established the molecular formula C19H11N3O2 (m/e = 313.08547, + 0.35 mass error) for amphimedine. In mass spectrum very few fragments were observed corresponding to loss of CH, CO, CHO and HCN. The UV spectra of compound (26) in absolute ethanol showed absorption at λmax 210 nm (19690), 233 nm (39393), 281 nm (9099), 341 nm (6060). Significant changes were observed upon addition of NaBH4 [λmax 235 nm (12879), 280 nm (9090)], indicating the presence of α,β-unsaturated ketone, which was further supported by the strong absorbtion at 1690 cm-1. Further presence of amide functionality was confirmed by IR and 13C NMR. No OH or NH absorptions were observed in the IR and due to low solubility of compound (26) in common organic solvents, NMR spectral data were obtained in trifluoroacetic acid-d4 and CDCl3 (2:1). The 2D NMR techniques (1H-1H correlation and 13C-13C INADEQUATE NMR) were also used to confirm the structure of amphimedine (26). In 1999, Ireland et al. reported the isolation of a new pyridoacridine, neoamphimedine (27) along with amphimedine (26) from Xestospongia sp. from the Philippines and Xestospongia cf. carbonaria from Micronesia [35]. He deduced the molecular formula for neoamphimidine as C19H11N3O2 by high-resolution fast atom bombardment (FAB) mass spectral analysis. Both amphimedine and neoamphimedine have the same molecular formula hence they are isomers. Recently, deoxyamphimedine (28) along with two known compounds (26 and 27) was isolated from two tropical Xestospongia sponges [36]. Amphimedine, neoamphimedine and deoxyamphimedine have the same skeleton, but they differ in biological activities and this is probably due to the the differences in their structures. Literature servey reaveled that amphimidine relatively inactive compared to neoamphimedine and deoxyamphimedine. Neoamphimedine inhibits topoisomerase II while amphimedine is relatively nontoxic at the same dose level [37] and deoxyamphimedine damages DNA independent of topoisomerase enzymes through the generation of reactive oxygen species [38].

O N

N O 26

N O

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Schmitz et al. reported the isolation of three new alkaloids 29-31 from two ascidians. The meridine (29) and a relatively stable tautomer of meridine i.e. 30 were isolated from Amphicarpa meridiana collected at Stenhouse bay, South Australia [39]. The structure of meridine (29) was determined by X-ray analysis while that of 31 was established by spectral analysis. The third alkaloid, 11hydroxyascididemin (31) was isolated from a Leptoclinides sp. from Truk Lagoon. All three alkaloids (29-31) were found to be cytotoxic. Recently, Menendez et al. synthesized a regioisomer of meridine named as 9 Hydroxybenzo[b]pyrido[4,3,2-de](1,10)-phenantrolin-8-one (32) from 5,8dimethoxy-6-nitro-4(1H)-quinolinone in eight steps with 23% overall yield [40]. Compound (32) was tested for cytotoxicity against different tumor cell lines and exhibited mild to strong cytotoxic activity against P-388, A-549, HT-29 and MEL-28 with IC50 values of 4.18, 0.03, 0.40 and 0.17, whereas IC50 values for meridine were 0.08, 0.08, 0.84 and 0.08, respectively. Compound 32 and the natural meridine (29) were also tested in vitro for Topoisomerase II inhibitory activity. Meridine showed mild activity (IC50 = 3 mM), whereas compound 32 was found to be inactive even at the highest concentration (33 mM). In 1988, a novel pentacyclic alkaloid, ascididemin (33) was isolated from brown colored tunicate Didemnum sp. collected at Kerama Islands, Okinawa [41]. The structure of compound was elucidated on the basis of spectroscopic data. Ascididemin (33) was found to be cytotoxic against L-1210 murine leukemia cells in vitro with IC50 value of 0.39 µg/mL. Delfourne et al. synthesized an isomer of ascididemin, named as 9H-quino[4,3,2-de][1,7]phenanthroline-9-one (34) starting from 1,4-dimethoxyacridine with an overall yield of 12% along with other derivatives (35-39) of compound 34 [42]. These compounds were tested in vitro at six different concentrations on 12 different human cancer cell lines such as glioblastomas, breast, colon, lung, prostate and bladder cancers. Almost all the compounds showed significant cytotoxic activity and compound 34 was found as much potent or slightly less potent as the natural ascididemin (33). Ascididemin (33) and the isomer (34) exhibited cytotoxicity against U-87MG (0.07, 0.8 µM), U-373MG (0.5, 0.8 µM), SW1088 (0.6, 3 µM), T-47D (0.6, 0.7 µM), MCF-7 (0.07, 0.9 µM), Lovo (0.9, 0.7 µM), HCT-15 (0.06, 0.4 µM), A-549 (0.2, 7 µM), A-427 (0.06, 0.08 µM), PC-3 (0.008, 0.09 µM), T-24 (0.8, 0.1 µM) and J-82 (0.3, 1 µM), respectively. A new pentacyclic alkaloid, cystodamine (40) was isolated from a mediterranean ascidian Cystodytes dellechiajei collected near the bay of Gabes, at Skhira, Tunisia [43]. The structure was determined by extensive 2D NMR data analysis and was found to contain a phenanthroline unit fused with 7 aminopyridine moiety. Cystodamine (40) showed cytotoxic activity against CEM human leukemic lymphoblasts with IC50 value of 1.0 µg/mL. Later, Delfourne et al. revised the structure of cystodamine (40) to 11-hydroxyascididemin (31) by comparison of the spectroscopic data with those of synthetic cystodamine, meridine and 11hydroxyascididemin [44]. 11-Hydroxyascididemin had been previously isolated by Schmitz et al. from the other marine source Amphicarpa meridian.

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In 1988, Scheuer et al. reported the isolation of a new pentacyclic alkaloid, shermilamine A (41) from purple colonical tunicate Trididemnum sp. [45]. After one year, shermilamine B (42) was reported by two groups simultaneously Scheuer [46] and Kashman [47]. In 1994, McDonald et al. isolated shermilamine C (43) from a Fijian ascidian Cystodytes sp. [48]. Shermilamine A (41) contains a pentacyclic pyridoacridine thiazinone system while shermilamine B (42) is a debromo analogues of shermilamine A (41). Two novel shermilamine alkaloids, shermilamine D (44) and E (45) were isolated from the Indian Ocean tunicate Cystodytes violatinctus collected at the Mayotte Lagoon, Comoros Islands, northwest of Madagascar [49]. Shermilamine D (44) exhibited cytotoxicity against P-388, A-549, HT-29 and MEL-28 cancer cell lines with IC50 values of 0.53, 0.27, 2.66 and 0.53 µM, respectively [50]. A new member of shermilamines, cycloshermilamine D (46) was isolated from the same marine tunicate Cystodytes violatinctus [51]. The structure of cycloshermilamine D (46) was established mainly on the basis of NMR spectroscopic data and was found to be closely related to shermilamine D (44) having hexacyclic structure. Kuanoniamines A-D (47-50) were isolated along with the known shermilamine B (42) from a tunicate and its prosobranch mollusc predator Chelynotus simperi [52]. The structures were established by extensive NMR analysis and correlations spectroscopy. Kuanoniamines C (49) and D (50) were also isolated from another tunicate of the genus cystodytes collected in Pohnpei [53]. Kuanoniamines B (48) and D (50) are homologues of kuanoniamine C (49) having isovaleramide and acetamide side chains, respectively. Kuanoniamine A (47) is structurally different from the other three alkaloids and lacks the 2-amidoethyl side chain and contains an iminoquinone moiety. In 1994,

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McDonald et al. isolated dehydrokuanoniamines B (51) from a Fijian ascidian Cystodytes sp. [54]. More recently, the N-deacyl derivative (52) was isolated from the sponge Oceanapia sp. collected at Truk Lagoon, Micronesia along with its two parent molecule (49) and (50) [55]. Kuanoniamines A-D (47-50) showed weak cytotoxicity. Kuanoniamine A (47) was found to be the most active compound of the group and inhibits the proliferation of KB (human pharyngeal cancer) cell lines in vitro with IC50 value of 1 µg/mL. Dehydrokuanoniamine B (51) and kuanoniamines D (50) were found to have comparative potentials in vitro against HCT (IC50 = 8.3 and 7.8 µM) and xrs-6 cells (IC50 = 80 and 88.9 µM). The N-deacyl derivative (52), kuanoniamine C (49) and D (50) were tested in vitro against two human cancer cell lines, HeLa cells and MONO MAC-6 cells. Kuanoniamine C (49), D (50) and N-deacyl derivative (52) exhibited IC50 values of 5.1, 1.4 and 1.2 µg/mL (HeLa) and values of 1.2, 0.8 and 2.0 µg/mL (MONO MAC-6).

H N

N H

H N

N N

N H

O R2

41, R1 = Br, R2 = NHCOMe 42, R1 = H, R2 = NHCOMe 43, R1 = H, R2 = NHCOCH=C(Me)2 44, R1 = H, R2 = NMe2 45, R1 = H, R2 = N(O)Me2

NMe2 R

48, R = NHCOCH2CH(CH3)2 49, R = NHCOCH2CH3 50, R = NHCOCH3 51, R = NHCOCH=C(CH3)2 52, R = NH3

In 1988, Gunawardanda et al. isolated dercitin (53) from the deep water marine sponge Dercitus sp. collected from Bahamas [56]. The structure of dercitin was assigned on the basis of spectroscopic data. This structure (53) was subsequently revised to structure (54) by the interpretation of the magnitude of long range protoncarbon coupling constants. Dercitin (54) exhibited in vitro antitumor activity against P-388 (IC50 = 0.05 µg/mL) and human tumor cells (HCT 8, A-549, T47D) with IC50 value of 1.0 µg/mL. Dercitin (54) also showed in vivo activity against P-388 (T/C 170%, 5 mg/kg). One year later, the same group isolated three new pentacyclic pyridoacridine alkaloids, nordercitin (55), dercitamine (56) and dercitamide (57) from the extract of a red coloured sponge Stelletta sp. collected in Bahamas [57]. Later dercitamide (57) was found to be identical to kuanoniamine C (49). Compounds (5557) inhibited the proliferation of P-388 murine leukemia cells in vitro with IC50 values of 4.79, 26.7 and 12.0 µM, respectively. Two new pyridoacridine alkaloids, arnoamines A (58) and B (59) were isolated from the ascidian Cystodytes sp. collected in the vicinity of Arno Atoll, Republic of Marshall Islands [58]. They were supposed to be the first members of

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pentacyclic pyridoacridine alkaloids having a pyrrole ring fused with the pyridoacridine ring system. The structures of 58 and 59 were established on the basis of spectroscopic data, particularly those obtained from HMBC and NOE NMR experiments. The arnoamines A (58) and B (59) displayed a much unexpected chemical reactivity. The pyrrole ring hydrogens labelled as Ha and Hb showed duterium exchange, when NMR were recorded in CDCl3/TFA-d4. Arnoamine A (58) exhibited cytotoxicity against the MCF-7, A-549 and HT-29 cell lines with GI50 values of 0.3, 2.0 and 4.0 µg/mL, respectively, whereas Arnoamine B (59) showed GI50 values of 5.0, 2.0 and 3.0 µg/mL against the MCF-7, A-549 and HT-29 cell lines, respectively. The methanol extract of the ascidian Cystodytes dellechiaijei, collected in Brazil yielded two novel alkaloids, sebastianine A (60) and B (61) [59]. The structures of both the compounds were established by analysis of spectroscopic data. Sebastianine A (60) was found comprising of a pyridoacridine system fused with a pyrrole unit and sebastianine B (61) is having a pyridoacridine system fused with a pyrrolidine system condensed with R-hydroxyisovaleric acid. Sebastianine A (60) and B (61) showed cytotoxic activity against a panel of HCT-116 colon carcinoma cells. N

Hb Y

N H N R

NMe2

55, R = N(CH3)2 56, R = NHCH3 57, R = NHCOCH2CH3

53, X = S, Y = N 54, X = N, Y = S

58, R = H 59, R = Me O

N H

N OH

N H

H N

CF3

62, R = H 63, R = OH

Recently, Davis et al. isolated two new pyridoacridine alkaloids, ecionines A (62) and B (63) from Australian sponge Ecionemia geodides [60]. Both the compounds were found to contain an imine moiety, which is very rarely found in pyridoacridine class of compounds. Both the compounds were tested against a panel of human bladder cancer cell lines (TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2) and the superficial bladder cancer cell line 5637. Compound (63) showed moderate cytotoxicity against all the cell lines, with IC50 values

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of 6.48 mM (TSU-Pr1), 6.49 mM (TSU-Pr1-B1), 3.55 mM (TSU-Pr1-B2) and 3.66 mM (5637), whereas Compound (64) showed cytotoxic effect on 5637 and TSU-Pr1-B2 cells at 10 mM, with cell growth inhibitions of 54% and 51% cells, respectively, but did not have any effect on TSU-Pr1-B1 cells at 10 mM.

1.3. Hexacyclic alkaloids The extracts of a deep violet sponge Dercitus sp. collected in the Bahamas yielded a hexacyclic alkaloid cyclodercitin (64). The sixth ring in cyclodercitin (64) is formally derived by cyclization of the 2-aminoethyl side chain to the acridine nitrogen, while the pyridine ring is substituted with an N-methyl group. Cyclodercitin (64) inhibited the proliferation of P-388 murine leukemia cells in vitro with IC50 value of 1.9 µM. Recently, stellettamine (65) was isolated from a deep water marine sponge Stelleta sp. [61]. The molecular formula, C20H14N4S was determined by high resolution FAB mass spectroscopy. The structure of the compound was established on the basis of 1H-13C correlation spectroscopy except the orientation of thiazole ring. Therefore complete structure of stellettamine (65) was determined by a single-crystal X-ray diffraction experiment.

1.4. Heptacyclic alkaloids Eilatin (66) is the only known heptacyclic pyridoacridine alkaloid of the marine origin [62]. Molecular formula of eilatin (66) was determined as C24H12N4 by high-resolution EIMS. 1H NMR spectrum showed only six aromatic protons that could agree with the common six protons of the benzodiazaphenanthroline system. The 13C NMR spectrum exhibited only 12 carbon lines (6 for monoprotonated carbons and 6 nonprotonated carbons). This suggests a symmetrical dimeric structure for eilatin (66). Various 2D NMR experiments such as 1H-13C correlations and a HETCOSY experiment were failed to deduce the structure and finally it was determined by a single-crystal X-ray analysis. Eilatin (66) was found to exhibit cytotoxic activity against HCT cell line with IC50 value of 5.3 µM.

N N NMe2

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1.5. Octacyclic alkaloids In 1991, Faulkner et al. isolated two novel optically active octacyclic alkaloids, eudistones A (67) and B (68) from the Seychelles tunicate Eudistoma sp. [63]. Eudistone A (67) was obtained as an amorphous yellow powder. The molecular formula C27H19N5O for eudistone A (67) was determined by high resolution mass spectroscopy, which implies 21 degrees of unsaturation. The 13 C NMR signal at 191.8 ppm and an IR band at 1660 cm-1 indicated the presence of an unsaturated ketone and the broad bands at 3360 and 3220 cm-1 attributed for primary or secondary amines. The complete structure of the compounds was determined on the basis of other correlations NMR techniques such as COSY, NOE, HMBC and HMQC. Eudistone B (68) was obtained as a white amorphous powder. The molecular formula C27H17N5O for eudistone B (68) was determined and has one more degree of unsaturation than that present in eudistone A (67). Therefore eudistone B (68) is a dehydrogenation product which was also supported by air oxidation of eudistone A (67) to eudistone B (68). When air is bubbled through a solution of eudistone A (67) in DMSO at 60oC for 48 hrs, the dihydropyridine ring of eudistone A (67) is aromatized to yield eudistone B (68). N OCH2CH3 O HN

N N

N H

N N

Recently, Demeunynck et al. synthesized an octacyclic analogue (69) of eilatin [64]. The compound (69) was tested against two cancer cell lines, HT-29 (human colon adenocarcinoma) and A-431 (human epithelial carcinoma). Unfortunately due to its low solubility in water, the compound could only be tested at low concentration (5 µM) and did not show any activity against HT-29 and 85% survival on A-431 cell lines.

2. Indole alkaloids Indole-containing alkaloids have frequently been isolated from diverse marine invertebrates including bryozoans, coelenterates, sponges, tunicates, algae, symbiotic bacteria and fungi [65-72]. Indole alkaloids show different type of biological activities such as cytotoxic, antitumor, antiviral, antimicrobial,

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225

antiparasitics, antiserotonin and anti-inflammatory activities [73]. Due to the interesting biological activities and unique structural features, the indole series have become an attractive research field for the development of new pharmacological lead compounds. In the past few years, some of the isolated natural organic compounds and their derivatives have been synthesized by chemists and evaluated for their biological activity to find new lead compounds against different infectious diseases [74-79].

2.1. Bisindole alkaloids In 1988, Kohmoto et al. isolated a bisindole alkaloid, dragmacidin (70) from a deep water marine sponge Dragmacidin sp. [80]. Dragmacidin was found to contain two indole groups joined by a piperazine ring system which had not been found before in marine natural products. The molecular formula of dragmacidin was deduced as C21H19Br3N4O from FAB HRMS data analysis. Several 2D NMR experiments such as COSY, HETCOR, COLOC and HETCOSY were performed in order to determine the structure of the compound. Dragmacidin (70), when treated with excess acetic anhydride and pyridine overnight at room temperature yielded the triacetate derivative (71). An ethanolic solution of dragmacidin (70) on treatment with 10% Pd/C at room temperature under 20 psi of hydrogen gives tridebromodragmacidin (72). Dragmacidin (70) exhibited in vitro cytotoxicity with IC50 values of 15 µg/mL against P-388 cell lines and 1-10 µg/mL against A-549 (human lung), HCT-8 (human colon) and MDAMB (human mammary) cancer cell lines. The pacific sponge Hexadella sp. collected from the coast of British Columbia yielded two other members of dragmacidin family, dragmacidon A (73) and dragmacidon B (74) along with a new alkaloid, topsentin C (75) [81]. The structures of the compounds 73-75 were proposed on the basis of spectroscopic analysis. Dragmacidon A (73) showed in vitro cytotoxicity in the L-1210 assay with ED50 value of 10 mg/mL, whereas topsentin C (75) and dragmacidon B (74) were found to be inactive. In 1995, Capon et al. reported the isolation of dragmacidin D (76) from a deep water marine sponge Spongosorites sp. collected from the southern Australian coast [82]. Dragmacidin D (76) was found to be active against human lung tumor cell lines and inhibited in vitro growth of the P-388 murine and A-549 with IC50 values of 1.4 and 4.5 μg/mL, respectively. Four new bisindole alkaloids, nortopsentins A-D (77-80) were isolated from the Caribbean deep sea sponge Spongosorites ruetzleri [83]. The structures of nortopsentins A-D (77-80) were established mainly on the basis of NMR spectroscopic data and were found to contain an imidazole ring between two indole units. Compounds (77-80) exhibited cytotoxic activity against P-388 cells with IC50 values of 7.6, 7.8, 1.7 and 0.9 µg/mL, respectively.

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The sponge Topsentia genitrix, collected from Banyuls (France) yielded two bisindole alkaloids, topsentin (81) and bromotopsentin (82). They were found to contain 2-acyl imidazole moiety inserted between two indole units with different substitution on benzene rings [84]. In 1995, Capon et al. reported the isolation of isobromotopsentin (83) from the deep water sponge Spongosorites sp. collected from the coast of southern Australia [85].

R4 N

R3 R2

N H

N R5

Br N H

N H

70, R1 = OH, R2 = R3 = Br, R4 = Me, R5 = H 71, R1 = OAc, R2 = R3 = Br, R4 = Me, R5 = OAc 72, R1 = OH, R2 = R3 = H, R4 = Me, R5 = H 73, R1 = H, R2 = R3 = Br, R4 = Me, R5 = H 74, R1 = H, R2 = R3 = Br, R4 = Me, R5 = Me H N O

H N

N HN

Br N HN

H2N

77, R1 = R2 = Br 78, R1 = Br, R2 = H 79, R1 = H, R2 = Br 80, R1 = R2 = H

Topsentin (81) inhibited proliferation of cultured human and murine tumor cells. It exhibited in vitro activity against P-388 with IC50 value of 3 μg/mL, human tumor cell (HCT-8, A-549, T47D) with IC50 value of 20 μg/mL and in vivo activity against P-388 (T/C 137%, 150 mg/kg) and B16 melanoma (T/C 144%, 37.5mg/kg) [86]. Bromotopsentin (82) showed antiproliferative activity against human bronocopuemonary cancer cells (NSCLC-N6) with an IC50 = 12 μg/mL [87]. Deoxytopsentin (84) was isolated from the sponge Hexadella sp collected in Jervis Inlet, British Columbia [88]. In 1999, bromodeoxytopsentin (85) and isobromodeoxytopsentin (86) were isolated from sponge Spongosorites genitrix collected from Jaeju Island Korea by Shin et al. [89]. Structurally topsentin (81) and deoxytopsentin (84) are the same except the indole ring which is unsubstituted in case of deoxytopsentin (84). Deoxytopsentin (84) showed the antiproliferative activity against human bronocopulmanary cancer cells (NSCLC-N6) with an IC50 value of 6.3 μg/mL. It also displayed moderate activity against breast cancer and hepatoma (HepG2) with an IC50 of 10.7 and 3.3 μg/mL, respectively.

Marine natural alkaloids as anticancer agents O

N H

R3 R1

N H

227

R3 N H

N H

84, R1 = R2 = R3 = H 85, R1 = Br, R2 = R3 = H 86, R1 = R2 = H, R3 = Br

81, R1 = R2 = H, R3 = OH 82, R1 = Br , R2 = H, R3 = OH 83, R1 = H , R2 = OH, R3 = Br

Recently, Kobayashi et al. isolated a new cytotoxic bis-indole alkaloid, hyrtinadine A (87) from Okinawan marine sponge Hyrtios sp. [90]. The structure elucidation was achieved on the basis of spectroscopic data. Hyrtinadine A (87) was supposed to be the first example of a bisindole alkaloid with a 2,5-disubstituted pyrimidine ring between two indole units. Hyrtinadine A (87) exhibited in vitro cytotoxicity against murine leukemia L-1210 and human epidermoid carcinoma KB cells with IC50 values of 1.0 and 3 µg/mL, respectively. Hyrtiosins A (88) and B (89) were also isolated together with known 5-hydroxyindole-3-aldehyde (90) from the Okinawan marine sponge Hyrtios erecta [91]. Compound (90) exhibited cytotoxic activity against human epidermoid carcinoma KB cells in vitro with IC50 value of 4.3 µg/mL, while hyrtiosins A (88) and B (89) were less cytotoxic than 5-hydroxyindole-3-aldehyde (90) and showed 21% and 16% inhibition, respectively, at 10 µg/mL against KB cells. HO

N H

OH HO

HO N H 89

N H

N H 90

2.2. Indolocarbazoles Staurosporine (91) was first isolated from Streptomyces staurosporeus Awaya (AM-2282) [92,93] and subsequently from other actinomycetes e.g. Streptomyces actuosus [94] and Streptomyces species strain M-193 [95]. The structure and stereochemistry of the compound in its MeOH-H2O solvate form was deduced by X-ray crystallography. Staurosporine (91) exhibited in vitro activity against several different type of tumors such as human neuroblastoma cell line (NB-1), HeLa S3 cells, B16 melanoma cells and P-388 leukemia cells [96,97]. Cordell et al. evaluated the cytotoxicity of staurosporine (91) towards the murine P-388 lymphocytic leukemia and human carcinoma KB cell lines. Staurosporine (91) showed potent cytotoxic activity with ED50 value of 0.0024 µg/mL for the KB system and <0.08 µg/mL for the P-388 system.

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Schupp et al. isolated two new indolocarbazole alkaloids, 3-hydroxy-3´demethoxy-3´-hydroxystaurosporine (92) and 11-hydroxy-4´-N-demethylstaurosporine (93) from the marine ascidian Eudistoma toealensis and its predator, Pseudoceros sp. along with four known congeners (94-97) and staurosporine (91) in their protonated states [98]. Recently, a natural staurosporine analogue, ZHD-0501 (98) was isolated from the fermentation broth of a marine-derived Actinomadura sp. 007 through a bioassay-guided separation procedure [99]. ZHD-0501 (98) was supposed to be the first example of staurosporine analogue carrying a heterocycle fused to the pyran ring. Schupp et al. evaluated the potential of these staurosporine derivatives as inhibitors of cell proliferation and macromolecule synthesis [100]. Compound (94) was found to be the most active staurosporine derivative both as MONO-MAC-6 cells inhibitor and inhibitor of RNA and DNA synthesis. The IC50 values of staurosporine (91) and the derivatives, 94, 95 and 96 for inhibiting MONO-MAC-6 cells were 24.4, 13.3, 33.3 and 29.7 ng/mL, respectively, while those of 92 and 93 was >100 ng/mL each. The percentage inhibition of RNA and DNA synthesis of compounds 91 and 94 were 93 and >98, 98 and >98, respectively. Compound (98) inhibited the proliferation of human cancer A-549, BEL-7402, HL-60 cells and mouse leukemia P-388 cells with the percentage inhibition of 82.6%, 57.3%, 76.1%, 62.2% in the SRB assay [101]. It also inhibited the proliferation of mouse cancer tsFT210 cells with the inhibition rates of 28.3% at 21 μM and 20.5% at 2.1 μM in the SRB assay. Analysis of structure activity relationship demonstrated that hydroxylation of staurosporine at position 3 of the indolocarbazole moiety causes an increase in antiproliferative activity, while hydroxylation at 11th position resulted in a decrease in activity. All these data suggested that not only the presence or absence of hydroxyl group, but also the position of OH group is crucial to determine the antiproliferative properties of the various staurosporine analogues.

H N

R1 N O N H H H H

Me R4 R3

HH R2

91, R1 = H, R2 = CH3, R3 = OCH3, R4 = H 92, R1 = OH, R2 = CH3, R3 = OH, R4 = H 93, R1 = H, R2 = H, R3 = OCH3, R4 = OH 94, R1 = OH, R2 = CH3, R3 = OCH3, R4 = H 95, R1 = H, R2 = CH3, R3 = OH, R4 = H 96, R1 = H, R2 = H, R3 = OCH3, R4 = H

N H

N O

N Me

CHO N OH OH O

O 99

Marine natural alkaloids as anticancer agents

229

A novel carbazole alkaloid, coproverdine (99) was isolated from an unidentified ascidian Anchorina sp. collected from the north Island of New Zealand [102]. The structure of 99 was established on the basis of extensive spectroscopic data analysis. Coproverdine (99) was evaluated against a variety of murine and human tumor cell lines such as P-388, A-549, HT-29, MEL-28 and DU-145 exhibiting IC50 values of 1.6, 0.3, 0.3, 0.3 and 0.3 µM, respectively.

2.3. Ergoline alkaloids Makarieva et al. isolated pibocin A (100) from the far-eastern ascidian Eudistoma sp. [103]. Its structure and absolute stereochemistry were established on the basis of spectroscopic and X-ray data analysis and was supposed to represent the first example of marine ergoline alkaloids. Pibocin A (100) exhibited moderate cytotoxicity against mouse Ehrlich carcinoma cells with ED50 value of 12.5 µg/mL. Recently, pibocin B (101) was isolated from the colonial ascidian Eudistoma sp. [104]. Its structure was established as (8β)-2-bromo-N-Omethyl-6,8-dimethylergoline on the basis of NMR, FAB and MALDI TOF MS data and chemical means. Pibocin B (101) exhibited moderate cytotoxic activity against mouse Ehrlich carcinoma cell with an ED50 value of 25 µg/mL. H3C

CH3 H

H3C

CH3

H Br

Br N OCH3

N H

100

101

2.4. Peptidoindoles Styelin D, a 32-residue, C-terminally amidated peptide was isolated from the blood cells of the solitary ascidian Styela clava [105]. It was found to contain two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual amino acids including, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. Styelin D exhibited cytotoxicity against HCT-116 cells with IC50 value of 10.1 µg/mL, and human ME-180 cervical epithelial cells with ED50 value of 50 µg/ mL. Nakao et al. isolated kapakahine B (102) from the marine sponge Cribrochalina olemda collected at Pohnpei, Micronesia [106]. Kapakahine B (102) was found having a cyclic hexapeptide with an α-carboline ring system and showed moderate cytotoxicity against P-388 murine leukemia cells with an IC50 value of 5.0 µg/mL.

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O N

N H

O NH

O HN

NH O

O NH2 NH

NH N

N H O OH

O H

H NH

O HN

O NH O

O OH

O H

N O

N O N H

H NH

102

103

104

Two isomeric cycloheptapeptides, phakellistatin 3 (103) and isophakellistatin 3 (104), were isolated from the Western Indian marine sponge Phakellia carteri [107]. They were supposed to represent the first examples of photo-Trp serving as a natural peptide unit. A significant difference in the activity was also observed with the photo-Trp indole ring juncture. Phakellistatin (trans-ring juncture) exhibited inhibition of P-388 (ED50 = 0.33 µg/mL) while isophakellistatin (cis-ring juncture) showed no significant effects.

2.5. β-Carbolines Eudistomin K (105) was isolated from the Caribbean ascidian Eudistoma olivaceum and found to exhibit antitumor activity against L-1210, A-549, HCT-8 and P-388 cell lines with IC50 of 0.01 µg/mL against P-388 cell line [108]. Recently Kobayashi et al. reported the isolation and structure elucidation of a new β-carboline alkaloid, eudistomidin G (106) from the Okinawan marine tunicate Eudistoma glaucus [109]. Eudistomidins G (106) exhibited significant cytotoxic activity against L-1210 murine leukemia cells with IC50 value of 4.8 µg/mL in vitro. Adesanya et al. reported the isolation of two novel brominated β-carbolines, eudistalbin A (107) and B (108) from the marine tunicate Eudistoma album along with the known compound eudistomin E (109) [110]. The cytotoxicity of these compounds was tested using the human nasopharyngeal carcinoma KB cell lines. Eudistomin E (109) exhibited 100% cytotoxicity at seven concentrations ranging from 10 to 0.005 µg/mL (ED50 <5.0 ng/ml). Eudistalbin A (107) showed 100% cytotoxicity at 10, 92% at 5, and 0% at 1 µg/mL (ED50 = 3.2 µg/mL), whereas eudistalbin B (108) exhibited 0% cytotoxic activity at 10 and 1 µg/mL.

Marine natural alkaloids as anticancer agents R1

N O

N Me H

N H H HN

231

N H

105, R1 = H, R2 = H, R3 = Br, R4 = H 109, R1 = Br, R2 = OH, R3 = H, R4 = H

106

N H H N 2

N H

108

107

Three new alkaloids, hyrtioerectines A-C (110-112) were isolated from a red coloured marine sponge Hyrtios erectus [111]. The structure of the compounds 110-112 were established on the basis of their spectral data including 1D (1H and 13 C) and 2D (1H-1H COSY, NOESY, ROESY, HMQC and HMBC) NMR experiments and compound 110 was found to contain 6-hydroxy β-carboline and 6-hydroxyindole units linked through C3-C3´ carbon bond. Hyrtioerectines A-C (110-112) were evaluated for their cytotoxicity against HeLa cells and showed moderate cytotoxic activity with IC50 values of 10, 5.0 and 4.5 µg/mL, respectively.

H N

COOH

O N

N H

110

111

CH3

HO N H

112

Foderaro et al. reported the isolation of a new tetrahydro-β-carboline alkaloid, bengacarboline (113) from the Fijian ascidian Didemnum sp. [112]. The structure of the compound was determined by 1H, 13C NMR and HRMS-FAB data analysis and was found to contain one indole and one tryptamine units attached to C-1 of a tetrahydro-β-carboline system through C-3 and C-2 of the indole and tryptamine moieties. Bengacarboline (113) was found to be cytotoxic towards a 26 cell line human tumor panel in vitro with a mean IC50 value of 0.9 µg/mL and also inhibited the catalytic activity of topoisomerase II at 32 µM.

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More recently, a new 1-imidazoyl-3-carboxy-6-hydroxy-β-carboline alkaloid, named as hyrtiocarboline (114) was isolated from a marine sponge Hyrtios reticulates [113]. The structure was elucidated on the basis of spectroscopic data such as 1H-13C and 1H-15N HMBC NMR experiments. Hytriocarboline (114) was tested for antiproliferative activity against 13 cancer cell lines and showed selective activity against three cancer cells lines, non-small cell lung (H522-T1), melanoma (MDA-MB-435) and lymphoma (U937) with IC50 values of 1.2, 3.0 and 1.5 μg/mL, respectively. Hyrtiocarboline (114) also exhibited 57% inhibition of HeLa cells at 230 μM. Two new β-carboline alkaloids, 6-hydroxymanzamine A (115) and 3,4dihydromanzamine A (116) were isolated from the marine sponge Amphimedon sp collected from the Kerama Islands, Okinawa, Japan [114]. The structures of the compounds were elucidated on the basis of NMR spectral data. Compounds 115 and 116 were found to be cytotoxic in vitro against L-1210 with IC50 values of 1.5 and 0.48 µg/mL, respectively and KJ3 cells with IC50 values of 2.5 and 0.61 µg/mL, respectively. O

H2N

OH N

N H NH

N H

113

N H

N H H

N H

114

N H

115

116

Edrada et al. reported the isolation of four new manzamine congeners 117-120 and four known compounds 121-124 from the marine sponge Xestospongia ashmorica collected from the shores of Mindoro Island, Philippines [115]. The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data analysis. The N-oxide structures for compounds 118-120 were confirmed by conversion to the corresponding tertiary bases by reduction with Zn/HCl. All compounds (117124) were tested for their in vitro cytotoxicity against L-5178 mouse lymphoma cells using the microculture tetrazolium (MTT) assay at different concentrations ranging from 0.3 to 20 µg/mL. All the compounds, except 121 were found to be active against L-5178 cell lines. From the activity profile, structure activity relationship between the different manzamine derivatives was also established. The N-oxide compounds 119 and 120 were the most active compounds with ED50 value of 1.6 µg/mL followed by compounds 117 and 122

Marine natural alkaloids as anticancer agents

N H

233

N H

N H OH

N H

N H OH

118

117

N H

N H OH

N H

120

119

N H

122

121

N H

124

123

(ED50 = 1.8 µg/mL each). The other compounds 118, 123 and 124 also exhibited significant cytotoxic activity with ED50 values of 3.2, 6.6 and 2.3 µg/mL, respectively. Two years later, three new manzamine congeners, manzamine M (125), 3,4dihydromanzamine J (126) and 3,4-dihydro-6-hydroxymanzamine A (127) were isolated from the Okinawan marine sponge Amphimedon sp. [116]. The structures and relative stereochemisty were determined on the basis of spectroscopic data. Manzamine M (125), 3,4-dihydromanzamine J (126) and 3,4-dihydro-6hydroxymanzamine A (127) showed cytotoxicity against murine leukemia L-1210 cells with IC50 values of 1.4, 0.5 and 0.3 µg/mL, respectively. OH

N H

N H OH

125

126

N H

N H OH

127

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2.6. Trisindole alkaloids In 1994, Bifulco et al. reported the isolation of two tris-indole alkaloids, Gelliusines A (128) and B (129) from a deep water new Caledonian sponge Gellius or Orina sp. [117]. Gelliusin A (128) and B (129) were found to be diastereomeric compounds made up by the coupling of three indole units. In compounds 128 and 129, two 6-bromo tryptamine units are linked through their aliphatic chains to the C-2 and C-6 position of a central serotonin moiety. The coupling of the indole unit appears to be non stereoselective giving two enantiomeric pairs, having different relative configuration at C-8 and C-8" named (±) Gelliusines A (128) and B (129). Gelliusines A (128) and B (129) showed cytotoxicity with an IC50 value of between 10 and 20 μg/mL against KB, P-388, P-388/dox, HT-29 and NSCLCN-6 cell lines. NH2 HN

NH2 N H

N H

H2N

128 and 129,

Gelliusine A and B

2.7. Miscellaneous indole alkaloids Kondo et al. reported the isolation of two new indole alkaloids, isoplysin A (130) and D6-bromohypaphorine (131) from the Okinawan marine sponge Aplysina sp. [118]. The structures of both the compounds were established by spectral and chemical means. Isoplysin A (130) was found to be weakly cytotoxic against murine lymphoma L-1210 (IC50 = 11.5 µg/mL) and human epidermoid carcinoma KB cells (31% inhibition at 20 µg/mL), while D-6-bromohypaphorine (131) showed no significant effects. In 2007, four new prenylated indole alkaloids, notoamides A-D (132-135) were isolated from marine-derived fungus Aspergillus sp. which was separated from the mussel Mytilus edulis collected off Noto Peninsula in the Sea of Japan [119]. The structures and absolute stereochemistry of the compounds were determined mainly on the basis of spectroscopic data analysis and were found comprising of pyranoindole ring system. Compounds (132) and (133) contain the bicyclo[2.2.2]diazaoctane ring system also. Notoamides A-C (132-134) exhibited weak cytotoxicity against HeLa and L-1210 cells with IC50 values in the range of 22–52 µg/mL but the IC50 value of notoamide D (135) was greater than 100 µg/mL and it was believed that the dihydroxypyrano-2-oxindole ring system, that is common to compounds 132-134 is responsible for the remarkable differences in cytotoxic activity. Notoamide D (135) contains a pyrroloindole

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instead of dihydroxypyrano-2-oxindole ring system. Recently, six new prenylated indole alkaloids, notoamides F-K (136-141) were isolated from a marine-derived Aspergillus sp [120]. Notoamide I (139) showed weak cytotoxicity against HeLa cells with an IC50 value of 21 μg/mL, whereas for notoamides F (136), J (140) and K (141), the IC50 values were more than 50 μg/mL. Three new indole alkaloids, shearinines D-F (142-144) along with the known shearinine A (145) were isolated from marine-derived fungus Penicillium janthinellum [121]. The structures of all the compounds were established by 1D and 2D NMR such as HSQC, HMBC, COSY, NOESY and HREIMS data analysis. Shearinines A, D, E and F were tested for cytotoxicity against mouse epidermal JB6 P+ Cl 41 cells using the MTS method. The compounds displayed no cytotoxicity up to 200 µM, whereas some of the compounds showed cancer preventive and antileukemic properties. Shearinine E (143) inhibited EGF-induced malignant transformation of JB6 P+ Cl 41 cells in a soft agar with INCC50 (inhibition of number of the colonies) value of 13 µM. The Shearinines A (145), D (142) and E (143) induced apoptosis in human leukemia HL-60 cells at 100 µM concentration by 10%, 39% and 34% of the apoptotic cells when compared to control cells, respectively. O

O H3C

N O

N(CH3)

COO

N NH

N(CH3)3 N H

130

O H

N H

N R1

136, R1 = R2 = H, R3 = OMe 137, R1 = OH, R2 = H, R3 = OMe 139, R1 = H, R2 = R3 = O

N O H

N N

132, R1 = OH, R 2 = H 133, R1 = R2 = H 138, R1 = OH, R 2 = OH

131

N R1

N H

O N H

N H

N H O

H O

N H

135, R = α H 141, R = β OH

N O

134

N H O

140

H1 OH OH N H

O N H

O O

142, H and H1 trans 143, H and H1 cis

144

OH O

O O

N H

O O

145

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Deepak Kumar & Diwan S. Rawat

Reyes et al. reported the isolation and structure elucidation of six new bromoindole alkaloids, aplicyanins A-F (146–151) from CH2Cl2/MeOH extract of the tunicate Aplidium cyaneum collected in Antarctica [122]. Aplicyanins A-F (146-151) were tested for cytotoxicity against three human tumor cell lines, including colon (A-549), lung (HT-29) and breast (MDA-MB-231). Compounds 147, 149, 150 and 151 showed cytotoxicity against these cell lines, whereas compounds 146 and 148 were found to be inactive. Compounds 147, 149, 150 and 151 demonstrated IC50 values of 0.66, 0.63, 8.70 and 1.31 (A-549), 0.39, 0.33, 7.96 and 0.47 (HT-29) and 0.42, 0.41, 7.96 and 0.81 (MDA-MB-231). From the activity profile it is clear that compound 150 shows the least activity and this was explained on the basis of presence of the acetyl group at N-16 in compounds 147, 149 and 151 which is crucial to exhibit the activity. Amade et al. reported the isolation and structure elucidation of new bromine containing oxindole alkaloid, matemone (152) along with a known compound, 6-bromoindole-3-carbaldehyde from the Indian Ocean sponge Iotrochota purpurea [123]. Compound 152 showed weak cytotoxicity against NSCLC-N6 L16 strain18 (lung cancer), Mia PaCa-2 cell line (pancreas cancer) and DU145 cell line (prostatecancer) with IC50 values of 30, 24 and 27 µg/mL, respectively. Dendridine A (153), a unique C2-symmetrical 4,4´-bis(7-hydroxy)indole alkaloid was isolated from an Okinawan marine sponge Dictyodendrilla sp. [124]. The structure of compound was elucidated by spectroscopic data including 2D NMR data such as the 1H-1H COSY, ROESY and HMBC spectra. Dendridine A (153) exhibited moderate cytotoxicity against murine leukemia L-1210 cells with IC50 value of 32.5 µg/mL. R1 H N N HN O Br R3

H N

OH N R2

146, R1 = R2 = R3 = H 147, R1 = Ac, R2 = R3 = H 148, R1 = R3 = H, R2 = OMe 149, R1 = Ac, R2 = OMe, R3 = H 150, R1 = H, R2 = OMe, R3 = Br 151, R1 = Ac, R2 = OMe, R3 = Br

N OMe H

152

H2N

NH2

N H

153

Four novel brominated indole alkaloids, arborexidines A-D (154-157) were isolated from the extract of a marine tunicate Pseudodistoma arborescens [125]. Out of four, only arborescidine D (157) showed in vitro cytotoxic activity against the growth of KB human buccal carbinoma cells with IC50 value of 3 µg/mL.

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N Me H

N N H

N Me H N

Br R1

154

156, R1 = H, R2 = OH 157, R1 = OH, R2 = H

155

3. Pyrrole alkaloids 3.1. Bromopyrrole alkaloids Kuramoto et al. isolated two novel alkaloids, cylindradines A (158) and B (159) from the marine sponge Axinella cylindratus collected at the Seto inland sea near Sada Cape in Ehime prefecture [126]. The chemical structures and absolute stereochemistry of these compounds were assigned by spectroscopic and X-ray data analysis. Cylindradines A (158) and B (159) displayed moderate cytotoxicity against the murine leukemia cell line P-388 with IC50 value of 7.9 and 33 μg/mL, respectively. In 1993, a novel alkaloid, agelastatin A (160) was isolated from the deep water marine sponge Agelas dendromorpha collected in the Coral Sea near New Caledonia [127]. Agelastatin A (160) showed significant in vitro activity against L-1210 and KB tumor cells [128]. He also studied the structure activity relationship of agelastatins and found that the C-8a hydroxyl group and both NH Br H H N

HO N

H H N

H2N

Br Br

H2N

H H

N H

O H

158

R2 Br R1

R3 O N H

H N

O NH2 NH

161, R1 = H, R2 = Me, R3 = Me 162, R1 = Br, R2 = H, R3 = H

NH Br

NH2 N

N H

160

159

N H

H N O

NH2 O

163

164

HN N

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Deepak Kumar & Diwan S. Rawat

groups are necessary for optimal activity. Alkylation or acylation of these functional groups, as well as removal of the C-1 pyrrole bromine, leads to a significant loss of potency. Recently, Tilvi et al. isolated three related pyrrole-imidazole alkaloids, named agelastatins E (161), F (162) and benzosceptrin C (163) along with agelastatin A (160) from marine sponge Agelas dendromorpha [129]. The structures of the compounds were established on the basis of spectroscopic data interpretation. The compounds 160-163 were evaluated for cytotoxic activity against the KB cell lines. All the compounds lacked significant bioactivity at 30 μM except for agelastatin A (160) which showed 100% activity at 30 and 3 μM. A new pyrrole alkaloid, clathrodin (164) was isolated from the MeOH extract of the Caribbean sea sponge Agelas clathrodes [130] and showed significant cytotoxicity against CHO-K1 cells with ED50 value of 1.33 µg/mL. The tetracyclic pyrrole-imidazole alkaloid, dibromophakellstatin (165) was isolated from the marine sponge Phakellia mauritiana [131]. The structure and absolute stereochemistry of the compound was determined by interpretation of NMR and X-ray crystal data analysis. Dibromophakellstatin (165) showed inhibitory activity against a panel of human cancer cell lines, ovary (OVCAR-3), brain (SF-295), kidney (A-498), lung (H-460), colon (KM20L2) and melanoma (SK-MEL-5) with ED50 values of 0.46, 1.5, 0.21, 0.62, 0.11 and 0.11 µg/mL, respectively. Br

Br H H N N

H2 N

O N H

N H

Br N

H2N X

N H

O O

O Me

165

166, R = H 167, R = Br

168

169

Four new alkaloids 3-bromomaleimide (166), 3,4-dibromomaleimide (167), 12-chloro-11-hydroxydibromoisophakellin (168) and N-methylmanzacidin C (169) were isolated from the marine sponge Axinella brevistyla collected in western Japan [132]. Their structures were determined on the basis of spectroscopic data analysis. Compounds 166-168 exhibited cytotoxicity against L-1210 cells with IC50 values of 1.1, 0.66 and 2.5 µg/mL, respectively, whereas N-methylmanzacidin C (169) was found to be inactive. Umeyama et al. reported the isolation and structure elucidation of a novel bromopyrrole alkaloid (170) along with (±)-171 and (±)-longamide (172) from the marine sponge Homaxinella sp. collected in japan [133]. Compounds 170 and (±)-171 showed mild cytotoxic activity in vitro against P-388 lymphocytic leukemia cells with ED50 values of 21.5 and 30 µg/mL, respectively, while compound (172) was inactive (ED50 = >100 µg/mL).

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Br O

Br Br

Br H N

N H

O Br

OCH3

Br O

H3CO

171

170

H3CO

N HO

171

172

More recently, Hertiani et al. reported the isolation of 11 new brominated pyrrole alkaloids from the Indonesian marine sponge Agelas linnaei [134]. These alkaloids includes a new dibromophakellin derivative (173), 4-(4,5-dibromo-1methylpyrrole-2-carboxamido)-butanoic acid (174), agelanin A and B (175 and 176), agelanesins A–D (177-180) and mauritamide B-D (181-183). NH 2

HN HO

NH 2

N Br

N O 173

N H

O N

174

175 N

O N H

H N

O N H

O 177, R1 = H, R2 = Br 178, R1 = H, R2 = I 179, R1 = Br, R2 = Br 180, R1 = Br, R2 = I

176

Br Br

O N H

O S OH O

O N H

N R

O HN

181, R = H 182, R = CH 2CH3

O OH S O

NH 183

All the compounds (173-183) were tested for cytotoxicity against the murine L-1578Y mouse lymphoma cell line. Agelanesins A–D (177-180) showed prominent activity while others were found to be inactive. The IC50 values for agelanesins A–D (177-180) were 9.55, 9.25, 16.76 and 13.06 µM, respectively. Compounds 177 and 178 were the most potent concluding that cytotoxicity of the

240

Deepak Kumar & Diwan S. Rawat

agelanesins is related to the degree of bromination of the pyrrole ring. Increase in bromination decreases the activity as observed for 179 and 180 compared to 177 and 178. While the presence of an iodide substituent on the tyramine moiety causes a small differences in activity as 177 and 179 have similar activity compared to 178 and 180.

3.2. Pyrroloquinones A new dipyrroloquinone, zyzzyanone A (184) was isolated from the Australian marine sponge Zyzzya fuliginosa [135]. Zyzzyanone A (184) showed mild cytotoxic activity against mouse Ehrlich carcinoma cells with IC50 value of 25 µg/mL. One year later Zyzzyanones B-D (185-187), three related dipyrroloquinones were isolated from the same sponge Zyzzya fuliginosa along with the known zyzzyanone A (184) [136]. The structures of the compounds 185-187 were established by extensive NMR spectroscopic data. Zyzzyanones B-D (185-187) also pronounced weak cytotoxicity against mouse Ehrlich carcinoma cells with IC50 value of 25 µg/mL. R N

R N

H N

O N

H2N OH

184, R = Me 185, R = H

CHO

186, R = Me 187, R = H

3.3. Pyrroloquinoline alkaloids In 1986, Landini et al. reported the isolation of discorhabdins C (190) from the extract of red-brown sponge Latrunculia du [137]. The structure of the compound was determined by a single crystal X-ray diffraction study and it was found to contain a new tetracyclic iminoquinone chromophore with a spiro 2,6dibromocyclohexadienone. Two years later, discorhabdins A (188) and (189) B along with known discorhabdins C (190) were isolated from the three species of Latrunculia sponge collected in New Zeeland [138]. A related compound, discorhabdins D (191) was isolated from Latrunculia brevis collected in New-Zeeland [139]. Discorhabdins A (188), B (189) and C (190) showed in vitro cytotoxicity against P-388 assays with ED50 values of 0.05, 0.1 and 0.03 µg/mL. Discorhabdin A (188) and discorhabdin C (190) showed no cytotoxicity against P-388 system in vivo but were found to be toxic to mice at about 2 mg per kg of

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body weight. Discorhabdin B (189) showed some antitumour effect with a T/C of 117% at a dose of 0.25 mg/kg, but this did not reach the significance level of 120%. Discorhabdins C (190) was also found to be active toward L-1210 tumor cells at very low levels (ED50 < 100 ng/mL). Discorhabdin D (191) exhibited mild cytotoxicity against P-388 in vitro with IC50 value of 6 µg/mL, however in vivo it showed significant activity against P-388 (T/C 132% at 20 mg/kg).

H N

H NH

188

190

189

191

Two other members of discorhabdin family, discorhabdins L (192) and I (193) were isolated from Latrunculia brevis and their structures were assigned on the basis of spectroscopic data analysis and comparison with the known discorhabdins A (188) and B (189) [140]. Discorhabdins L (192) and I (193) were tested against a panel of 14 tumor cell lines including prostate (DU-145 and LN-caP), ovary (SK-OV-3, IGROV and IGROV-ET), breast (SK-BR3), melanoma (SK-MEL-28), endothelio (HMEC1), NSCL (A549), leukemia (K562), pancreas (PANC1) and colon (HT29, LOVO and LOVO-DOX). Both the compounds exhibited potent cytotoxic activity in most of the cases. The HT-29 colon cell line was found to be the most sensitive with GI50 values of 0.12 and 0.35 µM for compounds 192 and 193, respectively. Recently, Lang et al. isolated a novel alkaloid, discorhabdin W (194) from a marine sponge Latrunculia sp [141]. It is a symmetrical dimer of discorhabdin in which two discorhabdin units are linked by a disulfide linkage. The structure and stereochemistry were assigned by 1D and 2D NMR experiments and mass spectrometry. Discorhabdin W (194) exhibited potent cytotoxicity against P-388 cells with IC50 value of 0.09 µg/mL. H N

H N

S S N HO H

192

NH O

193

N Br

N Br O

194

H N

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Deepak Kumar & Diwan S. Rawat

Sun et al. reported the isolation of three highly functionalized pyrroloquinoline alkaloids, batzelline A-C (195-197) from the deep water sponge Batzella sp collected in Bahamas [142]. The structure of 195 was determined by X-ray and those of 196 and 197 by comparison of their spectral data with that of 195 and by chemical transformations. One year later same group isolated four related alkaloids, isobatzellines A-D (198-201) from the sponge Batzella sp. [143]. Isobatzellines A-D (198-201) were found to exhibit in vitro cytotoxicity against P-388 leukemia cell lines, whereas batzellines (195-197) were inactive and it was explained on the basis of difference in their structure. Both, isobatzellines and batzellines have the same pyrrolo[4,3,2 de] quinoline ring system but isobatzellines contain an aminoiminoquinone moiety that is different from the aminoquinone moiety in the batzellines which could be responsible for the activity of isobatzellines. In 1999, two novel batzelline analogues, named as secobatzellines A (202) and B (203) were isolated from a deep water marine sponge Batzella sp. The structure of the compounds were determined by NMR, HR FABMS data analysis and chemical analysis and was found to contain pyrroloaminoiminoquinone moiety previously reported in isobatzellines. Secobatzellines A (202) and B (203) exhibited in vitro cytotoxicity against the cultured murine P-388 tumor cell line with IC50 values of 0.06, 1.22 µg/mL and against human lung carcinoma A-549 cell line with IC50 values of 0.04, 2.86 µg/mL, respectively. R

R2 N

O R

O Cl

N H

195, R = Me, R2 = SMe 196, R = H, R2 = SMe 197, R = Me, R2 = H

H2N

N R4

198, R3 = SMe, R4 = Cl 199, R3 = SMe, R4 = H 200, R3 = H, R4 = Cl 201, R3 = H, R4 = H

H2N

R1 Cl

202, R1 = NH, R2 = R3 = H 203, R1 = O, R2 = R3 = H

Kobayashi et al. reported the isolation and structure elucidation of a sulphur containing alkaloid, prianosin A (204) from the Okinawan marine sponge Prianos melanos [144]. Prianosin A (204) was found to be cytotoxic against L-1210 and L-5178Y murine leukemia cells with IC50 values of 37 and 14 ng/mL in vitro. In 1988, same group reported the isolation of three related alkaloids, prianosins B-D (205-207) from the same sponge Prianos melanos [145]. All the alkaloids were found having the same tetrahydrothiophene ring as prianosin A (204). Prianosins B-D (205-207) were evaluated for cytototoxic activity in vitro against murine lymphomas L-1210 and L-5178Y cells and human epidermoid carcinoma KB cells. The IC50 values were found to be 2.0, 1.8 and >5.0 µg/mL (24% inhibition at 5.0 µg/mL) for prianosins B (205), 0.15, 0.024 and 0.57 µg/mL for prianosins C (206) and 0.18, 0.048 and 0.46 µg/mL for prianosins D (207).

Marine natural alkaloids as anticancer agents

H N

S N Br

H N

N O

205

H N S

S N Br

204

243

206

H O

207

Radisky et al. reported the isolation and structure elucidation of seven novel pyrroloiminoquinones, the makaluvamines A-F (208-213) from the Fijian sponge Zyzzya cf. marsailis [146]. The makaluvamines A-F (208-213) exhibited potent in vitro cytotoxicity against the human colon tumor cell line HCT-116, topoisomerase II sensitive CHO cell line xrs-6, and also inhibited the catalytic activity of topoisomerase II. Makaluvamine A (208) and C (210) also exhibited in vivo antitumor activity against the human ovarian carcinoma ovcar-3 implanted in athymic mice. Makaluvamine F (213) was found to be the most active compound followed by makaluvamine E and A. Makaluvamine D and C were less potent than A, E and F, whereas Makaluvamine B was found not active against HCT-116. The same activity pattern was also observed against xrs-6, a Chinese hamster ovary (CHO) cell line being makaluvamine F (213) the most potent compound, while makaluvamine B least active. However, the metabolite cytotoxicity trends are substantially different than the hypersensitivity factors (HF) obtained by comparison of the cytotoxicity against xrs-6 versus BR1 (a DNA-repair proficient CHO line). Makaluvamine A (208) exhibited the largest hypersensitivity factor of 9, followed by makaluvamines F, E, C, and D. These results give a clue about the mechanism of action of Makaluvamines that involves DNA double-stranded breakage, an activity characteristic of topoisomerase II inhibitors. Makaluvamine G (214) was isolated from a sponge of the genus Histodcnnella collected in Indonesia [147]. The structure of the compound was determined on the basis of 1D and 2D NMR experiments. Makaluvamine G (214) pronounced significant cytotoxicity to several tumor cell lines exhibiting an IC50 value of 0.50 µg/mL against P-388 (murine leukemia), A-549 (human nonsmall cell lung cancer), HT-29 (human colon cancer) and MCF-7 (human breast cancer) and value of 0.35 µg/mL against KB (human oral epidermoid carcinoma). It was also found to be a moderate inhibitor of topoisomerase-I (IC50 = 3.0 µM) and did not significantly inhibit topoisomerase-II. It also inhibited RNA (IC50 = 15 µM), DNA (15 µM) and protein (21 µM) synthesis. In 1997, a new related alkaloid, makaluvamine N (215) was isolated from the Philippine sponge Zyzzya fuliginosa [148]. Compound 215 showed in vitro cytotoxicity against the human colon tumor cell line HCT-116 with LC50 value of 0.6 µg/mL. Makaluvamine N (215) also demonstrated an ability to inhibit the

244

Deepak Kumar & Diwan S. Rawat O

O NH2

NH2

208 H N

H N

NH OH

211 H N

H N S

NH OH

212 H N

NH2

210 H N

209 O

H N

213 Br O

O NH2

H N

Br N

214

NH OH

215

216

catalytic activity of topoisomerase II and exhibited 90% inhibition of topoisomerase II unwinding of pBR-322 at 5 µg/mL. Casapullo et al. reported the isolation of a new member of makaluvamine family, makulavamine P (216) from the sponge Zyzzya cf. fuliginosa collected in the Vanuatu Islands [149]. The compound was characterized on the basis of its spectral data and comparison with the other related compounds. Makulavamine P (216) exhibited moderate cytotoxicity to KB tumor cells (64% inhibition of cell growth at 3.2 µg/mL). Recently Shinkre et al. synthesized two series of makaluvamine analogs 217 (a-g) and 218 (c-g) by introducing different substituents at the 7-position of the pyrroloiminoquinone ring present in makaluvamines. These compounds were obtained in two steps by treatment of the methoxypyrroloiminoquinone with different primary amine derivatives and subsequent removal of tosyl protecting group [150]. Compounds 217 (a-g) and 218 (c-g) were evaluated for their cytotoxicity against human breast cancer cell lines MCF-7 and MDA-MB-468 and human colon cancer cell line HCT-116 using etoposide and m-AMSA as standard drugs. HCT-116 cells were shown to be the most sensitive to etoposide and m-AMSA with IC50 values of 1.7 and 0.7 µM, respectively and MDA-MB-468 cells showed IC50 values of 13.6 and 8.5 µM for etoposide and m-AMSA, respectively, whereas MCF-7 cells were found to be the least sensitive with IC50 values of 35.6 and 21.7 µM for etoposide and m-AMSA, respectively. Most of the makaluvamine analogs have shown significantly better inhibition than the control drugs in these assays. Compounds (217c, 218d, 218f, and 218g)

Marine natural alkaloids as anticancer agents

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exhibited better activity (IC50 = 1.3, 0.5, 1.0 and 0.8 µM, respectively) against HCT-116 as compared to control drug etoposide (IC50 = 1.7 µM). Compound 218d exhibited better IC50 value against HCT-116 as compared to m-AMSA (IC50 = 0.7 µM). All the compounds exhibited better IC50 values against MCF-7 and MDA-MB- 468 as compared to etoposide as well as m-AMSA. Compounds 217 (a-g) and 218 (c-g) were also evaluated for their ability to inhibit topoisomerase II enzymatic activity and found that five makaluvamine analogs (217c, 217d, 217f, 218c and 218e) exhibited inhibition of topoisomerase II comparable to etoposide and m-AMSA. Three of these compounds (217f, 218c and 218e) showed the strongest inhibition of catalytic activity of topoisomerase II. In 1997, the methanol extract of the Fijian sponge Zyzzya fuliginosa yielded a new pyrroloiminoquinone derivative, veiutamine (219) [151]. The structure of the compound was determined by 1D and 2D NMR experiments and was found bearing a p-oxy benzyl substituent at carbon 6 of the basic pyrroloiminoquinone system. Veiutamine (219) exhibited cytotoxicity against the human colon tumor cell line HCT-116 with IC50 value of 0.3 µg/mL. Wakayin (220) was isolated from the ascidian Clauelinu sp [152]. It was supposed to represent the first example of pyrroloiminoquinone alkaloid to be isolated from an ascidian. Wakayin (220) exhibited in vitro cytotoxicity against the human colon tumor cell line (HCT-116) with IC50 value of 0.5 µg/mL. Preliminary studies such as Inhibition of topoisomerase II enzyme (250 µM) and the observation of a 3-fold differential toxicity toward the CHO cell line EM9 (sensitive to DNA-damaging genotoxic agents) versus BR16 (resistant to BCNU) provided evidences that the activity of wakayin could be related to interfering with or damaging DNA. H N

Ts N

H N R

217 (a-g)

218 (c-g)

CH3

CH2CH3

H N

H2CH2C

H2C

d Br

H2CH2C

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Deepak Kumar & Diwan S. Rawat

Two new bispyrroloiminoquinone alkaloids, tsitsikammamme A (221) and tsitsikammamine B (222) were isolated from the South African Latrunculid sponge Tsitsikamma favus [153]. Reinvestigation of the extracts of the sponge Tsitsikamma favus yielded two N-18 oxime analogues of tsitsikammamine A and B, 223 and 224 [154]. Compounds 223 and 224 exhibited significant cytotoxic activity against human colon tumor (HCT-116) cell line with IC50 values of 128.2 and 16.5 μM, respectively, when compared with their parent alkaloids 221 and 222 (IC50 = 1.4 and 2.4 mM, respectively). Recently, two aza-analogs, 225 and 226 of tsitsikammamine and wakayin were synthesized based on a 1,3-dipolar cycloaddition reaction between indole 4,7-dione and a diazo-aminopropane derivative in which the pyrrole ring of the pyrroloquinoline moiety was replaced by a pyrazole ring [155]. The ability of the compounds 225 and 226 to inhibit the DNA cleavage activities of human topoisomerases I and II was assayed in a cell-free assay. Both the compounds exhibited 0% inhibition of topoisomerase II. Compound 225 inhibited partially topoisomerase I at 100 µM, whereas no inhibitory activity was observed for compound 226. O

H N

NH2

H N

220

H N

219 O

H N

NH N H

R2 N

R N

H N

221, R = H 222, R = CH3 H N

N N

N H R1

223, R1 = OH, R2 = H OH 224, R1 = OH, R2 = Me

225

N H

226

3.4. Pyrroloacridine Two novel alkaloids, plakinidine A (227) and B (228) were isolated from Vanuatuan red sponge Plakortis sp. [156]. Their structures were determined by 1D and 2D NMR experiments and were found to contain a pyrrolo (2,3,4-kl) acridine system. In the same year, IreIend et al. reported the isolation and structure elucidation of a new compound plakinidine C (229) together with plakinidine A (227) and B (228) from the MeOH extract of Plakortis sp. collected

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in Fiji [157]. Plakinidine A-C (227-229) exhibited cytotoxic activity towards L-1210 murine leukemia cell lines with IC50 values of 0.1, 0.3 and 0.7 µg/mL, respectively. R N

N NH

227, R = H 228, R = CH3 229, R = H, 9,10-didehydro

3.5. Miscellaneous pyrrole alkaloids Ircinamine B (230) was isolated from the marine sponge Dactylia sp. collected at Cape Sada in Japan and showed moderate cytotoxic activity against the murine leukemia cell line P-388 with IC50 value of 0.28 µg/mL [158]. A novel tetracyclic alkaloid, perinadine A (231) was isolated from the cultured broth of the fungus Penicillium citrinum separated from the gastrointestine of a parrot fish Scalus ovifrons collected at Hedo Cape, Okinawa Island [159]. Perinadine A (231) exhibited mild cytotoxicity against murine leukemia L-1210 cell line with IC50 value of 20 µg/mL. In 1994, Perry et al. isolated a new alkaloid, Variolin B (233) from the Antarctic sponge Kirkpatrickia varialosa. The structure was determined by X-ray crystallography and interpretation of spectral data [160]. Variolin B (233) was supposed to be the first examples of natural products with a pyridopyrrolopyrimidine moiety. In the same year two other pyridopyrrolopyrimidine alkaloids, variolin A (232) and N(3´)-methyl tetrahydrovariolin B (234) were isolated from the same sponge Kirkpatrickia varialosa [161]. Variolins (232-234) were tested in vitro against P-388 cell lines. Variolin A (232) and variolin B (233) showed in vitro activity against P-388 cell lines with IC50 value of 3.8 ng/mL and 210 µg/mL, respectively. Compound 234 was found to be inactive against P-388 but showed in vivo activity against P-388 leukemia (T/C 125% at 10 mg/Kg). Compound 234 also showed significant in vitro activity against the HCT-116 cell line with IC50 value of 0.48 µg/mL. Kashman et al. isolated a novel bisquinolinylpyrrole alkaloids, halitulin (235) from a marine sponge Haliclona tulearensis collected in Sodwana Bay, Durban, South Africa [162]. Its structure was established mainly on the basis of spectroscopic data and chemical means. Halitulin (235) was considered as the first natural

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compound to be discovered that has a 7,8- dihydroxyquinoline system and found to be cytotoxic against several tumor cell lines such as P-388 murine leukemia, A-549 human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human melanoma with IC50 value of 0.025, 0.012, 0.012 and 0.025 µg/mL, respectively. O

NH2

N H OH

O N H

232

231

NH2

N NH2

NH2 N

230

N N N

NH2

233

234

235

4. Pyridine alkaloids In 1999, Kobayashi et al. isolated a novel pyridine alkaloid, pyrinodemin A (236) from the Okinawan marine sponge Amphimedon sp. [163]. The structure of compound 236 was assigned from 2D NMR data and EIMS fragmentation and was found to contain two 3-alkyl-substituted pyridine rings with a cis-cyclopent[c]isoxazolidine moiety. Pyrinodemin A (236) demonstrated potent cytotoxicity in vitro against murine leukemia L-1210 and KB epidermoid carcinoma cells with IC50 values of 0.058 and 0.5 µg/mL, respectively. One year later, three new bis-pyridine alkaloids, pyrinodemins B-D (237-239) were isolated together with pyrinodemin A (236) from the same sponge Amphimedon sp. [164]. Pyrinodemins B-D (237-239) exhibited potent cytotoxicity in vitro against murine leukemia L-1210 with IC50 values of 0.07, 0.06 and 0.08 µg/mL, respectively and KB epidermoid carcinoma cells (IC50 = 0.5 µg/mL each).

Marine natural alkaloids as anticancer agents

249

A novel pyridine alkaloid, pyrinadine A (240) was isolated from the marine sponge Cribrochalina sp. collected from the Unten Port, Okinawa [165]. The structure was established by spectroscopic data and chemical conversions. When treated with zinc/acetic acid, pyrinadine A yielded compound (241), generated by cleavage at the azoxy moiety of pyrinadine A. Pyrinadine A (240) exhibited in vitro cytotoxicity against L-1210 murine leukemia (IC50 = 2 µg/mL) and KB human epidermoid carcinoma cells (IC50 = 1 µg/mL).

H H

236 H

H O

237 H

H O

238

N H

H O

239

N N

240 NH2 N

241

In 2006, Takekawa et al. reported the isolation of amphimedosides A-E (242-246) from a marine sponge Amphimedon sp. [166]. The structures of compounds 242-246 were determined by NMR, FABMS data interpretation. The site of glycosylation in compound 242 was confirmed by the 1H-15N HMBC experiment and the location of the double bond in 246 was assigned on the basis of tandem FABMS data. Amphimedosides (242-246) were the first examples of β-D-glucosylated 3-alkylpyridine alkaloids till the date and exhibited mild to strong cytotoxicity against P-388 murine leukemia cells with IC50 values of 11, 11, 5.0, 0.45 and 2.2 µg/mL, respectively.

250

Deepak Kumar & Diwan S. Rawat HO HO HO

O OH

OCH3 N n

242, m = 3, n = 9 243, m = 3, n = 7 244, m = 1, n = 9

m N

HO HO HO

OCH3 N

245

HO HO HO

OCH3 N

246

Echinoclathrines A-C (247-249), a new class of pyridine alkaloids having 4-aryl-2-methylpyridine unit, were isolated from an Okinawan sponge, Echinoclathria sp. [167]. The structures of compounds were established by interpretation of spectral data. Only echinoclathrine A (247) displayed weak cytotoxicity (IC50 = 10 µg/mL) against P-388, A-549 and HT-29 cell lines, while others were found to be inactive. OH O

OR O

N H

247 N

SR1

248, R = H, R1 = Ac 249, R = R1 = H

5. Isoquinoline alkaloids Two new isoquinolinequinones alkaloids, cribrostatins 1 (250) and 2 (251) were isolated from a deep blue colored sponge Cribrochalina sp. [168]. The structures of the compounds were determined by extensive NMR data analysis and single-crystal X-ray diffraction experiment. Cribrostatins 1 and 2 were found to be active against lymphocytic leukemia cell line (P-388) with ED50 values of 1.58 and 2.73 µg/mL, respectively. Pettit et al. reported the isolation of cribrostatins 3 (252), 4 (253) and 5

Marine natural alkaloids as anticancer agents

251

(254) from the same sponge Cribrochalina sp. [169]. Compounds 251-254 were evaluated for cytotoxicity against several cancer cell lines. Mouse leukemia P-388 cell line was found to be the most sensitive to Cribrostatins 3 (252), 4 (253) and 5 (254) exhibiting with ED50 values of 2.5, 2.2 and 0.045 µg/mL, respectively. Cribrostatin 6 (255) was also isolated from the same marine sponge Cribrochalina sp. [170]. The structure of compound was assigned on the basis of 1 H, 13C, 15N NMR and HRMS data interpretation and finally structure was confirmed by X-ray crystal data analysis. Cribrostatin 6 (255) was found to inhibit the growth of murine P-388 lymphocytic leukemia (GI50 = 0.29 µg/mL) and a panel of human cancer cell lines. Among human cancer cell lines, the best activity in terms of potency was obtained against MCF-7 (GI50 = 0.21) followed by SF-268 (GI50 = 0.24) and DU-145 (GI50 = 0.38), whereas GI50 value of >1µg/mL was observed against BXPC-3, NCI-H460 and KM20L2 cell lines. A new isoquinoline alkaloid, jorumycin (256) was isolated from the mantle and the mucus of the pacific nudibranch Jorunna funebris [171]. The structure of compound was established on the basis of ESIMS data and of an extensive 2D NMR analysis. Jorumycin (256) showed very interesting activity against NIH 3T3 tumor cells (100% of inhibition at 50 ng/mL) and also exhibited promising cytotoxic activity against P-388, A-549, HT-29 and MEL-28 with IC50 value of 12.5 µg/mL each. O

Me N

H2N O

250

251

HO O

OCH3 CH3

N H

N O

H O

252, R = H O 254, R = CH3

253

255

O H

OH O

N O

H NCH3

H3CO

OCH3 CH3

H NCH3

H3CO O

H O

256

6. Guanidine alkaloids In 1989, Kashman et al. reported the isolation of a novel guanidine alkaloid ptilomycalin A (257) from the Caribbean sponge Ptilocaulis spiculifer and the red sea sponge Hemimycale sp. [172]. Ptilomycalin A (257) consists of a pentacyclic guanidine unit and a spermidine unit linked by a linear long-chain fatty acid.

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Ptilomycalin A (257) exhibited significant cytotoxic activity against P-388, L-1210 and KB cell lines with IC50 values of 0.1, 0.4 and 1.3 mM, respectively. H

N N O H

O N H O CH3

CH3 H2N H2N

N O

257

Recently, Black et al. synthesized three novel analogues, 258, 259 and 260 of ptilomycalin A (257) [173]. Compounds 258-260 were tested against four cancer cell lines including human chronic myelogenous leukaemia (K-562), human ovarian carcinoma (A-2780), human large cell carcinoma (H-460) and mouse lymphoid neoplasm (P-388). Compound 258 showed the best activity against all the cell lines comparable to the parent compound (257). The IC50 values of 0.52, 0.92, 0.52 and 0.69 µg/mL were obtained against K-562, A-2780, H-460 and P-388, respectively for compound 258, whereas compound 259 was found to be less potent than compound 258. Compound 260 was the least active compound of the three, which indicated that the presences of a spacer chain and spermidine residue are essential for the compounds to demonstrate the biological activity.

H2N H2N

O NH Cl 2CF3COOH NH O

O NH Cl 2HCl NH O

N O

258

H2N H2N

O O H

259

O NH

H N H

NH O

260

BF4

Marine natural alkaloids as anticancer agents

253

Seven new tricyclic guanidine alkaloids, netamines A-G (261-267) were isolated from the extract of the poeciloscleridae sponge Biemna laboutei collected near the Sainte-Marie Island on the east coast of Madagascar [174]. The structures of compounds were determined on the basis of 1D, 2D NMR and HRFABMS data interpretation. All the compounds 261-267 were evaluated for cytotoxicity against three human tumor cell lines: NSCL (A-549), colon (HT-29) and breast (MDA-MB-231). Only netamines C (263) and D (264) showed promising activity against A549 (GI50 = 4.3 and 6.6 µM) HT29 (GI50 = 2.4 and 5.3 µM) and MDA-MB-231 (GI50 = 2.6 and 6.3 µM), whereas other compounds were found to be inactive or very less toxic. NH HN

CH3

261

CH3

263

262

NH HN

CH3

265

CH3

264

CH3

CH3 CH3

CH3

266

267

7. Aminoimidazole alkaloids Ralifo et al. reported the isolation and structure elucidation of two novel alkaloids, leucosolenamines A (268) and B (269) from the marine sponge Leucosolenia sp. [175]. Compound 268 was found to contain a 2-aminoimidazole unit substituted at C-4 and C-5 by an N,N-dimethyl-5,6-diaminopyrimidine-2,4dione and a benzyl group, respectively. Although, compound 269 has the same core structure but C-4 is substituted by a 5,6-diamino-1,3- dimethyl-4-(methylimino)3,4-dihydropyrimidin-2(1H)-one moiety. This substitution pattern is unique and had never been observed in imidazole alkaloid chemistry. Leucosolenamine A (268) exhibited mild cytotoxicity against the murine colon adenocarcinoma C-38 cell line, whereas compound 269 was inactive. In the same year the other group isolated two new imidazole alkaloids, naamidines H (270) and I (271) from the marine sponge Leucetta chagosensis collected in North Sulawesi, Indonesia [176]. The compounds 270 and 271 demonstrated weak cytotoxicity against HeLa cells with IC50 values of 5.6 and 15 μg/mL, respectively.

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Deepak Kumar & Diwan S. Rawat

O H2N H N

CH3 CH3 N N O

CH3 N O

H2N

CH3

H N

CH3 N

N H

OMe

MeO

N R

O O

268

269

OMe

270, R = O 271, R = NMe

8. Steroidal alkaloids Four novel steroidal alkaloids, plakinamine G (272), plakinamine H (273), 4Rhydroxydemethylplakinamine B (274) and tetrahydroplakinamine A (275) were isolated from the marine sponge Corticium sp. [177]. The structures of these compounds were established spectroscopically mainly by 1D, 2D NMR and mass spectrometry (HR-EIMS). Compounds 272-275 were tested for cytotoxicity against rat glioma (C6) and murine macrophages (RAW-264) cell lines. Compounds 272 and 275 found to be the most active against C6 cells with IC50 values of 6.8 and 1.4 µg/mL, respectively, whereas they showed no activity against RAW-264 cell line. Compounds 273 and 274 were cytotoxic against both the cell lines with compound 273 being more active against C6 cells (IC50 = 9.0 µg/mL) than to RAW-264 (IC50 = 61 µg/mL), while compound 274 showed greater value of IC50 (16.2 µg/mL) against RAW-264 cell line than to C6 cells (IC50 = 26.1 µg/mL). One year later four new related steroidal alkaloids, plakinamine I-K (276-278) and dihydroplakinamine K (279) were isolated from the same sponge Corticium niger [178]. Compounds (276279) as their hydrochloride salts were evaluated for cytotoxicity against the human colon tumor cell line (HCT-116). Compounds 278 and 279 were found to be the most active in terms of potency with an IC50 value of 1.4 µM each. Compounds 276 and 277 were moderately active with IC50 values of 10.6 and 6.1 µM, respectively. Ritterazines B (280) and C (281), two dimeric steroidal alkaloids were isolated from the tunicate Ritterella tokioka collected off the Izu Peninsula [179]. Their structures including absolute stereochemistry were assigned by spectral and chemical methods. Ritterazines B (280) and C (281) displayed potent cytotoxicity against the P-388 murine leukemia cells with IC50 values of 0.018 and 9.4 ng/mL, respectively. Three novel steroidal alkaloids, cortistatins J-L (282-284) were isolated from the Indonesian marine sponge Corticium simplex [180]. The structures of compounds 282-284 were established by 1D and 2D NMR (COSY, HMQC and HMBC) data analysis. Cortistatin J (282) demonstrated potent cytostatic anti-proliferative activity

Marine natural alkaloids as anticancer agents

255

against human umbilical vein endothelial cells (HUVEC) with IC50 value of 8 nM and also inhibited migration and tubular formation of HUVEC induced by VEGF or bFGF, whereas cortistatins K (283) and L (284) were less potent than cortistatin J (282) with IC50 values of 40 and 23 nM, respectively. NH HN O H2N

N O

272

273

N HN

H2N

H2N OH

274

275

H N

H NH2

276

277

HN H

H N H

N H

H OAc

278

279

OH H H

280

282 O

OH H H

H O

H N

N R O

H N

281

283, R = H 284, R = OH

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9. Miscellaneous alkaloids Four novel alkaloids 285-288, related to aaptamines were isolated from the MeOH extract of the Indonesian marine sponge Xestospongia sp. collected from Jakarta along with the known aaptamine (289), isoaaptamine (290), demethyl(oxy)aaptamine (291) and its dimethylketal (292) [181]. Their structures were determined on the basis of 1D and 2D NMR spectroscopic data. All the compounds 285-292 were evaluated for cytotoxic activity against KB cell lines. Compounds (289-292) exhibited moderate cytotoxicity against KB cells with ID50 values of 3.7, 0.5, 1.8 and 3.5, respectively, while compounds (285-288) were less potent with ID50 value of >10 µg/mL. Four tetracyclic alkyl-piperidine alkaloids, Haliclonacyclamie E (293) arenosclerins A (294), B (295) and C (296) were isolated from the marine sponge Arenosclera brasiliensis [182]. All the compounds were tested for their cytotoxicity against HL-60, B-16, U-138 and L-929 cancer cell lines. Compound 293-296 exhibited almost the same range of cytotoxicity with IC50 values of 4.23, 4.31, 4.07, 3.65 (HL-60), 1.82, 1.77, 1.76, 1.71 (B-16), 6.06, 3.83, 3.62, 3.60 (U138) and values of 3.89, 2.34, 2.24, 2.17 (L-929), respectively. R2 O R1

OCH3

H3CO

H3CO N

285, R1 = H, R2 = CH3 286, R1 = CH3, R2 = H

287

288

OCH3

OCH3 HO

OCH3

N R

N N

289

N N

290

291, R = CH(CH3)2 292, R = H

Four bis-piperidine alkaloids, madangamine F (297), haliclonacyclamine F (298), arenosclerins D (299) and E (300) were isolated from the marine sponge Pachychalina alcaloidifera [183]. Compounds 297-300 were evaluated for cytotoxicity against SF-295 (human CNS), MDA-MB-435 (human breast), HCT8 (colon) and HL-60 (leukemia) cancer cell lines. Haliclonacyclamine F (298) and arenosclerin D (299) were found to be the most active compounds with IC50 values of 4.5 and 5.9 µg/mL (SF-295), 1.0 and 1.2 µg/mL (MDA-MB-435), 8.6 and 6.2 µg/mL (HCT-8), 2.2 and 6.2 µg/mL (HL-60), whereas compounds 297

Marine natural alkaloids as anticancer agents

257

and 300 showed IC50 values of 19.8 and 8.7 µg/mL (SF-295), 16.2 and 3.1 µg/mL (MDA-MB-435), 16.7 and 6.9 µg/mL (HL-60), >25 and >25 µg/mL (HCT-8) cell lines.

H N

N H H

H H

H H H

297

295

N N OH

298

294

H H

HO 293

296

N H H

N HO

299

H N H HO

300

Matsunaga et al. reported the isolation and structure determination of two new 3-alkylpiperidine alkaloids, tetradehydrohalicyclamine A (302) and 22 hydroxyhalicyclamine A (303) along with a known halicyclamine A (301) from a marine sponge Amphimedon sp. collected in southern Japan [184]. Compounds 301, 302 and 303 were found to be cytotoxic against P-388 cells with IC50 values of 0.45, 2.2 and 0.45 µg/mL, respectively. Three new diketopiperazine alkaloids, 6-methoxyspirotryprostatin B (304), 18-oxotryprostatin A (305) and 14-hydroxyterezine D (306) along with other metabolites were isolated from the ethyl acetate extract of a marine-derived fungal strain Aspergillus sydowi [185]. All the compounds were evaluated for Cytotoxicity against A-549 and HL-60 cell lines. Compounds 304-306 exhibited weak cytotoxicity against A-549 cells with IC50 values of 8.29, 1.28 and 7.31 μM, respectively. In addition, compound 304 also demonstrated significant cytotoxicity against HL-60 cells with an IC50 value of 9.71 μM. Two novel pyrazine alkaloids botryllazine A (307) and botryllazine B (308) along with the new imidazole alkaloid 2(p-hydroxybenzoyl)-4-(phydroxyphenyl)-imidazole (309) were isolated from the red ascidian Botryllus leachi [186]. The structures of compounds 307-309 were elucidated by interpretation of spectral data and botryllazine A (307) was supposed to represents the first example of a marine alkaloid containing a pyrazine nucleus derived from three tyrosine precursors. All the compounds (307-309) were tested in vitro for cytotoxicity against P-388 mouse lymphoma, A-549 human lung carcinoma, HT-29 human colon carcinoma and MEL-28 human melanoma. Botryllazine A (307) was inactive with ED50 value of 10 µg/mL each.

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Deepak Kumar & Diwan S. Rawat

NH H

NH N 2CHF3CO2

NH 2CHF3CO2 H

NH 2CHF3CO2

H OH

301

302 O

MeO

303 H

O NH

HN O

N HO H

N O

MeO

304

305

306

Botryllazine B (308) exhibited weak cytotoxicity (ED50 = 5 µg/mL) against A-549 and MEL-28 cell lines, whereas compound 309 was mildly active against all the four tumor cell lines with ED50 of 5 µg/mL. OH

OH N

O O

HO OH

307

308

309

Kobayashi et al. isolated novel bromotyrosine alkaloids, maedamines A (310) and B (311) from Okinawan marine sponge Suberea sp. [187]. Structures were elucidated on the basis of spectroscopic data and these compounds were found containing a 2(1H)-pyrazinone moiety between two bromotyrosine units. Maedamines A (310) and B (311) exhibited in vitro cytotoxicity against murine leukemia L-1210 cells with IC50 values of 4.3 and 3.9 µg/mL, respectively and epidermoid carcinoma KB cells with IC50 values of 5.2 and 4.5 µg/mL, respectively. Maedamine A (310) also demonstrated inhibitory activity against c-erbB-2 kinase with IC50 value of 6.7 µg/mL, while compound 311 being inactive against c-erbB-2 kinase (IC50 >10 µg/mL).

Marine natural alkaloids as anticancer agents Br H3CO

259 H N Br

310, R = CH3 311, R = H

N R

Two new bromotyrosine alkaloids, purealidin S (312) and purpuramine J (313) were isolated from the Fijian marine sponge Druinella sp. [188]. Compound 313 contains a bromotyrosine N-oxide unit which is very rarerly found in marine natural products. Both the compounds were tested for cytotoxicity against A-2780 (Ovarian tumor) and K-562 (leukaemia) cell lines. Compounds 312 and 313 showed mild cytotoxicity against these two cell lines with IC50 values of 7.44 and 6.77 µg/mL (A-2780) and values of 6.02 and 1.24 µg/mL (K-562), respectively. OMe Br HO

O N

H N O

H2 N

OH H N

O Br

312

NH OH

313

Two new dimeric polysulfide alkaloids, lissoclinotoxins E (314) and F (315) were isolated from the MeOH extract of a Philippine didemnid ascidian [189]. The polysulfide structures for compounds 314 and 315 were determined by interpretation of spectroscopic data and chemical means. Computational chemistry studies suggested the trans- and cis- orientations of N-alkyl chains about the tricyclic systems of lissoclinotoxins E (314) and F (315), respectively. Compounds 314 and 315 exhibited significant cytotoxicity against PTENdeficient human breast carcinoma, MDA-MB-468 cell lines with IC50 values of 2.3 and 1.5 µg/mL, respectively. Williams et al. isolated motuporamines A-C (316-318) from the marine sponge Xextospongia exigua [190]. The crude mixtures of motuporamines A-C could not readily be separated and they were obtained as a mixture of three (316318). The mixture of motuporamines A-C (316-318) showed significant cytotoxicity against a panel of human solid tumor cancer cell lines with IC50 value of 0.6 µg/mL.

260

Deepak Kumar & Diwan S. Rawat N OMe

OMe MeO

SMe

MeO

S S

MeS

OMe

MeS

OMe OMe SMe

314

315

X N H

N R

H2N

N H

316, R = H, X = CH2 317, R = H, X = (CH2)2

318

Two novel alkaloids, pterocellins A (319) and B (320) were isolated from the New Zealand marine bryozoans Pterocella vesiculosa [191]. The structures were assigned by NMR and mass spectral data analysis and finally structure was confirmed by single-crystal X-ray diffraction experiments. Pterocellins A (319) and B (320) were evaluated for cytotoxicity against P-388 murine leukemia cell lines and exhibited relatively potent activity with IC50 values of 477 and 323 ng/mL, respectively. O

O O

N N

319

320

The cytotoxicity of pterocellins A (319) and B (320) was also evaluated by the NCI in their 60 cell line panel, which represents a variety of human tumor cell types such as leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, ovarian, renal, prostate and breast cancers. Compounds 319

Marine natural alkaloids as anticancer agents

261

and 320 exhibited potent cytotoxicity with panel average values of GI50 = 1.4 µM, TGI = 4.8 µM, LC50 = 17.0 µM for pterocellin A (319) and GI50 = 0.7 µM, TGI = 2.1 µM, LC50 = 6.9 µM for pterocellin B (320). The leukemia cell line (CCRF-CEM) was found to be the most sensitive cell line to pterocellin A (319) with GI50 value of 0.05 µM and TGI value of 0.8 µM, although the high LC50 value of >100 µM implied that pterocellin A (319) is cytostatic rather than cytotoxic to this cell line. The most sensitive cell line to pterocellin B (320) was the melanoma cell line MALME-3M with GI50 value of 0.03 µM and TGI value of 0.1 µM, whereas cell lines such as NCI-H23, melanoma MALME-3M, M14, SK-MEL-5, breast MDA-MB-435 and MDA-N were found to be sensitive to both the compounds.

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Bhakuni, D.S., Rawat, D.S. Bioactive Marine Natural Products, Springer Newyork, ISBN: 1-4020-3472-5, 2005. 2. Proksch, P., Ebel, R., Edrada, R.A., Schupp, P., Lin, W.H., Sudarsono, Wray, V., Steube, K. Pure Appl. Chem., 2003, 75, 343. 3. Ojima, I. J. Med. Chem., 2008, 51, 2588. 4. Newman, D. J. J. Med. Chem., 2008, 51, 2589. 5. Thomas, T.R.A., Kavlekar, D.P., LokaBharathi, P.A. Mar. Drugs, 2010, 8, 1417. 6. Singh, R., Sharma, M., Joshi, P., Rawat, D.S. Anti-Cancer Agents-Med. Chem., 2008, 8, 603. 7. Morris, J.C., Phillips, A.J. Nat. Prod. Rep., 2010, 27, 1186. 8. Gochfeld, D.J., ElSayed, K.A., Yousaf, M., Hu, J.F., Bartyzel, P., Dunbar, D.C. Mini. Rev. Med. Chem., 2003, 3, 401. 9. Rawat, D.S., Joshi, M.C., Joshi, P., Aethaya H. Anti-Cancer Agents-Med. Chem., 2006, 6, 33. 10. Mayer, A.M.S., Hamann, M.T. Mar. Biotechnol., 2004, 6, 37. 11. Pauletti, P.M., Cintra, L.S., Braguine, C.G., Da Silva Filho, A.A., Silva, M.L.A., Cunha, W.R., Januário, A.H. Mar. Drugs, 2010, 8, 1526. 12. Blunt, J.W., Copp, B.R., Munro, M.H.G., Northcote, P.T., Prinsep, M.R. Nat. Prod. Rep., 2010, 27, 165. 13. Urban, S., Hickford, S.J.H., Blunt, J.W., Munro, M.H.G. Curr. Org. Chem., 2000, 4, 765. 14. Garcia, M.M., Valdes, M.D., Espla, A.R., Salvador, N., Lopez, P., Larriba, E., Anton, J. Mar. Drugs, 2007, 5, 52. 15. Haefner, B. Drug Discovery Today, 2003, 8, 536. 16. Arif, J.M., Al-Hazzani, A.A., Kunhi, M., Khodairy, F.A. J. Biomed. Biotech., 2004, 2, 93. 17. Patterson, A.M., Capell, L.T., Walker, D.F. The Ring Index, 2ndEd., 1960, American Chemical Society: Washington, DC. 18. Delfourne, E., Bastide, J. Med. Res. Rev., 2003, 23, 234. 19. Schmitz, F.J., Deguzman, F.S., Hoseain, M.B., Vanderhelm, D. J. Org. Chem., 1991, 56, 804.

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 269-282 ISBN: 978-81-308-0448-4

8. Microtubule binding natural substances in cancer chemotherapy Ram C. Mishra Department of Biology, Georgia State University, P O Box 4010, Atlanta, Georgia 30302-4010, USA

Abstract. Microtubules constitute the major part of the cytoskeleton and play active role in cell division. Their dynamic instability and role in spindle formation during mitosis makes them an interesting target for anti-cancer drug development. Natural products are well known to be utilized for improving the human health. There are many natural products currently in use for providing cure to all kinds of diseases including cancer. Taxol and Vinblastine are examples of such natural products which interact with tubulin and are used in chemotherapy of cancer. This article briefly describes the microtubule binding natural substances and their use as anticancer agents.

1. Introduction Natural products have shown to be the major source of anticancer drugs. In the last 25 years more than 60 % of the anticancer drugs are either natural products or have natural product origin [1]. Microtubules are one of the major components of the cytoskeleton which are essential for many cellular processes including maintenance of cell structure, protein transportation and mitosis. These are also referred to as conveyer belts inside the cell [2]. The microtubules are composed of a group of cylindrical proteins know as tubulins and perform many of their functions by binding to MAPs i.e. Correspondence/Reprint request: Dr. Ram C. Mishra, Department of Biology, Georgia State University P O Box 4010, Atlanta, Georgia, 30302-4010, USA. E-mail: mishra@rcmishra.in

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microtubule associated proteins. Microtubules are directly involved in the formation of mitotic spindle which helps in segregating the replicated chromosomes towards two daughter nuclei at the end of mitosis. Involvement of microtubules in this particular cell cycle event makes them an important target in cancer chemotherapy [3]. The anticancer activity of taxanes and vinca alkaloids is attributed to their affinity and binding ability to tubulin units of the microtubules.

2. Microtubule chemotherapy

structure

and

target

for

cancer

During the cell division i.e. mitosis, microtubules play an important role in segregation of chromosomes via spindle formation. Microtubules as the name suggests are the hollow tube like structures with a diameter of 15-25 nanometer and form the major part of the cytoskeleton. Their length may vary from 200 nm to 25 micrometers. This hollow structure is formed by an imperfect helix like arrangement of the protofilaments. The protofilaments in turn, are the product of end to end polymerization of the tubulin heterodimers namely alpha tubulin and beta tubulin. Polarity is another feature of the microtubule structure as during the end to end polymerization process alpha subunit of one tubulin dimer is attached to beta subunit of the other. This leads to the formation of protofilaments with beta subunits exposed at one end and alpha subunit at the other. These are designated as plus (+) and minus (–) ends respectively. In a microtubule the protofilaments bundle parallel to each other so that there is one end with beta tubulin subunit (plus end) exposed and the other with alpha (minus end) unit exposed. The minus end is capped, so that elongation occurs from the plus end [4]. The mitotic spindles are formed by attachment of GTP-tubulin to the growing end of the protofilament. The microtubules undergo rapid assembly and disassembly leading to their dynamic instability [5,6]. This dynamic instability along with their involvement in mitotic spindle formation helps in the metaphase to anaphase transition of the mitosis. This continued assembly and disassembly process in microtubules are crucial to the normal cell division and any interference in this leads to cell death via apoptosis. Usually the anti-mitotic agents arrest the metaphase to anaphase transition in mitosis. The defective spindles formed due to disturbances in dynamics of microtubules at low concentrations of the anti-mitotic agent are unable to cross the mitotic spindle checkpoint and initiate the anaphase stage. This leads to prolonged mitotic arrest and finally cell death by apoptosis.

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Figure 1. Microtubule architecture. Table 1. Diverse origin of taxane-domain binding drugs. ORIGIN

DRUG

SOURCE

PLANT

Paclitaxel Docetaxel

Taxus brevifolia (Yew tree bark) Taxus baccata (semi-synthetic)

10-deacetylbaccatin III BACTERIAL Epothilones Cyclostreptin MARINE

Discodermolide Dictyostatin Laulimalide

CORAL

Peloruside Eleutherobin Sarcodictyins

Taxus brevifolia (Yew tree leaves) Sporangium cellulosum (myxobacterium) Streptomyces sp. Discoderma dissolute (marine sponge) Spongia (marine sponge) Hyattella sp. and Fasciospongia rimosa (marine sponges) Mycale hentscheli (marine sponge) Eleutherobia sp. (soft coral) Sarcodictyon roseum (soft coral)

The beta unit of the tubulin heterodimer has the priority over the alpha unit in interaction with the drugs. Its structure has been solved by electron diffraction [7]. Beta-tubulin has the binding sites for both the taxane drugs and the vinca alkaloids at different locations. The taxane drug, paclitaxel binds on two sites of the beta subunit, the N-terminal unit and the region between the amino acids 217-231 [8]. The vinca alkaloid drugs also bind to same beta subunit but in the region bound by amino acids 175 and 213 [9].

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The extensively studied natural ligand of the tubulin, colchicine, binds between the two subunits and is not used clinically as anticancer drug. Another group of the natural products known as epothilones also bind to tubulin at its taxane binding site [10]. The natural products binding to the tubulin can affect its dynamics either by promoting or by inhibiting the polymerization process. Based on this general characteristic the tubulin binding natural products have been classified as under inhibitors or promoters of the tubulin polymerization.

3. Promoters of tubulin polymerization The microtubule polymerization promotors can be broadly classified in to the taxanes and the epothilones both of which bind to same domain of the beta subunit of the tubulin heterodimer. Apart from these two classes, there are few more compounds which are known for their tubulin polymerization properties. In the following table the diverse origins of the drugs binding to taxane domain and their source has been presented.

3.1. The taxanes Paclitaxel has been the main chemotherapeutic agent for the various types of cancers including breast, ovarian and the prostate cancer. This compound was first isolated and reported from the pacific yew tree bark in 1960 and named as Taxol [11]. Its mechanism of action was discovered in 1980s. The new and currently used generic name Paclitaxel was given when the drug was developed commercially by Bristol-Mayers Squibb and sold under the trade name Taxol. The drug is also used in chemotherapy of NSCLC in combination with Cisplatin [12].

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The success of the paclitaxel led to the development of many of its analogs which are currently in clinical trials. The only analog approved in USA is the Docetaxel, which is a semi-synthetic analog and was developed in France [13]. Apart from the paclitaxel and docetaxel which are the only approved taxanes in therapeutic use there are many analogs in different phases of in clinical trials which are mostly the semi-synthetic analogs starting from 10-deacetylbaccatin III [14].

10-Deacetylbaccatin-III

3.2. Epothilones Epothilones belong to macrolide class of the drugs and act as microtubule stabilizers. They are produced by Myxobacterium Sorangium cellulosum and initially found to have antifungal and cytotoxic activity [15]. Later, the cytotoxic activity of these epothilones A (R = H) and B (R = Methyl) was found to be associated with mitotic arrest, which occurs via over polymerization of microtubules. Patupilone which is a natural epithilone B derivative is in phase III clinical trials by Novartis for the ovarian cancer. It has been found to be many times more effective than paclitaxel and also crosses the blood-brain barrier [16,17]. Initially another epothilone B derivative, Ixabepilone [18] has shown to be of clinical use however later it was dropped from further development.

3.3. Other compounds Apart from the two major classes of the compounds described above with tubulin polymerization promoter activity, there are some other recently discovered compounds which have been shown to possess tubulin polymerization properties. These include Discodermolide [19], Laulimalide

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[20] and Eleutherobin [21,22]. Discodermolide along with Dictyostatins was isolated from marine sponges. The sponges producing them use the microtubule toxins as part of their self-defense mechanism. Although development of the Discodermolide has been stopped there is a possibility of its derivatives to become a clinical candidate in near future. Eleutherobon was isolated [22] from corals and have similar binding properties as that of paclitaxel. A total synthesis has been developed for this molecule, although it is not yet in clinical trials [23]. Laulimalide which also stabilizes the microtubules has a different binding site on tubulin in contrast to Paclitaxel. It has potential to kill paclitaxel and epothilone resistant cells and a total synthesis for this molecule has also been reported.

HO O

OH OH

H HO

O O

O H

NH2

Discodermolide

O Me

O H

N Me

O O

O Me

OMe O AcO OH

Laulimalide

O Eleutherobin

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4. Tubulin polymerization inhibitors Vinca alkaloids constitute the major class of the compounds that inhibit the polymerization of tubulins. Other important compounds in this category include the Combretastatins, Dolastatins, Noscapine analogs, Hemisterlin and Rhizoxins. The Table 2 gives a summary of the compounds with tubulin destabilizing activity along with their natural origin and chemical nature. Table 2. Vinca-domain binding drugs of diverse origin. Name of the drug

Source

Chemical nature

Plant origin Vinca alkaloids Alkaloids

Vinblastine Vincristine Vinorelbine

Catharanthus roseus (Vinca rosea) and analogs

Vinflunine Vindesine Maytansinoids Maytansine

Maytenus ovatus

Macrolide

Ansamitocins

Nocardia

Macrolide

Marine origin Dolastatin 10 Dolastatin 15 Halichondrin Spongistatin 1

Dolabella auricularia

Pseudo peptide

Halichondira okadai Kadota

Lactone polyether

Hyrtios altum

Macrocyclic lactone

Fungal origin Rhizoxin

Rhizopus chinensis

Macrocyclic lactone

Phomopsin A

Phomopsis leptostomiformis

Peptide

Ustiloxin

Ustilaginoidea virens

Peptide

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4.1. Vinca alkaloids The vinca alkaloids vinblastine and vincristine were the first natural products to enter in the clinical use for cancer chemotherapy. These compounds were isolated by two different research groups in late 1950’s and early 1960’s from Madagscar periwinkle known as Vinca rosea or Catharanthus roseus [24]. One of the groups working on them was interested in finding a substance affecting the blood glucose levels. However, at the same time they also noticed the effect of the extract on the white blood cell counts. This lead to the discovery of its antileukemic activity and finally the isolation and structure elucidation of vincaleukoblastin which was later shortened to vinblastine [25]. OH N H N H O

O O

N H N O O

Vinblastine

H OH O O

The vinca groups of alkaloids binding to the beta subunit of tubulin are constituted by several closely related compounds. These include vincristine, vindesine, vinorelbine and vinflumine, which are the semisynthetic vinca alkaloids. Vinblastine and vincristine are in clinical use as anticancer drugs since last 50 years. They are also used in combination therapy of acute leukemias and lymphomas, bladder and breast cancers [26].

4.2. Combretastatins and derivatives Although the vinca alkaloids are the only tubulin polymerization inhibitor compounds which are in clinical use, there are several other groups of compounds which bind to same domain of the tubulin and have similar mechanism of action. Many of these analogs are in advanced stages of clinical trials e.g. Combretastatins, which were isolated from the root bark of Combretum caffrum [27].

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OH OMe MeO

OMe OMe Combretastatin A4

They are well-known as antimitotic agents and Combretastatin A2 (CA2) & Combretastatin A4 (CA4) are the most potent members of this family. CA4 is highly cytotoxic than its tubulin destabilizing activity [28]. The phosphorylated CA4 known as CA4P has anti-angiogenic properties via the disruption of the endothelial cytoskeleton [29]. This compound is in phase III trials for treatment of cervical, colorectal, NSCLC, prostate and ovarian cancers [30,31].

4.3. Dolastatins Pettit isolated Dolastatin 10 from Dolabella auricularia, the most potent member of a big family of dolastatins [32]. It has a distinct binding site on tubulin where usual antimitotic peptides bind [33]. In 1990 it entered the clinical trials by NCI for solid tumor treatments. Another peptide, Dolastatin 15 is also as potent as Dolastatin 10 but in contrast to the later, it is not involved in the nucleotide exchange inhibition and aggregation induction. One of the Dolastatin 15 derivatives is in phase II clinical trials [34].

H H N H

O N H3C

N H

O H3CO

N O

Dolastatin 10

N H

4.4. Noscapinoids The phthalideisoquinoline alkaloid from Papaver somniferum, Noscapine is in medicinal use since long for its antitussive activity [35]. Currently this molecule is in phase I-II clinical trials for the treatment of multiple myeloma. Noscapine and its derivatives are different from other microtubule binding drugs in the fact that they keep the total polymer mass of the tubulin unaltered [36].

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O OCH3

CH3

H OCH3

O O

OCH3

S,R-(Alpha)-Noscapine

Stoichiometric binding of Noscapine induces a conformational change in tubulin and interrupts the cell cycle in mitosis. It inhibits the dynamic instatbility of tubulins by extending the relaxation time. Several analogs of the parent molecule have been prepared and evaluated against diverse cancer cell lines. It has been concluded that noscapinoids are the gentlest molecules involved in the microtubule dynamics creating mitosis checkpoints without any significant toxicity profile [37].

4.5. Eribulin and halichondrins These complex natural products of marine origin were isolated from western pacific sponge Halichondria okadai and also from Axinell sp [38,39]. This class of molecules have shown to be highly cytotoxic specially the Halichondrin B and homohalichondrin. These compounds were shown to bind to tubulin and inhibit their polymerization. They have shown subnanomolar activity in NCI’s 60 cell anti-cancer screening panel along with promising activity in many animal models [39]. The attempts towards the total synthesis of Halichondrin B resulted in the discovery of Eribulin [40]. Similar to its parent, it also inhibits tubulin polymerization and is currently in phase III clinical trials for several cancer types [41]. OH MeO H3N

H O

O O

Eribulin

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Microtubule binding natural substances in cancer chemotherapy

4.6. Hemiasterlin Hemiasterlin is a tripeptide of marine origin. It was first isolated from Hemiasterella minor and found to be active against murine leukemia cell lines [42]. Later, its antitubulin and antimitotic activity was discovered by Anderson [43]. The phenyl alanine derivative of the parent compound, HTI-286 [44] has been found to be more potent and more synthetically accessible. Both these molecules are in clinical trials [45].

N Me

N H

Me N

O OH

Hemiasterlin

4.7. Rhizoxin Rhizoxin was isolated from a plant pathogenic fungus Rhizopus chinensis and was discovered to be inhibitor of tubulin polymerization [46]. It is a macrocyclic lactone, although very similar to maytansine [47], it is comparatively more potent against human and murine tumor cells. It has been synthesized and gone through the clinical trials. The molecule is yet to be approved for clinical use.

O O

N O

O OMe Rhizoxin

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5. Challenges and future prospects Since last two decades the importance of tubulin dynamics as a target for anticancer drug development has been increased significantly. The established tubulin interactive drugs include the two vinca alkaloids and the taxens, paclitaxel and docetaxel. Epothilones have just been made available clinically. There are many candidates in phase II-III trials as described earlier. It is now well know that almost all tubulin interactive agents are the natural products. The supplies of these compounds for clinical use in near future should be guaranteed. In future apart from the discovery of the new tubulin interactive agents, the development of the novel noscapinoids, taxanes, epothilones and other compounds will continue towards finding the new and improved drug candidates. Next, the targeted drug delivery approach would augment the current situation e.g. the use of nanoparticles for the targeted delivery of Noscapine is under investigation [49]. Design of simpler synthetic compounds taking the clue from molecular modeling studies and better understanding of the tubulin interactions with known molecules would also play a major role in development of new and improved anticancer agents in future [50].

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 283-311 ISBN: 978-81-308-0448-4

9. Natural products: Anti-fungal agents derived from plants Tasleem Arif, T. K. Mandal and Rajesh Dabur National Research Institue of Basic Ayurvedic Sciences Nehru Garden, Kothrud, Pune-411038, India

Abstract. As new spectrums of human fungal infections are increasing due to increased cancer and AIDS patients. The increased use of antifungal agents also resulted in the development of resistance to these drugs. It makes necessary to discover new classes of antifungal compounds to treat fungal infections. The research on natural products and natural products derived compounds has accelerated in recent years due to their importance in drug discovery. Plants are rich source of bioactive secondary metabolites of wide variety such as tannins, terpenoids, alkaloids, and flavonoids, reported to have in vitro antifungal properties. A series of molecules with antifungal activity against different strains of fungus have been found in plants, which are of great importance to humans and plants. These molecules may be used directly or considered as a model for developing better molecules. This review attempts to summarize the current status of reported antifungal compounds from plants.

1. Introduction The prevalence of resistance to antifungal agents significantly increased in the past decade. Resistance to antifungal agents has important implications for morbidity, mortality and health care in the community. Until recently, Correspondence/Reprint request: Dr. Rajesh Dabur, National Research Institue of Basic Ayurvedic Sciences Nehru Garden, Kothrud, Pune, MH-411038, India. E-mail: rajeshdabur@yahoo.com

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fungi were not recognized as important pathogens because the annual death rate due to candidiasis was steady from 1950 to 1970 [1, 2]. Since 1970, this rate increased significantly due to more widespread use of immunosuppressive therapies, indiscriminate use of broad-spectrum antibacterial agents, the common use of indwelling intravenous devices and immunosuppressive viral infections such as AIDS. These developments and the associated increase in fungal infections [3] necessitated the search for new, safer, and more potent agents to combat serious fungal infections. For nearly 30 years, amphotericin B, which causes significant nephrotoxicity, was the sole drug available to treat serious fungal infections. The imidazoles and the triazoles in late 1980s and early 1990s were major advances in safe and effective treatment of local and systemic fungal infections. The high safety profile of triazoles, in particular fluconazole, has led to their extensive use. Fluconazole has been used to treat in excess of 16 million patients, including over 300,000 AIDS patients, in the United States alone since the launch of this drug [4]. Due to selective pressure and widespread use of these few antifungal drugs, there have been increasing reports of antifungal resistance [5]. Medicinal plants have been a source of wide variety of biologically active compounds for many centuries and used extensively as crude material or as pure compounds for treating various disease conditions. Relatively 1-10 % of plants are used by humans out of estimated 250,000 to 500,000 species of plants on Earth [6]. The plants are relatively cheap source of biological material having a vast variety of metabolites, primary or secondary, available in them for selecting the molecule of desired biological activity. Mainstream medicine is increasingly receptive to the use of antimicrobial and other drugs derived from plants, as traditional antibiotics become ineffective. Another driving factor for the renewed interest in plant antimicrobials in the past 20 years is due to the rapid extinction rate of (plant) species [7]. The scientific discipline, ethno botany, is utilizing the impressive array of knowledge assembled by indigenous peoples about the plant and animal products they have used to maintain health [8,9]. Lastly, the ascendancy of the human immunodeficiency virus (HIV) has spurred intensive investigation into the plant derivatives, which may be effective, especially for use in underdeveloped nations. Few of the compounds isolated from plants such as 2-decanone, hydroxydihydrocornin-aglycones [10], various indole derivatives [11] and isoflavanone are reported to have antifungal activities. However, development of useful antifungal drugs from these compounds has not yet been possible.

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2. Major groups of antifungal compounds from plants Plants have an almost limitless ability to synthesize aromatic substances of different functional groups, most of which are phenols or their oxygensubstituted derivatives [12]. Most are secondary metabolites, of which at least 13,000 have been isolated that is less than 10% of the total [13]. In many cases, these substances serve as plant defense mechanisms against predation by microorganisms, insects, and herbivores. Some plants used for their odors (terpenoids), pigment, (quinones and tannins) and flavor (terpenoid capsaicin from chili peppers) were found to be endowed with medicinal properties. Some of the herbs and spices used by humans as season food yield useful medicinal compounds.

2.1. Simple phenols and phenolic acids In recent years, numbers of studies have been reported on the antifungal activity of phenolic compounds from natural sources. Some of the simplest bioactive phytochemicals consist of a single substituted phenolic ring (Fig 1). The common herbs tarragon and thyme both contain caffeic acid, a representative of a wide group of phenylpropane-derived compounds which is effective against fungi [14]. The site(s) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms, with evidence that increased hydroxylation results in increased toxicity [12]. In addition, it was also reported that more highly oxidized phenols are more inhibitory [15, 16]. The mechanisms thought to be responsible for phenolic toxicity to microorganisms include enzyme inhibition by the oxidized compounds, possibly through reaction with sulfhydryl groups or through more nonspecific interactions with the proteins [17]. OH

1 HO

O HO

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Tannins and salicylic acid are polyphenol compounds extracted from Gaullher procumbens, Rhammus purshiand and Anacardum pulsatilla showed antifungal activity [18,19]. Piper crassinervium, Piper aduncum, Piper hostmannianum and Piper gaudichaudianum, contain phenolic acid derivatives crassinervic acid (1), aduncumene, hostmaniane and gaudichaudanic acid, respectively, were reported for fungitoxic activity [20]. Phenolic compound Eriosemaones A–D (2, MIC ¼ 20 mg/mL) are reported to have good antifungal activities [21]. Phenolic compound from Croton hutchinsonianus [22], and pinosylvin (3), a constituent of pine, showed growthinhibitory activity against C. albicans and Saccharomyces cerevisiae [23]. Four phenolic amides, dihydro-N-caffeoyltyramine, trans-Nferuloyloctopamine, trans-N-caffeoyltyramine, and cis-N-caffeoyltyramine isolated from Lycium chinense reported to have anti-fungal activity in a range of 5-10 μg/mL [24a]. Three phenolic compounds, 1-galloyl-β-D-glucopyranosyl-(1->4)-β-D-galactopyranoside, 2-methoxy-5-(1’,2’,3'-trihydroxypropyl)-phenyl-1O-(6"-galloyl)-β-D-glucopyranoside and 2-methoxy-5-hydroxymethyl-phenyl-1O-(6"-galloyl)-β-D-glucopyranoside together with the known compounds from the leaves of Baseonema acuminatum were reported for antifungal activity against Candida albican strains with inhibitory concentration to 50% microorganism (IC50) values in the range of 25-100 μg/mL [24b].

2.2. Flavonoids Flavones are phenolic structures containing one carbonyl group and the addition of a 3-hydroxyl group yields a flavonol (Fig 2). Flavonoids are hydroxylated phenolic substances synthesized by plants in response to microbial infection. They have been found to be effective antimicrobial substances against a wide array of microorganisms. Their activity is probably due to their ability to complex with extracellular and soluble proteins and to complex with fungal cell walls. More lipophilic nature of flavonoids may also disrupt fungal membranes [25]. Flavonoids isolated from the stem bark of Erythrina burtii [26] were reported for antifungal activity. 4-methoxy-5,7-dihydroxyflavone 6-Cglucoside (isocytisoside) from the leaves and stems of Aquilegia vulgaris showed activity against the mould A. niger [27]. Pelalostemumol (4) from Pelalostemium had strong antifungal activity against many pathogenic fungi [28]. Galangin (5), derived from the perennial herb Helichrysum aureonitens, seems to be a particularly useful compound, since it has shown activity against wide range of fungi [29]. A flavonoid from rhizome of Alpinia officinarum had strong antifungal activity against variety of pathogenic fungi. Minimum inhibitory concentration (MIC) against the fungi varied from

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3 μg/mL [30,31]. A flavon 3,4',5,7-tetraacetyl quercetin isolated from heartwood of Adina cordifolia exihibited moderate antifungal activity against A. fumigatus and Cryptococcus neoformans [32]. Flavonoid derivative phloretin from Malus sylvestris have antifungal properties [33]. Isopiscerythrone (6), allolicoisoflavone A (7), piscisoflavones A (8) and B (9) from different plants were reported to be endowed with antifungal activity [34].

HO HO

OH O

HO O

OH 5

O HO

OH OH

4 HO

OH 8

O O

HO O

HO OHO

OH 7

Heartwood extracts of Acasia auriculiformis and Acasia mangium were reported to have antifungal activity due to the compounds 3,4',7,8tetrahydroxyflavanone and tetrracidin [35]. The four compounds eupomatenoid-3, eupomatenoid-5, conocarpan and orientin, from Piper solmsianum exhibited antifungal action against all the dermatophytes tested, with MIC values in range of 2 to 60 μg/mL and with a potency as high as the standard antifungal drug ketoconazole [36]. Flavonoids, azulenes, sesquiterpenes and essential oils from Inula viscosa were proved to have a significant antifungal activity against dermatophytes even at low concentrations (0.01 mg/mL). The high concentration of the sesquiterpene

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(carboxyeudesmadiene), occurring in the leaf extracts, is reported to have greater antifungal activity [37]. The 95% ethanol extract of the bark of Swartzia polyphylla afforded the flavonoids biochanin A and dihydrobiochanin A as antifungal constituent and another plant from the Fabaceae family Teramnus labialis was also reported for antifungal flavonoid [38]. The antifungal activity of a series of prenylated flavonoids purified from five different medicinal plants of moraceae family was reported antifungal against C. albicans and S. cerevisiae [39]. These results also support the use of prenylated flavonoids in traditional medicine to treat fungal infections. A. nobilis furnished several flavonoids which showed good fungicidal activity against C. cladosporioides [40]. Amentoflavone from Selaginella tamariscina exhibited potent antifungal activity against several pathogenic fungal strains but had a very low hemolytic effect on human erythrocytes [41].

2.3. Coumarins Coumarins have been reported to stimulate macrophages which could have an indirect negative effect on infections. Coumarins are phenolic substances made of fused benzene and α-pyrone rings (Fig 3). Their fame has come mainly from their antithrombotic50, anti-inflammatory [42] and vasodilatory [43] activities and their use to prevent recurrences of cold sores caused by HSV-1 in humans 48. Hydroxycoumarin scopoletin (10) was isolated from seed kernels of Melia azedarach [44] reported to be antifungal against Fusarium verticillioides.

O HO

O O

O OH

10 O

HOOC

O OCH3

H3CO O

N O 13

H 14

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Tithoniamarin is a new isocoumarin dimer isolated from Tithonia diversifolia [45] showed antifungal and herbicidal activities. Deng and Nicholson [46] reported the antifungal properties of surangin B (11), a coumarin from Mammea longifolia. Phytoalexins, which are hydroxylated derivatives of coumarins, are produced in carrots in response to fungal infection and can be presumed to have antifungal activity [47]. A coumarin namely, 6,7-dimethoxycoumarin (12), isolated from P. digitatum-infected Valencia fruit confers resistance against the mycotoxigenic fungi A. parasiticus [48]. Clausenidin (13), dentatin, nor-dentatin, and carbazole alkaloid clauszoline J (14) isolated from Clausena excavata showed antimycotic activity (MIC 50 μg/mL). Methylated clausenidin (MIC 50 μg/mL), a synthetic coumarin, also exhibited moderate antimycotic activity [50]. Data about specific antibiotic properties of coumarins are scarce, although many reports give reason to believe that some utility may reside in these phytochemicals [51].

2.4. Quinones Quinones are aromatic rings with two ketone substitutions and characteristically highly reactive. Fig 4 shows some of the important antifungal quinones. They can switch between diphenol (or hydroquinone) and diketone (or quinone) easily through oxidation and reduction reactions. These compounds, being colored, are responsible for the browning reaction in cut or injured fruits and vegetables [52]. In addition to providing a source of stable free radicals, quinones are known to complex irreversibly with nucleophilic amino acids in proteins [53]. Therefore the quinone inactivate the protein and impair there function. Quinones bind with surface-exposed adhesins, cell wall polypeptides, membrane-bound enzymes and form complex which inactivate the enzymes. In the anthraquinone group, there are only a few reports concerning their antifungal activity. Schmidt et al. [54] reported the antifungal activity of the major anthraquinone aglycones, alizarin (15) and emodin (16) of Rubia tinctorum and Rhamnus frangula. Hypericin (17), from Hypericum perforatum, known as an antidepressant and Duke reported in 1985 that it had general antimicrobial properties [14]. Examples of other antifungal anthraquinones from medicinal species also included a new 1,3-dihydroxy-2methyl-5,6-dimethoxyanthraquinone (18) from the roots of Prismatomeris fragrans [55]. The naphthoquinones kigelinone (19), isopinnatal, dehydroalpha-lapachone, and lapachol from Kigelia pinnata were reported for antifungal activity [56].

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CH3 O

16 O

CH3

O OH

OH O

HO O

A novel compound 11-hydroxy-16-hentriacontanone isolated from Annona squamosa was reported for its antifungal potential [57]. Hypericum an anthraquinone extracted from Hypericum perforatum showed antifungal activity [58]. The 2-hydroxy-1,4-naphthoquinone (Lawsone) (20) from Lawsonia inermis were found to exhibit strong fungitoxicity [59]. Emodin, physcion and rheins were isolated from Cassia tora showed strong fungicidal activity against the microorganism tested [60]. Hopeanolin MIC value range 0.1-22.5 μg/mL, an unusual resveratral trimer with an O-quinone nucleus, from the stem bark of Hopea exalata is reported to have antifungal activity [61].

2.5. Saponins Saponins are secondary metabolites that occur in wide range of plant species (Fig 5). They are stored in plant cells as inactive precursors but are readily converted into biological active antibiotics by enzymes in response to pathogen attack. Saponins are glycosylated compounds widely distributed in plant kindom and can be divided into three major groups, a triterpenoid, a steroid or a steroidal glycoalkaloid. CAY-1, a triterpene saponin from the Capsicum frutescens was found to be active against sixteen different fungal strains, including Candida spp, A. fumigatus and C. neoformans [62]. Importantly, CAY-1 appears to act by disrupting the membrane integrity of fungal cells. Recently, steroidal saponins ypsilandroside B, ypsilandroside A, iso ypsilandroside A, iso ypsilandroside B and isoypsilandrogaine isolated from Ypsilandra thebetica were reported for antimicrobial activities by [63]. Two new spirostanol saponins were isolated from the roots of Smilax medica,

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together with the known smilagenin 3-O-β-D-glucopyranoside (21) exhibited antifungal activity against the human pathogenic yeasts C. albicans, C. glabrata and C. tropicalis in a range of 6.25-50 μg/mL [64]. Mollugo pentaphylla, a tropical herb, contains an antifungal saponin, mollugogenol-A (22) [65]. Phytolaccosides B (23) and E (24) from Phytolacca tetramera showed antifungal activities against a panel of human pathogenic opportunistic fungi [66]. Novel spirostanol saponins together with three known saponins were reported for antimycotic activity. The most active compound was found to be 6α-O-[β-D-xylopyranosyl-(1→3)-β-Dquinovopyranosyl]-(25,S)-5α-spirostan-3β-ol, with IC50 values of 25 μg/mL against T. mentagrophytes and T. rubrum [67]. From Solanum species, Solanum chrysotrichum five new spirostan saponins showed antimycotic activity against T. mentagrophytes, T. rubrum, A. niger and C. albicans. Another compound isolated from same plant, 6-α-O-β-D-xylopyranosyl(1-->3)-β-D-quinovopyranosyl-(25R)-5α-spirostan-3β,23α-ol was reported to be active in a rage of 12.5 to 200 μg/mL against T. mentagrophytes, T. rubrum, A. niger and C. albicans [68]. Recently, saponins isolated from Alternanthera tenella is reported to have strong antifungal in the range varied from (MIC) 50-500 μg/mL [69]. Bioassay-guided fractionation of the ethanol extracts of the aerial parts of the Tibetan medicinal herb Clematides tangutica led to the isolation of two new antifungal triterpene saponins 3-O-α-L-arabinopyranosyl hederagenin 28- O-α- L-rhamnopyranosyl ester and 3-O-β-D-glucopyranosyl-(1-->4)-αL-arabinopyranosyl hederagenin 28-O-α-L-rhamnopyranosyl ester (MIA 2.5 μg/disc) [70]. Two dammarane saponins from the stems of Anomospermum grandifolium jujubogenin 3-O-α-l-arabinofuranosyl(1-->2)[β-D-glucopyranosyl(1-->6)β-D-glucopyranosyl(1-->3)]-α-l-arabinopyranoside, ujubogenin 3-O-α-l-arabinofuranosyl(1-->2)-[6-O-[3-hydroxy-3-methylglutaryl]β-D-glucopyranosyl(1-->3)]-α-l-arabinopyranoside and a new lupane saponin, 3β-hydroxylup-20(29)-en-27,28-dioic acid 28-O-β-D-glucopyranosyl(1-->2)-[βD-xylopyranosyl(1-->3)]-β-D-xylopyranosyl(1-->2)-β-D-glucopyranoside ester, jujubogenin 3-O-α-l-arabinofuranosyl(1-->2)-[β-D-glucopyranosyl(1-->3)]-α-larabinopyranoside and 3β-hydroxylup-20(29)-ene-27,28-dioic acid revealed antifungal properties against C. albicans [71]. From the rhizomes of Dioscorea cayenensis, the dioscin (25) exhibited antifungal activity against the human pathogenic yeasts C. albicans, C. glabrata and C. tropicalis [72]. Three antifungal steroidal saponins were isolated from the root of Smilax medica [73, 74]. Saponins, named minutoside A, minutoside B, minutoside C, sapogenins, alliogenin and neoagigenin, were isolated from the bulbs of Allium minutiflorum showed promised antifungal activity [75].

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H O

OH HO HO

OH H O

OH OH 22

21 HO HO

O HO H HO

HO HO

23 O

O HO

HO O

OH HO

O 24

Nineteen saponins from Medicago sativa, M. murex, M. arabica and M. hybrida, were were reported to be active against three dermatophytic fungi Microsporum gypseum, T. interdigitale and T. tonsurans [76]. OH HO

O HO

HO O

O OH

O O

O OH O

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Two saponins from Tribulus terrestris were reported for promised antifungal activity against fluconazole resistant Candida strains (MIC 0.15 mg/mL) [77,78]. Tigogenin-3-O-β-D-xylopyranosyl (1-->2)-[β-Dxylopyranosyl (1-->3)]-β-D-glucopyranosyl (1-->4)-[α-L-rhamnopyranosyl (1-->2)]-β-D-galactopyranoside was reported to have in vivo activity in C. albicans vaginal infection model. Another saponin from same plant, tigogenin-3-O-β-D-glucopyranosyl (1-->2)-[β-D-xylopyranosyl (1-->3)]-βD-glucopyranosyl (1-->4)-β-D-galactopyranoside was found to be in vitro very effective against several pathogenic Candida species (MIC80 = 4.4, 9.4 μg/mL), C. neoformans (MIC80 =10.7, 18.7 μg/mL) and inherently resistant C. krusei (MIC80 = 8.8, 18.4 μg/mL [79]. The Avenacin obtained from the Avena sativa showed varying degree of in vitro antifungal activity [80]. Other antifungal saponins from medicinal plants also included Astragalus verrucosus [81], A. suberi [82], A. auriculiformis [83] and Hedera taurica which possessed in vitro antifungal activity against C. albicans, C. krusei and C. tropicalis [84]. Saponins from several plants i.e Hedera colchica [85], Kalopanax pictus [86], Dracaena mannii and D. arborea [87], Trillium grandiflorum [88] and Solidago virgaurea [89] were reported for antifungal activity.

2.6. Xanthones Xanthones are a restricted group of plant polyphenols, biosynthetically related to the flavonoids. These are planar-six carbon molecules in a conjugated ring system consisting of a backbone molecule and various chemical groups attached to it. Xanthone backbone consists of two benzene rings attached through a carbonyl group and oxygen not allowing free rotation about the carbonZcarbon bonds. The unique backbone along with type and position of the attached chemical groups defines specific properties of xanthones. Xanthones possess numerous bioactive capability including antifungal properties (Figure 6). Caledonixanthone E (26) isolated from the stem bark of Calophyllum caledonicum was reported for strong antifungal activity (MIC80 ¼ 8 mg/mL) [90]. Isoprenylated xanthones, toxyloxanthone C (27), and wighteone (28) showed antifungal activity against C. albicans with MIC values of 25 and 12.5 mg/mL, respectively [91]. The dichloromethane extract of Securidaca longepedunculata yielded 1,7-dihydroxy- 4-methoxyxanthone (29) which exhibited antibacterial activity against Staphylococcus aureus and antifungal activity against A. niger, A. fumigatus, and a Penicillum species [92]. 1,3,6Trihydroxy-2,5-dimethoxyxanthone (30) isolated from the aerial part of Monnina obtusifolia was reported to have antifungal potential [93]. Seven xanthanolides from Xanthium macrocarpum were reported to be effective

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OH O

OH 27

26 HO

28 HO

O O HO

O HO

OH O

against C. albicans, C. glabrata, and A. fumigatus [94,95]. Two new 2-hydroxy-3-methylbut-3-enyl-substituted xanthones, -caledol and -dicaledol, were isolated from a dichloromethane extract of the leaves of Calophyllum caledonicum and have been reported for antifungal activity against A. fumigatus [96]. Xanthones from the green fruits of Garcinia mangostana were reported to have strong antifungal activities [97]. Cudrania fruticosa yielded an isoprenylated xanthone, cudrafrutixanthone which showed antifungal activity against C. albicans [98, 98]. Xanthone analogues bearing the basic chain of butenafine were reported for significant activity against C. neoformans (1.5 mg/mL) [100].

2.7. Terpenoids and essential oils A large number of studies have been done in recent years on the antifungal activity of terpenoids of natural origin (Fig 7). These reports concern mainly sesquiterpenes and sesquiterpene lactones. The fragrance of plants is carried in essential oil fraction. These oils are secondary metabolites that are highly enriched in compounds based on an isoprene structure. They are called terpenes, their general chemical structure is C10H16, and they occur

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as diterpenes, triterpenes, and tetraterpenes (C20, C30, and C40), as well as hemiterpenes (C5) and sesquiterpenes (C15). The mechanism of action of terpenes is not fully understood but is speculated to involve membrane disruption by the lipophilic nature. Mendoza et al. [101] found that increasing the hydrophilicity of kaurene diterpenoids by addition of a methyl group drastically reduced their antimicrobial activity. A number of terpenes or terpenoids are reported active against fungi [102-104]. In 1977, it was reported that 60% of essential oil derivatives examined to date were inhibitory to fungi while 30% inhibited bacteria [105]. The antifungal activities of the essential oil from Agastache rugosa and its main component, estragole (31), combined with ketoconazole, were reported to show significant synergistic effects [106]. A known sesquiterpene lactone, encelin (32), isolated from the Mexican species Montanoa speciosa has a determining action on growth and the morphogenetic process of fungal cells [107]. The roots of Delphinium denudatum have yielded 8-acetylheterophyllisine, panicutine, and 3-hydroxy-2-methyl-4H-pyran-4one (33) has shown antifungal activity against a number of human pathogenic fungi [108]. Khaya ivorensis afforded methyl angolensate and 1,3,7trideacetylkhivorin displayed antifungal activity, with 62.8 and 64% mycelial growth inhibition at 1000 mg/L, respectively [109]. Estragole and the essential oil of A. rugosa exhibited strong activities against the tested fungi and showed synergism with ketoconazole against B. capitatus [110]. From other Centaurea species, C. thessala and C. attica, two eudesmanolides, 4-epi-sonchucarpolide, 8-(3-hydroxy-4-acetoxy-2-methylene-butanoyloxy) derivative and eudesmane derivative named atticin (35) showed a considerable antifungal effect against nine fungal species [111]. Two new dammarane type triterepenes, ailexcelone and ailexcelol from Ailantus excelsa were reported to be endowed with antifungal activities [112]. Amesterol, isolated from Amaranthus viridis strongly inhibited a growth of pathogenic fungus [113,]. Two diterpenes isolated by Batista et al. [114] were found to be active against Candida spp. Terpenoid isolated from Atrus sinenisis, Armoracia rusticana, Metha peperita [115] and grapefruit showed antifungal activity [116]. A. sativum oil exhibited the strongest inhibition of growth of T. rubrum, T. erinacei, and T. soudanense with MIC of 4.0 μg/mL, while the activities of A. cepa and A. fistulosum were relatively mild [117]. The bark extract of Drimys brasiliensis led to the isolation of the sesquiterpene polygodial, 1-β-(p-methoxycinnamoyl)-polygodial (36), drimanial and 1-β-(p-cumaroyloxy)-polygodial which were selectively active against fungi [118]. An antimicrobial diterpene 8β−17-epoxylabd-12-ene-15, 16-dial from Alpinia galanga synergistically enhanced the antifungal activity

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of quercetin and chalcone against C. albicans [119]. Triterpenoid glycosides obtained from Solidago virgaurea and Bellis perennis inhibit the growth of human-pathogenic yeasts (Candida and Cryptococcus species) [120].

O O O

O HO O 31

O 33

32 O O

O O O

O 34

O O

OH H

OH HO

The oil from leaves of J. oxycedrus spp. oxycedrus was reported antifungal with MIC and MLC values ranging from 0.08-0.16 μM/L and 0.08-0.32 μM/L, respectively. The chemical constituents of the essential oil extracted from the fruits of Lindera glauca have epishyobunol acetate, caryophyllene oxide and 3,6,6-trimethyl-2-norpinene exhibited more manifest antifungal properties with MIC between 0.03-0.5 mL/L for pathogenic fungi species [121]. The essential oil from the leaves of Litsea cubeba have α−cis-ocimene,3,7-dimethyl-1,6-octadien-3-ol and ntransnerolidol had manifest antifungal activities with MIC between 0.03-0.4 μL/mL for utilized pathogenic fungi and 1.0-2.0 μL/mL for moulds [122,]. The oil of the leaves, clemateol, and the alcohol from Calea clematidea showed a moderate antifungal activity [123]. Daucus carota (Apiaceae), afforded four sesquiterpene daucane esters [124] found to contain a range of low antifungal activity against Fusarium oxysporum and A. niger. Carotol, which was observed to be the main constituent of carrot seed, inhibited the radial growth of fungi by 65%. The Vernonanthura tweedieana afforded one

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antifungal active sesquiterpene, 6-cinnamoyloxy-1-hydroxyeudesm-4-en-3one [125]. Barrero et al. [126] investigated six Centaurea species: C. bombycina, C. granatensis, C. monticola, C. incana, C. maroccana and C. sulphurea of Astraceae family. The sesquiterpene lactones costunolide and dehydrocostunolide showed noticeable IC50 values. Other antifungal sesquiterpene lactones from the Asteraceae family also included those isolated from Ajania fruticulosa [127]. A fruit pulp extract of Detarium microcarpum endowed with four new clerodane diterpenes which showed antifungal activity [128]. The diterpenoids 16α-hydroxy-cleroda-3,13-(14)-Z-diene-15,16-olide and 16oxo-cleroda-3,13-(14)-E-diene-15-oic acid isolated from the hexane extract of the seeds of Polyalthia longifolia demonstrated significant antifungal activity [129]. Five new diterpenoids from Casimirella namely, humirianthone, 1-hydroxy-humirianthone, 15R-humirianthol, patagonol and patagonal showed activity against pathogenic fungi [130]. Antifungal activity of oxygenated pimarane diterpenes from Kaempferia marginata was reported by Thongnest et al. [131]. The triterpenoids pristimerin and celastrol isolated from the roots of Celastrus hypoleucus exhibited inhibitory effects against diverse pathogenic fungi [132]. Oleanane triterpenoid, triterpenetetrol isolated from the chloroform extract of the aerial parts of Leontodon filii were reported to have antifungal prop [133]. Carvone, dinydrocarvone, limonene, dillapiole and dillapional from Anethum sowa revealed antifungal activity at a concentration of 1:100 and 1:250 [134,135,136]. A derivative of dillapiole, isodilapiole tribromide found to more active [137]. The steam distillate of fresh mature leaves containing odorous oil rich in cyclic tri-and tetra-sulphides of C3, C5 and C9 units exihibited antifungal activity at 125 μg/mL in vitro [139]. The active compounds, 1'-acetoxychavicol acetate from Alpinia galanga strong inhibitory affects at a MIC 0.024 μg/mL against several fungal pathogens [140 a,b]. Most of the species of Oscimum showed in vitro antifungal activities against a broad range of fungi as well as bacteria. An Indian chemotype Ocimum gratissimum, with a high level of ethyl cinnamate, presents, in vitro, an interesting spectrum of antifungal properties [141].

2.8. Alkaloids Heterocyclic nitrogen compounds are called alkaloids (Fig 8 A-B). The first medically useful example of an alkaloid was morphine; isolated in 1805 from the opium poppy Papaver somniferum [142,], Codeine and heroin are both derivatives of morphine. Diterpenoid alkaloids, commonly isolated

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from the plants of the Ranunculaceae [143, 144] are found to have antimicrobial properties [145]. While alkaloids have been found to have microbiocidal effects including against Giardia and Entamoeba species [146], the major antidiarrheal effect is probably due to their effects on transit time in the small intestine. Recently, a novel alkaloid, 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)1-methylethyl pentanoate (38) was isolated from the plant Datura metel showed in vitro as well as in vivo activity against Aspergillus and Candida species [147]. Another novel alkaloid, 6,8-didec-(1Z)-enyl-5,7-dimethyl-2,3dihydro-1H-indolizinium (37) from Aniba panurensis demonstrated the activity against a drug-resistant strain of C. albicans [148]. The antifungal alkaloids β-carboline, a tryptamine- and two phenylethylamine-derived alkaloids and N-methyl-N-formyl-4-hydroxy-beta-phenylethylamine (39) from Cyathobasis fruticulosa [149] and haloxylines A and B, new piperidine from Haloxylon salicornium displayed antifungal potentials [150]. Jatrorrhizine (40) from Mahonia aquifolium was found to be the most effective against all fungal species tested (MIC ranges from 62.5 to 125 μg/mL), while the crude extract, berberine , and palmatine exhibited only marginal activity (MIC 500 to >/= 1000 μg/mL) [151]. Cocsoline (43), a bisbenzylisoquinoline alkaloid from the Epinetrum villosum displayed antifungal activities [152]. The alkaloids N-methylhydrasteine hydroxylactam and 1-methoxyberberine chloride from Corydalis longipes showed high efficacy individually [153]. Four alkaloids, dicentrine (41), glaucine (42), protopine, and alpha-allocryptopin (46) from Glaucium oxylobum exhibited good activity against Microsporum gypseum, Microsporum canis, T. mentagrophytes and Epidermophyton floccosum [154].

C8H17

O N

O C8H17

OH 39

O O

OH N

O O

O 40

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Flindersine (45) and haplopine from Haplophyllum sieversii were growth-inhibitory compounds against various fungi [155]. Canthin-6-one and 5-methoxy-canthin-6-one of Zanthoxylum chiloperone var. angustifolium exhibited antifungal activity against C. albicans, A. fumigatus and T. mentagrophytes [156]. Frangulanine, a cyclic peptide alkaloid and waltherione A, a quinolinone alkaloid from leaves of Melochia odorata were reported to exhibit antifungal activities against a broad spectrum of pathogenic fungi [157]. The indole alkaloid venenatine exhibited antifungal activity against all the 10 tested fungi, showed an especially high sensitivity towards this compound, exhibiting germination levels below 10% [158]. From the root bark of Dictamnus dasycarpus two antifungal furoquinoline alkaloids were isolated. 3-methoxysampangine from Cleistopholis patens exhibited significant antifungal activity against C. albicans, A. fumigatus, and C. neoformans [159]. O

NH O O O

O HO

44 O

N O

O N H 45

2.9. Lectins and polypeptides Peptides, which are inhibitory to microorganisms, were first reported in 1942 [160]. They are often positively charged and contain disulfide bonds [161]. Their mechanism of action may be the formation of ion channels in the microbial membrane [162] or competitive inhibition of adhesion of microbial

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proteins to host polysaccharide receptors [163]. Recent interest has been focused mostly on studying fungi by these macromolecules, such as that from the herbaceous Amaranthus, has long been known [164]. Thionins are peptides commonly found in barley and wheat and consist of 47 amino acid residues. Thionins AX1 and AX2 from sugar beet were active against fungi but not bacteria [165, 166]. Raw Allium Sativum extract showed antifungal activity beyond 48h in extract stored at room temperature. Antifungal activity was stable upto 8h under these conditions [167]. A novel lectin was isolated from the roots of Astragalus mongholicus [168] and a protein with a novel N-terminal sequence from ginger rhizomes exerted antifungal activity towards various fungi [169]. An antifungal peptide was reported from fresh fruiting bodies of the mushroom Agrocybe cylindracea [170]. Two antifungal peptides from seeds of Pharbitis nil exhibited potent antifungal activity against both chitincontaining and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/mL and from 0.6 to 75 μg/mL [171]. From Phaseolus species, mung bean (Phaseolus mungo) seed, chitinase with antifungal activity was isolated [172]. This species also yielded a novel lysozyme exhibiting antifungal activity toward Botrytis cinerea [173]. Another Fabaceae species, Trigonella foenum-graecum, yielded defensins, small cysteine rich peptides, which exhibited antifungal activity against the broad host range fungi [174]. A novel antifungal peptide, cucurmoschin, was isolated from the seeds of the black pumpkin inhibited mycelial growth in the fungi [175]. A peptide designated cicerarin from seeds of the green chickpea Cicer arietinum showed antifungal activity. The antifungal activity was preserved after exposure to 100 degrees C for 15 min [176]. Two other antifungal peptides cicerin and arietin were reported from seeds of the chickpea (Cicer arietinum). Arietin manifested a higher antifungal potency toward Mycosphaerella arachidicola, Fusarium oxysporum and Botrytis cinerea [177]. An antifungal peptide designated angularin was isolated from the adzuki bean exhibited antifungal activity against a variety of fungal species [178]. Two novel antifungal peptides, designated alpha- and betabasrubrins, respectively, were isolated from seeds of the Ceylon spinach Basella rubra [179]. An antifungal protein, AFP-J, was purified from potato tubers, Solanum tuberosum strongly inhibited yeast fungal strains, including C. albicans, Trichosporon beigelii and S. cerevisiae [180]. Pineapple leaf chitinase-B from Ananas comosus exhibits strong antifungal activity toward Trichoderma virida [181]. Another chitinase with antifungal activity was also purified from the bulbs of the plant Urginea indica, known as Indian squill.

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The protein was an active growth inhibitor of the fungal pathogens in an in vitro assay [182]. A novel protein was isolated from the Chinese herb Astragalus mongholicus Bunge. It exerted selective antifungal activity against various fungi [183]. Recently, a new protein from E. coli, having anti-Aspergillus activity was reported by Yadav et al. [184]. Antifungal peptides and proteins from medicinal species also included two chitin-binding proteins from spindle tree Evonymus europaeus [185], a thaumatin-like protein from banana Musa acuminate [186], and a protein from ginger rhizomes Zingiber officinalis (Zingiberaceae), which exerted antifungal activity toward various fungi [187].

2.10. Other compounds Many phytochemicals not mentioned above have been found to exert antifungal properties. This review has attempted to focus on reports of chemicals, which are found in multiple instances to be active. There are reports of chemical having antifungal properties associated with several different classes not covered above (Figure 9). OH O HO

O HO

N H

O 49

O O

O 52

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N-trans-feruloyl-4-methyldopamine (47) recently isolated from Achranthes ferruginea was reported to be active against a broad range of fungi [188]. Leaves of Piper aduncum accumulate the anti-fungal chromenes, methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate [189]. Iridodial β-monoenol acetate, from Nepeta leucophylla, and actinidine from N. clarkei were found to be endowed with antifungal activities [190]. Anofinic acid and fomannoxin acid (48) from Gentiana algida were found to be active against fungi [191]. The antifungal activity of Artemisia herba-alba was found to be associated with two major volatile compounds as carvone and piperitone [192]. The phenylpropanoids p-coumaric acid and ferulic acid from Kigelia pinnata were observed to have antifungal activity [193]. Piper cubeba afforded the compounds (8R,8'R,9'S)-5methoxyclusin, (-)-clusin, (-)-yatein, ethoxyclusin, and (-)-dihydroclusin showed very potent and selective inhibitory activity against CYP3A4 with IC50 values (0.44-1.0 μM) identical to that of the positive control, ketoconazole (IC50, 0.72 μM) [194]. Demethoxyageratochromene (49) from Ageratum conyzoides showed antifungal activity [195,196] at a concentration of 2000 ppm. The leaf extract was found to be active and showed strong toxicity against the fungi causing ringworm [197]. 2’,4’-Dihydroxy-3’-methoxychalcone (50) and 2’,4’-dihydroxychalcone (51) in the dichloromethane extracts of Zuccagnia punetata was found to have antifungal properties [198]. Plumbagin from Plumbago zeylanica was also reported to inhibit the fungel species viz. Trichophyton, Epidermophyton, Microsporum in a range of 30-40 μg/mL [199]. Hexane extracts of the stem bark as well as (-)-kaur-16-en-19-oic acid of Annona glabra revealed antifungal activity [200]. Isoalantolactone (52) and alantolactone (53), the major constituents of the roots of Inula racemosa showed in vitro antifungal activity againest T. mentagrophytes and Microsporum canis [201]. Withafarin A and amemonine isolated from Withania somniferm and Anemone pulsatillea, salicin a phenolic glucoside compound from Salix alba showed antifungal activity [202]. The psoralen, 8-methoxypsoralen and imperatorin in extracts of leaf, fruit, stem, bark and root of Zanthoxylum americanum demonstrated a broad spectrum of antifungal activity and inhibited fungal species in a disk diffusion assay [203]. Myrothecium roridum contains macrocyclic trichothecenes of the verrucarin type, 16-hydroxyverrucarin A and verrucarin X exhibited moderate antifungal activity. Both compounds were reported to preferentially inhibit in vivo protein biosynthesis [204].

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3. Conclusion Phytomedicines are major component of traditional system of healing in developing countries, which have been an integral part of their history and culture. Besides wide spread use of botanicals as medicinal products in developing countries, such products are becoming part of the integrative healthcare system of industrialized nations, known as complementary and alternative system of medicines (CAM). Existing costly therapy is not affordable well for the millions of individuals particularly in the developing world. Plant extracts are the cheap and easily available source to poor people. Plants are great source of thousands new useful phytochemicals of great diversity, which have inhibitory effects on all types of microorganisms in vitro. Till date more than 600 plants have been reported for their antifungal properties, however a few of them were explored for the active components. The current pharmaceutical armoury of antifungal is a clear cause for satisfaction, not from gloom. However, we still do not have agents that fulfil every one of the criteria that a physician would set as desiterata for antifungal drugs. They need to be active against those fungai causing infections which we can’t yet depend on eradacting. They need to be formaluted for both oral and parenteral administration, they need to be extremly safe and as cheep as possible. The search for new antifungal agents therefore must go on. Identification of new chemotypes for drug development remains an urgent need in antifungal therapeutics. Simultaneously, a number of antifungal compounds reported till date, are tested for their in vitro activities not for in vivo acitivities. In vivo and in vitro activities of a compound may be different and a very small number of plants extracts or components have been studied for their in vivo activity. Therefore these should be subjected to animal and human studies to determine their effectiveness in whole-organism systems. Also in vitro testing and method of extraction should be standardized so that the search could be more systematic. The current set of clinically available antifungal agents includes three classes of natural product and four classes of synthetic chemicals. We therefor can’t abandon intrest in biodiversity as a source of natural antifungal products. Furthermore, the inactive plant extracts may be subjected to chemical diversification of their components to increase the activity. The transformation of chemical groups in natural products into rare chemical groups is possible which are rarely produced by secondary metabolism. Therefore, biosynthesis machinery can be complemented to produce a whole range of new semisynthetic compounds in one step which may become an alternative source of compounds to feed the discovery process for new intresting compounds.

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The study of alternative mechanisms of infection prevention and treatment is essential. Plant products furthermore may be structurally modified to increase their in vivo activity. For example isodilapiole tribromide, a derivative of dillapiole was found to more active. Another example include echnicandin type peptide FR901379, chemical modification of which lead toward more active FK463 compound. Therefore, attention toward the plant derived principles, their chemical modification and chemotherapeuitic potential is needed.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 313-334 ISBN: 978-81-308-0448-4

10. Sesquiterpene lactones: Structural diversity and their biological activities Devdutt Chaturvedi Natural Products Chemistry Division, North-East Institute of Science and Technology (CSIR) Jorhat-785006, Assam, India

Abstract. Sesquiterpenes lactones (SLs) have been isolated from numerous genera of the family Asteraceae (compositae) and can also be found in other angiosperm families. They are described as the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases. They are known to possess wide variety of biological and pharmacological activities such as antimicrobial, cytotoxic, antiinflammatory, antiviral, antibacterial, antifungal activities, effects on the central nervous and cardiovascular systems as well as allergenic potency. Their wide structural diversity and potential biological activities have made further interest among the chemists. The present chapter will be highlighted on the recent developments on the SLs and their diverse biological activities.

1. Introduction Sesquiterpene lactones (SLs) constitute a large and diverse group of biologically active plant chemicals that have been identified in several plant families such as Acanthaceae, Anacardiaceae, Apiaceae, Euphorbiaceae, Lauraceae, Magnoliaceae, Menispermaceae, Rutaceae, Winteraceae and Hepatideae etc [1]. However, the greatest numbers are found in the Compositae Correspondence/Reprint request: Dr. Devdutt Chaturvedi, Natural Products Chemistry Division, North-East Institute of Science and Technology (CSIR), Jorhat-785006, Assam, India E-mail: ddchaturvedi@rrljorhat.res.in

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(Asteraceae) family with over 3000 reported different structures [2]. Sesquiterpene lactones are a class of naturally occurring plant terpenoids that represent a diverse and unique class of natural products and are important constituent of essential oils, which are formed from head-to-tail condensation of three isoprene units and subsequent cyclization and oxidative transformation to produce a cis or trans-fused lactone. These secondary compounds are primarily classified on the basis of their carbocyclic skeletons into pseudoguainolides, guaianalides, germanocranolides, eudesmanolides, heliangolides and hyptocretenolides etc (Figure 1). The suffix "olide" refers to the lactone function and is based on costunolide, a germanacranoride which is related to the ten-membered carbocyclic sesquiterpene, germacrone. However, SLs exhibit variety of other skeletal arrangements. An individual plant species generally produces one skeletal type of SLs concentrated primarily in the leaves and flower heads. The percentage of SLs per dry weight may vary from 0.01% to 8%. Losses of livestock intoxicated by plants containing SLs are well known. In fact, they have been shown to exhibit a wide range of biological activities.

O O

Guaianolides

Pseudoguaianolides

O O

O O O

Germacronolides

O O

O Heliangolides Eudesmanolides

O Hypocretenolides

Figure 1. Basic skeletons of sesquiterpenes lactones.

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An important usual feature of the SLs is the presence of a γ-lactone ring (closed towards either C-6 or C-8) containing in many cases, an α-methylene group. Among other modifications, the incorporation of hydroxyls or esterified hydroxyls and epoxide ring are common. A few SLs occur in glycoside form and some contain halogen or sulfur atoms [3]. Majority of SLs have shown cytotoxic activity (KB and P388 leukemia in vitro) and activity against in vivo P388 leukemia. Structure activity relationship studies showed that various cytotoxic SLs react with thiols, such as cystiene residues in the protien, by rapid Michael type of addition. These reactions are mediated chemically by α,β-unsaturated carbonyl system present in the SLs. These studies support the view that SLs inhibit tumor growth by selective alkylation of growth regulatory biological macromolecules such as key enzymes, which controls cell division, thereby inhibiting a variety of cellular functions, which directs the cell into apoptosis. Differences in activity between individual SLs may be explained by different number of alkylating structural elements. However, other factors, such as lipophilicity, molecular geometry, and chemical environment or the target sulfhydryl may also influence the activity of sesquitepenes lactones. OH O

O OH

O O

HO O

Costunolide (1)

Tagitinin C (3)

Tagitinin A (2)

OH O

HO CH2OH

O O

CH2OH O

O O

O Cynaropicrin (4)

O Parthenin (6)

Eupatoriopicrin (5)

H O O O

O O O H

O O

CH2

O O

O OH Helenalin (7)

O Artemisinin (8)

Vernodalin (9)

Figure 2. Structurally diverse sesquiterpenes lactones (SLs 1-9).

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Some of structurally diverse sesquiterpenes lactones have been shown in Figure 2 and 3. Distribution of different structural classes of sesquiterpene lactones have been depicted in Table 1.

2. Biological activity of sesquiterpene lactones [A] Anticancer activity In recent years, many researchers over the world have reported that sesquiterpenes lactones possess potential anticancer activity. Some of the important compounds of this class have been discussed below: O H O O

H O O

HO O

Parthenolide (10)

Dehydrocostus lactone (11)

Vernolide (12)

Hanphyllin (13) O

O O

O Epoxy(4,5α)- 4,5-dihydrosantonin (15)

Alantolactone (14) O

OCOCH3 OH O

OCOCH3 OiVal

O O

HO O

OH OAc

H HO

O O

11,13-dihydrovernodalin (19)

Vernodal (18)

OH CH2

OH MeO

O Tagitinin C (3)

O Neurolenin B (17)

CH2

O O

O 4,(5) α− Epoxy-4,5-dihydrosantonin (16)

Rupicolin A 8-Acetate (20)

Ridentin (21)

Figure 3. Structurally diverse sesquiterpenes lactones (10-21).

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Biologically active sesquiterpene lactones

Table 1. Distribution of different structural classes of sesquiterpene lactones in the family-Compositae. Tribes (No. of genera)

No. of genera with sesquiterpene lactones

Eupatorieae (50)

Vernonieae (50)

Astereceae (100)

Inuleae (100)

Heliantheae (250)

Type of lactones present Germacranolides Elemanolides Guaianolides Ambrosanolides Seco-Ambrosanolides Germacranolides Elemanolides Guaianolides Germacranolides Guaianolides Elemanolides Guaianolides Xanthanolides Ambrosanolides Helenanolides Seco-Eudesmanolides Seco-Ambrosanolides Germacranolides Elemanolides Guaianolides Eudesmanolides Xanthanolides Ambrosanolides Helenanolides Seco-Eudesmanolides Seco-Ambrosanolides Seco-Helenanolides

Germacranolides Xanthanolides Eremophilanolides Helenanolides Bakkenolides

Anthemideae (50)

Germacranolides Elemanolides Guaianolides Helenanolides Cadinanolides Chrymoranolides

ArcototeaeCalenduleae (50)

Guaianolides

Senecioneae (50)

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Table 1. Continued

Cynareae (50)

Mutisieae (55)

Lactucae (75)

Germacranolides Elemanolides Guaianolides Eudesmanolides Eudesmanolides Germanocranolides Eudesmanolides Guaianolides

I. Costunolide Costunolide (1, Figure 2) is an active component from the crude extract of Saussurea lappa roots, a traditional Chinese medicinal herb [3]. The anticancer property of costunolide was first reported in a rat intestinal carcinogenesis model induced by azoxymethane and supported by a subsequent study using a DMBA induced hamster buccal pouch carcinogenesis model [4]. Following these two in vivo experiments, considerable efforts have been devoted to understad the mechanism responsible for the anti-cancer activity of costunolide. First, costunolide is a potent apoptotic inducer in cancer cells, via multiple pathways. It has been reported that costunolide readily depletes intracellular GSH and disrupts the cellular redox balance [5]. It triggers an intracellular reactive oxygen species (ROS) burst which leads to mitochondrial dysfunction: loss of mitochondrial membrane potential, onset of mitochondrial membrane transition, and release of mitochondrial pro-apoptotic proteins [6]. The apoptosis-inducing activity of costunolide was found to be closely associated with Bcl-2, based on observations that costunolide treatment decreased the anti-apoptotic Bcl-2 protein expression [7], while over expression of Bcl-2 protein attenuated costunolide-induced apoptosis [12]. Second, costunolide suppresses NF-kB activation via prevention of IkB phosphorylation [8], a process also responsible for the strong anti-inflammatory activity of costunolide [9]. Third, costunolide is capable of promoting leukemia cell differentiation [10], inhibiting endothelial cells angiogenesis [11], and disrupting nuclear microtubule architecture in cancer cells [12].

II. Parthenolide Parthenolide (10, Figure 3), is the major SL responsible for bioactivity of feverfew (Tanacetum parthenium), a traditional herb plant which has been used for the treatment of fever, migraine and arthritis for centuries [13]. One well-explored bioactivity of parthenolide is its potent anti-inflammatory

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effect, which is mainly achieved through its strong inhibitory effect on NF-kB activation. It has been well established that parthenolide acts on multiple steps along the NF-kB signaling pathway [14]. By suppressing NF-kB parthenolide inhibits a group of NF-kB regulated pro-inflammatory cytokines, such as interleukins and prostaglandins [15]. The anticancer activity of parthenolide has been pursued in a number of laboratories. A large number of studies have been undertaken to investigate the mechanism of action of parthenolide at molecular levels in the different phases of carcinogenesis. The data were obtained using different tumor cell systems. Parthenolide induced apoptosis in pre-B acute lymphoblastic leukemia lines, including cells carrying chromosomal translocations [16]. Parthenolide induced rapid apoptotic cell death distinguished by loss of nuclear DNA, externalization of cell membrane phosphatidyl-serine, and depolarization of mitochondrial membranes at concentrations ranging from 5 to 100 μM. Steele et al. investigated the in vitro actions of parthenolide on cells isolated from patients with chronic lymphocytic leukemia. Brief exposure to the sesquiterpene lactone (one to three hours) was sufficient to induce caspase activation and commitment to cell death. The mechanism of cell killing was via parthenolide induced generation of ROS, resulting in turn in a proapoptotic Bax conformational change, release of mitochondrial cytochrome C and caspase activation. Other studies also demonstrated that parthenolidemediated apoptosis correlated well with ROS generation. Parthenolide strongly induced apoptosis in four multiple myeloma cell lines, although there are considerable differences in susceptibility to the sesquiterpene lactone. KMM-1 and MM1S sensitive to parthenolide possess less catalase activity than the less sensitive KMS-5 and NCI-H929 cells. These findings indicate that parthenolide-induced apoptosis in multiple myeloma cells depend on increased ROS and that intracellular catalase activity is a crucial determinant of their sensitivity to parthenolide. Chen et al. also reported the anti-proliferative and apoptosis-inducing effects of parthenolide on human multiple myeloma cells, mediated by an enhancement of caspase-3 activity [17].

III. Helenalin Helenalin (7, Figure 2) is another SL, from Arnica species, which has been reported to possess cytotoxicity and anti-cancer activity [18]. Earlier studies demonstrated its potent activity to inhibit nucleic acid and protein synthesis [19]. Similiar to other anticancer SLs, mechanism of action mainly involve: (i) thiol depletion, (ii) inhibition of NF-kB, and (iii) induction of apoptosis [20]. These prominent bioactivities make helenalin another potential anti-cancer agent.

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IV. Artemisinin and its derivatives Given the high accumulation of iron in cancer cells, researchers Henry Lai and Narendra Singh became interested in possible artemisinin (8, Figure 2) activity against malignant cells and have used artemisinin against numerous cancer cells in vitro [21]. There are a number of properties shared by cancer cells that favor the selective toxicity of artemisinin against cancer cell lines and against cancer in vivo. In addition to their high rates of iron flux via transferrin receptors when compared to normal cells, cancer cells are also particularly sensitive to oxygen radicals. Artemisinin becomes cytotoxic in the presence of ferrous ion. Since iron influx is naturally high in cancer cells, artemisinin and its analogs can selectively kill cancer cells in vivo [22]. Furthermore, it is possible to increase or enhance iron flux in cancer cells by supplying conditions that lead to increased intracellular iron concentrations. However, intact in vivo systems do not need holotransferrin, since the body provides all the necessary iron transport proteins. In recent years, in order to search for potential anticancer agents many researchers have directed their efforts in synthesizing various kinds of artemisinin dimers, trimers, tetramers wherein several of which have shown potential anticancer activity and are in the various phases of clinical trials [23]. [B] Anti-inflammatory activity Sesquiterpenes lactones have displayed potential anti-inflammatory activity through NF-kB pathway. Since NF-kB plays a central role in most disease processes, and since it can regulate the expression of many key genes involved in inflammatory as well as in a variety of human cancers [24], NF-kB represents a relevant and promising target for the development of new chemopreventive and chemotherapeutic agents. Some of the important SLs have displayed anti-inflammatory activity are as follows:

I. Costunolide Costunolide (1, Fig. 2) is a closely related sesquiterpene lactone analogue of parthenolide present in several plants such as Magnolia grandiflora, Tanacetum parthenium. Koo et al. showed that costunolide also dosedependently inhibited LPS-induced NF-kB activation. In this assay system, costunolide even exhibited more potent inhibitory activity than parthenolide. Detailed mechanism studies revealed that, similar to parthenolide, costunolide also significantly inhibited the degradation of IkB-α and IkB-β. In addition, costunolide also inhibited the phosphorylation of IkB-α. These

Biologically active sesquiterpene lactones

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accumulative results indicate that costunolide inhibits NF-kB activation by preventing the phosphorylation of IkB, and therefore, sequestering the complex in an inactive form [8].

II. Parthenolide Parthenolide (10, Figure 3) is a sesquiterpene lactone present in several medicinal plants that have been used in folk medicine for their antiinflammatory and analgesic properties. Several in vitro studies have shown that a great part of the anti-inflammatory action of this compound appears to be related to its ability to inhibit the NF-kB pathway. In vitro studies have proven that the sesquiterpene lactone parthenolide does not interfere with the generation of oxygen radicals [25], whereas it specifically inhibits activation of the NF-kB pathway by targeting IKK [26] and/or preventing the degradation of IkB-α and IkB-β [25]. Furthermore, parthenolide has recently been reported to exert beneficial effects during endotoxic shock in rats through inhibition of NF-kB DNA binding in the lung [27]. These effects of parthenolide may also accounts for its inhibition of proinflammatory mediator genes, such as the gene for the inducible nitric oxide synthase after endotoxin stimulation in rat smooth muscle cells [28] and the gene for IL-8 in immune-stimulated human respiratory epithelial cells [29]. In addition, parthenolide has also been demonstrated to protect against myocardial ischemia and reperfusion injury in the rat by selective inhibition of IKK activation and IkBα degradation [30].

III. Helenalin Since different types of sesquiterpene lactones showed inhibition of NF-kB activation at similar concentrations, this effect seems to be characteristic for many of the sesquiterpene lactones with an exomethylene group like parthenolide and costunolide. Exomethylene groups of α,β-unsaturated carbonyl compounds can react by Michael type addition to sulfhydryl groups of cysteine residues in the DNA binding domain of the NF-kB subunit [31]. Recently, Lyu et al. provided evidence that a sesquiterpene lactone, helenalin (7, Fig. 2), containing two functional groups, namely α,β-unsaturated carbonyl group and α-methylene-δ-lactone ring, exerts its effect by direct alkylation of the p65 subunit of NF-kB without inhibition of IkB degradation [32]. In vitro studies also demonstrated that helenalin selectively modifies the p-65 subunit of NF-kB at the nuclear level, therefore inhibiting its DNA binding [33]. However, costunolide differs from helenalin

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in a number of functional groups and inhibits degradation of IkB by inhibiting phosphorylation of IkB. Therefore, another functional group other than the exomethylene group and the molecular geometry of sesquiterpene lactone compounds appear to be important factors to determine the mode of NF-kB inhibition. However, the epoxide group in parthenolide is not likely important because parthenolide is at least less effective to inhibit both NF-kB activation and NO production.

[C] Anti-malarial activity I. Artemisinin and its derivatives In 1972, a group of Chinese researchers isolated a new anti-malarial drug (+)- artemisinin (8, Figure 4), a sesquiterpene lactone of the amorphene sub-group of cadinene from the hexane extract of a traditional Chinese medicinal plant Artemesia annua (Asteraceae) - a plant which has been used for the treatment of fever and malaria since ancient times [23]. Artemisinin is a sesquiterpene lactone containing an endoperoxide linkage in it. This highly oxygenated sesquiterpene lactone peroxide, unlike most other anti-malarials, lacks nitrogen containing heterocyclic ring systems and was found to be superior plasmocidal and blood schizontocidal agent to conventional antimalarial drugs, such as chloroquine, quinine etc against malaria strains, without obvious adverse effects in patients. Artemisinin is active at nanomolar concentrations in vitro both against chloroquine sensitive and resistant P. falciparum strains. However, the practical values of artemisinin, nevertheless, are impaired (i) poor solubility either in oil or water; (ii) high rate of parasite recrudescence after treatment; (iii) short-plasma half life (3-5h) and poor oral activity. However, a low level of resistance has

O O

22 R = H (Dihydroartemisinin) 23 R = Me (Artemether) 24 R = Et (Arteether) 25 R = COCH2CH2COONa (Sodium artesunate) 26 R = COCH2C6H4COONa (Sodium artelinate)

O O O 12

11 13

O OR

8 (Artemisinin)

Figure 4. Structure of artemisinin and its analogs.

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recently been observed using artemisinin, which disappeared as soon as the drug-selection pressure has been withdrawn. However, artemisinin with an endoperoxide linkage is a sensitive molecule for large scale derivatization. Fortunately, it was found that the carbonyl group of artemisinin 8, can be easily reduced to dihydroartemisinin 22 in high yields using sodium borohydride, which has in turn led to the preparation of a series of semi-synthetic first-generation analogues included oil soluble artemether 23, arteether 24, water soluble sodium artesunate 25, and sodium artelinate 26. These three analogs become very potent anti-malarial drugs effective against chloroquine-resistant strains of P. falciparum. Artemether 23 has been included in the WHO lists of Essential Drugs for the treatment of severe MDR malaria. In this family, the Walter Reed Institute of research has patented a stable, watersoluble derivative called artelinic acid 26 which is now being tested in animals. A key advantage of these endoperoxides containing anti-malarial agents, which have been used for nearly two decades, is the absence of drug resistance. It has been realized through the structure-activity relationship (SAR) of artemisinins that mainly endoperoxide affects the antimalarial activity. In order to increase antimalarial potency of these molecules, researchers around the world become interested to synthesize artemisinin dimers, trimers and tetramers in recent years. Many of them have shown promising antimalarial activity than artemisinin and their first generation analogs.

II. Miscellaneous antimalarials Antimalarial activity of sesquiterpenes lactones from Neurolena lobata has been documented (Figure 5) [34]. Germacranolide sesquiterpenes lactones like neurolenin B (17, IC50 = 0.62 μM) more potent than furanoheliangolides lobatin B (IC50 = 16.51 μM). Among the germacranolides, the shift of the double bond from the 2,3-position (neurolenin B) into the 3,4-position (lobatin A) led to dramatic decrease in the activity suggesting that one of the structural requirements is the presence of α/β-unsaturated keto function. Additionally, a free hydroxyl group at C-8 increased the antiplasmodial activity, while a free hydroxyl group at C-9 decreased the activity. Goffin et al. investigated the antiplasmodial properties of Tithonia diversifolia against three strains of P. falciparum, and sesquiterpene lactone (Fig. 5/Fig 3) Tagitinin C (3) was found to be active against FCA strain (IC50 = 0.33 μg/mL) [35]. Jenett-Siems et al. reported four sesquiterpenes, vernodalol (18), 11β,13-dihydrovernodalin 11β,13-dihydrovernolide (19) and 11β,13,17,18tetrahydrovernolide from Vernonia colorata. Among these, vernodalol (18) and 11β,13-dihydrovernodalin (19) exhibited the strongest antiplasmodial activity (IC50 = 4.8 and 1.1 μg/mL) respectively). Among the sesquiterpene lactones obtained from Artemisia afra, 1-desoxy-1α-peroxy-rupicolin A-8-O-acetate

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OCOCH3 OH O

OCOCH3 OiVal

OH MeO

O Tagitinin C (3)

O Neurolenin B (17) HO O

Vernodalol (18)

OH OAc

CH2

O H

CH2

O O

Helenalin (7)

Rupicolin A 8-Acetate (20)

11,13-dihydrovernodalin (19)

O OH

R O O HO

O O

Ridentin (21)

O Hanphyllin (13)

27, 28

R = H, CH3 O

Figure 5. Structures of some of anti-malarials sesquiterpene lactones.

(20), 1β,4β-dihydroxy-bishopsolicepolide and rupicolin A-8-O-acetate (20) possessed in-vitro antiplasmodial activity (IC50 = 10.8-17.5 μg/mL) [36]. Passreiter et al. have isolated sesquiterpene lactones of the pseudoguaianolide type from Arnica Montana, helenalin (7), dihydrohelenalin and their acetates showing activities against P. falciparum in vitro (IC50 = 0.23 to 7.41 μM) [37]. Inhibitory effect upon the growth of P. falciparum has been reported for sesquiterpene lactones (27) and (28) isolated from Camchaya calcarea (IC50 = 1.2 and 0.3 μg/mL) respectively [38]. [D] Antiviral activity In spite of an effective and safe vaccine therapy against hepatitis B virus (HBV), viral infection by HBV caused a global health problem in the world, especially the third world. Moreover, because direct antiviral therapy against HBV infection is not yet perfectly developed, it is important to discover the lead compounds for novel anti-HBV agents from the potential library. Recently, there was a report about anti-HBV activity of artemisinin (8) and artesunate (25) based on the screening by using HBV-transferred HepG2 2.2.15 cell [39], which is derived from hepatoblastoma HepG2 cell

Biologically active sesquiterpene lactones

325

[40]. This screening method is a useful in vitro model for evaluation of novel anti-HBV drugs, as well as to study several steps of the HBV biology [41]. Artemisinin (8), artesunate (25), and a variety of purified compounds from traditional Chinese medicine remedy were investigated by measuring the release of surface protein (HBsAg) and HBV-DNA after drug exposure (0.01-100 μM) for 21 days [39]. As a result, artesunate (25) strongly inhibited the HAsAg secretion with an IC50 of 2.3 μM and IC90 of 16 μM, respectively, whereas artemisinin (8) had a mild inhibition activity. To evaluate an enhancement in viron production, the amount of the HBV-DNA release to the HepG2 2.2.15 culture medium during different treatments was measured, and it was significantly reduced. In addition, it was discovered that, for artesunate (25), toxicity in host cell was shown in drug concentration of 20 μM and therapeutical index (TI) calculated from IC50 of HBV-DNA release was 40. When comparing to TI value (500) of lamivudine as positive control, the value of artesunate (25) is quite low, but reasonable value for further investigation. Finally, artesunate (25) was tried in combination treatment with lamivudine. When both compounds were administered together in concentration of 20 nM each, no toxicity was observed, but a synergic inhibitory effect in HBsAg release was found. It means that it is possible to be potential antiviral agent against infection of lamivudine-resistance HBV strains, frequent problem in clinical treatment [42]. This result was quite similar to potency previously reported for human cytomegaloviruses [39]. Anti-viral activity of various sesquiterpene lactones was reported by Hsieh and their coworkers against hepatitis C virus (Fig. 6) [43]. They have tested a series of 10 compounds such as parthenolide (10, EC50 = 2.21 μM), costunolide (1, EC50 = 2.69 μM), dehydrocostus lactone (11, EC50 = 3.08 μM), Helenalin (7, EC50 = 1.25 μM), alantolactone (14, EC50 = 2.03 μM), Epoxy-dihydrosantonin (15, EC50 = >10 μM), artemisinin, and two other conjugated lactones. Wherein they found the best anti-HCV activity was shown by helenalin. They have further derivatized a series of parthenolide analogs 29 wherein they found that best activity was realized while putting a piperidine moiety (R = piperidine, EC50 = 1.64 μM). [E] Antibacterial activity There has been an overwhelming amount of evidence indicating that certain SLs are effective in exerting antibacterial activity. Rabe et al. showed that Vernonia colorata, a member of the Compositae found in west, central and South Africa possess SLs with antibacterial activity primarily against Gram-positive species and lower activity towards Gram-negative species [44]. The SLs vernodalin (30), vernolide (12) (Fig. 7) and 11β,13dihydroovernolide were isolated and screened against Staphylococcu aureus

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Devdutt Chaturvedi H

O parthenolide (10)

Costunolide (1)

O Dehydrocostus lactone (11)

Helenalin (7)

O O O O

O O

O Epoxy(4,5 α) - 4,5-dihydrosantonin (15) 4,(5)α− Epoxy-4,5-dihydrosantonin (16)

Alantolactone (14)

R = N(CH3)2, N(Et)2, Pyrrolidine, Piperidine, Morpholine

O 29 Parthenin derivatives

Figure 6. Antiviral sesquiterpene lactones.

and Bacillus subtilis (Gram-positive species) and Escherichia coli and Klebsiella pneumoniae (Gram-negative species). 11β,13-Dihydroovernolide is a novel SLs in that it has never been isolated from a Vernonia species before. All three of the compounds screened had very low inhibitory action against the Gram-negative bacteria. However, S. aureus and B. subtilis showed the most sensitivity towards all of the SLs screened. It needs to be noted, however, that although 11β,13-dihydrovernolide is a novel SL, it had the lowest activity against the Gram-positive species compared to vernolide and vernodalin which had MIC values of 0.1-0.5 mg/mL. Taylor and Towers isolated, characterised and screened three SLs belonging to the pseudoguaianolides class of SLs from Centipeda minima, a member of the Compositae [45]. This plant is used throughout Southeast Asia to treat colds, coughs, and sinus infections. Three SLs, 6-O-methylacrylylplenolin (31), 6-O-angeloylplenolin (32), and 6-O-isobutyroylplenolin (33) (Figure 8) were isolated, with 6-O-methylacrylylplenolin being novel, and were then screened for antibacterial activity against B. subtilis and S. aureus. All three of the SLs screened had significant activity against the bacteria with 6-Oisobutyroylplenolin being the most bioactive. Both 6-O-isobutyroylplenolin and 6-O-methylacrylylplenolin exhibited MIC value of 150 μg/mL against B. subtilis. 6-O-Angeloylplenolin was less active with a MIC of 300 μg/mL. All three

327

Biologically active sesquiterpene lactones O HO O

O CH2 O

O OH

O O

Vernodalin (30)

Vernolide (12)

Figure 7. Antibacterial sesquiterpenes lactones (30, 12).

SLs showed activity against both methicillin-resistant and methicillinsensitive strains of S. aureus. Both 6-O-isobutyroylplenolin and 6-O-methylacrylylplenolin had a MIC of 300 μg/mL against methicillinresistant S. aureus, while 6-O-angeloylplenolin was less active with a MIC of 600 μg/mL against this strain. With respect to the methicillin-sensitive strain of S. aureus, 6-O-methylacrylylplenolin and 6-O-angeloylplenolin had MIC values of 75 μg/mL while 6-O-isobutyroylplenolin had a MIC of 38 μg/mL indicating that this SL is more bioactive against methicillin-sensitive S. aureus than the other SLs screened. Further amplifying the possibility for the use of SLs found in plant oils, Wang and coworkers recently discovered four new SLs in a plant species known as Ligulariopsis shichuana, which is a new genus of the Compositae [46]. The four SLs isolated and characterised were: (a) 3β-acetoxy-9β-angeloyloxy-1β, 10β-epoxy-8α-hydroxyeremophil-7(11)-en-8β-(12)-olide (34); (b) 3β-senecioyloxy-1β,10β-epoxy-8α-hydroxyeremophil-7(11)-en-8β-(12)-olide (35); (c) 6βangeloyloxy-8α-hydroxyeremophil-1(10),7(11)-dien-8β-(12)-olide (36); and (d) 1-oxo-6β-senecioyloxy-8α-hydroxyeremophil-7(11),9(10)-dien-β-(12)-olide (37)

O O

6-O-Methylacrylylphenolin (31) 6-O-Angeloyphenolin (32) 6-O-Isobutyroylphenolin (33)

Figure 8. Antibacterial sesquiterpene lactones (31-33).

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Devdutt Chaturvedi

O-Ang OH

H OH

O O

AcO

SenO 34

35 OH

OSen

OAng

Figure 9. Antibacterial sesquiterpenes lactones (34-37).

(Fig. 9). Only compounds 34 and 35 were screened for their antibacterial activity. Compound 34 showed moderate activity towards both the Gram-positive and Gram-negative species B. subtilis and E. coli, respectively. Compound 35, while exhibiting moderate activity towards E. coli, showed much stronger activity against B. subtilis at MIC concentrations up to 100 μg/mL. Finally, there have been reports that other SLs, such as helenalin 7, showed inhibitory action against Mycobacterium tuberculosis as well as activity against Corynebacterium diptheriae [47]. Helenalin 7, a mixture of alantolactone 14 and isoalantolactone 38, is derived from the plant species Inula helenium (Fig. 10). Helenalin 7 has primarily been utilised as an antiseptic for the urinary tract [47]. However, helenalin 7 was also shown to inhibit both Gram-positive and Gramnegative bacterial growth, with the former showing more sensitivity [48]. As one can see, there is certain hope for those essential oils containing SLs in therapeutics. The preclinical data implicates that SLs are effective in reducing bacterial growth which gives strength to the idea that SLs could be potentially used in the medical treatment of both Grampositive and Gram-negative bacterial infections. O

O O

Alantolactone (14)

Isoalantolactone (38)

Figure 10. Antibacterial sesquiterpene lactones (14, 38).

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Biologically active sesquiterpene lactones

[F] Antifungal activity There certainly exists a vast amount of empirical data supporting that certain SLs found in essential oils have the potential to act as antibacterial agents. It also needs to be shown that certain SLs also possess antifungal activities. The following section will focus on studies implicating the SLs for probable use as antifungal agents. Calera et al. isolated, characterised, and screened two bioactive SLs from the roots of yellow flowered perennial herb Ratibida mexicana. This plant is found primarily along the Sierra Madre Occidental in the northwestern part of Mexico [49]. Indian tribes find that the roots are useful in alleviating headaches, colds and rheumatism. The two SLs isolated from this plant are isoallolantolactone (38) and elema-1,3,11-trien-8,12-olide (39) (Fig. 11). The in vitro antifungal screen revealed that both SLs inhibited the radial growth of Helminthosporium with the MIC being 650 μg/mL for both SLs. Pythium growth was far more sensitive to isoallolantolactone (38) with a MIC of 125 μg/mL. Fusarium was also screened for sensitivity against isoallolantolactone (38) and elema-1,3,11-trien-8,12-olide, with again isoallolantolactone (39) showing the most bioactivity by inhibiting 45% of radial growth at 200 μg/mL for this particular fungus. O

O O

Isoalantolactone (38)

Elema-1, 3,11-trien-8,12-olide(39)

Figure 11. Antifungal activity of sesquiterpene lactones (38, 39).

Two new eudesmanolides were isolated from the aerial parts of Centaurea thessala spp. drakiensis and C. attica spp. attica, plants which are primarily used in folk medicine in the Mediterranean region [50]. The two novel eudesmanolides isolated were 8α-hydroxy-4-epi-sonchucarpolide (40) and the 8α-(4-acetoxy-3hydroxy-2-methylenebutanoyloxy) derivative (41) of the 8α-hydroxy-4-episonchucarpolide, also known as 40-acetoxymalacitanolide (Fig. 12). A variety of fungal species showed sensitivity towards 8α-hydroxy-4epi-sonchucarpolide (40) and 40-acetoxymalacitanolide (41) [50]. 8α-Hydroxy-4-epi-sonchucarpolide 40, when compared to 40acetoxymalacitanolide 41, showed higher activity against all the fungal

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Devdutt Chaturvedi

OH OH

OAc

O O

H CHO

O O

Figure 12. Antifungal activity of sesquiterpene lactones (40-41).

species screened, with the exception of one species. The MIC values for 8α-hydroxy-4-epi-sonchucarpolide 40 were considerably lower indicating that sensitivity is much higher for this compound. The exception was for Cladosporium cladosporioides; this species showed higher sensitivity towards 40-acetoxymalacitanolide, with a MIC value of 0.06 μg/mL while the MIC value for 8α-hydroxy-4-epi-sonchucarpolide was 0.5 μg/mL. In addition, both SLs had indentical MICs against Penicillium funiculosum, showing no disparity between these two SLs in their inhibitory action against this particular species. The authors of this paper speculated that the differences in activity between these two SLs could be attributed to the different skeletal types and functional groups present on the compounds. Finally, it needs to be mentioned that both SLs, possessed greater antifungal activity that miconazole, a commercial fungicide used as the positive control.

3. Structural-activity relationships (sar) of sesquiterpene lactones It is generally believed that the bioactivity of SLs is mediated by alkylation of nucleophiles through their α, β- or α, β, γ-unsaturated carbonyl structures, such as α-methylene-γ-lactones or α,β-unsaturated cyclopentenones. These structure elements react with nucleophiles, especially the cysteine sulfhydryl groups by Michael-type addition. Therefore, it is widely accepted that thiol groups such as cysteine residues in proteins, as well as the free intracellular GSH, serve as the major targets of SLs. In essence, the interaction between SLs and protein thiol groups or GSH leads to reduction of enzyme activity or causes the disruption of GSH metabolism and vitally important intracellular cell redox balance. The relationship between chemical structure and bioactivity of SLs has been studied in several systems, especially with regards to cytotoxicity,

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anti-inflammatory and antitumor activity. It is believed that the exomethylene group on the lactone is essential for cytotoxicity because structural modifications such as saturation or addition to the methylene group resulted in the loss of cytotoxicity and tumor inhibition. However, it has also been shown that the factor responsible for the cytotoxicity of SLs might be the presence of the O=C-C=CH2 system, regardless of lactone or cyclopentenone. It was latter demonstrated that the presence of additional alkylating groups greatly enhanced the cytotoxicity of SLs. Furthermore, it was established that the α-methylene-γ-lactones and α,β-unsaturated cyclopentenone ring (or αepoxycyclopentenone) present in SLs essential for their in vivo anti-tumor activity. It has been confirmed through various published reports that the various kinds of biological activities displayed by SLs is due to presence of either αmethylene-γ-lactones and α,β-unsaturated cyclopentenone ring. In summary, the differences in activity among individual SLs may be explained by differences in the number of alkylating elements, lipophilicity, molecular geometry, and the chemical environment of the target sulfhydryl group.

n O

O O

Figure 13. General structure of sesquiterpene lactones.

4. Conclusions Sesquiterpene lactones are an important group of natural products obtained from many species of medicinal plants. Their structural diversity and diverse potential biological activities such as anticancer, antiinflammatory, anti-tumor, anti-malarial, antiviral, antibacterial, antifungal etc. have made further interest among the chemists to the drug discovery research. Although, the exact mechanism of action of SLs are not well known but it has been documented through the various published reports that the biological activity displayed by majority of sesquiterpene lactones is due to the presence of α-methylene-γ-lactones and α,β-unsaturated cyclopentenone ring. The present chapter deals an overview on the various kinds of biologically activity of structurally diverse sesquiterpene lactones

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which may be useful for the chemists/pharmacologists working in the area of drug discovery of the relevant subject.

Acknowledgements Author is thankful to the Director, North-East Institute of Science and Technology (CSIR), Jorhat, Assam, for providing the necessary facilities during the preparation of this book chapter.

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Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 335-365 ISBN: 978-81-308-0448-4

11. A review on natural products with mosquitosidal potentials Navneet Kishore, Bhuwan B. Mishra, Vinod K. Tiwari and Vyasji Tripathi Department of Chemistry, Faculty of Science, Banaras Hindu University Varanasi-221005, India

Abstract. Mosquito, a flying insect of family Culicidae, serves as crucial vector for a number of arboviruses (arthropod-borne viruses) and parasites that are maintained in nature through biological transmission between susceptible vertebrate hosts by blood feeding arthropods (mosquitoes, psychodids, ceratopogonids, and ticks) responsible for inflammation/encephalitis, dengue, malaria, rift valley fever, yellow fever and others. Despite of a direct human affliction, they are also known to transmit several diseases and parasites that are lethal to dogs and horses, i.e., dog heartworm (Dirofilaria immitis), West Nile virus (WNV) and Eastern equine encephalitis (EEE) with ability to affect the central nervous system and cause severe complications and death. Vector control is by far the most successful method for reducing the incidences of diseases, but the emergence of widespread insecticide resistance and the potential environmental issues associated with some synthetic insecticides (such as DDT) has indicated that additional approaches to control the proliferation of mosquito population would be an urgent priority research. In concern to quality & safety of life on controlling mosquito vectors has shifted steadily from the use of conventional chemicals toward alternative insecticides that are target-specific, biodegradable, environmentally safe, and botanicals in origin. In present article, we have discussed Correspondence/Reprint request: Dr. Vyasji Tripathi, Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India. E-mail: vyas_45@rediffmail.com

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the local and traditional uses of plants in mosquito control and have reviewed 185 phytochemicals of paramount importance for the development of efficient chemical entities to control mosquito population by direct as well as indirect inhibitions. In order to highlight any possible mechanism based action for promising mosquitosides, the review has been organized according to chemical structural classes.

1. Introduction Mosquito serves as crucial vector for a number of arboviruses (arthropod-borne viruses) and parasites that are maintained in nature through biological transmission between susceptible vertebrate hosts by blood feeding arthropods responsible for inflammation/encephalitis, dengue, malaria, rift valley fever, yellow fever and others. The Word Health Organization (WHO) estimates that each year 300-500 million cases of malaria occur and more than 1 million people die of malaria. About 1,300 cases of malaria are diagnosed in the United States each year. In addition, some 2500 million people (two fifth of the world's population) are now at risk from dengue [1]. One can imagine the dangers of these mosquitoes with all the other diseases that it can transmit. Vector control is by far the most successful method for reducing incidences of mosquito born diseases, but the emergence of widespread insecticide resistance and the potential environmental issues associated with some synthetic insecticides (such as DDT) has indicated that additional approaches to control the proliferation of mosquito population would be an urgent priority research. Currently, numerous products of botanical origin, especially the secondary metabolites, have received considerable renewed attention as potentially bioactive agents used in insect vector management. However, there is a little other than anecdotal, traditional or cultural evidence on this topic [2]. The Greek natural philosopher Pliny the Elder (1’st century AD) recorded all the known pest control methods in ‘‘Natural History’’. The use of powdered chrysanthemum as an insecticide comes from Chinese record. The other natural products like pyrethrum, derris, quassia, nicotine, hellebore, anabasine, azadirachtin, d-limonene, camphor and turpentine were among some important phytochemical insecticides widely used in developed countries [3]. The discovery of DDT’s and the subsequent development of organochlorines, organophosphates and pyrethroids suppressed natural product research as the problem for insect control were thought be solved. However, high cost of synthetic pyrethroids, environment and food safety concerns, the unacceptability and toxicity of many organophosphates and organochlorines, and increasing insecticide resistance on a global scale argued for stimulated research towards potential botanicals [4].

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Mosquitoes in the larval stage are attractive targets for pesticides because they breed in water and, thus, are easy to deal with them in this habitat. Some of new significant larvicidal insect growth regulators such as methoprene, pyriproxyfen, diflubenzuron and endotoxins obtained from Bacillus thuringiensis israelensis and B. sphaericus have been developed. The plant Azardichita indica has gained wide acceptance in some countries as an antifeedant [5] while many essential oils from plant origin such as citronella, calamus, thymus, and eucalyptus are reportedly promising mosquito larvicides [6-10]. The use of herbal products is one of the best alternatives for mosquito control. The search for herbal preparations that do not produce any adverse effects in the non-target organisms and are easily biodegradable remains a top research issue for scientists associated with alternative vector control [11]. Many plant species are known to possess biological activity that is frequently assigned to the secondary metabolites. Among these, essential oils and their constituents have received considerable attention in the search for new biopesticides. Many of them have been found to possess an array of properties, including insecticidal activity, repellency, feeding deterrence, reproduction retardation and insect growth regulation against various mosquito species [12-16].

2. Traditional mosquito repellents and usage custom There are several reports particularly in Africa describing about the burned plant materials effective to drive away mosquitoes. Thirteen percent of rural Zimbabweans use plants and 15% using coils [17] while 39% of Malawians burn wood dung or leaves [18]. Up to 100% of Kenyans burned plants to repel mosquitoes [19], and in Guinea Bissau 55% of people burned plants or hung them in the home to repel mosquitoes [20]. The local communities adapt various methods to repel the insects/ mosquitoes. Application of smoke by burning the plant parts is one of the most common practices among the local inhabitants. Other types of applications are spraying the extracts by crushing and grinding the repellent plant parts, hanging and sprinkling the repellent plant leaves on the floor etc. The leaf of repellent plant is one of the commonly and extensively used plant parts to repel the insects and mosquitoes, followed by root, flower and remaining parts of repellent plants [21]. Various traditional repellent plants used by the local inhabitants in order to avoid mosquito bites have been listed in Table 1.

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Table 1. Traditional plants as mosquito repellents [21]. Traditional Names Tinjut Woira Neem Wogert Kebercho Waginos Eucalyptus Ades Gemmero Tej-sar Ats-faris Endode Azo-hareg Berberra Gullo

Scientific Names Ostostegia integrifolia Olea europaea Azadirachta indica Silene macroserene Echinops sp. Brucea antidysenterica Eucalyptus camaldulensis Myrtus communis Capparis tomentosa Cymbopogen citrates Datura stramonium Phytolacca dodecandra Clematis hirsuta Millettia ferruginea Ricinus communis

Family Lamiaceae Oleaceae Meliaceae Caryophyllaceae Asteraceae Simaroubaceae Myrtaceae Myrtaceae Capparidaceae Rutaceae Solanaceae Phytolaccaceae Ranunculaceae Fabaceae Euphorbiaceae

3. Natural products as potential antimosquito agents The plant world comprises a rich untapped pool of phytochemicals that may be widely used in the place of synthetic insecticides. Plant-based products have been used to control domestic pests for a very long time. The search for and investigation of natural and environmentally friendly insecticidal substances are ongoing worldwide [22-24]. Insecticidal effects of plant extracts vary not only according to plant species, mosquito species and plant parts, but also to extraction methodology [25]. A brief delve into the literature reveals many laboratory and applied investigations [26-28] into the biological activity of many plant derived components against a large number of pathogens and arthropods but the lack of reviews in this area is somewhat surprising since much effort been invested in locating mosquitocidal phytochemicals from edible crops, ornamental plants, herbs, grasses, tropical and subtropical trees and marine angiosperms. A review by Roark, 1947 [29] highlights about 1200 plant species with a wide spectrum of bioactive insecticides. A relevant effort to present context comes from a review by Sukumar et al., 1991 [30] who listed 344 insecticidal botanical agents. Reviews by Schmutterer, 1990 [31] and Mulla & Su, 1999 [32] did not cover significant topics such as structure-activity relationship based activity, mode and site of action and joint action of botanical extracts with other phytochemicals and synthetic insecticides. This review is focused to cover the entire formal and constant research on mosquitocidal natural

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products from the 1947 to early 2010 with special attention on structureactivity relationship (SAR) based activity and mechanism of action for most of natural products, in addition to a number of bioassay procedures and toxicities of crude plant extracts on different species of mosquitoes reported in literature Table 2. Table 2. Mosquitocidal activity of crude plant extracts against different mosquito larvae as well as adults. Plant Family

Plant species

Parts

Mosquitoes

Acoraceae

Acorus calamus [91,92]

Rhizome

Agavaceae Alliaceae Annonaceae

Agave sisalana [93] Allium sativa [94] Annona squamosa [95]

Fiber Bulb Leaf

Apiaceae

Mkilua fragrans [96] Xylopia caudata [96] Xylopia ferruginea [96] Calotropis procera [97] Catharanthus roseus [98] Daucus carota [92]

Aerial Part Leaf Leaf Root Whole Seed

Acanthaceae

Rhinocanthus nasutus [99]

Leaf

Hygrophila auriculata [98]

Shoot

Cx. quinquefasciatus Ae. aegypti Ae. albopictus An. tessellates An. subpictus Cx. fatigans Cx. pipiens Cx. pipiens Ae. aegypti Cx. quinquefasciatus An. gambiae Ae. aegypti Ae. aegypti An. labranchiae Cx. quinquefasciatus Ae. Aegypti, Cx. fatigans Ae. aegypti An. Stephensi Cx. quinquefasciatus Cx. quinquefasciatus

Justicia adhatoda [98] Anthemis nobilis [100] Baccharis spartioides [101] Cotula cinerea [97] Sassurea lappa [92]

Leaf Flower Aerial Part Whole Plant Leaf

Tagetes minuta [102]

Whole Plant

Araceae Betulaceae

Homalomena propinqua [92] Alnus glutinosa [103]

Rhizome Old Litter

Cucurbiataceae Caesalpinaceae

Bryonopsis laciniosa [104] Cassia tora [105]

Whole Plant Seed

Apocynaceae

Asteraceae

Cx. quinquefasciatus Cx. pipiens Ae. aegypti An. labranchiae Ae. Aegypti Cx. fatigans Ae. aegypti An. stephensi Aedes aegypti Cx. pipiens Ae. rusticus Ae. albopictus Ae. aegypti Cx. quinquefasciatus Ae. aegypti, Cx. pipiens pallens

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Table 2. Continued Cupressaceae

Callitris glaucophylla [106]

Wood

Clusiaceae

Calophyllum inophyllum [99]

Leaf and Seed

Cannabaceae

Cannabis sativa [107]

Leaf

Caulerpaceae

Caulerpa scalpelliformis [108] Cleome viscosa [109] Dictyota caryophyllum [110] Dictyota dichotoma [109] Codiaeum variegatum [95]

Whole Plant Whole Plant Flower Whole Plant Leaf

Jatropha curcus [111] Ricinus communis [112] Abrus precatorius [98] Cassia obtusifolia [105]

Leaf Whole Plant Shoot Seed

Croton bonplandianum [98] Vicia tetrasperma [105] Pelargonium citrosum [96] Endostemon tereticaulis [113] Lavandula afficinalis [112] Leucas aspera [98] Mentha arvensis [112] Mentha piperita [114]

Shoot Seed Whole Plant Aerial Parts Whole Plant Whole Whole Plant Aerial Parts

Minthostachys setosa [115]

Whole Plant

Moschosma polystachyum [96] Ocimum basilicum [116]

Leaf

Cx. quinquefasciatus Ae. aegypti Ae. aegypti Ae. Aegypti, Cx. quinquefasciatus Cx. quinquefasciatus An. stephensi Cx. quinquefasciatus Ae. aegypti, Cx. pipiens pallens Cx. quinquefasciatus Ae. Aegypti, Ae. aegypti An. gambiae An. stephensi Cx. quinquefasciatus An. stephensi Ae. aegypti An. Tessellatus Cx. quinquefasciatus Ae. Aegypti An. Tessellatus Cx. quinquefasciatus

Aerial Parts

An. stephensi

Origanum majoranal [100] Plectranthus longipes [116] Pogostemon cablin [117] Rosmarinus officinalis [118] Thymus capitatus [119] Cinnamomum iners [96] Cinnamomum kuntsleri [96]

Leaf Aerial Parts Leaf Shoot Whole Plant Leaf Leaf

Cx. pipiens An. gambiae Ae. aegypti An. stephensi Cx. Pipiens Ae. aegypti Ae. aegypti

Cinnamomum pubescens [96]

Leaf, Bark and Twig Bark

Ae. aegypti Ae. aegypti

Bark and Leaf

Ae. aegypti

Capparidaceae Caryophyllaceae Dictyotaceae Euphorbiaceae

Fabaceae

Geraniaceae Labiatae

Lauraceae

Cinnamomum scortechinii [96] Cinnamomum sintoc [96]

Ae. aegypti, Cx. annulirostris Cx. quinquefasciatus, An. Stephensi Ae. aegypti An. Stephensi, Cx. quinquefasciatus Ae. aegypti Ae. aegypti

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Table 2. Continued Cinnamomum zeylanicum [118]

Bark and Leaf

Liliaceae Lythraceae

Gloriosa superb [98] Pemphis acidula [120]

Whole leaf

Menispermaceae Meliaceae

Abuta grandifolia [115] Azadirachta indica [95]

Fruit Leaf and Seed

Khaya senegalensis [106] Lansium domesticum [95]

Seed Leaf

Melia azadirachta [112]

Whole Plant

Melia volkensii [121]

Seed and Fruit

Eucalyptus camaldulensis [122] Eugenia caryophyllus [112] Eucalyptus globules [100]

Fruit

Syzygium aromaticum [117]

Leaf

Oleaceae Papaveraceae Pinaceae Piperaceae

Jasminum fructicans [100] Argemone mexicana [111] Cedrus deodara [112] Piper longum [123] Piper nigrum [124]

Leaf Leaf Whole Plant Fruit Fruit

Plumbaginaceae

Plumbago dawei [125]

Root

An.gambiae

Plumbago stenophylla [125] Plumbago zeylanica [125] Cymbopogon citratus [96] Cymbopogon flexuosus [112] Cymbopogon martini [112] Sorghum bicolour [126] Vetiveria zizanioides [100]

Root Root Whole Plant Whole Plant Whole Plant Seedling Rhizome

An.gambiae An.gambiae Cx. quinquefasciatus An. stephensi An. stephensi Cx. pipiens Cx. pipiens

Spermacoce hispida [98] Citrus limon [94] Zanthoxyllum acanthopodium [96]

Whole Peel Stem

Cx.quinquefasciatus Cx. pipiens Ae. aegypti

Myrtaceae

Poaceae

Rubiaceae Rutaceae

Whole Plant Whole Plant

An. stephensi Ae. aegypti Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Ae. aegypti Ae. aegypti Ae. aegypti Cx. quinquefasciatus Cx. annulirostris Ae. Aegypti Cx. quinquefasciatus An. stephensi Cx. pipiens molestus Cx. pipiens molestus Ae. aegypti An. arabiensis Cx. pipiens An. stephensi An. stephensi Cx. pipiens Ae. albopictus Ae. aegypti Cx. quinquefasciatus An. dirus Cx. pipiens Cx. quinquefasciatus An. stephensi Cx. pipiens pallens Cx. pipiens pallens Ae. aegypti Ae. togoi

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Table 2. Continued Solanaceae

Simaroubaceae Thymelaeaceae Umbelliferae

Valerianaceae Verbenaceae

Zingiberaceae

Solanum elaeagnifolium [97] Solanum indicum [98] Solanum sodomaeum [97] Withania somnifera [111] Solanum xanthocarpum [127] Quassia amara [128] Aquilaria malaccensis [96] Dirca palustris [129] Angelico glauca [92]

Berry Shoot Seed Leaf Leaf Whole Plant Wood Seed Aerial Parts

Pimpinella anisum [122] Valarian wallichii [92] Aloysia citriodora [101] Clerodendrun inerme [98] Stachytarpheta jamaicensis [98] Vitex nequrdo [109] Curcuma domestica [91] Kaempferia galangal [98] Zingiber officinalis [130]

Seed Rhizome Whole Plant Leaf Shoot

An. labranchiae Cx. quinquefasciatus An. labranchiae Cx. quinquefasciatus Cx. quinquefasciatus Cx. quinquefasciatus Ae. aegypti Ae. aegypti Ae. aegypti Cx. fatigans Cx. pipiens Ae. Aegypti Ae. aegypti Cx. quinquefasciatus Cx. quinquefasciatus

Whole Plant Rhizome Whole Tubers

Cx. quinquefasciatus An. culicifacies Cx. quinquefasciatus Cx. quinquefasciatus

4. Alkanes, alkenes, alkynes and simple aromatics The hydrocarbon, octacosane (1) isolated from Moschosma polystachyum shows significant larvicidal activity against Culex quinquefasciatus mosquito with LC50 value of 7.2±1.7 mg/L [33]. The (E)-6-hydroxy-4,6-dimethyl-3heptene-2-one (2) isolated from Ocimum sanctum exhibit toxicity against fourthinstar larvae of Aedes aegyptii with LD100 value of 6.25 μg/mL in 24 h [34]. Among the acetylenic compounds, falcarinol (3) and falcarindiol (4) isolated from Cryptotaenia canadensis display strong activity against Culex pipiens larvae [35,36]. The more lipophilic 3 with LC50 values of 3.5 and 2.9 ppm in 24 h and 48 h, respectively exert strong toxicity than the more polar acetylene 4 with LC50 values of 6.5 and 4.5 ppm in 24 and 48 h, respectively [37]. The volatile aromatics, 4-ethoxymethylphenol (5), 4-butoxymethylphenol (6), vanillin (7), 4-hydroxy-2-methoxycinnamaldehyde (8), and 3,4dihydroxyphenylacetic acid (9) isolated from Vanilla fragrans show very efficient mortality against mosquito larvae. The compunds 5-8 display 100% larval mortality at 0.5, 0.4, 2.0 and 1.0 mg/mL concentrations, respectively while 9 shows 17% larval mortality at a concentration of 1.0 mg/mL [38]. The hexane extract of Delphinium cultorum shows significant mosquitosidal activity (100% mortality at a concentration of 10 mg/mL) against Ae. aegyptii larvae at 2 h. A literature report comprising GC-EIMS

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analysis of hexane extract of D. cultorum resulted into isolation of six volatiles, ethylmethylbenzene (10), 1-isopentyl-2,4,5-trimethylbenzene (11), 2-(hex-3-ene-2-one)phenyl methyl ketone (12), E and Z isomers of 3-butylidene-3H-isobenzofuran-1-one (13 and 14) and 2-penten-1-ylbenzoic acid (15) [39]. The trans-asarone (16) isolated from seeds of Daucus carota shows 100% mortality at a concentration of 200 μg/mL against fourthinstar larvae of Ae. Aegyptii [40]. The compound (17) isolated from rhizomes of Curcuma longa display 100% mortality against Ae. aegyptii larvae with LD100 value of 50 μg/mL in 18 h [27]. Similarly, 18 isolated from leaf and stem of Ocimum sanctum display mosquitocidal activity against fourth-instar larvae of Ae. aegyptii with LD100 value of 200 μg/mL in 24 h, respectively [34]. The 5-allyl-2-methoxyphenol (19) isolated from seeds of Apium graveolens exhibit 100% mortality on fourth-instar Ae. aegyptii larvae at 200 μg/mL concentration [41]. The trans-anethole (20), methyl eugenol (21) and iso-methyl eugenol (22) isolated from Myrica salicifolia display 100% mortality with LD100 value of 20, 60 and 80 ppm in 24 h against 4th instar larvae of Ae. aegypti [42]. The stilbenes (23-29), isolated from the root bark of Lonchocarpus chiricanus possess larvicidal activities Ae. aegypti mosquito larvae. Among these, 27 at a concentration of 3.0 ppm exhibits highest activity while 24 and 25 with minimal concentration of 6.0 ppm each, display pronounced affect by kill all the larvae in 24 h. The compounds 23, 26, 28 and 29 with ≈50 ppm concentrations show moderate activity against larvae of Ae. aegypti [43].

5. Lactones The lactones 30 and 31, isolated from Hortonia floribunda, H. angustifolia and H. ovalifolia, exhibit potent larvicidal activity against the second instar larvae of Ae. aegypti with LC50 values of 0.41 and 0.47 ppm, respectively [44]. The 3-n-butyl-4,5- dihydrophthalide (32) isolated from seeds of Apium graveolens show 100% mortality on fourth-instar Ae. aegyptii larvae at a concentration of 25 μg/mL [41]. The sedanolide (33) isolated from seeds of same species exhibits 100% mortality at 50 μg/mL concentrations against fourth-instar larvae of Ae. aegyptii [45].

6. Essential oils and fatty acids The essential oils, α-phellandrene (34), limonene (35), p-cymene (36), γ-terpinene (37), terpinolene (38) and α-terpinene (39) isolated from leaves of Eucalyptus camaldulensis possess significant larvicidal activity against

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CH3

H2C

H 3C

CH3

R CH3 CH3

3R=H 4 R = OH COOH

CHO

CHO OCH3 CH3 OCH3

C2 H5

C4H9

CH3

O CH3 H3 C O

CH3

H 3C

12 CH3

CH 3

CH3

OCH3

H3CO

OH OCH3 H 3C

H3 C

O CH2

CH2

OH H3CO

H3CO H3CO H3CO

H3CO

CH 3

CH2

H 3C

CH3 OH

H3C

CH3 CH3

OCH3

OH H3C

H3C

CH3

23 H3C O

CH3 OH

OCH3

H3C HO

CH3 H3C

CH3 O

CH3

OCH3

CH3

H3C 28

CH3

Mosquitosidal natural products H3C

345

O H

H3C

C4H9

fourth-instar larvae of Ae. aegypti and Ae. albopictus. The compound 39 exert the strongest activity against Ae. aegypti larvae with LC50 value of 14.7 μg/mL (LC90 = 39.3 μg/mL) in 24 h, following the compounds 34 (LC50 = 16.6 μg/mL, LC90 = 36.9 μg/mL), 35 (LC50 = 18.1 μg/mL, LC90 = 41.0 μg/mL), 36 (LC50 = 19.2 μg/mL, LC90 = 41.3 μg/ mL), 38 (LC50 = 28.4 μg/mL, LC90 = 46.0 μg/mL) and 37 (LC50 = 30.7 μg/mL, LC90 > 50.0 μg/mL) [46]. Similarly, 40-49 isolated from leaves of different Cinnamomum osmophloeum exhibit strong activity against Ae. aegypti larvae. CH3

H3C

CH3

H3C

CH2

H3C

CH3

H3C

CH3

H3C

CH3

H3C

CH3 38

CH3 39

CHO

OH CHO

COOH

HO 41

42 O O

OCOCH3

CH3

H2C

H3C H

CH3 CH3 49

CH3

H3C H H2C 47

46 O

CH3

H2C

CH3 CH3

H2C CH3

CH3 O 48

CH3

CH3 51

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Among these volatiles, benzaldehyde (40) 4-hydroxybenzaldehyde (41), benzenepropanal (42), cinnamic acid (43), cinnamyl alcohol (44), bornyl acetate (45), β-caryophyllene (46), caryophyllene oxide (47) and linalool (48) possess strong activities with LD50 value of 50 μg/mL while 49 with LD50 value of 33 μg/mL produce significant larvicidal effect [47]. Likewise, among the 2,2-dimethyl-6-vinylchroman-4-one (50) and 2-senecioyl-4vinylphenol (51) isolated from the roots of Eupatorium betonicaeforme, 50 shows efficient larvicidal potential, causing 84% larval mortality at a concentration of 12.5 μg/mL in compared to 51 exhibiting 40-100% mortality at 5-100 μg/mL concentrations [48]. The fatty acid constituents, linoleic acid (52) and oleic acid (53) isolated from Dirca palustris exhibit mosquitocidal activity against fourth instar Ae. aegyptii larvae with LD50 values of 100 μg/mL at 24 h, each [49]. H3C(H2C)6H2C H3C(H2C)4(HC

CHCH2)2(CH2)5CH2COOH

CH2(CH2)5CH2COOH C

H 53

7. Terpenes 7.1. Monoterpenes The monoterpenoids, thymol (54), cholorothymol (55), carvacrol (56), β-citronellol (57), cinnamaldehyde (58) and eugenol (59) isolated from a number of plant species possess mosquitocidal activity against forth instar larvae of Culex pipiens with LC50 values of 37.95, 14.77, 44.38, 89.75, 58.97 and 86.22 μg/mL, respectively. The N-methyl carbamate derivatives of 54-57, i.e. 60-63 display high toxicities against forth instar larvae of Cx. pipiens with LC50 values of 7.83, 11.78, 4.54, 15.90 μg/mL, respectively. Moreover, the N-methyl carbamate derivatives of geraniol (64) and borneol (65) also exhibit significant activity against forth instar larvae of Cx. pipiens with LC50 values of 24.08 and 33.00 μg/mL, respectively [50]. Likewise, 1,8-cineole (66) isolated from leaves of Hyptis martiusii display pronounced insecticidal effect against Ae. aegypti larvae at concentrations 25 (10%), 50 (53%), 100 (100%) mg/mL [51]. Other monoterpenoids, geranial (67) and neral (68) isolated from Magnolia salicifolia show 100% mortality with LD100 value of 100 ppm in 24 h against 4th instar Ae. aegypti [42].

Mosquitosidal natural products CH3

347

CH3

CH3 OH

OCH3

CHO

OH CH2

H3C

CH3

H3C

CH3 H3C

54 CH3

CH3

H3C

OCNHCH3

H3C

CH3

H3C

60 CH3

O OCNHCH3

H3C

CH3

CH3 CHO

O CHO

H3C 65

CH3 63

CH3

CH3 64

O OCNHCH3

OCNHCH3

H3C

CH3

O OCNHCH3

CH3

CH3 66

CH3

H3C 67

H3C

CH3 68

7.2. Sesquiterpenes The β-selinene (69) isolated from seeds of Apium graveolens show 100% mortality against fourth-instar larvae of Ae. aegyptii at a concentration of 50 μg/mL [41]. The pregeijerene (70), geijerene (71), and germacrene D (72) isolated from leaves of Chloroxylon swietenia possess activity against An. gambiae, Cx. quinquefasciatus and Ae. aegypti. The results of SAR indicate that 72 with LD50 values of 1.8, 2.1 and 2.8×10-3 exert highest activity followed by 70 with LD50 values of 3.0, 3.9 and 5.1×10-3 while 71 with LD50 values of 4.2, 5.4 and 6.8×10-3 display lowest activity against An. gambiae, Cx. quinquefasciatus and Ae. aegypti, respectively [52]. The sesquiterpene lactones, 73 and 74 isolated from leaves, stem bark, flowers and fruits of Magnolia salicifolia exhibit significant toxicity against Ae. aegypti larvae. The lactone 73 with LD100 value of 15 ppm kills all the mosquito larvae of Ae. aegypti in 24 h while 74 possess 100% mortality with LD100 value > 50 ppm in 24h [53]. The sesquiterpene, 74 does not show mosquitocidal activity at 50 ppm, thus suggesting the presence of a double bond rather than an epoxide at C-4 and C-5 in 73 is essential for mosquitocidal activity [42].

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CH3

CH2 CH2

CH2

CH3

CH2

CH3

CH3 71

70 CH3

CH3

H3C CH3

CH2

CH3

CH2 H3C

CH2 O O

7.3. Diterpenes Among the diterpenes, 75-77 isolated from Pterodon polygalaeflorus exhibit significant larvicidal activity against fourth-instar larvae of Ae. aegypti with LC50 values of 50.08, 14.69 and 21.76 μg/mL, respectively [54]. Similarly, hugorosenone (78) isolated from the Hugonia castaneifolia display larvicidal activity against mosquito larvae An. gambiae with LC50 values of 0.3028 and 0.0986 mg/mL at 24 and 48 h, respectively [55]. O

CH3

O O H3C

H CH3 OH 75

H3C

H CH3 OH 76

CH2 CH3

CH3

OH H3C

H CH3 OH 77

CH3

OCH3 HO H3C

H CH3 78

7.4. Triterpenes The triterpenes, 3β,24,25-trihydroxycycloartane (79) and beddomeilactone (80) isolated from Dysoxylum malabaricum and D. beddomei possess strong larvicidal, pupicidal and adulticidal activity and also affect the reproductive potential of adults by acting as oviposition deterrents. Among these, the 79 at a concentration of 10 ppm kills more than 90% of pupae and 85% of adults. Similarly, 80 at the same concentration results in more than 95% of pupal and larval mortality and more than 90% mortality in case of adult An. Stephensi [56].

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349 OH

H3C CH3 H2C

HO H3C

CH3

OH CH3

CH3

H CH3

H3C 79

CH3

H3C

CH3

H2C

CH3

H CH3

H COOH 80

7.5. Tetranortriterpenoids The limonin (81), nomilin (82) and obacunone (83) isolated from the seeds of Citrus reticulate [57] exhibit mosquitocidal activity against fourth instar larvae of Cx. quinquefasciatus at 59.57, 26.61 and 6.31 ppm concentrations, respectively [58]. The limonoids 84-86, isolated from the root bark of Turraea wakefieldii exhibit activity against late third or early fourth-instar larvae of An. gambiae. In SAR, the strong larvicidal activities of 84, 85 and 86 with LD50 values of 7.83, 7.07 and 7.05 ppm, respectively indicate that the epoxidation of the C-14, C-15 double bond or deacetylation of the 11-acetate group does not alter the larvicidal activity [59]. Other limonoids, azadirachtin (87), salannin (88), deacetylgedunin (89), 17-hydroxyazadiradione (90), gedunin (91) and deacetylnimbin (92) isolated from Azadirachta indica possess significant activity against An. stephensi larvae. Among these, 87 with EC50 value of 0.014, 0.021, 0.028 and 0.034 ppm, 88 with EC50 value of 0.023, 0.036, 0.047 and 0.061 ppm, 89 with EC50 value of 0.028, 0.041, 0.0614 and 0.078 ppm, 90 with EC50 value of 0.047, 0.054, 0.076 and 0.0104 ppm, 91 with EC50 value of 0.058, 0.073, 0.095 and 0.0117 ppm and 92 with EC50 value of 0.055, 0.067, 0.091 and 0.0113 ppm, show activity against first, second, third and fourth instar larvae of An. stephensi, respectively. The metabolite 87 exerts 100% larval mortality at 1 ppm concentration thus demonstrates that the A. indica (Neem) products may have benefits in mosquito control programs [60]. Likewise, 93-95 isolated from Turraea wakefieldii and T. floribunda exhibit toxicity against An. gambiae larvae with LD50 values of 7.1, 4.0, and 3.6 ppm, respectively and display more potency than azadirachtin (87; LD50 value of 57.1 ppm), a commonly used positive control [61].

350

Navneet Kishore et al. O O

CH3 O

OAc CH3

H3C

81 OAc OAc CH 3

CH3

H3C

H CH3

H3C

H3C 84

O H3C

CH3

H3C O

CH3

H3C O CH3

CH3

AcO

CH3

CH3OH

CH3 O H3C

CH3

CH3 OAc CH3

H3C

AcO OAc CH3

CH3

HO H3C

H O

(H3C)2HCOCO CH3 AcO OAc CH3 CH3

CH3

OAc CH3 94

H OH

O (H3C)2HCOCO

H O

CH3

OAc

H3C

CH2

CH3 O

OAc

CH3

O OH

H3C

CH3

OH CH3

O HO

CH3 O H 3C

H3C

CH3

H3C

OCH3

OAc

H CH3

H 3C

H3C

H3C AcO AcO

OAc OH CH3

H CH3

CH3

OAc CH3 OH O

O O

O H3C

CH3

CH3 H

CH3

OAc OAc CH 3

CH3 H

CH3

O O

CH3

O O

CH3

HO H3C

H O

OH CH3

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8. Alkaloids 8.1. Alkamides The alkamides, undeca-2E-4Zdien- 8,10-diynoic acid isobutylamide (96), undeca-2Z,4E-dien-8,10-diynoic acid isobutylamide (97), dodeca2E,4Z-dien-8,10-diynoic acid isobutylamide (98), undeca-2E,4Z-dien8,10-diynoic acid 2-methylbutylamide (99), dodeca-2E,4Z-dien-8,10diynoic acid 2-ethylbutylamide (100), and a mixture of dodeca2E,4E,8Z,10E-tetraenoic acid isobutylamide (101) and dodeca2E,4Z,8Z,10Z-tetraenoic acid isobutylamide (102) isolated from the dried roots of Echinacea purpurea and other plant species of family Asteraceae [62] display significant mosquitocidal activity against Ae. aegyptii. The mixture of 101 and 102 exert most effective mosquitocidal activity at 100 μg/mL concentration with 87.5% mortality of mosquito larvae in 15 min while 96 display 100% mortality at same concentration in 2 h. The alkamides, 97 and 98 exhibit 50% mortality at the end of 9 h with 100 μg/mL while 99 and 100 show least activity with 10% mortality at a concentration of 100 μg/mL in 24 h [63]. Among isobutyl amides, pellitorine (103), guineensine (104), pipercide (105), and retrofractamide-A (106) isolated from fruits of Piper nigrum exhibit toxicity against Cx. Pipiens larvae. The toxicities against Cx. pipiens larvae falls in the order: 105 (0.004 ppm)> 106 (0.028 ppm) > 104 (0.17 ppm) > 103 (0.86 ppm). These compounds also display larvicidal activity against Ae. aegypti larvae in which 106 exerts pronounced activity at a concentration of 0.039 ppm than 105 (0.1 ppm), 104 (0.89 ppm) and 103 (0.92 ppm). Also, the amides 105, 106 and 104 exhibit 255, 31 and 5 times more toxicity than 103, respectively. The SAR indicates that the N-isobutyl amine moiety might play a crucial role in the larvicidal activity, but the methylenedioxyphenyl moiety does not appear essential for toxicity [64].

8.2. Carbazole alkaloids Among carbazoles, mahanimbine (107), murrayanol (108) and mahanine (109) isolated from leaves of Murraya koenigii display promising mosquitocidal activity against Ae. Aegyptii [65]. The alkaloid 107 exhibits 100% mortality at a concentration of 100 μg/mL while 108 and 109 at 12.5 μg/mL concentration display 100% mortality [66,67].

352

Navneet Kishore et al. O 96

H2 C CH2

C NH C

C H

H2 C CH2

C C

CH3 O

H3C

H H

O C

C C

H2 C

N H

H2 C CH2

101 H

H C

102

C H

CH3 CH3

C H2C CH2

H H

C C

103

H2 C CH

H C

H3C

H N

H H

CH3

H2C H2 C CH

CH2

H2C

H C

N H

H H3C

C C

H N

H3C O

CH3

O H3C

H2C

100

CH3 CH3

H2 C CH2

H2 C CH

H 99

H N

C C

CH3

H2 C CH

H N

H2 C CH2

CH3 CH3

H2 C CH

H N

H2 C CH

CH3 CH3

O CH3 CH3

CH3

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O 104

CH3

H N

CH3

H N

105 O

CH3

O O

CH3

H N

106 O

CH3

O H3C

CH3 R

N H

H3CO CH3

CH3

OH CH3

N H

CH3 CH3

CH3 108

107: R = H 109: R = OH

8.3. Naphthylisoquinoline alkaloid The alkaloid, dioncophylline-A (110) isolated from Triphyophyllum peltatum [68] possess promising activity against different larval stages of An. stephensi with LD50 values of 0.5, 1.0 and 2.0 mg/L concentrations at 3.33, 2.66 and 1.92 h, respectively. In each instar larval stage, the LC50 values decrease as a function of time indicating that 110 continues to exert its action during at least 48 h [69]. CH3 CH3 N OH

CH3

H3CO H3CO

110

8.4. Piperidine alkaloids The alkaloid, pipernonaline (111) isolated from fruits of Piper longum exhibits activity against the fourth-instar larvae of Ae. aegypti [70] and Cx. Pipiens [71] with LC50 values of 0.25 and 0.21 mg/L, respectively in 24 h.

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The larvicidal potential of 111 against the Ae. aegypti, is comparable to that of pirimiphos-methyl, a commonly used insecticide and may be useful for development of new mosquito larvicides [70]. Similarly, N-methyl-6β-(decal',3',5'-trienyl)-3-β-methoxy-2-β-methylpiperidine (112) isolated from stem bark of Microcos paniculata shows significant insecticidal activity against second instar larvae of Ae. aegypti with MC50 value of 1.0 ppm and LC50 value of 2.1 ppm at 24 h against second instar larvae of Ae. aegypti [72]. O O

111 O O CH3 112

H3C

CH3

Insecticidal activity evaluation of piperidine derivatives (113-145) against female adults of Ae. aegypti, along with the structure-activity relationships (SAR) using piperine (E,E)-1-piperoyl-piperidine as standard insecticide (LD50 value of 8.13 μg per mosquito) reveals that different moieties (ethyl-, methyl-, and benzyl-) attached to the piperidine ring are responsible for different toxicities (i.e. 113, 1.77; 114, 2.74; 115, 8.76; 116, 1.20; 117, 1.09; 118, 1.13; 119, 4.14; 120, 1.92; 121, 2.07; 122, 1.80; 123, 4.90; 124, 4.25; 125, 2.63; 126, 6.71; 127, 1.22; 128, 1.67; 129, 0.94; 130, 1.56; 131, 1.83; 132, 0.84; 133, 29.20; 134, 14.72; 135, 19.22; 136, 12.89; 137, 0.80; 138, 1.38; 139, 3.59; 140, 1.32; 141, 2.07; 142, 7.43; 143, 1.54; 144, 2.72 and 145, 14.72 μg) against Ae. aegypti. The 3-methylpiperidines (119-122) exhibit slightly lower toxicities than that of 2-methyl-piperidines (113-118) with LD50 values ranging from 1.80 to 4.14 μg. However, there is no significant difference found between the toxicities of 3-methyl piperidines (119-122) and 4-methyl piperidines (123127), whose LD50 values range from 1.22 to 6.71 μg while the saturated long chain derivatives of 4-methyl-piperidine (123 and 126) show lower toxicity than others with LD50 values of 4.90 and 6.71 μg, respectively [73]. Further, SAR among the piperidines with two different moieties (ethyl- and benzyl-) attached to the carbons of the piperidine ring against Ae. aegypti establishes that 2-ethyl-piperidines (128-132) show higher toxicity than the benzylpiperidines (133-136) with LD50 values ranging from 0.84-1.83 and 12.8929.20 μg, respectively. The results of SAR suggest that ethyl-piperidines

Mosquitosidal natural products

355 O

O R

H3C

O CH3

CH3

119

O N

121

O N

CH3

122

CH3

125

O N

CH3

H3C

127

O N

CH3

129

130

O C8H17

CH3 132

H3C

N 134

H2C

135

136

O H2C

N 137 R = CH3 139 R = C6H5

N 138

H3C O

O H2C

H2C

N 141

140

CH3

O H2C

N CH3 143

O N

144

CH3

H2C

142

H2C

CH3 131

133

CH3

124

CH3

O (H2C)2

CH3

128

H3C 118

CH3

120

123 R = C9H19 126 R = C11H23

H3C

117

C6H13

H3C

114 R = C9H19 115 R = C11H23 116 R = C6H13

113

O N

H2C CH3

N 145

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Navneet Kishore et al.

generally exhibit higher toxicities than methyl-piperidines, followed by benzyl-piperidines whose toxicities are lowest. Among the three 1-undec-10-enoyl-piperidines (134-139) with the three different moieties at the second carbon of the piperidine ring, the 137 displays highest toxicity with LD50 value of 0.80 μg, in compared to 138 (LD50 value of 1.38 μg) and 139 (LD50 value of 3.59 μg). Similarly, among the three 1-undec-10-enoyl-piperidines (140-142) with the three different moieties attached to the third carbon of the piperidine ring, the 140 exhibit highest toxicity (LD50 value of 1.32 μg), followed by the 141 and 142 with LD50 values of 2.07 and 7.43 μg, respectively. Likewise, among the three 1-undec-10-enoyl-piperidines (143-145) with the three different moieties attached to the fourth carbon of the piperidine ring, the 143 shows highest toxicity (LD50 value of 1.54 μg), following 144 (LD50 value of 2.72 μg) and 145 (LD50 value of 14.72 μg).

8.5. Stemona alkaloids The Stemona alkaloids, stemocurtisine (146), stemocurtisinol (147) and oxyprotostemonine (148) isolated from roots of Stemona curtisii exhibit potency against mosquito larvae An. minimus with LC50 values of 18, 39 and 4 ppm, respectively. Among these, 148 display highest potency with LC50 value of 4 ppm [74]. H

H H3C

H H

OCH3 H3C

H N

H3C O

147

146 CH3 H HC OCH3 3

O N

H3C O

OCH3H3C

O 148

O N

CH3 OH

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9. Phenolic derivatives 9.1. Naphthoquinones The cordiaquinones (149-152), isolated from the roots of Cordia curassavica show toxic properties against larvae of the yellow fever-transmitting Ae. aegypti. The quinones 149 and 151 with 25.0 μg/mL concentration result in 100% larval mortality while 150 and 152 with 12.5 μg/mL concentrations kill all the Ae. aegypti larvae in 24 h [75]. Likewise, the alkaloids 153-155 isolated from the roots of Cordia linnaei exhibit larvicidal potency against Ae. aegypti at 12.5, 50.0 and 25.0 μg/mL concentrations, respectively [76]. The naphthoquinone, plumbagin (156) isolated from Plumbago zeylanica [77] and other plant species [78,79] exhibit mosquito larvicidal activity against An. gambiae with LC50 value of 1.9 μg/mL [80,81]. Some of natural and synthetic naphthoquinones e.g. lapachol (157) and its synthetic derivatives (158-160) possess toxicity against fourth instar larvae of Ae. aegypti. The quinone 159 with LC50 value of 15.24 μM exerts higher activity in compared to 160 (19.45 μM), 158 (33.94 μM) and 157 (108.7 μM). Likewise, juglone (161) and its synthetic derivatives (162-170) display significant toxicity against fourth instar larvae of Ae. aegypti. The bromonaphthoquinone 167 with LC50 value of 3.46 μM exhibits the best larval toxicity in compared to 164 (4.64 μM), 165 (3.98 μM), 166 (36.48 μM), 167 (3.46 μM), 168 (24.79 μM) and 169 (21.62 μM) while 161 and derivatives 162, 163 and 170 display relatively weak toxicity with LC50 values of 20.61, 21.08, 42.12 and 86.93 μM, respectively [82]. The shikonin (171), alkannin (172) and shikalkin (173) isolated from root of Lithospermum erythrorhizon [83], Alkanna tinctoria [84] and young leaves and stems of L. officinale [85] exhibit toxicities against mosquito larvae. The quinone 171 at a concentration of 3.9 mg/L show high toxicity against mosquito larvae followed by 173 and 172 with 8.73 and 12.35 mg/L concentrations, respectively. Results of SAR indicate that naphthoquinones, compared with other natural compounds with larvicidal activity, are very toxic against mosquito larvae and would be a potential source of natural larvicidal substances [86].

9.2. Coumarins Coumarin, pachyrrhizine (174), isolated from Neorautanenia mitis exhibits activity against An. gambiae adults with LC50 value 0.007 mg/mL. The marmesin (175), isolated from Aegle marmelos exhibits toxicity against An. gambiae adults with LC50 and LC90 values of 0.082 and 0.152 mg/L, respectively [87].

358

Navneet Kishore et al. CH3

H3C

CH3 O O CH3

H3C O

CH3 O

149

O CH3

H3C

150

O CH3

CH3

CH3 H2C O

151

CH3

153

CH3 CHOH

CH3

H3C O O

152

H3C

CH3

154

CH3

H3C

OH O

O O

155

OAc

CH3

159

CH3

158

157

OLi

CH3

CH3 O

OH O

156

O Br

CH3 CH3

OH O

OAc O

161

162

160

OCH3O

OH O

163

164

Br OAc

OCH3 O

165

166

OCH3 O

168

169

OAc

167

Br O

CH3 CH3

CH3 O 170

O 171

CH3 OH 172

CH3

CH3 OH

O 173

CH3

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H3C O

H3C

OCH3

O 175

174

9.3. Isoflavonoids The isoflavonoids neotenone (176), neorautanone (177) isolated from Neorautanenia mitis display activity against adult An. gambiae mosquitoes with LD50 values of 0.008 and 0.009 mg/mL, respectively [88]. O

OCH3 OCH3 OCH3

OCH3

O 177

176

9.4. Pterocarpans The pterocarpans, neoduline (178), 4-methoxyneoduline (179), and nepseudin (180) isolated from tubers of Neorautanenia mitis exhibit mosquitocidal activity against An. gambiae and Cx. quinquefaciatus larvae with LD50 values 0.005, 0.011 and 0.003 mg/mL, respectively [87,89]. O

O O

O H

H O

178

H3CO

OCH3

179

180

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Navneet Kishore et al.

9.5. Lignans The lignans, conocarpan (181), eupomatenoid-5 (182), eupomatenoid-6 (183) and decurrenal (184) isolated from Piper decurrens possess significant mortality at 10 μg/mL concentrations against mosquito larvae [90]. O

OCH3

O OH

H3C 181 O H

H3C

CH3

182

CH3

O OH

OH C O

H 183

CH3

H3C

CH3 184

10. Conclusive remarks Our ancestors exclusively depended on the use of plant-derived products to repel or kill mosquitoes and other blood sucking insects. Modern synthetic chemicals could provide immediate results for the control of insects/mosquitoes; on the contrary they bring irreversible environmental hazard, severe side effects and pernicious toxicity to human being and beneficial organisms. In concern to the quality and safety of life and the environment, the emphasis on controlling mosquito vectors has shifted steadily from the use of conventional chemicals toward alternative insecticides that are target-specific, biodegradable, and environmentally safe, and these are generally botanicals in origin. Therefore, right now use of eco-friendly and cost-free plant based products for the control of insects/mosquitoes is inevitable. Efforts should be made to promote the use of easy accessible and affordable traditional insect/mosquito repellent plants.

Acknowledgement The author sincerely acknowledged Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, India, for infrastructural facilities.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 367-383 ISBN: 978-81-308-0448-4

12. Soybean constituents and their functional benefits Ajay K. Dixit1, J. I. X. Antony1, Navin K. Sharma1 and Rakesh K. Tiwari2 1

Biosciences, ITC R & D Center, Peenya Industrial Area, Peenya, Banglore-560058, India Dept. Biomed. & Pharma. Res., University of Rhode Island, Kingston, RI 02881 0809, USA

Abstract. Soybean [Glycine max (L.)] is in use for more than 5000 years in China and South East Asia as food. Epidemiological studies show its importance in prevention of several diseases. Recently, an upsurge of consumer interest in the health benefits of soybean and soy products is not only due to its high protein (38%) and high oil (18%) content, but also due to the presence of physiologically beneficial phytochemicals. Past several years of clinical and scientific evidences have revealed the medicinal benefits of the soy components against metabolic disorders (cardio-vascular diseases, diabetes and obesity etc.) as well as other chronic diseases (cancer, osteoporosis, menopausal syndrome and aneamia etc.). Many of the health benefits of soy are derived from its secondary metabolites, such as, isoflavones, phyto-sterols, lecithins, saponins etc. In this review we discuss the bioactive components of soybean and their role in prevention, maintenance, and/or curing of diseases.

1. Introduction The term ‘functional foods’ was first introduced in Japan in the mid1980s and refers to processed foods containing ingredients that aid specific bodily functions in addition to being nutritive [1]. The soybean [Glycine max(L.) Merrill] a native of China, have been extensively used as important Correspondence/Reprint request: Dr. Ajay K. Dixit, Biosciences, ITC R & D Center, Peenya Industrial Area Peenya, Banglore-560058, India. E-mail: ajay.dixit@itc.in

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source of dietary protein and oil throughout the world. Though, soybean is a widely cultivated crop, most of it is used as the raw material for oil milling, and the residue (soy meal) is mainly used as feedstuff for domestic animals [2]. Dry soybean contain 36% protein, 19% oil, 35% carbohydrate (17% of which dietary fiber), 5% minerals and several other components including vitamins [2]. Several years of rigorous scientific and clinical research has established that most of the components of soybean have beneficial health effects as characterized by its preventive potential for the so-called life-style-related diseases. The impact of most of the nutritionally and physiologically functional components of soybean [3] have been summarized in Table 1. Table 1. Functional components of soy and their impact [3].

α-Linolenic acid Isoflavones

Lecithins Lectins Linoleic acid Peptides Phytosterols Protein Saponin

Essential fatty acid, hypotriglyceridemic, improves heart health Estrogenic, hypocholesterolemic, improves digestive tract function, prevents breast, prostate, and colon cancer, bone health, improve lipid metabolism Improve lipid metabolism, improve memory and learning abilities Anti-carcinogenic, immunostimulator Essential fatty acid, hypocholesterolemic Readily absorbed, reduce body fat, anticancer Hypocholesterolemic, improves prostate cancer Hypocholesterolemic, antiatherogenic, reduces body fat Regulates lipid metabolism, antioxidant

2. Constituents of soybean 2.1. Proteins Soybean contains 35–40% protein on a dry-weight basis, of which, 90% is comprised of two storage globulins, 11S glycinin and 7S β-conglycinin [2,4]. These proteins contain all amino acids essential to human nutrition, which makes soy products almost equivalent to animal sources in protein quality but with less saturated fat and no cholesterol. Soybean also contains the biologically active protein components hemagglutinin, trypsin inhibitors, α-amylase and lipoxygenases [2]. As per the FDA’s ‘Protein Digestibility Corrected Amino Acid’ source method, soybean is not only high quality protein, but it is now thought to play preventive and therapeutic roles for several diseases [5].

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2.2. Oil Soybean contains roughly ~19% oil, of which the triglycerides are the major component. Soy oil is characterized by relatively large amounts of the polyunsaturated fatty acids (PUFA), i.e., ~55% linoleic acid and ~8% α-linolenic acid, of total fatty acids [6] (Fig. 1). Linoleic acid in soy oil is an essential fatty acid (EFA) belonging to the ω-6 family of PUFAs, which exerts important nutritional and physiological functions. Even the α-linolenic acid is also an EFA belonging to ω-3 fatty acid family, and plays an important role in the regulation of a number of metabolic pathways. However, due to the presence of lipoxygenases in soybean, linoleic acid renders the soybean oil prone to rancidification [2]. The minor components of crude soybean oil are phospholipids, collectively called lecithin, as well as phytosterols, and tocopherols. O HO Linoleic acid O HO α-Linolenic acid Figure 1. Two EFAs present in soy oil.

2.3. Carbohydrates Soybean contains ~35% carbohydrates, most of which is nonstarch polysaccharides. It also contains oligosaccharides [5] such as, stachyose (4%), and raffinose (1.1%). Stachyose is a tetraose with a galactosegalactose-glucose-fructose structure, while raffinose is a triose with a structure of galactose-glucose-fructose. Polysaccharides are composed mainly of insoluble dietary fiber. Soybean curd refuse (Okara) contains soluble polysaccharides with galacturonic acid as its underlying structure. In addition to use as a dietary fiber supplement, soluble polysaccharides have been used to modify the physical properties of various foods [7].

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2.4. Vitamins and minerals Soybean is a better source of B-vitamins [2] compared to cereals, although it lacks B12 and vitamin C. Soybean oil also contains tocopherols [2,3], which are excellent natural antioxidants. Soybean oil contains α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol in trace amount (mg/kg). Soybean also contains ~5% minerals [3]. It is relatively rich in K, P, Ca, Mg, and Fe. Soy ferritin can supplement reasonable quantities of iron.

2.5. Isoflavones Isoflavones is a sub-group of heterocyclic plant phenolic category called flavonoids. Besides isoflavones, the other subclasses of flavonoids include flavones, flavonols, flavanols, aurones, red and blue anthocynin pigments, and chalcones. In isoflavones the phenyl ring B is connected at position 3 of 1,4-benzopyrone ring (Fig. 2). The soybean is most abundant source [8] of isoflavones (up to 3 mg/g dry weight) in the nature. Soybean contain three types of isoflavone aglycone viz., daidzein, genistein and glycitein; each of them present in three glycosidic forms in addition to their aglycone form (Fig. 3). Daidzein, genistein and their glycosides contribute to >90% of total isoflavone; whereas glycetein and its glycoside are present as minor component (<10%), only. Isoflavones are structurally similar [6] to mammalian estradiol as shown in Fig. 4, and can bind to both α and β isoforms of estrogen receptor (ER), thus called phytoestrogens. Though, the isoflavones are not essential nutrients that are required to support life, still they exert many beneficial health effects, therefore, are of immense help for maintaining healthy life.

8 7

A 6

4' 5'

Figure 2. Basic structure of isoflavones.

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Figure 3. Chemical Structures of 12 isoflavones found in soybean. OH

O Isoflavone (Equol)

O Estrogen (Estradiol)

Figure 4. Comparison of equol (isoflavone metabolite) and estradiol structure.

2.6. Phytosterols Soybean oil contains about 300 to 400 mg of plant sterols per 100 g. The major components of soy sterols are β-sitosterol (53 to 56%), campesterol (20 to 23%), and stigmasterol (17 to 21%) [9]. These phytosterols differ from cholesterol only in the structure (Fig. 5) of their side chains; sterols differ from stanols in being unsaturated versus saturated at the C5-C6 double bond in their B ring. These sterols are proven to have cholesterol-lowering activity, though the mechanism is not completely understood [10].

2.7. Phospholipids Soybean oil contains 1-3% phospholipids [2,3], of which ~35% phosphatidyl choline, ~25% phosphatidyl ethanolamine, ~15% phosphatidyl inositol, ~5-10% phosphatidic acid. The phospholipids are removed from the

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HO HO

β -sitosterol

Campesterol

HO Stigmasterol

Cholesterol

Figure 5. Soy phytosterols and their structural similarity with cholesterol. Table 2. Soy phospholipids and their structure. O RO R'O

O O

O O P OR'' R and R' O same or different fatty acids O

Name Phosphatidyl choline Phosphatidyl ethanolamine Phosphatidyl inositol Phosphatidic acid

R’’ -CH2CH2NH3 -CH2CH2N(CH3)3 (CH(OH))6 H

oil mainly during the ‘degumming’ process and are used as a natural food emulsifier. They are polar lipids and contribute to the structure of cell membrane. The structure of soy phospholipids are given in the Table 2.

2.8. Saponins Soybean also contains ~2% soy saponins (triterpene glycosides) which are currently attracting lot of scientific attention. Soybean saponins have unique chemical structures and physiological functions. Soy saponins are oleonane type triterpene glycosides. They can be classified according to type

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of aglycon, the moiety attached at the C-22 position on the aglycon, and the carbohydrate sequences at the C-3 position on the aglycon [11,12]. A representative structure is shown in Fig. 6. So far, total 30 soy saponins are reported, but their presence and quantity differ from genetic and agronomical variation. Soy saponins are found to have several biological activities [13] such as hepatoprotective, anti-hyperlipidemic, anti-cancer, anti-oxidative, and anti-HIV etc.

R1 21 22

HOOC

O O

CH2OH

HO OH O O

Group A saponin

Disaccharide

Group B saponin

Group E saponin

DDMP saponin

Maltol

CH3 OH OH

Figure 6. The chemical structure and types of soy saponins.

2.9. Ferritins Soybean contains ferritin, a multimeric iron storage protein [14]. It is now well proven that the iron from soybean ferritin is as much absorbed and bio-available as much it is from the animal products [14]. Therefore, soybean is recommended to be incorporated in the diet of people suffering from anemia.

3. Health benefits of soybean The health effects of soy components have been extensively studied through human clinical trials, experimental animal studies, and in vitro cell culture studies. Going further, we will confine our discussion to various

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scientifically validated/supported heath benefits of soybean consumption to ameliorate various human health issues, though there are few studies which present less promising and unfavorable role of soybean in human health.

3.1. Soybean and cardiovascular disease Cardiovascular disease (CVD) includes all diseases that affect the heart and blood vessels, such as coronary heart disease (CHD), coronary artery disease, dyslipidemia, and hypertension [15]. Looking at present scenario CVD has become one of the major health problems around the world including developing countries. The role of soy in the prevention of CVD, particularly LDL cholesterol– lowering effects, has been the subject of numerous controlled clinical studies [15]. In 2006, a study [16] reported findings from a 1-year trial in which 66 individuals who adhered well to the portfolio diet (31.8% of participants) experienced reduced serum LDL cholesterol levels by 29.7%. In response to increased interest and the expanding body of knowledge in soy and health [17], the U.S. Food and Drug Administration (FDA) approved in 1999 a health claim for use on food labels [18] which stated that daily diet containing 25 g/day of soy protein, which is also low in saturated fat and cholesterol, may reduce the risk of heart disease. Modest reductions in serum LDL cholesterol levels have been achieved with soy intake, especially for subjects with hypercholesterolemia [15]. Soy protein consumption in several human controlled clinical trials ranged between 14 and 113 g/day (with a median of 36 g/day). The beneficial effects which have been documented include decreased low-density lipoprotein (LDL) concentrations, triglycerides, lipoprotein, C-reactive protein, homocysteine, oxidized LDL, and blood pressure, and increased high-density lipoprotein (HDL) concentrations [17,19-24]. In these studies, the amount of isoflavones prescribed was up to 185 mg/day with a median of 80 mg/day. The lack of understanding of the mechanism remains an obstacle for a better acceptance of soybean protein by clinical community. There are different hypothesis to explain these mechanisms. One of these hypothesis is that amino acid composition or distribution in soybean change the cholesterol metabolism, possibly, due to changes in endocrine status, because there are alterations in insulin: glucagon ratio and thyroid hormone concentrations [25], as well as an increase in plasma thyroxin concentrations which is related to reduced plasma cholesterol [26]. Another hypothesis proposes that nonprotein components such as saponins, fibre, phytic acid, minerals and isoflavones associated with soybean protein affect cholesterol metabolism. Several in vitro and in vivo studies have shown favourable effects of

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isoflavones in a variety of atherosclerosis models [27]. These include the reduction of homocysteine levels in plasma [28], prevention of LDL oxidation [29], improvement of vascular reactivity [30], inhibition of proinflammatory cytokines, or cell adhesion proteins [31,32], inhibition of reactive nitrogen species [33], as well as reduction of platelet aggregation [34]. Legumes are relatively rich in soluble fiber, which may play an important role in the prevention of heart disease [35]. The major effects of soybean soluble fibers on serum lipoproteins appear to be related with bile acid binding and with a decrease in the reabsorption of bile acid [36]. Therefore, there is an increase in the cholesterol used to synthesize bile acids [37]. Also the fermentation of soluble fibers in the colon produces short-chain fatty acids that contribute to reduce hepatic cholesterol synthesis [38]. It has been shown that propionic acid, one of the short-chain fatty acids, decreases the hepatic cholesterol [39]. Moreover, the attenuation in the synthesis of cholesterol in the liver leads to reduction in serum insulin concentrations, which in turn, reduces the activation of an enzyme that participates in cholesterol synthesis. Perhaps, on the other hand, it might also be due to an alteration of the bile acid profile in the liver [37]. There is also a hypothesis that isoflavones may inhibit atherosclerotic development, because they have antioxidant properties against LDL oxidation, which generates a cascade of events producing atherosclerotic plaques. Additionally, isoflavones possess a hypocholesterolemic effect, due to the interaction of isoflavones with estrogenic receptors. Serum cholesterol concentrations may decrease by this mechanism, also. Another component of soybean is phytosterols. The mechanism underlying the capability of plant sterols/stanols to reduce plasma LDL cholesterol levels relates to their structural similarity to cholesterol (Fig. 5). This enables them to compete with cholesterol for incorporation into micelles, which are translocated over the brush border membrane, from the gut into the plasma, via intestinal cholesterol transporters, known as NPC1L1 and SR-BI [40,41]. Moreover, free phytosterols are assumed to be taken up by enterocytes directly and to prevent cholesterol from being esterified by blocking the ACAT system and subsequent transport to the mesenteric lymph. Free sterols/stanols are hypothesized to stimulate the ABC-ATP binding cassette mediated enterocytic cholesterol transfer back to the intestinal lumen for excretion from the body. Consequently, sterols/stanols will significantly down regulate the cholesterol influx and stimulate the efflux over the brush border membrane [42]. In brief, various components of soybean might be contributing independently towards its overall cardiac health benefits. To overemphasize the importance of one and/or another component may not be able to justify the total effect of soybean on cardiovascular system.

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3.2. Soybean and cancer In the last two decades, many groups of researchers have suggested that the regular consumption of soybean is associated with the relatively lesser risk of different cancers in countries that include soybean in their diets [6,43,44]. Researchers have evaluated dietary differences between Japan and the Western nations to try to explain variations in death rates from cancer [45]. A number of soy components have been investigated for potential anticancer activity. Soybean contains several components with anticancer activity, such as, isoflavones, protease inhibitors, phytosterols, saponins, phenolic acids, and phytates. Most of the data support that predominantly isoflavones are responsible for the anticancer effects of soybean [6,43,45]. Based on the estrogenic activity of isoflavones, they can possibly be used for prevention and treatment of hormone dependent cancers [47]. Prevailing hypothesis is that isoflavones may act like antiestrogen when they are in a high estrogen concentration, and like estrogen when they are in a low estrogen environment. Genistein, one of the two primary isoflavones, may be contributing its anticancer effects due to its very good antioxidant properties. The anticancer effects of genistein are also due to the fact that it is a specific inhibitor of protein tyrosine kinase, MAP kinase, ribosomal S6 kinase, topoisomerase II, which form part of growth factorstimulated signal transduction cascades in normal and transformed cancer cells. It has also been proved in vitro, that genistein increases concentrations of TGF-β, which may inhibit the growth of cancer cells. Moreover, genistein has an important role as a potent inhibitor of angiogenesis in vitro [43]. Japanese people with high phytoestrogens (isoflavone) plasma levels, have low incidence of breast, prostate, and colon cancer. This also indicates the safety aspect of isoflavones and soybean consumption [47]. 3.2.1. Breast cancer Several studies suggest that consumption of soy foods during childhood and adolescence in women reduces the risk of breast cancer in later part of life. The growth of both estrogen-dependent and estrogen independent breast cancer cells in vitro has been inhibited by genistein, but it is not clear if the concentrations reached in vitro could be reached in vivo. Studies have shown that soybean intake may help in preventing the initiation of breast cancer cells [6,43].

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3.2.2. Prostate cancer It is known that estrogens cause programmed cell death of prostate cancer cells and inhibit enzymes associated with different processes in the development of cancer [47]. Soybean foods may be a factor contributing to the diminution of prostate cancer mortality of the soy consuming populations. Genistein has been shown to reduce DNA synthesis in human prostate cells in vitro and inhibit testosterone effect in prostate cancer development in rats [48]. Prostate cancer is also reported to be associated with increased levels of dihydrotestosterone, and soybean isoflavones are known to inhibit 5α-reductase, which is involved in the conversion of testosterone to dihydrotestosterone [49]. However, the mechanism by which soybean may prevent prostate cancer still remains unclear and controversial [50]. 3.2.3. Colon cancer Epidemiological evidences show protective effects of soybean products on colon cancer. In vitro studies on soybean products have shown an antiproliferative effect even on cells of gastrointestinal tract among the various other cell types [51]. An important role in colon cancer is attributed to dietary fiber from soy, which also reduces the risk of other chronic diseases in digestive system. The fermentation of soy fiber in colon produces an increase of short-chain fatty acids that present a potential protective effect against colon cancer and bowel infections through inhibition of putrefactive and pathogenic bacteria, respectively [52]. Nonetheless, scientific evidence in support of the protective effect of soybean isoflavones on colon cancer is limited [47].

3.3. Soybean and menopause Menopause is a natural stage of life all women experience as they age. Thermoregulatory disturbances like hot flashes (HF), night sweats, mood swings and lack of energy can make menopause one of the most physically and emotionally miserable times in a woman's life [53]. HF arises as a sudden feeling of heat in the face, neck, and chest [54]. Menopausal hormone therapy (MHT) is the most effective therapy for vasomotor symptoms [55]. However, current data have indicated adverse effects of MHT by increasing the risk of e.g. stroke, breast cancer and gallbladder disease [56]. Dietary soy has gained much attention since reports of reduced menopausal discomfort and reduced morbidity incidence of several hormone-

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dependent diseases in soy consuming Asian compared with non-soy consuming Western populations. Epidemiological studies in Japanese women suggest that consumption of soy products has a protective effect against menopausal symptoms [57-59]. In Japan, the total isoflavone intake from soy food averages from 25 to 50 mg per day. A small proportion of the Asian population (<10%) seems to consume more than 100 mg isoflavones per day [60]. Isoflavones bind to the estrogen receptors in certain cells in the body and produce weak estrogenic effects, especially when an inadequate amount of estrogen is present in the body [61]. Over 20 human studies have tested the hypothesis that soy products alleviate post menopausal symptoms [62,63]. In these studies, perimenopausal women as well as postmenopausal women who suffered menopausal symptoms consumed soy proteins for 4 weeks or longer. These studies show that the soy isoflavone do have the beneficial health effects on menopausal symptoms. In summary, though the available human studies seem to show a few conflicting results in terms of the consistent efficacy of soy isoflavones in alleviating post menopausal symptoms, from the epidemiological studies it can be stated that isoflavone do help the regular soybean consumer to better manage their post menopausal symptoms.

3.4. Soybean and osteoporosis The loss of estrogen during menopause puts women at greater risk to develop weaker bones and joints as they age. Menopause leads to rapid decrease in estrogen levels, which causes bone breakdown and loss of more calcium via urine. Over time, bones may become weak and brittle with tiny holes inside, this condition is called osteoporosis. Phytoestrogens help prevent osteoporosis in the presence of subnormal endogenous estrogen [64]. Studies in Asia reveal that women in Shanghai, China, who ate sumptuous amount of soy foods, were onethird less likely to experience a fracture than Chinese women who consumed lower amount of soy [65]. This observation has led to the hypothesis that soybean or soybean isoflavones are a possible alternative option for the prevention of osteoporosis. Randomized controlled studies [66,67] that used isoflavone extracts or pure genistein reported that soy isoflavones have a mild but significant and independent effect on the maintenance of bone mineral density at doses ranging between 35 to 54 mg of aglycone equivalent. The mechanism of isoflavones on bone health is yet to be understood.

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3.5. Soybean and diabetes Soybean diet may be a good option in type 2 diabetes individuals due to its effect on hypertension, hypercholesterolemia, atherosclerosis and obesity, which are very common diseases in diabetic patients [68]. Furthermore, substituting animal protein for soybean or other vegetable protein may also decrease renal hyperfiltration, proteinuria, and renal acid load and therefore reduces the risk of renal disease in type-2 diabetes [69]. Soluble fiber from soybean may be useful because of its insulin-moderating effect. It is generally accepted that a high fiber diet, particularly soluble fiber, is useful to control plasma glucose concentration in diabetics. Soybean fiber intake has also been implicated for the improvement of the blood glucose levels of diabetics [43]. It also increases fecal excretion of bile acid and therefore may cause a low absorption of fat [20,69]. Though further research is needed, it can be suggested that diabetic patients with soybean diets show several potential advantages, such as, reduced insulin resistance, renal damage, and fatty liver, thereby improving their quality of life.

3.6. Soybean and obesity Obesity poses a major public health challenge since it is a well recognized independent predictor of premature mortality [70]. The dramatic increase in the occurrence of overweight and obesity over the past several decades is attributed in part to changes in dietary and lifestyle habits, such as rapidly changing diets, increased availability of high-energy foods, and reduced physical activity of peoples in both developed and developing countries [71]. Ingestion of foods with high protein content is well known to suppress appetite and food intake in humans [72]. Several nutritional intervention studies in animals and humans indicate that consumption of soy protein reduces body weight and fat mass in addition to lowering plasma cholesterol and triglycerides. In obese humans, dietary soy protein also reduces body weight and body fat mass in addition to reducing plasma lipids [73]. Several lines of evidence suggest that soy protein may favorably affect lipid absorption, insulin resistance, fatty acid metabolism, and other hormonal, cellular, or molecular changes associated with adiposity. It is well established that soy protein consumption reduces serum total cholesterol, LDL cholesterol, and triglycerides as well as hepatic cholesterol and triglycerides. Studies in animals indicate that soy protein ingestion exerts its lipid-lowering effect by reducing intestinal cholesterol absorption and

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increasing fecal bile acid excretion, thereby reducing hepatic cholesterol content and enhancing removal of LDL [74,75]. Dietary soy protein has also been shown to directly affect hepatic cholesterol metabolism and LDL receptor activity [76].

4. Conclusion Several nutritional advantages could be obtained by incorporating soybean based foods in the diet. Soybean represents an excellent source of high quality protein with a low content in saturated fat, with no cholesterol, and a great amount of dietary fiber. Therefore, the possible use of soybean in functional food design is very promising, since the consumption of soybean protein and dietary fibre seems to reduce the risk of cardiovascular diseases and to improve glycemic control. Furthermore, soybean and several of its components have shown in various in vitro, in vivo, and human clinical studies their effectiveness and potential role in the prevention and treatment of different diseases. The use of soybean in food form for several centuries assures us of its safety and nutritive value for human health. Consequently, it is imperative for all the conscious societies to incorporate this abundantly available ‘treasure of functionality’ in their daily diet and harness the complete benefit of this yellow ‘miracle’ seed.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 385-409 ISBN: 978-81-308-0448-4

13. Mutasynthesis of medicinally important natural products through manipulation of gene governing starter unit Deepak Sharma, Syed Khalid Yousuf and Debaraj Mukherjee Natural Product Chemistry (Microbes), Indian Institute of Integrative Medicine Jammu-180001, India

Abstract. Many of our most valuable drugs are natural products or are, in some way, inspired by their structures. However, the complex molecular architecture that lies behind the biological activity makes it extremely difficult and time consuming to modify these metabolites by semisynthesis, or to assemble analogues in stereospecific way from scratch. One way to circumvent this problem is mutasynthesis, which couples the power of chemical synthesis with molecular biology to generate derivatives of medicinally important natural products. Mutasynthesis grew from precursordirected biosynthesis (PDB). Some recent developments in this area citing literature from 2001 to 2009 will be covered in this review.

1. Introduction Since the dawn of medicine, natural products have played an important role throughout the world in treating and preventing human diseases. In fact history of natural product based medicine dates back practically to the existence of human civilization. Plants, animals and microorganisms are the different sources of these important materials. Nearly 28% of all new chemical entities (NCEs) launched onto the market are natural products, and Correspondence/Reprint request: Dr. Debaraj Mukherjee, Natural Product Chemistry (Microbes), Indian Institute of Integrative Medicine, Jammu-180001, India. E-mail: dmukherjee@iiim.ac.in

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24% of the NCEs launched during 1981-2002 were synthetic or natural mimic compounds. The data (52% of all NCEs are natural products or are derived from them) suggests that natural products are important sources for new drugs and are also good lead compounds suitable for further modification during drug development. This can be attributed to diverse structures and the intricate carbon skeletons of natural products. As secondary metabolites from natural sources have been elaborated within living systems, they are often perceived as showing more “drug-likeness and biological friendliness than totally synthetic molecules,” making them good candidates for further drug development. Recently Newman and Cragg have reviewed the continuing value of natural products as sources of potential chemotherapeutic agents [1].

2. Different approaches used to obtain bioactive natural compounds Natural compounds are commercially developed and modified to generate a collection of chemically related structures to fulfill the needs of high throughput screening (HTS). The newly developed strategies in modern organic chemistry has played a crucial role in optimization of the drug leads, but this approach is limited in applicability for complex, high molecular weight natural products that contain a great number of reactive groups, some of which require multistep orthogonal protection/deprotection steps during the reaction process. Even though Nicolaou and others have convincingly demonstrated that highly complicated molecules such as vancomycin, everninomycin, or epothilone can be synthesized de novo [2], still there is a pressing need for alternative techniques for the production of large amounts of the compounds needed for pharmaceutical purposes. Some of the recently devised techniques being practiced for achieving the growing needs of HTS and the development of new chemical entities include the utilization of genes from different natural product-producing microbes to create novel hybrid compounds [3]. This combinatorial biosynthetic approach was adapted by Madduri and coworkers for the synthesis of epirubicin (4-epidoxorubicin) by introducing a desoxysugar biosynthesis gene into the doxorubicin producer S. peucetius [4]. Over last few years, combinatorial biosynthesis has been shown to be a powerful tool for modifying several natural compounds with highly diverse structure [5]. Another important technique for the generation of novel secondary metabolites is precursor-directed biosynthesis. This requires the basic knowledge concerning the cultivation of the producer organisms and deep understanding of biosynthetic pathway of the target natural product.

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Again this is the earliest example of combining chemical and biological approaches for the generation of novel natural products [8]. However, one drawback of this approach is that, under normal circumstances, the alternative precursors must compete with the natural precursors, so the yield of novel derivatives is rather low. This disadvantage can be overcome by blocking the synthesis of the natural precursor, either by mutating key genes [6] in the respective synthetic pathway or by adding specific inhibitors of biosynthetic enzymes.

3. Precursor-directed biosynthesis (PDB) In PDB, synthetic chemistry is used to construct analogues of the early intermediates in the pathway, leaving the producing organism to accomplish the remaining, more difficult, transformations. In its simplest form, this technique involves feeding the producing organisms with precursor analogues, which get incorporated into a new product [7] (Fig. 1). This approach can be effective at generating new analogues of complex natural products; for example the orally active β-lactam antibiotic penicillin V (phenoxymethylpenicillin) is produced by adding phenoxyacetic acid to fermentation broths of Penicillium chrysogenum [9]. A comprehensive example of PDB is that recently applied by Zeeck, de Meijere and coworkers who generated new analogues of hormaomycin, a peptide lactone from Streptomyces griseoflavus with a broad spectrum of biological activities, including antibacterial as well as antimalarial activities. Preliminary feeding experiments [10] with standard deuterium-labelled building blocks pointed to 2-nitrocyclopropylalanine as a suitable precursor for the cyclopropane units. Interestingly, 2-aminocyclopropylalanine is neither an intermediate nor an acceptable substrate for the multienzyme complex. Subsequent feeding experiments [11] with a variety of building blocks resulted in the incorporation of unnatural amino acids, thus leading to novel hormaomycins. However, the authors noted difficulties in separation of the analogues from the competitively produced hormaomycin, a principal problem for PDB. Nonetheless, structure–activity relationship (SAR) studies were conducted and this revealed the unexpected antibacterial activity for one derivative against the opportunistic fungal pathogen Candida albicans by an order of magnitude equivalent to the antimycotic agent nystatine. Other remarkable studies exemplifying the potential of PDB include the generation of soraphen-derivatives employing its natural producer Sorangium cellulosum [12] and formation of novel rhamnopyranosides exploiting Streptomyces griseoviridis [13].

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However, there are drawbacks to precursor-directed biosynthesis; a mixture of natural and unnatural products with similar physical properties are often produced, leading to complex downstream purification procedures; high concentrations of synthetic precursor are often required to compete with the preferred natural precursor and a limited range of intermediates are efficiently incorporated into the final product [7]. Once a suitable type of precursor has been chosen for supplementation experiments, several uncertainty factors remain, like whether these analogues will (a) be assimilated by the organism, (b) exhibit non-predictable toxic side-effects, (c) be accepted by the biosynthetic machinery in the presence of the natural building block, and finally (d) be possible separable from the natural variants with a reasonable amount of work.

4. Mutational biosynthesis (MBS) An important technique that combines chemical synthesis with metabolic engineering is mutasynthesis (mutational biosynthesis; MBS), which developed from precursor-directed biosynthesis (PDB) (Fig. 1). Both techniques are based on the cellular uptake of modified biosynthetic intermediates and their incorporation into complex secondary metabolites. Mutasynthesis utilises genetically engineered organisms in conjunction with feeding of chemically modified intermediates. From a synthetic chemist’s point of view the concept of mutasynthesis is highly attractive, as the method combines chemical expertise with nature’s synthetic machinery and thus can be exploited to rapidly create small libraries of secondary metabolites. MBS was first demonstrated 40 years ago using mutants of the neomycin producer Streptomyces fradiae [14]. Mutasynthesis, a term coined by Rinehart (1977) and originally proposed as an alternative method to PDB, is a means of obtaining new structural diversity (Birch 1963). According to Rinehart, the mutasynthesis approach consists of five central steps: (1) generation of the biosynthetic block mutant; (2) generation of the mutasynthon; (3) integration of the mutasynthon; (4) isolation of the target compound (metabolite); and (5) evaluation of biological activity [15]. Two advances have recently converged to make MBS an even more attractive prospect. Although the original PDB experiments relied on laborious chemical synthesis to generate suitable precursor compounds, tens of thousands of complex small molecules are now available from commercial suppliers not only as racemic mixtures but also in stereochemically pure forms. When the desired analogues cannot be purchased, commercial compounds can still be elaborated into the target

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Figure 1. Diagrammatic representation of precursor-directed biosynthesis (PDB) and mutational biosynthesis (MBS). (a) Biosynthesis of natural product by the wild type strain. (b) Precursor-directed biosynthesis: the culture medium is supplemented with an analogue of the natural building block (mutasynthon), which competes for incorporation into the natural product. Both original and required (modified) products are formed. (c) Mutasynthesis: biosynthesis of only the required product by mutant strain by inactivating gene which regulate synthesis of a precursor. Only novel analogues are produced.

molecules using state-of-the-art chemical synthesis. The second important development is the immense growth in sequence information for many natural product pathways. In silico analysis of the proteins encoded by the gene clusters, coupled with gene inactivation in vivo, often enables each gene product to be assigned, unambiguously, to a particular role in the biosynthesis. Together, these experiments can identify suitable candidate genes for constructing metabolic nonproducers, a much more efficient alternative to screening vast numbers of randomly generated mutants for the desired phenotypes [15]. Some early examples of precursor mutational biosynthesis have been summarized in table 1 and schematically described in trailing diagrams (Fig. 2-6).

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Table 1. Some early examples of precursor mutational biosynthesis. Year Mutants 1969 1977 1984 1991

Streptomyces fradiae S. fradiae Streptomyces tendae Tu901

1997

Pseudomonas aeruginosa S. coelicolor CH999

1998

S. avermitilis

1999

S. tendae Tu901

Active mutasynthons Streptamine and epistreptamine Streptamines Pyrimidines Salicylic acid analogues N-acetylcysteamine thioesters Cyclohexanecarb oxylic acid Benzoic acid derivatives

Product

Activity

Fig.

Hybrimycins Aminoglycoside antibiotics Neomycins Antibiotics Nikkomycin Antifungal, X and Z insecticidal, acaricidal Pyochelins Iron transporter 6Precursor for Deoxyerythr erythromycin onolide antibiotics Doramectin Antiparasitic Antibiotic

Nikkomycin Bx/Bz

2 3 4 5 6

5. Antiproliferative and immunosuppressant Streptomyces species produce three anticoumarins (aminocoumarin antibiotics) which inhibit DNA gyrase - novobiocin, clorobiocin and coumermycin. Among them novobiocin is the best-known and most important. However, aminocoumarins possess considerable toxicity towards eukaryotic cells, so that less toxic derivatives of this class of antibiotics are required. H 2N O HO HO

H2 N H2 N

S. fradiae mutant

R NH2

O HO

H2N R NH2

HO HO

HO R=H, deoxystreptamine R=OH, streptamine

HO HO H2N

NH2

R=H, neomycin B/C R=OH, neomycin analog

Figure 2. Mutasynthesis of neomycins usng streptamine mutasynthone in S. fradiae mutant cuture [16].

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Streptomyces tendae Tu901

NH2

HO N

COOH

H N O

N OHC

OH N H

N H

Nikkomycin Z

Nikkomycin X

Figure 3. Mutasynthesis of nikkomycin Z and nikkomycin X from Streptomyces tendae Tu901 supplemented with substituents uracil and 4-formyl-imidazolin-2-one [17]. Ar

COOH

P. aeruginosa mutan A602 S Ar-COOH OH

Ar = F

Figure 4. Mutasynthesis of siderophore analogs 5-fluoropyochelin, 4-methyl pyochelin and 6-azapyochelin using mutant P. aeruginosa IA 602 [18]. O O

OH O

O O

S. avermitilis mutant

O O

O H

H Doramectin

Avermectin A1

Ivermectin unsaturated

O H

Figure 5. Mutasynthesis of doramectin by using a mutant strain of the avermectin producing organism Streptomyces avermitilis supplemented with cyclohexanecaroxylic acid [19]. S. tendae mutant nikC::aphII

OH HO COOH

NH2 H N

nikkomycin Bx/Bz

COOH O

O OH

Figure 6. Mutasynthesis of nikkomycin Bx and Bz using mutant S. tendae supplemented with benzoic acid [20].

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The aminocoumarin antibiotic chlorobiocin consists of three components: an aminocoumarin moiety, a noviose sugar, and a 3-dimethylallyl-4-hydroxybenzoic acid (DMAHB) moiety. Mutasynthesis of this class of antibiotics was achieved by employing a mutant deficient in the biosynthesis of the 3-dimethylallyl-4-hydroxybenzoyl moiety (DMAHB, ring A), which is coupled via an amide synthetase to the aminocoumarin ring (ring B, Galm et al. 2004). In a biochemical study, the amide transferases of all three coumarin antibiotics (CloL, NovL and CouL) were heterologously expressed in Escherichia coli and screened for promiscuity in substrate conversion. Among these three enzymes the amide synthetase CloL was found to be most promising and therefore subsequent mutasynthesis experiments were preferentially performed with the prenyltransferasedeficient cloQ-mutant unable to synthesize the DMAHB precursor (Fig. 7). A key step in the biosynthesis of DMAHB is catalysed by CloQ, a dimethylallyl transferase. In order to produce analogues of chloroiocin, a strain of the producing organism Streptomyces roseochromogenes was constructed in which the cloQ gene was inactivated [21]. Streptomyces hygroscopicus produces polypeptide-polyketide nature compound rapamycin. Rapamycin inhibits the signals required for cell cycle progression, growth and proliferation by binding protein mTOR (the mammalian target of rapamycin), a downstream protein kinase within the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling pathway [22]. Thus, rapamycin is useful as a lead structure for development of anti-cancer, anti-inflammatory and immunosuppressive agents. All rapamycin analogues currently in clinical trials have been altered by semi-synthesis in the starter unit, 4,5-dihydroxycyclohex-1-enecarboxylic acid (DHCHC) [23]. In rapamycin biosynthesis, DHCHC acts as a starter unit for polyketide chain extension on a modular polyketide synthase (PKS) [24], resulting in the release of ‘pre-rapamycin’. Finally, pre-rapamycin is modified through regio and stereospecific oxidation and methylation by a series of post-PKS enzymes. As recently reviewed by Weissman, researchers at Biotica were able to generate a strain of Streptomyces hygroscopicus that was unable to make the DHCHC starter unit. Supplementing the strain with different analogues of DHCHC resulted in novel rapamycin analogues (Fig. 8). Borrelidin is a polyketide derivative inhibiting angiogenesis [25]. Natural starter unit in borrelidin is trans-cyclopentane-(1R,2R)-dicarboxylic acid. As recently reviewed by Weissman, researchers at Biotica and their collaborators at the University of Oviedo cloned and sequenced the borrelidin gene cluster from Streptomyces parvulus Tu¨4055. This advance enabled them to mutate a key gene involved in starter unit production, using insertional gene

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393 OH HO O

3-dimethyllyl-4-hydroxybenzoic acid OH

OH O

H N

Streptomyces roseochromogenes inactivated gene cloQ

H N

O Cl

Chlorobiocin NH2

OH HO

OH O

HO O

OH HO

3-dimethyllyl-4-hydroxybenzoic acid analogues supplemented in feed

Figure 7. Mutasynthesis of chlorobiocin analogues using supplement of DMAHB (Galm et al. 2004). HO

O COOH

COOH

COOH COOH

COOH

COOH COOH

DHCHC analogues supplemened in feed

H3CO

S. hygroscopicus MG2–10 gene inactivated rapK

N O

O O

O H3CO

HO O

OCH3

Rapamycin

Figure 8. Mutasynthesis of rapamycin analogues using supplement of DHCHC.

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Streptomyces parvulus Tu¨4055 O NC COOH

Feed supplemented with cyclobutane analogues Borreldine COOH

COOH

COOH H

COOH H3CS

Figure 9. Mutasynthesis of borrelidin analogues using supplement of different dicarboxylic acid.

inactivation. More than 40 commercially available mono and dicarboxylic acids were fed to BIOT-1302 cultures to evaluate their ability to initiate the biosynthesis of modified borrelidins [26]. Although the monocarboxylic acids were not incorporated, use of cyclobutane-trans-1,2-dicarboxylic acid resulted in a novel compound at 50% of the normal borrelidin titre. Because the trans isomer of the cyclobutane dicarboxylic acid was accepted, and not the cis (meso) form, the researchers surmised that the particular geometrical arrangement of carboxylic acids in the precursor was vital for substrate recognition (Fig. 9). Kalaitzis et al. (2003) reported the generation by MBS of wailupemycin and enterocin analogs from Streptomyces maritimus, which is the first example of the use of MBS for type-II PKS (Fig. 10) [27]. Another unusual starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), is required for the biosynthesis of ansamycin antibiotics, a distinctive class of polyketide-based metabolites. Ansamitocins are cytotoxic compounds known to inhibit different leukemia cell lines as well as human solid tumors at very low concentrations. The natural producer is Actinosynnema pretiosum.

Mutasynthesis of medicinally important natural products OH

395 O

Streptomyces maritimus encP-mutant

OH R OH HO

O O R-COOH HO

wild type

O Enterocin

Waliupemycin

S R= S

Figure 10. Mutasynthesis of waliupemycin and enterocin analogues by feeding culture with carboxylic acid derivatives.

The different members of this group differ in the nature of the acyl sidechain, with ansamitocin P-3 (AP-3) being an important example [29]. Ansamitocins are highly potent and are currently being evaluated in phase I studies for target-directed antibody conjugates [29]. Total synthesis has not provided new AP-3 derivatives, while semisynthesis has mainly addressed ester side chain modifications and dehalogenation [28]. Meanwhile, work towards generation of novel ansamitocin analogues employing a mutant blocked in the biosynthesis of the unique starter unit AHBA has been conducted [30]. By using different benzoic acid derivatives as a supplement in culture of A. pretiosum mutant, novel AP-3 could be generated in amounts suitable for structural identification and activity analysis (Fig. 11) [31]. The analogues exhibited strong antiproliferative activity against several tumor cell lines (IC50 values in pg mL−1 range). MeO

NH2

wild type

O R

O O

COOH

Actinosynnema pretiosum mutant

Feed supplemented with benzoic acid analogues

N OH H OMe

R NH2

R= F, Cl, Br COOH

Figure 11. Mutasynthesis of ansamitocin analogues using AHBA supplement.

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The soil bacterium Streptomyces thioluteus produces auerothin. Starer unit for this is p-nitro benzoate (PNBA) [32,33]. Aureothin exhibits a variety of pharmacological properties, which include weak cytotoxic, antifungal, and antiviral activities [34]. Expression of the mutated gene clusters in the S. lividans host appeared suitable for mutasynthesis, since the Aur pathway could only be restored with exogenously supplied PABA or PNBA. To explore the substrate specificity of the Aur PKS, at first p-, m-, and o-PNBA were administered to a growing culture of S. lividans ZX1::pHJ79. Not surprisingly, only the p-substituted acid was used as substrate, while the regioisomers were not incorporated. In a series of further feeding experiments a variety of p-substituted PNBA surrogates were tested, either as free acids or as the corresponding N-acetyl cysteamine (NAC) thioesters, which were synthesized using the dicyclohexyl carbodiimide/4-dimethylaminopyridine (DCC/DMAP) method [35,36] The NAC adducts serve as activated acyl CoA mimics [37] that may diffuse into the bacterial cells and bypass a potential bottleneck, the putative acyl CoA ligase AurE. Of the various p-substituted benzoic acids, p-iodo, p-bromo, p-chloro, and p-fluoro benzoate, as well as p-N-acetamido anthranilic acid and p-dimethylamino benzoate were probed on a 100-mL fermentation scale. Unfortunately, novel aureothin derivatives could not be detected in the crude extracts of these feeding experiments. Also, toluic acid and terephthalic acid monomethyl ester failed to incorporate. Strikingly though, addition of p-cyano benzoic acid to a culture of the aurF null mutant yielded a novel metabolite, which was detected by ESI-MS in the positive mode (m/z 378). As only relatively low quantities of the new compound were produced, p-cyano benzoyl-SNAC was tested to find if it would provide higher yields, as it does not need to be activated as an acyl CoA adduct by the ligase. However, the yield did not exceed that obtained with the free acid, which reveals that in this case acid activation by the ligase AurE is not a bottleneck (Fig. 12) [38]. COSCoA

COOH

CoASH

NAC DCC DMAP

PCBA-CoA O S

AurABCHI

AurE

H N

PCBA-SNAC

O OMe

Aureonirile O

Figure 12. Mutasynthesis of aureonitrile with PCBA and PCBA-SNAC.

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Salinosporamide A, a chlorinated natural product from the marine bacterium Salinispora tropica, is a potent proteasome inhibitor currently in development for the treatment of multiple myeloma and other cancers [39-41]. Besides the major analogue 2, S. tropica also accumulates the deschloro compound salinosporamide B. Administration of synthetic 5’-FDA [42] to a salL-knockout mutant of S. tropica [43] devoid of 2 led to the production of a new salinosporamide derivative (Fig. 13). NH2

N O

N Cl

L-Met Cl-

N OOC

N N

se na i r lo Ch lL NH 2 Sa

NH3 -

5'-CIDA

N N

Salinosporamide A(2)

NH2 N

N S

L-Met

N F

N HO

Fluorinase

SAM

5'-FDA O NH -

salL mutant of S. tropica

OH O

Fluoriosalinosporamide (1)

Figure 13. Fluorosalinosporamide was generated from 5’-FDA in a salL-mutant of S. tropica. SAM: S-adenosyl-l-methionine; 5’-ClDA: 5’-chloro-5’-deoxyadenosine; 5’-FDA: 5’-fluoro-5’-deoxyadenosine [44].

Geldanamycin is a potential antitumor drug [45] that binds to the N-terminal ATP-binding domain of heat shock protein 90 (Hsp90) and inhibits its ATP-dependent chaperone activities [46]. Most geldanamycin derivatives reported to date are 17-aminated compounds and were obtained by semisynthesis [47]. Related to geldanamycin is reblastatin, which is saturated across C4-C5 and has a benzene chromophore instead of a benzoquinone or a hydroquinone moiety [46]. Importantly, reblastatin shows lower cytotoxicity than geldanamycin but has a higher affinity for Hsp90 [48].

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The producing microorganism Streptomyces hygroscopicus var. geldanus NRRL 3602 creates geldanamycin through biosynthetic machinery based on a polyketide synthase (PKS) and additional post-PKS enzymes. The starter unit for geldanamycin is 3-amino-5-hydroxybenzoic acid (AHBA), which originates from a shikimate-type biosynthetic pathway. New geldanamycin derivatives were obtained by using mutational biosynthesis with an AHBAblocked mutant of the geldanamycin producer, Streptomyces hygroscopicus K390-61-1 [49]. Twenty different 3-aminobenzoic acids were chosen and individually added to cultures of strain K390-61-1. Mutasynthons 1, 2, 3 (fig. 14) and aminonicotinic acid turned out to be the most promising candidates with respect to yields [50]. O

MeO

1 7 21 O

N H

21 4

MeO

1 7

MeO

O Geldanamycin NH2

O Reblastatin Reblastatin analogues

S. hygroscopicus K390-61-1

Successfull precursor added OMe F O OH

NH2

O OH

NH2

Br N O

O OH

NH2

NH2 OH Aminonicotinic acid

Figure 14. Mutasynthesis of reblastatin analogues.

O NH2

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5.1. Antimicrobial A series of triketide analogues were synthesized and shown to be processed by supplemented culture of S. venezuelae BB138 strain with N-acetyl cysteamine thioester of the triketide (Fig. 15). Four of them were shown to be processed into new biologically active 14-membered macrolide products. The levels of production of these new macrolides varied, but in all cases were at least tenfold lower than seen for pikromycin production from the natural triketide. Preliminary analysis of one new product, 15,16dehydropikromycin, indicated slightly improved antibacterial activity [51]. Studies integrating sophisticated methods of molecular biology and chemical synthesis were carried out with the aim of elucidating the acceptance of advanced intermediates by the 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, the producer of the broad spectrum antibiotic erythromycin B [54]. The DEBS system represents the most extensively characterised modular polyketide synthase and for a more detailed description of the insights gained into its utilization of advanced intermediates, the reader is directed to the work of Ward et al. [52]. Studies were carried out with mutants of the natural producers, due to the selectivity of the DEBS loading module for standard biosynthetic building blocks such as propionyl-CoA, the elimination of internal precursor competition is not possible by blocking the respective starter unit biosynthesis. To make the DEBS PKS suitable for a mutasynthesis approach, a different strategy was employed. A point mutation was introduced into the active site of the first PKS ketosynthase (KS1) domain; thereby blocking diketide formation based on the available internal starter units [53]. These engineered KS10 DEBS systems were shown to convert the natural diketide as well as modified diketides and triketides into analogues of 6-deoxyerythronolide B [54,55]. These advanced precursors could be further modified O

S. venezuelae (BB138) PikAl mutated gene

SNAC

a OH

Synthetic triketide SNAC

+ R1 R2 N

R1 R2

N OH

1a R1=CH3, R2=CH2CH3,pikromycin 2a R1=CH3, R2=CH=CH2, 15,16-dehydropikromycin 1b R1=CH3, R2=CH2CH3,narbomycin 2b R1=CH3, R2=CH=CH2, 15,16-dehydronarbomycin

Figure 15. Mutasynthesis of pikromycin, dihydropikromycin, nabromycin and dihydronabromycin using synthetic triketide SNAC.

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Streptomyces coelicolor mutant

OH OH

O R

R= Bu,Bn,Et , 56-58% Diketide feeded

25%

SNAC

Streptomyces coelicolor mutant

OH O O O

SNAC R1

OH OH

R1=H R2=Me R1=Me R2=H R1=H R2=H Triketide incorporated

HO O O

O O OH OH O

O O

Post-PKS modification

OH OH OH

6-Deoxy-erythronolide B

NMe2 OH

O O

OMe

Erythromycin B

Figure 16. Mutasynthesis of 6-deoxy-erythronolide and formation of erythromycin B in post PKS modification.

into novel erythromycins by application of a S. erythrea mutant unable to synthesise the core polyketide, but equipped with the full set of post-PKS tailoring enzymes. Remarkably, when SNAC-esters of 2,3-unsaturated triketide derivatives were administered, polyketide elongation and macrolactonisation yielded 16-membered lactones which spontaneously formed the corresponding lactols (fig. 16) [56].

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The triketide analogues were apparently accepted as surrogates for the absent diketide precursors and treated accordingly by the biosynthetic machinery, finally resulting in an increased ring size. In a more recent study it was demonstrated that removal of the DEBS loading domain and the first module, rather than catalytic inactivation of the latter, resulted in an increased utilisation of supplemented diketide precursors by the engineered PKS system [52]. Vancomycin is a glycopeptidal antibiotic used for treatment of lifethreatening infections with methicillin-resistant Staphylococcus aureus (MRSA) [57]. Due to resistance development in enterococcal and staphylococcal against vancomycin, treatments require the novel vancomycin analogues. Amycolatopsis balhimycina is the producer of the vancomycintype glycopeptide antibiotic balhimycin [58]. The first series of mutasynthesis experiments were performed with an in-frame deletion mutant of the bhp perhydrolase gene (Puk et al. 2002) inactivated in Hty biosynthesis. Other mutasynthetically generated fluoro-balhimycines were obtained by feeding 2-fluoro- or 3,5-difluoro-β-hydroxytyrosine, respectively (Fig. 17). All novel fluorobalhimycines were found to be active and showed antibiotic activity against the strain Bacillus subtilis. The DL-Tyrosine or phenyl serines lacking the 4-OH group were not accepted as substrates [59]. The lantibiotics are a class of polycyclic peptide natural products that possess antimicrobial activity [60, 61]. They are also used in food preservation [62]. The major problem associated with lantibiotics is solubility and stability. Lantibiotics are ribosomally synthesized as linear prepeptides and are extensively post-translationally modified to their active forms. These modifications introduce thioether containing cross linked amino acids called lanthionine (Lan) and methyllanthionine (MeLan) as well as dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues. The latter motifs result from enzymatic dehydration of Ser or Thr residues, respectively. (Me)Lan crosslinks are then installed through enzymatic intramolecular Michael type addition of Cys thiols onto the unsaturated amino acids [63]. Lacticin 481 is a lantibiotic produced by Lactococcus lactis CNRZ 481 that contains three Me(Lan) rings and one Dhb residue [64]. Lacticin 481 synthetase (LctM) introduces these post-translational modifications by catalyzing both dehydration and cyclization of its substrate prepeptide, LctA [65]. LctA is a 51 amino acid peptide consisting of a C-terminal structural peptide that undergoes posttranslational modification and an N-terminal leader peptide that is required for efficient processing by LctM [66,67]. Several recent reports have characterized the promiscuous substrate specificity of LctM toward truncated LctA mutant substrates [68-70]. A significant challenge for the introduction of nonproteinogenic amino acids

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A.balhimycin

Fluoro balhmycin

bhp deleted mutant

NH2 HO COOH

OH NH2

NH2

COOH

NH2

COOH

NH2

COOH

HO COOH

F OH

Accepted mutasynthone

Non-accepted mutasynthone

OH OH HO

CH2OH O

H2 N

H3C

CH3

O F O

H N

HH O

H N

NH2 O

H HOOC

OH OH

HO Fluoro balhimycin

Figure 17. Mutational biosynthesis of fluorobalhimycin analogues by using mutated strain of A. balhimycin.

into lacticin 481 is the preparation of full-length LctA prepeptides. For this purpose, a triazole-linked LctA peptide analogue (3) has been synthesized via Cu(I)-catalyzed 1,3-dipolar cycloaddition of an alkyne functionalized LctA leader peptide (1) and an azide modified LctA structural region (2) [71]. This strategy

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was utilized to prepare triazole-linked LctA substrates containing several nonproteinogenic amino acids in the structural peptide, including β-amino acids, D-amino acids, and N-alkylglycine (peptoid) residues. Additional two mutations, Asn15Arg and Phe21His, were included in substrate 3 to improve solubility [71]. Amino acids that are tolerated by LctM,14 substrates including the following nonproteinogenic amino acid mutations were prepared: sarcosine (Sar) and aminocyclopropanoic acid (Acpc) in place of Gly5, D-valine at position 6, 4cyanoaminobutyric acid (Cba) in place of Glu13, β3- homoarginine (β3-Arg) at position 15, N-butylglycine (N-Nle) and β-Ala replacing Met16, naphthylalanine (Nal) at Trp19, 4-pyridynylalanine (Pal) at position 21, and homophenylalanine (hPhe) in place of Phe23. A final analogue contained three concomitant replacements of Gly2, Gly3, and Gly5 with β-Ala (Fig. 18). The triazole linked

Figure 18. In vitro mutasynthesis of lacticin 481 analogues. Synthetic substrate analogues were prepared using copper-catalyzed [2+3] cycloaddition of two fragment peptides. The resulting LctA analogues were treated with LctM, which dehydrated the underlined Ser and Thr residues and incorporated the thioether rings shown. Mutant analogues generated are indicated with arrows. Lan residues are shown in red, and the MeLan crosslink is shown in blue [72].

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LctA substrate analogues (0.5-1.5 mg) are incubated with 0.5 μM LctM in the presence of 10 mM Mg2+ and 1 mM ATP. Assay progress was monitored by MALDI-TOF MS. Each of the unnatural LctA substrates was dehydrated four times by LctM. To produce bioactive lacticin 481 analogues, the leader peptide and the triazole linker are removed by proteolysis using commercially available endoproteinase LysC, which efficiently cleaved the modified substrates C-terminal to Lys1. Thus, lacticin 481 analogues are produced in high purity. To ensure that complete cyclization had occurred, the analogues were incubated with a thiol modifying reagent, demonstrating that no free thiols remained in the LctM-treated peptides [72].

5.2. Insecticidal The insect pathogen Beauveria bassiana produces several secondary metabolites, including the cyclooligomer nonribosomal depsipeptides beauvericin and bassianolide, the diketomorpholine bassiatin, the cyclic peptides beauverolides, the 2-pyridone tenellin, and the dibenzoquinone oosporein [73-77]. The cyclooligomer depsipeptides beauvericin and bassianolide represent rich pharmacophores with diverse biological activities. Bassianolide is a tetramer of the dipeptidol monomer d-Hiv-N-methyl-lleucine. Bassianolide causes smooth muscle contraction by inhibiting acetylcholine activity [78]. It is toxic to insect larvae [75], and exerts antimycobacterial, antiplasmodial, and cytotoxic activity [79]. Beauvericin is a cyclic trimer assembled from three d-Hiv-N-methyl-l-phenylalanine dipeptidol monomers the main cyclooligomer depsipeptide product of B. bassiana, transports monovalent ions across membranes; this uncouples oxidative phosphorylation [80]. Beauvericin is insecticidal [81], displays moderate antifungal and antibiotic activity [73], reverses multidrug resistance in Candida albicans [82,83] possesses broad spectrum antiproliferative activity (activating calcium-sensitive cell apoptotic pathways) [84] and is a potent inhibitor of haptotactic motility [85]. As a consequence of the oligomeric nature of beauvericin, precursordirected biosynthesis with the wild-type strain and an appropriate d-Hiv analogue yields beauvericin and a series of three beauvericin analogues in which one, two, or all three d-Hiv moieties are replaced by the externally supplied precursor [86,87] (Fig. 19). In contrast, the kivr mutant strain is unable to produce any beauvericin-like compounds unless the fermentations are supplemented with an appropriate d-Hiv analogue. Moreover, upon d-Hiv analogue supplementation, this strain biosynthesizes only a single

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beauvericin compound, in which all the d-Hiv positions are fully substituted by the externally supplied precursor. For mutasynthetic approach, kivr mutant B. bassiana strain was supplemented with d-2-hydroxybutyrate (d-Hbu) instead of d-Hiv and the major product obtained is beauvericin G3. To produce a larger variety of beauvericin analogues, combinatorial simultaneous feeding of pairs of precursor analogues was also used during mutasynthesis. O O OH D-2-Hydroxyisovaleric acid (D-Hiv)

R O N OH

Dipeptidol monomer 4x

N O

O O N

O O

Beauvericin R= CH2-C6H5 Enniatins R= iPr, sBu or iBu

Bassianolide

Figure 19. Mutational biosynthesis of beauvericin analogues by using kivr mutant B. bassiana strain supplemented with d-2-hydroxybutyrate (d-Hbu).

6. Summary Mutasynthesis seems to have a healthy future in lead optimization and drug discovery. In the current review we have demonstrated with example that using mutasynthesis one can not only synthesize a very complex bioactive natural product but also construct subset analogues obviating

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several multistep laborious synthetic protocols. Despite these, mutasynthesis has clear limitation that only the portion of structure can be modified. However, such a minor modification in structure can result a drug e.g. Doramectin is commercially available insecticidal differ from avermectin only in starter unit [88].

Acknowledgements Authors are thankful to Dr Ram. A. Vishwakarma, Director IIIM Jammu for his keen interest and support.

References 1. 2.

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21. Galm, U., Heller, S., Shapiro, S., Page, M., Li, M.S.M. Heide, L. Antimicrob. Agents Chemother., 2004, 48, 1307. 22. Brown, E.J., Albers, M.W., Shin, T.B., Ichikawa, K., Keith, C.T., Lane, W.S. Schreiber, S.L. Nature, 1994, 369, 756. 23. Gregory, M.A., Petkovic, H., Lill, R.E., Moss, S.J., Wilkinson, B., Gaisser, S., Leadley, P.F., Sheridan, R.M. Angew. Chem. Int. Ed. 2005, 44, 4757. 24. Aparicio, J.F., Molnár, I., Schwecke, T., Konig, A., Haydock, S.F., Khaw, L.E., Staunton, J., Leadlay, P.F. Gene, 1996, 169, 9. 25. Wilkinson, B., Gregory, M.A., Moss, S.J., Carletti, I., Sheridan, R.M., Kaja, A., Ward, M., Olano, C., Mendez, C., Salas, J.A., Leadlay, P.F., Ginckel, R., Zhanga, M.-Q. Bioorg. Med. Chem. Lett., 2006, 16, 5814. 26. Moss, S.J., Carletti, I., Olano, C., Sheridan, R.M., Ward, M., Math, V., Nur-EAlam, M., Brana, A.F., Zhang, M.-Q., Leadley, P.F., Mendez, C., Salas, J.A., Wilkinson, B. Chem. Commun., 2006, 2341. 27. Kalaitzis, J.A., Izumikawa, M., Xiang, L., Hertweck, C., Moore, B.S. J. Am. Chem. Soc., 2003, 125, 9290. 28. Cassady, J.M., Chan, K.K., Floss, H.G., Leistner, E. Chem. Pharm. Bull., 2004, 52, 1. 29. Kovtun, V.V., Audette, C.A., Ye, Y., Xie, H., Ruberti, M.F., Phinney, S.J., Leece, B.A., Chittenden, T., Blattler W.A., Goldmacher, V.S. Cancer Res., 2006, 66, 3214. 30. Yu, T.-W., Bai, L., Clade, D., Hoffmann, D., Toelzer, S., Trinh, K.Q., Xu, J., Moss, S.J., Leistner, E., Floss, H.G. Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 7968. 31. Taft, F., Brunjes, M., Floss, H.G., Czempinski, N., Grond, S., Sasse, F., Kirschning, A. ChemBioChem, 2008, 9, 1057. 32. Hirata, Y., Nakata, H., Yamada, K., Okuhara, K. Naito, T. Tetrahedron, 1961, 14, 252. 33. He, J. Hertweck, C. J. Am. Chem. Soc., 2004, 126, 3694. 34. Schwartz, J.L., Tishler, M., Arison, B.H., Shafer, H.M., Omura, S. J. Antibiot., 1976, 29, 236. 35. Neises, B., Steglich, W. Angew. Chem., 1978, 90, 556. 36. Wilkinson, C.J., Frost, E.J., Staunton, J., Leadlay, P.F. Chem. Biol., 2001, 8, 1197. 37. Staunton, J., Sutkowski, A.C. J. Chem. Soc. Chem. Commun., 1991, 1110. 38. Ziehl, M., He, J., Dahse, H.-M. Hertweck, C. Angew. Chem. Int. Ed., 2005, 44, 1202. 39. Feling, R.H., Buchanan, G.O., Mincer, T.J. Kauffman, C.A., Jensen, P.R., Fenical, W. Angew. Chem., 2003, 115, 369; Angew. Chem. Int. Ed., 42, 355. 40. Maldonado, L.A., Fenical, W., Jensen, P.R., Kauffman, C.A., Mincer, T.J., Ward, A.C. Bull, A.T., Goodfellow, M. Int. J. Syst. Evol. Microbiol., 2005, 55, 1759. 41. Chauhan, D., Catley, L., Li, G., Podar, K., Hideshima, T., Velankar, M., Mitsiades, C., Mitsiades, N., Yasui, H., Letai, A., Ovaa, H., Berkers, C., Nicholson, B., Chao, T.H., Neuteboom, S.T., Richardson, P., Palladino, M.A., Anderson, K.C. Cancer Cell, 2005, 8, 407. 42. Ashton, T.D., Scammells, P.J. Bioorg. Med. Chem. Lett., 2005, 15, 3361. 43. Eustaquio, A.S., Pojer, F., Noel, J.P., Moore, B.S. Nat. Chem.Biol., 2008, 4, 69.

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Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Opportunity, Challenge and Scope of Natural Products in Medicinal Chemistry, 2011: 411-431 ISBN: 978-81-308-0448-4

14. Carbohydrate-containing natural products in medicinal chemistry 1

Hongzhi Cao1, Joel Hwang2 and Xi Chen2

National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250012 P. R. China; 2Department of Chemistry, University of California-Davis One Shields Avenue, CA 95616, USA

Abstract. Carbohydrates are essential components of many natural products known for great medicinal importance. The carbohydrate moieties can increase drug water solubility, decrease toxicity, and/or contribute to the bioactivity of the natural products. This review provides a short summary of diverse carbohydratecontaining natural products, recent advances in introducing glycan diversity to natural products, and their potential application in medicinal chemistry.

1. Introduction Carbohydrates are the most abundant biomolecules. They are presented as free monosaccharides, oligosaccharides, polysaccharides, and as essential components of glycoconjugates, including glycolipids, glycoproteins or glycopeptides, and glycosylated natural products. Glycosylated natural products have been commonly used as antimicrobial drugs and now as emerging anti-cancer drug candidates. The sugar moieties in many bioactive natural products do not only increase water solubility thus the bioavailability of the compounds, but also decrease toxicity. Some glycans are also the essential components for the bioactivity of the natural products. This review Correspondence/Reprint request: Dr. Hongzhi Cao, National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250012, P. R. China. E-mail: hzcao@sdu.edu.cn; Prof. Xi Chen, Department of Chemistry, University of California-Davis, One Shields Avenue, CA 95616, USA E-mail: chen@chem.ucdavis.edu

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summarizes the diverse carbohydrate-based and glycosylated natural products that have been used as drugs and those have great drug potential. Recent advance in developing novel glycosylated natural products by glycorandomization/glycodiversification is also included in the review.

2. Iminosugars, aminoglycosides and carbohydrate mimics Iminosugars, also known as azasugars or polyhydroxylated alkaloids, are a family of naturally occurring carbohydrate mimics. These sugar mimics in which the ring oxygen is replaced by nitrogen are classified into five structural classes: polyhydroxylated piperidines, pyrrolidines, indolizidines, pyrrolizidines, and nortropanes [1]. Some representative iminosugar structures are shown in Figure 1. Nojirimycin (1, Figure 1) [2] was the first natural iminosugar isolated from Streptomyces roseochromogenes R-468 and S. lavendulae SF-425 and was shown to be a potent inhibitor of α- and β-glucosidases from various sources [3]. The chemically more stable 1-deoxy nojirimycin (DNJ, 2) was also isolated shortly after the discovery of nojirimycin. So far, many iminosugars have been isolated from natural source and most of them exhibit specific potent inhibition against different glycosidases, a class of carbohydrate catabolic enzymes [1-4]. Aminoglycosides are an important group of carbohydrate-based antibiotics typically containing one aminocyclitol linked to two or more uncommon monosaccharides. Streptomycin (9, Figure 2) from Streptomyces griseus was the first aminoglycoside natural product discovered [5]. Streptomycin and many other aminoglycosides (Figure 2) isolated from natural sources have been widely used as antibacterial agents, especially against mycobacterium tuberculosis. Recently, aminoglycosides have been demonstrated to inhibit catalytic RNAs in vitro as well as to interfere with HIV replication by disruption of essential protein-RNA contacts [6-8].

Figure 1. Structures of representative iminosugars.

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H2N

NH2

NH HN OH OH

HO NH

H2N

NH CHO O H3C HO HO

HO HO

H2N

NH2

O OH NHMe

H2N O

H2N NH2

O OH

NH2

H2N

Streptomycin, 9

H2N

NH2

H2N

H2N O HO

NH2 O

Kanamycin B, 12

H2N NH2 O

H2N O HO

NH2 O

OH O

H2N O HO

Paromomycin, 11

NHCH3

NH2

O OH

Neomycin B, 10

NH2 HO HO

H2N

HO HO

OH NH2

O HO

CH3HN

Gentamicin C1, 13

CH3 OH

O HO

CH3HN

CH3 OH

Sisomicin, 14

Figure 2. Structures of representative aminoglycosides.

Many natural occurring and synthetic carbohydrate mimics including iminosugar- and aminoglycoside-based glycosidase inhibitors have been used as drugs to treat diabetes, viral infections, cancers, and Gaucher disease (Figure 3) [1-4]. For example, acarbose (15), a pseudotetrasaccharide isolated from the fermentation broth of the Actinoplanes strain SE 50, was the first marketed α-glucosidase inhibitor. It was introduced in early 1990s under the name of GlucobayTM [3]. Two other synthetic carbohydrate mimics, miglitol (16) and voglibiose (17), have also been introduced to the market as α-glucosidase inhibitors for the treatment of type II diabetes [4,9]. As α-glucosidase inhibitors, acarbose (15), miglitol (16) and voglibiose (17) can decrease the carbohydrate digestion rate and reduce postprandial hyperglycaemia (PPHG). Neu5Ac2en (20), the dehydrated Neu5Ac, is a transition state analog inhibitor of sialidases (also known as neuraminidases). To overcome the low efficiency and poor selectivity of Neu5Ac2en in inhibiting influenza virus neuraminidases, Relenza (18) and Tamiflu (19) were developed in recent years as competitive inhibitors against influenza viral neuraminidase [1,4]. These two blockbuster “flu drugs” have played important roles in combating the recent flu pandemic and epidemics.

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Figure 3. Some glycosidase inhibitor-based drugs.

The iminosugar Miglustat (22) was the first market azasugar anticancer drug. It was also used to treat Gaucher disease by inhibiting the glycosyltransferase involved in the biosynthesis of glucosylceramide [4,9]. Other iminosugars, such as naturally occurring swainsonine (5), castanospermine (6), and a DNJ synthetic derivative (NMDNJ, 21), are current anticancer drug candidates in ongoing clinic trails. These iminosugars are inhibitors against catabolic glycosidasees associated with cancer progresses [1,4]. During the course of synthesizing novel inhibitors against glycosidases and glycosyltransferases, many new synthetic approaches and methods for iminosugars have been developed. For example, Wong and co-workers developed a one-pot chemoenzyamtic approach for the synthesis of a library of iminocyclitols using fructose-6-phosphate aldolase (FSA) [10]. Most recently, the Wong group also developed a two-step chemical synthesis of iminocyclitols using Petasis-type aminocyclization as the key step [11]. The organocatalytic aldo reaction was also intensively investigated in recent years for the synthesis of iminosugars [12,13].

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3. Saponins Saponins are a class of glycosylated secondary metabolites that have been found in various plant species and some marine organisms. Thousands of saponins have been characterized and they usually can be classified into steroidal glycosides and triterpenoid glycosides according to their aglycones. As natural surfactants, saponins have not only been used as detergents or foaming agents for many years, they have also been used in Africa to kill infected snails and prevent the transmission of schistosomiasis. Plant saponin extracts from ginseng, liquorice, horse chestnuts, ivy leaves, quillaia barks, primula roots, senega roots, sarsaparilla roots and others have been used as folk medicines [14-16]. The cardiac glycosides include well-known drugs such as digoxin (23) has been used for many years to treat congestive heart failure. O OH

HO OH

O OH

OH O

Digoxin, 23

Some recent studies showed that digoxin also has anti-cancer activity and can be used as a novel cancer therapeutic agent [17,18]. Antimicrobial, especially antifungal, activities of many steroidal saponins (e.g. 24-29 in Figure 4) have also been reported [19-25]. Ginseng (Panax genus) is a family of slow-growing perennial plants belonging to the family Araliaceae. Its root has been used to increase the quality of life in China and East Asia since ancient time. So far, more than 30 different ginsenosides (Figure 5) have been isolated, and these triterpene saponins are considered to be the main active compounds in the ginseng products [26,27]. Accumulated evidences have shown that ginsenosides also have anti-inflammation [28], anticancer [29-31] anti-diabetic [32,33] activities, and can prevent neurodegeneration [34,35]. OSW-1 (Figure 6, 34) is a high potent anticancer cholestane glycoside. OSW-1 and its four natural analogues (35-38) have been isolated from the bulbs of Ornithogalum saundersiae, a perennial grown in southern Africa where it is cultivated as a cut flower and garden plant [36]. These cholestane

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HO O HO O

HO HO

HO O

HO HO

TTS-12, 24 HO O HO O

HO HO HO

HO O

HO HO

TTS-15, 25 O O

OH O

HO HO

HO O

HO HO

Dioscin, 26

29 O

OH O

HO O HO O HO HO

HO HO

OH O

O HO O

O OH

OH O

Aginoside, 27 HO

HO HO

O HO O OH

HO HO O O OH HO HO

OH O

HO O

CAY-1, 28

Figure 4. Structures of representative antimicrobial saponins.

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HO HO

O HO

HO OH

OH O O

HO HO

HO O

O HO HO

30, R = Rha, Ginsenoside Re, HO 31, R = Glc, Ginsenosied Rg1

32, R = H, Ginsenoside Rh2 33, R = Glc, Ginsenoside Rg3

Figure 5. Structures of some representative ginsenosides (30–33).

glycosides exhibited extremely potent cytotoxicity against human promyelocytic leukemia HL-60 cells with IC50 between 0.1 and 0.3 nM. OSW-1, the major constituent, exhibited high potent activity against various malignant tumor cells, including leukemia, mastrocarcinoma, lung adenocarcinoma, pulmonary large cell carcinoma and pulmonary squamous cell carcinoma. The cytotoxicity is 10-100 fold more potent than some wellknown anticancer agents in clinical use, such as mitomycin C, cisplatin, camptothecin, adriamycin and taxol [37]. Due to its unique structure and exceptional highly potent anticancer activity, OSW-1 has been an attractive synthetic target for organic chemists. Three groups have reported the total synthesis of OSW-1. All of these synthetic approaches utilized a convergent strategy that glycosylated the aglycone acceptor with disaccharide donor to realize the coupling (Figure 7). The most challenging part of the total synthesis was the synthesis of aglycone O

OH O AcO R1O

HO HO

34, R1 = H, R2 = p-methoxybenzoyl, OSW-1 35, R1 = H, R2 = 3,4-dimethoxybenzoyl O 36, R1 = H, R2 = (E)-cinnamoyl O O OH 37, R1 = Glc,R2 = p-methoxybenzoyl 38, R1 = Glc,R2 = (E)-cinnamoyl OR2

Figure 6. OSW-1 and its analogs.

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35% 39

O OH OH

3 steps

9 steps HO

54%

30% 40

TBSO

2 steps

TBSO

OSW-1

(b) Jin's synthesis (2001) O

66% 39

TBSO

O OH OH 3 steps

OAc 2 steps

7 steps HO

58%

74%

TBSO

OSW-1

4 steps

3 steps

37%

41%

TBSO

O OH OH

3 steps 41%

OSW-1

Figure 7. Total synthesis of OSW-1 (34).

acceptor. It was achieved from commercially available 5-androsten-3β-ol-17one (39) in all three reports. The Hui and the Yu groups reported the first total synthesis of OSW-1 (34) in 1999 (Figure 7, path a) [38]. In their synthesis, the side chain elongation was realized by employing sequentially Wittig olefination, Ene reaction, Dess-Martin oxidation, Grignard addition, PDC oxidation, and protection of keto with ethylene glycol to give the key diene intermediate 40. The diene 40 was subjected to OsO4 to afford the corresponding 16α,17α diol intermediate in moderate yield, which was converted to the natural aglycone as the acceptor for the next glycosylation step by reversing the 16α-OH to 16β-OH through an oxidation-reduction process. Finally, the OSW-1 (34) was constructed from commercially available dehydroisoandro-sterone, L-arabinose, and D-xylose in 14 linear steps with a total yield of 6%. Jin and co-workers developed a new strategy for steroselective introduction of the aglycone side chain via 1,4-addition of α-alkoxy vinyl cuprate to 17(20)-en-16-one steroid to give the intermediate 42 (Figure 7, pathway b) [39,40]. In Jin’s synthesis, a new strategy was developed to introduce the 16β,17α diol to avoid using toxic OsO4. The total synthesis was finished in 10 linear steps with a 28% overall yield.

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Recently, Guo and co-workers reported gram-scale synthesis of OSW-1 (34) (Figure 7, pathway c) [41]. In their synthesis, an efficient new approach was developed to elongate the steroid side chain to give the key diene intermediate 40 in 4 steps with a 37% total yield. Then, following the similar approach as described by Hui and Yu, the OSW-1 was synthesized in 10 linear steps in a 6% overall yield. Structure-activity relationship studies of OSW-1 analogues revealed that a new 23-oxa-analogue of OSW-1 had much more potent antitumor activity than its parent OSW-1 [42]. Recently, a biotinylated OSW-1 was successfully synthesized for cell targeting studies [43]. Other than the anti-cancer activity of some saponins such as OSW-1 and its analogs described above, some saponins such as QS-21Aapi and QS-21Axyl has been used as the potent immunoadjuvants for vaccine. The saponin extracts of South American tree Quillaja saponaria Molina have long been used as foaming agents in beverages. Recently, the extracted saponins attract tremendous attention for its immunological adjuvant activities and have been used as a critical adjuvant component in many vaccine therapy trials [44]. QS-21A (the 21st fraction from Reverse Phase-HPLC) was the minor constituent which comprised two isomeric triterpene glycoside saponins QS-21Aapi (43) and QS-21Axyl (44) (Figure 8) [45]. These two complex triterpene-oligosaccharide-normonoterpene conjugates consist of a quillaic acid as a central lipophilic core, a branched trisaccharide, a linear tetrasaccharide, and an extended glycosylated diester side chain (Figure 8). QS-21Aapi (43) and QS-21Axyl (44) have a β-D-apiose and a β-D-xylose, respectively, as the terminal saccharide residue on the linear tetrasaccharide substructure. Unfortunately, obtaining sufficient quantities of these natural products in pure form is a daunting project due to their low abundance.

O HO O HO HO

O HO O OH HO OH O HO

CO2-

O HO

O CHO

OH HO O

HOO HO

O OH

43, QS-21Aapi R=

HOOOH

44, QS-21Axyl R= HO O

Figure 8. Structures of QS-21Aapi (43) and QS-21Axyl (44).

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The synthesis of the fully protected branched trisaccharide and the linear tetrasaccharide components of QS-21Aapi were reported by Zhu et al. [46]. The total synthesis of QS-21 Aapi (43) [47], QS-21Axyl (44) [48], QS-7-Api [49] was successfully accomplished by Gin and his co-workers. The total synthesis of QS-21 Aapi (43) was achieved in 2006 by judicious choice of the coupling protocols and protecting patterns (Figure 9) [47]. A convergent coupling strategy was used for conjugating four building blocks including a branched trisaccharide (45), a quillaic acid acceptor (46), a tetrasaccharide fragment (47), and an acyl chain (48) (Figure 9). The glycosyl acceptor, a 30-carbon triterpene quillaic acid ester (46), was prepared by acid-mediated hydrolysis of natural semipurified QS saponins followed by selective protection. In Gin’s total synthesis, most of the glycosidic linkages were constructed with sulphoxide-mediated dehydrative glycosylation (Ph2SO-Tf2O) method using hemiacetal donors. The steroselective coupling between branched trisaccharide α-imidate (45) and the quillaic allyl ester (46) was achieved using a less common B(C6F5)3 Lewis acid as the promoter. The coupling of linear tetrasaccharide (47) and acyl chain (48) under Yamaguchi conditions provided the complex sugar ester in 90% yield. This sugar ester was then converted to its α-imidate and coupled with the acid of glycosylated quillaic acid triterpene to give the fully protected QS-21Aapi. QS-21Aapi was finally achieved after global deprotection [47]. BnO BnO

O BnO BnO

CO2Me

AcO O

OBn O

CO2All OH

BnO CCl3

OBz

CHO

45 OH BnO TIPSO

HO OBn OBn

O O

O BnO O

TBSO O

TBSO O O

O TBSOO TBSO

OTBS

Figure 9. Structures of four building blocks for the synthesis of QS-21Aapi (43).

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Carbohydrate-containing natural products in medicinal chemistry

The total synthesis of the other two QS saponins, QS-21Axyl (44) [48] and QS-7-Api [49], were also accomplished by the same group using a similar approach. Most recently, Gin and his co-workers designed and synthesized several amide-modified, non-natural QS-21 analogs [50]. These synthetic saponins were chemically stable and exhibited similar or even better immunopotentiating effects in in vivo assays with GD3-KLH melanoma conjugate vaccine. The highly convergent synthesis of these novel non-natural saponins provides new avenues for searching and identifying improved molecular adjuvants for specifically tailored vaccine therapies. Some other saponins have been isolated and characterized with significant biological activities such as anticancer, anti-infection, anti-fungi etc. Because of the lengthy steps of protection/deprotection and sometimes low yields and low stereoselectivity in the glycosidic coupling processes suffered in saponin synthesis, only a small portion of these natural products have been synthesized. For example, two complex triterpene saponins, Lobatoside E (49) [51] and Candicanoside A (50) [52] both exhibited potent anticancer activity, have been recently synthesized by Yu’s group (Figure 10). However, many others, such as Avicin D (Figure 10, 51) [53] which also exhibited potent anticancer activity, are still attractive total synthetic targets that have not been synthesized.

O HO O

HO HO HO

O O O HO O

OH OH O

O HO O

O OH O

O O

HO HO OH

HO HO HO

OH O

HO HO

Anticancer Total Synthesized in 2008 Lobatoside E (49)

Anticancer Total Synthesized in 2007 Candicanoside A (50) O O

HO O HO HO

O O HO HO

O O NHAc

OH O

O O

HOO

OH HO

O HO HO OH

OH OH O

OH OH OH

Anticancer (To be synthesized) Avicin D (51)

Figure 10. Structures of three bioactive complex triterpene saponins.

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4. Glycosylated macrolides Many microlides produced by bacteria have sugar moieties and have excellent activity against Gram-positive bacteria. Many of them such as erythromycin A (52), oleandomycin, spiramycin, josamycin, tylosin, and midecamycin have been successfully used in clinic for years [54,55]. In addtion, cytotoxic tetraene macrolide CE-108 (53) [20-56], a secondary metabolite of Streptomyces diastaticus 108, and amphotericin B (54) [57] are good candidates for broad-spectrum antifungal drugs. Most recently, a new 18-membered macrolide glycoside, biselyngbyaside (55) from marine cyanobacterium Lyngbya sp., has been reported to exhibit uncommon broadspectrum antitumor activity in a human tumor cell line panel [58].

5. Glycosylated cyclic peptides Cyclic polypeptides have been considered as potential antimicrobial agents for pharmaceutical, preservation, cleaning, and disinfection uses [59]. Most of these cyclic polypeptides are of microbial origin and many have carbohydrate moieties. Many of these glycosylated cyclic peptides, such as O HO

HO HO

NMe2

HO O

O O

NH2

(Antifungal) CE-108, 53 OH

O OH

OMe

(antibacterial) Erythromycin A, 52

OH O

O HO

(Antifungal) Amphotericin B, 54

O NH2

HO HO MeO

OMe

(antitumor) Biselyngbyaside, 55

Figure 11. Structures of representative glycosylated macrolides.

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Carbohydrate-containing natural products in medicinal chemistry OH H2N

HO OH HO Cl O

O HO HO HO

O O

HO HO

Cl O

H N

N H

O O

HO NH2 H H N

H2N

O O H N

Vancomycin, 56

NH2

S+

H N

N H

H N

N H

NH O

NH2

O O

OH OH

H N

HO OH

Teicoplanin, 57

NH2 H

N H

Cl O

AcHN O

NH2

H N

N H O

OH O

O O

HO O

O H N O

O H

H H N

O HO H O H O HO N H H H N O H OH O O N H OH OH O OH OH O

O NH2

HO H N

S H N

NH N

Bleomycin, 58

HO H N

O N H

H2N O

HO HO HO

N O O

R OH

O NH2

H N

O HO

N H

O O

R = OH, Hassallidin A, 59a R = L-Rhamnose, Hassallidin B, 59b

Figure 12. Structures of some glycosylated cyclic peptides.

vancomycin (56), teicoplanin (57), bleomycin (58) and ristocetin etc., are very important antibiotics and some of them have been considered as the last resort for treating multiple resistant bacteria infections (Figure 12) [60]. Hassallidins A (59a) [61] and B (59b) [62] isolated from a cyanobacterium Hassallia sp., have shown broad-spectrum antifungal activity. Compared to hassallidin A, hassallidin B has an extra rhamnose attached to the 3-hydroxyl group of the acyl chain and was shown to have increased water solubility without decreasing its potent antifungal activity [20,62].

6. Cyanogenic glycosides Cyanogenic glycosides are secondary metabolites widely distributed in more than 2500 plant species. They comprise a sugar moiety, mostly D-glucose, beta-linked to an alpha-hydroxynitrile type aglycone. The sugar in some cases can also be gentibiose, primeverose or others, and the aglycones can be aliphatic or aromatic compounds (Figure 13) [63,64]. Cyanogenic glycosides can release hydrocyanic acid (HCN) upon hydrolysis. They are believed to participate in defense mechanisms of many plants against different phytopathogens [63,65].

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CH3

O OH

HO HO HO

O OH

CO2H

HO HO HO

Cynocardin, 62

HO HO HO

O O

NC O O OH

CO2H

Lithosperm, 64

Tryglochinin, 63

NC O

O OH

Aciapetalin, 61

O OH

CH3

Linamarin, 60 HO HO HO

HO HO HO

Figure 13. Structures of some cyanogenic glycosides.

7. Glucosinolates Glucosinolates are sulfur-rich secondary metabolites of plants which contain beta-D-thioglucose and sulpholated oxime moieties (Figure 14) [66-68]. The glucosinolates share some common features with cyanogenic glycosides, such as similar biosynthetic pathway at the early stages and both can be hydrolyzed to generate toxic degradation products in plant defense. The biosynthesis of glucosinolates comprised three steps, sidechain elongation of precursor amino acids, formation of the core glucosinolate structure, and side-chain decoration. The biological activity of glucosinolates is not limited to protection against various pathogens and weeds in case of plants, and recently studies demonstrated it has antifungal, antibacterial, antioxidant, antimutagenic and anticarcinogenic effects [69-73]. HO HO HO

N O S OH

Sinalbin, 66

OSO3-

HO HO OH HO

OSO3-

O S

H N

HO HO HO

OSO3-

Glucobrassicin, 67

Sinigrin, 68

Figure 14. Structures of some glucosinolates.

8. Other carbohydrate-containing natural products Other carbohydrate-containing major secondary metabolites which have recently been identified as potent antimicrobial agents or dietary supplements include antifungal glycosylated flavonoid 69, antimicrobial glycosylated iridoids (monoterpenoids) 71 and 73, antimicrobial glycosylated lignan 74, and antibacterial glycosylated terpenoid 72 (Figure 15) [20,74-76]. In addition, flavonol glycoside rutin (70, from buckwheat and rue) and the flavanone glycoside hesperidin from Citrus peels have been used as vitamin P in dietary supplements [5].

425

Carbohydrate-containing natural products in medicinal chemistry MeO2C O

HO HO

OH HO

HO HO HO

OH O

O H

O AcO O O

H CO2H

O O

OH OMe HO

O HO

H OH

AcO

OMe

HO O

H OH

OH HO HO HO

rutin, 70

69 O

OH O-Glc-Rha

OH OH

HO HO HO

MeO

OMe OH

OMe HO HO

OH OH

hesperidin, 75

Figure 15. Some glycosylated flavonoids, iridoids, lignans & terpenoids.

9. Current advances in glycosylated natural products: Glycorandomization/glycodiversification Many glycosylated naturally occurring antibiotics and its synthetic analogs are widely used in the clinic for the treatment of various human diseases such as antibacterial, anticancer, antifungal, antiparasite drugs, etc [77,78]. These antibiotics can be classified to macrolides, enediynes, anthracyclines, coumarins, non-ribosomal peptides, aminoglycoside, polyenes, aureolic acids, and others, according to their specific architectures. The emergence of pathogenic bacteria that are resistant to multiple antibiotics represents a growing threat to human health and has given additional driving force for the search for novel antibiotic drugs [79-81]. Fewer and fewer new drugs have been found in target screening programs during the past two decades, and scientists have started to look for new technologies to generate new compounds. Accumulating evidence has shown that the sugar moiety of many antibiotics play pivotal roles in drug targeting and activity. Alteration of the carbohydrate structures of drugs, therefore, will have profound effect to their molecular targeting and organism specificity. Recently, Thorson’s group reported a promising new glycoengineering (glycorandomization or glycodiversification) strategy for drug development by quick accessing a library of diverse natural product analogs [82,83]. One example is glycoengineering of vancomycin using a promiscuous glucosyltransferase GtfE and an expanded pool of NDP-sugars (Figure 16) [84,85]. Vancomycin (56), a glycosylated natural product from Amycolatopsis orientalis, is considered the last defense against infections caused by methicillin-resistant Gram-positive bacteria. Two glycosyltransferases, GtfE and GtfD, are involved in the vancomycin biosynthetic pathway to stepwisely add L-vancosaminyl-1,2-D-glucosyl disaccharide to the 4-hydroxyphenylglycine of the heptapeptide vancomycin aglycone (Figure 16a) [86].

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a) Glycosylation steps in the vancomycin biosynthetic pathway

O HO R OH

vancomycin aglycone

dTDP-Glc

dTDP-vancosamine

GtfE

HO HO

GtfD

vancomycin aglycone

O O

vancomycin aglycone

b) Glycoengineering and futher chemical diversification of vancomycin (Thorson, 2003) R1

NDP-sugars vancomycin aglycone

alkynes vancomycin aglycone

GtfE

OH O

vancomycin aglycone

N H

vancomycin aglycone

OH O

N H O

H N O

N H

H N

NH2

O HO

H N

OH O

HO O

Click-chemistry

HO HO N

OH OH

Figure 16. Glycoengineering of vancomycin.

Previous studies have shown that GtfD and GtfE have flexible substrate specificity [86,87]. Thorson and his co-worker further exploited these properties and found that 21 of the 23 TDP-sugars generated through chemoenzymatic synthesis were utilized by GtfE to give a library of novel vancomycin analogs (Figure 16, Path way B). The vancomycin analog which has an azidosugar moiety (6-azido-6-deoxy-glucopyranose) can be further modified in the presence of alkynes via “Click-Chemistry” to generate 39 additional vancomycin derivatives. One of the new compounds displayed improved antibiotic activity against Staphylococcus aureus and Enterococcus faecium (Figure 16) [84,85]. The glycodiversification strategy has been recently employed by the same group in generating calicheamicin analogs. A new reversible reaction mechanism catalyzed by the glycosyltransferases (GTs) was discovered during the course of their studies (Figure 17) [88]. Calicheamicin (Figure 17, 77) is a member of the enediyne family of antitumor antibiotics isolated from Micromonospora echinospora. Thorson and his co-workers demonstrated

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Carbohydrate-containing natural products in medicinal chemistry A) Glycosylation step catalyzed by CalG1 in the Calicheamicin biosynthetic pathway

O HO

O HO MeSSS

O I

H O N HO

OMe OH

HO OMe

NHAc HO MeO

OTDP

CalG1

B) Modification Calicheamicin by in vitro glycodiversification

S OMe OH

NHAc

MeSSS O H N HO 76

OMe

O HO MeO OH

OMe OH OMe

O H N HO

Calicheamicin, 77

OTDP O

CalG1

MeSSS

O I

NHAc

O O

MeSSS

OMe OH

O H N HO

NHAc

H O

OMe Calicheamicin derivatives with non-natural sugar moiety

C) Modification Calicheamicin by reverse glycosyltransferase reaction

S OMe OH

NHAc

MeSSS

O H N HO

O HO

O I

OTDP O

CalG1

MeSSS

OMe OH

O H N HO

NHAc

H O

OMe

Calicheamicin derivatives with non-natural sugar moiety

O OTDP HO MeO OH

CalG1 TDP

O HO MeSSS

O I

OMe OH

O O HO MeO OH

OMe

O H N HO

NHAc

H O

Figure 17. Glycoengineering of calicheamicin by in vitro glycodiversifation and reverse glycosyltransferase reaction.

that ten different TDP-sugars can be utilized by calicheamicin glycosyltransferase CalG1 to give calicheamicin derivatives with different sugars (Figure 17, Pathway B). Quite interestingly, when TDP-3-deoxy-α-Dglucose was incubated with CalG1 in the presence of calicheamicin (77), a calicheamicin derivative with the sugar moiety being replaced by 3-deoxy-αD-glucose was identified (Figure 17, Pathway C). Close investigation revealed that CalG1 catalyzed a reverse glycosyltransferase reaction in the presence of TDP to generate TDP-sugar and deglycosylated calicheamicin as a glycosyltransferase acceptor for producing the final derivative (76) [88]. More than 70 different calicheamicin derivatives were generated by this CalG1 catalyzed reverse reaction from 8 calicheamicin derivatives and 10 CalG1 recognized TDP-sugars. This GTs catalyzed reverse reaction was demonstrated to be a novel approach for the “aglycone exchange” reaction and can be applied for the synthesis of NDP-sugars [88]. Calicheamicin aminopentosyltransferase (CalG4) and vancomycin GTs (GtfD and GtfE) were also shown to catalyze reversible reactions in this study, suggesting that reversibility may be a general property of GTs involved in glycosylation of natural products in vitro [88].

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10. Prospective and conclusion Finding drugable carbohydrate-containing natural products remains an ongoing process. With the increasing interests in the field of carbohydrates and the rapid advance of the powerful tools including chemical synthetic strategies, cheomenzymatic methods, and glycodiversification strategies, it is now possible to expand the existing repertoire of carbohydrate-containing natural products to find new drugs that can be used to protect human health and to combat and treat diseases. Nevertheless, developing more efficient and more economic synthetic approaches for synthesizing carbohydratecontaining natural products remains to be a great challenge and thus an active area of research for years to come.

Acknowledgements We are grateful for financial supports from Shandong University (to H.C.), the National Science Foundation of China (No. 20902087 to H.C.), the University of California-Davis (to X.C.), the National Institutes of Health (R01GM076360 and U01CA128442 to X.C.), the National Science Foundation (CAREER Award 0548235 to X.C.), Alfred P. Sloan Foundation (to X.C.), and the Camille & Henry Dreyfus Foundation (to X.C.). X.C. is an Alfred P. Sloan Research Fellow, a Camille Dreyfus Teacher-Scholar, and a UC-Davis Chancellor’s Fellow.

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