Biology and Chemistry Research
Design and Synthesis of a Novel Thiolate Histone Deacetylase Inhibitor Maxwell Tucker ABSTRACT Histone deacetylase inhibitors are a class of chemotherapeutic epigenetic drugs that have recently been found to be quite effective in the treatment of a number of late stage carcinomas. The inhibitors interfere with gene expression by chelating the zinc ion found in class I histone deacetylases, which causes apoptosis in cancer cells. However, current commercial inhibitors suffer from either limited selectivity causing a host of side effects, or structural complexity which poses a significant synthetic challenge. Here, a novel thiolate histone deacetylase inhibitor with a small aromatic surface recognition region and an aliphatic linker is reported. This molecule was designed using the MolDock virtual docking method, and is anticipated to display significantly greater selectivity than current hydroxamic acid inhibitors by combining the thiolate chelating agent characteristic of depsipeptide inhibitors with a structurally simple linker and capping group. After design and modeling, this compound was synthesized with a simple three-step synthesis, verified by FT-IR and NMR, making production very simple when compared to current selective cyclic depsipeptide inhibitors. This compound shows significant promise as a novel histone deacetylase inhibitor for the treatment of cancer. 1 Introduction Histone deacetylase inhibitors are a class of drugs which have been used for many years to treat a number of disorders, including mood disorders and epilepsy. These inhibitors have only recently come into use as cancer treatments; although research into this application began in the midnineties, the FDA did not approve the first drug, vorinostat, until 2006 [1]. Currently only two histone deacetylase inhibitors are approved for cancer treatment, although many more are in development. This class of drugs is very promising due to its favorable cytotoxicity profile along with its high selectivity and potency [2]. However the most potent inhibitors feature complex structural components which make synthesis a challenge. In this project a novel inhibitor with high predicted selectivity and simple synthesis is proposed. Histone deacetylase (HDAC) is an enzyme which removes acetyl groups from histones. Histones are a class of proteins upon which DNA is coiled and are a major component of chromatin. The removal of acetyl groups allows for tighter packing of the DNA on the histone proteins, and as a result limits gene expression due to the physical inaccessibility of the genetic material. This function is opposite of that which is completed by the histone acetyltransferase enzyme, which acetylates certain histones and therefore promotes gene expression. Eighteen HDACs have been identified in humans, and they are divided into three classes based on structure. Class I and class II HDACs contain a metallic ion cofactor in their active site, Zn2+ [3]. While other HDACs do play important roles in the body, class I is of primary concern for cancer treatment [4]. This is because class I HDACs are found almost exclusively in the nucleus and have the most effect on gene 40 | 2013-2014 | Volume 3
expression, while class II inhibitors are believed to have tissue specific functions [5]. Thus selectivity for this class is a valuable trait in inhibitors, as it prevents unwanted side effects and yields better performance. Histone deacetylase inhibitors (HDACi) offer epigenetic treatment and act as antagonists, binding to the HDAC and preventing it from fulfilling its purpose. This is desirable because overexpression of HDACs is reported in a number of cancers, and inhibition can lead directly to apoptosis [5]. Structurally, HDACis consist of 3 parts: the chelating agent, the linker, and the capping group (Figure 1).
The chelating group has the largest effect on HDACi efficacy because it coordinates with the Zn2+ in the HDAC, thus preventing it from catalyzing deacetylation [6]. The linker length can have a significant impact on efficacy of a HDACi because it effects the ability of the chelating agent to reach the zinc ion. However, this length is easily adjustable and can be optimized simply. The capping group, which acts as a surface recognition section for the enzyme, is perhaps one of the most important parts of an inhibitor, as it has a large effect on the selectivity and activity of an inhibitor. HDACis are divided into categories based on the properties of their capping groups and chelating agents.
Biology and Chemistry Research 1.1 Hydroxamic Acid Inhibitors The most studied class of HDACis is the hydroxamic acids. These inhibitors contain a hydroxamic acid as a chelating agent, which forms a very stable 5 membered ring with the zinc ion [3]. One such inhibitor is suberoylanilide hydroxamic acid (SAHA), also known as vorinostat, which is now on the market under the name Zolinza for late stage cancer treatment [1,7]. Studies have even shown that SAHA may be effective for the treatment of Huntington’s disease, hinting at possible other applications for deacetylase inhibitors [8]. Hydroxamic acid inhibitors tend to have small, hydrophobic, and usually aromatic surface recognition groups, which are simple synthetically due to the easy availability of precursors. In figure 2, note these similarities across 3 common hydroxamic acid inhibitors [9]. This hydrophobicity is very important to activity, as it increases binding affinity by creating favorable interactions with the amino acid residues around the active site, and in fact nearly all HDACis have a hydrophobic surface recognition areas. Despite these favorable stuctural characteristics, hydroxamic acid inhibitors do feature a notable downside when compared to other inhibitor classes. These inhibitors do not show the high level of selectivity seen in other inhibitors, and therefore have less favorable cytotoxicity profiles. This manifests itself in uncomfortable side effects for the patients, as evidenced by clinical trials of vorinostat, in which some patients experienced pain, anorexia, fatigue, nausea, and vomiting [4].
body to form the active thiol [10]. Another naturally derived depsipeptide inhibitor is largazole, a compound that is still in preliminary stages but may one day be approved for cancer treatment. Largazole contains a thioester which is transformed within the body to form the thiol active metabolite. Largazole and romidepsin actually are very structurally similar, with identical chelating agents and linkers, along with very similar macrocycles [11].
In general, the depsipeptide inhibitors yield higher activity and selectivity than hydroxamic acid inhibitors [9]. However, depsipeptide inhibitors have large macrocycles which are challenging synthetically. For example, largazole has a 17 carbon macrocycle which contains two five membered heterocycles as well as a number of substituents. This creates a synthesis which consists of many steps and has a relatively low yield overall. When considering commercial production, this complexity could make such a drug prohibitively expensive, especially when compared to the structurally simple hydroxamic acid inhibitors. 1.3 Project Goals
1.2 Depsipeptide/Thiolate Inhibitors Another class of HDACi is the depsipeptide class. These inhibitors contain large cyclic depsipeptide capping groups and feature a thiol chelating agent. One inhibitor of this class, romidepsin (FK228), is one of only two HDACis to be approved by the FDA for cancer treatment. Romidepsin, like many depsipeptide inhibitors acts as a prodrug, with a disulfide bond that is reduced within the
The purpose of this project is to create a synthetically and structurally simple yet highly effective histone deacetylase inhibitor for use in cancer treatment. This can be accomplished by combining desirable traits from multiple classes of inhibitor, in particular by combining the thiolate chelating agent with the simple aromatic capping regions from hydroxamic acid inhibitors. Surprisingly, work in this area has been relatively limited. While a few attempts at thiolate analogues of SAHA have been made, few entirely engineered HDACis have been synthesized [12,13]. Therefore the goal is to create a structurally simple, easy to synthesize product that should be highly effective as an inhibitor, and in particular yields greater selectivity than current hydroxamic acid inhibitors. The design of this molecule can be carried out using computational models of protein-ligand binding to accurately predict most optimal structures.
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Biology and Chemistry Research 2 Materials and Methods 2.1 Computational Modeling and Structural Determination To determine an ideal molecular structure for a novel inhibitor, computational methods were utilized. Molegro Virtual Docker software was used to model the interactions between large number of potential structures and histone deacetylase proteins, as well as modeling several known HDACis as reference ligands. Molegro Virtual Docker uses the MolDock molecular docking method, which utilizes a heuristic method and an evolutionary algorithm. This particular docking method has been shown to yield significantly higher accuracy than other molecular docking methods, and allowed for easy relative comparison of many ligands, which was a valuable trait for these tests [14]. Additionally, MolDock supports metal cofactors, which is especially important for class I HDACs which contain zinc dependent binding sites. The crystal structure of HDAC8, determined by X-ray crystallography, was obtained from the worldwide Protein Data Bank [15]. This structure was imported into the Virtual Docker, and then an analysis of electrostatic forces was used to detect cavities which could be likely binding sites. The proper cavity was selected manually based on the location of the zinc ion, and ligands were subsequently docked with this cavity. In total, eight ligands were docked on the protein, 6 of which were novel structures, along with two reference molecules, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both visible in figure 2. Each of these novel compounds featured a similar surface recognition domain, derivative of the structure found in TSA. However, the tertiary amine was modified to a primary amine for the sake of synthetic simplicity. This surface recognition structure was decided upon due to the relatively high performance of TSA when compared to other hydroxamic acid inhibitors [16]. In these six novel ligand structures, changes were made to the linker domain, including esterification and varying aliphatic chain length. Figure 4 shows some of the capabilities of this software, including the graphical interface and output. The software was also instructed to calculate relative binding affinities
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of the top 5 poses for each ligand These poses were then viewed for structural accuracy, and then compared to yield the structure with most ideal inhibition properties. Results from this modeling can be seen in table 1. 2.2 Novel Thiolate Synthesis Based on binding affinity results from the computational modeling described above, the molecule visible in figure 5 was settled upon. Structurally, it consists of two identical molecules joined by a disulfide bond. The starting materials for this synthesis were 4-nitrobenzoyl chloride and 6mercapto-1-hexanol, as seen in figure 6.
The first synthetic step was the esterification reaction between the acid chloride and the alcohol. This procedure was adapted frvom Hubbard and Brittain [17, 18]. 7.45x10−4 moles of both 4nitrobenzoyl chloride and 6-mercapto-1-hexanol were combined in 10 mL dichloromethane with triethylamine catalyst. This reaction mixture was stirred for three and a half hours in an ice bath at 0â—ŚC under an argon atmosphere. The reaction was monitored via thin layer chromatography using a 2:3 ratio of hexane to ethyl acetate as the eluent, and then visualized using UV light. The product mixture was washed three times with a brine solution, then dried over sodium sulfate. This sample was purified using column chromatography with silica gel 60 and a solvent mixture consisting of 3:1 hexane and ethyl acetate. After purification, the sample yielded one spot on TLC and was deemed to be relatively pure. The solvent was extracted from the column fractions using rotary evaporation, and produced an oily yellow solid. An attenuated total reflectance (ATR) attachment on a Fourier transform infrared spectrophotometer (FT-IR) was used to verify structure. Strong absorbance at 2928 cm−1
Biology and Chemistry Research was indicative of an aliphatic CH stretch, and the carbonyl peak at 1720 cm−1 was indicative of the successful addition of the acid chloride. Strong absorbance at 1350 cm−1 was also indicative of the nitro group, which provides additional evidence for a successful synthesis. The next step in this synthetic plan was the reduction of the nitro group to an amine. This reduction was accomplished using stannous chloride dihydrate as described by Bellamy [18,19]. The purified product from the previous reaction was combined in a 1:5 molar ratio with SnCl2·2H2O in ethyl acetate. This reaction mixture was then heated to 70◦C and stirred for one hour. The majority of SnCl2·2H2O was removed using gravity filtration, and the remaining reaction mixture was washed with brine and then dried over sodium sulfate. TLC showed the sample to be relatively pure, and thus no further purification was necessary. Solvent was evaporated using a rotary evaporator, and the sample was then massed to calculate yield. IR spectroscopy was once again used for structural verification, and there was clear indication of a primary amine with absorbance at 3364 cm−1. This reaction yields the product seen in figure 8.
The final reaction involved the oxidation of the thiol to form a disulfide dimer of figure 8, thus producing the molecule seen in figure 5. This was accomplished by stirring the thiol in a solution of dichloromethane under an oxygen atmosphere at slight positive pressure for 20 hours, producing a near quantitative yield of the disulfide. Final structural verification was carried out using proton and carbon-13 NMR. After this first synthesis, a second scale-up synthesis was completed to verify results and test scale-up feasibility. For the benzoyl chloride esterification, all amounts were quintupled, and reaction time was increased to 4.5 hours. Column purification failed with a ethyl acetate:hexane eluent due to poor product solubility in this mixture. To rectify this, a 3:2 mixture of dichloromethane and hexane was used. The nitro reduction reaction was run using an identical procedure. However, the final oxidation of the thiol to a disulfide was done using a new procedure, using hydrogen peroxide catalyzed with potassium iodide, adapted from Kirihara et al [20]. Molar equivalents of the thiol and 30% H2O2 solution were combined in the presence of one mole percent KI and stirred for 4 hours in 10 mL of ethyl acetate at room temperature. Extraction was preformed using ethyl acetate, washed once with deionized water and three times with brine. The resulting organic layer was dried over sodium sulfate, and then solvent was removed using a rotary evaporator. 2.3 Instrumentation All infrared spectra were acquired using a Shimadzu FTIR 8400S. Eighty scans were run to yield accurate spectra. An attenuated total reflectance (ATR) adapter was used to allow for the analysis of the solid samples. Proton and C13 NMR were obtained using an Anasazi EFT-60 spectrometer. The proton spectra were collected running 128 scans at 60.01 MHz. Carbon-13 spectra were collected with 32,768 scans at 15.089MHz. Both scans were collected on approximately 30 mg of product dissolved in deuterated chloroform. These spectra were created using equipment at an off-site academic institution due to the lack of instrumentation available locally. 3. Results and Discussion 3.1 Molecular Modeling and Structure Determination The MolDock virtual docker engine generated hundreds of potential poses for each of the eight potential ligands modeled. For each pose generated, numerous parameters are considered, including hydrogen bond energies, electron affinity, cofactor interactions, and many more. All of these parameters are combined to create the rerank score, which is designed to accurately predict relative binding affinity for a number of different ligands. Table 1 shows the top pose for each of the eight tested ligands and the associated Volume 3 | 2013-2014 | 43
Biology and Chemistry Research values for a number of key factors. All energies on an arbitrary relative scale unless otherwise noted. 
Note the six potential inhibitors tested, with varying linker length and esterification, along with a methyl group akin to TSA’s linker domain. There are some interesting trends to note from these data. In general, longer linker chains tend to perform better than shorter, and ester linkages seem to perform better than the ketone equivalents. Additionally, a methyl group directly adjacent to the carbonyl seemed to yield better binding affinity. While SAHA did outperform the ultimate synthetic target in terms of binding affinity for HDAC8, it is expected that the synthetic target will yield better selectivity due to the thiolate chelating agent. While better performance might have been gained from addition of a methyl group, this was decided against in favor of synthetic simplicity. If the modeled ligand from figure 10 is compared to the synthetic target from figure 5, it may be noticed that the synthetic target is a disulfide dimer of the modeled molecule. This is because the free thiol is vulnerable to various reactions within the body, and if unprotected will have limited effects in vivo. Other thiolate inhibitors have a variety of protecting groups. Romidepsin features a disulfide bond which is reduced to a thiol in the body, while largazole utilizes a thioester which is also reduced in vivo [10,21]. In this case, a disulfide dimer seemed to be a convenient solution that protected the otherwise vulnerable thiol while also avoiding any wasteful protecting groups. 44 | 2013-2014 | Volume 3
3.2 Synthesis Results The reaction of 4-nitrobenzoyl chloride and 6-mecapto-1-hexanol was successfully carried out with a yield of 30.4%. After purification, the sample produced one spot on TLC, indicating relatively high purity. This yield was lower than hoped, likely due to the low catalytic activity of triethylamine. Next the reduction of the nitro group to a primary amine was successfully carried out with a yield of 42.9%. This was surprising, as TLC reaction monitoring seemed to indicate that the reaction had gone to completion. Because of this, it seems likely that product was lost in the extraction process. In particular, there may be a significant amount of product in the aqueous portion from extraction. The final thiol oxidation had poor yield and a long reaction time using the O2 gas method. To rectify this, the hydrogen peroxide procedure was adopted, and produced near quantitative yields in a much shorter time than the O2 procedure.
While intermediate products were verified using IR spectroscopy, final structural verification was carried out using NMR. Proton and carbon-13 shifts were predicted computationally using B3LYP/6-31G**calculations after structural optimization had been preformed using a 6-31G*basis set. Figure 11 shows the carbon-13 spectrum used for final verification. Noise along the baseline is due partially to impurities and partially to the low resolution of this 60 MHz NMR. This spectrum shows 11 distinct peaks in locations similar to those predicted by the computations. This is conclusive evidence for the presence of the expected disulfide product. 4. Conclusions This project was successful in synthesizing an entirely novel compound that shows promise as a highly selective histone deacetylase inhibitor. Modeling using the MolDock platform yielded insights into the functionality of the inhibitors, and allowed for the design of a previously
Biology and Chemistry Research unsynthesized molecule with favorable docking features. This compound was then synthesized in a simple three step synthesis that should be easily replicable and required minimal purification. This offers significant improvement over current highly selective inhibitors. Romidepsin is synthesized with an overall yield of 9.5%, and requires separate syntheses for many uncommon starting materials, making it a huge synthetic challenge overall [22]. The increased structural simplicity of the product reported above is key when considering the large scale production necessary of all pharmaceutical substances. 5. Future Work Future work for this project falls into three main categories. First, additional computational data should be obtained to determine more about the properties of this inhibitor design. Experimentation with varying hydrophobic regions and non-aliphatic linkages could produce interesting results. However, the main computational work lies in validating the assertion that the thiolate chelating agent does in fact yield improved selectivity. This can be done by modeling the same set of test ligands on a number of other HDACs, including HDAC1, 2, and 3, as well as several class 2 and 3 HDACs. This would produce compelling evidence for the increased selectivity of thiolate chelating agents. Another important aspect of future work is increased synthetic yield and validation. Currently, the esterification reaction has relatively low yield, likely due to the low catalytic activity of triethylamine. This yield may be improved by using a different tertiary amine catalyst, such as 4-dimethylaminopyridine (DMAP) or diethylmethylamine [17]. The next reaction in the synthesis is the nitro group reduction. This reaction also showed a relatively low yield, although the cause of this is not fully understood. Thin layer chromatography indicated that the reaction went to completion, yet final product mass was disappointingly low. This may be due to product losses in the extraction process, where some product may have been retained in the aqueous phase or in the stannous chloride from gravity filtration. A better workup process, including switching extraction solvents, may produce better yield. Due to the inability to access NMR or mass spectrometry equipment, product analysis, especially of intermediate products, is not particularly robust. In a future synthesis replication, it would be necessary to obtain accurate spectra for each product to verify structure. Additionally, reaction scale-up should be attempted to production on a multi-gram scale. The final step in the project development would be preliminary biological testing. Testing can be performed on a variety of histone deacetylases to calculate IC50 values. This will allow for accurate real world comparison of this drug to other inhibitors, and if this preliminary testing is promising, in vivo testing could take place using established cell lines. Ultimately, it is hoped that this compound
has the potential to reach clinical trials and perhaps one day serve as a chemotherapeutic epigenetic drug for the treatment of late stage carcinomas. Acknowledgments I would like to thank Dr. Myra Halpin of the North Carolina School of Science and Mathematics, for mentoring me throughout this entire project. I would like to thank Dr. Darrell Spells for his organic chemistry expertise, along with the entire research program at NCSSM. Finally, I would like to thank Dr. W. Andrew Tucker of Queens University for his assistance with NMR Spectroscopy. References [1] Merck, “Study of ZOLINZA ( vorinostat ) for Investigational Use for Advanced Malignant Pleural Mesothelioma Did Not Meet Primary Endpoint,” Merck Newsroom, pp. 1–2, 2011. [2] J. a. Souto, E. Vaz, I. Lepore, A.-C. Poppler,¨ G. Franci, R. Alvarez, L. Altucci, and A. R. de Lera, “Synthesis and biological characterization of the histone deacetylase inhibitor largazole and C7- modified analogues.,” Journal of medicinal chemistry, vol. 53, pp. 4654–67 , June 2010. [3] K. E. Cole, D. P. Dowling, M. A. Boone, A. J. Phillips, and D. W. Christianson, “Structural Basis of the Antiproliferative Activity of Largazole, a Depsipeptide Inhibitor of the Histone Deacetylases,” Journal of the American Chemical Society, no. 133, pp. 12474–12477, 2011. [4] X. Ma, H. H. Ezzeldin, and R. B. Diasio, “Histone deacetylase inhibitors: current status and overview of recent clinical trials.,” Drugs, vol. 69, pp. 1911–34, Oct. 2009. [5] M. Dokmanovic, C. Clarke, and P. a. Marks, “Histone deacetylase inhibitors: overview and perspectives.,” Molecular cancer research : MCR, vol. 5, pp. 981–9, Oct. 2007. [6] Y. Liu, L. A. Salvador, S. Byeon, Y. Ying, J. C. Kwan, B. K. Law, J. Hong, and H. Luesch, “Anticolon Cancer Activity of Largazole , a Marine-Derived Tunable Histone Deacetylase Inhibitor,” Journal of Pharmacology and Experimental Therapeutics, vol. 335, no. 2, pp. 351 – 36, 2010. [7] W. K. Kelly, O. a. O’Connor, L. M. Krug, J. H. Chiao, M. Heaney, T. Curley, B. MacGregoreCortelli, W. Tong, J. P. Secrist, L. Schwartz, S. Richardson, E. Chu, S. Olgac, P. a. Marks, H. Scher, and V. M. Richon, “Phase I study of an oral histone deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced cancer.,” Journal of clinical oncology : official journal of the American Society of Clinical Oncology, vol. 23, pp. 3923–31, June 2005. Volume 3 | 2013-2014 | 45
Biology and Chemistry Research [8] E. Hockly, V. M. Richon, B. Woodman, D. L. Smith, X. Zhou, E. Rosa, K. Sathasivam, S. Ghazi-Noori, A. Mahal, P. a. S. Lowden, J. S. Steffan, J. L. Marsh, L. M. Thompson, C. M. Lewis, P. a. Marks, and G. P. Bates, “Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, pp. 2041–6 , Feb. 2003. [9] T. a. Miller, D. J. Witter, and S. Belvedere, “Histone deacetylase inhibitors.,” Journal of medicinal chemistry, vol. 46, pp. 5097–116, Nov. 2003. [10] R. Furumai, A. Matsuyama, N. Kobashi, and H. Deacetylases, “FK228 ( Depsipeptide ) as a Natural Prodrug That Inhibits Class I Histone Deacetylases FK228 ( Depsipeptide ) as a Natural Prodrug That Inhibits Class I,” Cancer Research, vol. 228, pp. 4916–4921, 2002. [11] J. Hong and H. Luesch, “Largazole: from discovery to broad-spectrum therapy.,” Natural product reports, vol. 29, pp. 449–56, Apr. 2012. [12] T. Suzuki, A. Kouketsu, A. Matsuura, A. Kohara, S.-I. Ninomiya, K. Kohda, and N. Miyata, “Thiol-based SAHA analogues as potent histone deacetylase inhibitors.,” Bioorganic & medicinal chemistry letters, vol. 14, pp. 3313–7, June 2004. [13] a. Saito, T. Yamashita, Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando, T. Suzuki, T. Tsuruo, and O. Nakanishi, “A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, pp. 4592–7, May 1999. [14] R. Thomsen and M. H. Christensen, “MolDock: a new technique for high-accuracy molecular docking.,” Journal of medicinal chemistry, vol. 49, pp. 3315–21, June 2006. [15] J. R. Somoza, R. J. Skene, B. a. Katz, C. Mol, J. D. Ho, A. J. Jennings, C. Luong, A. Arvai, J. J. Buggy, E. Chi, J. Tang, B.-C. Sang, E. Verner, R. Wynands, E. M. Leahy, D. R. Dougan, G. Snell, M. Navre, M. W. Knuth, R. V. Swanson, D. E. McRee, and L. W. Tari, “Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.,” Structure (London, England : 1993), vol. 12, pp. 1325–34, July 2004. [16] M. Yoshida, M. Kijima, M. Akita, and T. Beppu, “Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A .,” The Journal of Biological Chemistry, 1990. [17] P. Hubbard and W. J. Brittain, “Mechanism of Amine-Catalyzed Ester Formation from an Acid Chloride and Alcohol,” The Journal of Organic Chemistry, vol. 63, pp. 677–683, Feb. 1998. [18] T. W. Bentley, G. Llewellyn, and J. A. McAlister, “S(N)2 Mechanism for Alcoholysis, Aminolysis, and Hydrolysis of Acetyl Chloride.,” The Journal of organic chemistry, vol. 61 , pp. 7927–7932, Nov. 1996. [19] F. D. Bellamy, “SELECTIVE REDUCTION 46 | 2013-2014 | Volume 3
OF AROMATIC NITRO COMPOUNDS WITH STANNOUS CRLORIDE IN NON ACIDIC AND NON AQUEOUS MEDIUM,” Tetrahedron Letters, vol. 25, no. 8, pp. 3–6, 1984. [20] M. Kirihara, Y. Asai, S. Ogawa, T. Noguchi, A. Hatano, and Y. Hirai, “A Mild and Environmentally Benign Oxidation of Thiols to Disulfides,” Synthesis, vol. 2007, pp. 3286–3289 , Nov. 2007. [21] K. Taori, V. J. Paul, and H. Luesch, “Structure and Activity of Largazole , a Potent Antiproliferative Agent from the Floridian Marine Cyanobacterium Symploca sp .,” Journal of the American Chemical Society, vol. 130, no. 6, pp. 1806–1807, 2008. [22] T. Greshock, D. Johns, Y. Noguchi, and R. Williams, “Improved total synthesis of the potent HDAC inhibitor FK228 (FR-901228),” Organic letters, vol. 10, no. 4, pp. 613–616, 2008.