ADC Case Study
Custom Synthesis of ADC Linker-payload SET Introduction The warhead of an antibody-drug conjugate (ADC) is comprised of a cytotoxic payload drug and a molecular linker that covalently bridges the antibody and the payload. With an extensive library of payload drugs and linkers, plus years of experience in synthetic and conjugation chemistry, Creative Biolabs is dedicated to helping our clients design and prepare highly customized linker and drug-linker complexes for the creation of ADCs using our featured “DrugLnk” services. From conventional payload-linkers bearing auristatin or maytansinoid derivatives to more innovated warheads such as topoisomerase inhibitors, we have created many unique compounds tailored to clients’ special projects. Presented here is a case study for the synthesis of two customized payload-linker complexes via the “DrugLnk” organic synthesis service. SN38 is the payload of choice and two cleavable linkers are used to formulate the SN38-linker complexes. For the protection of custom IP, the reaction conditions, catalysts, as well as solvents are omitted from the synthesis routes.
SN38: a novel payload for ADC development SN38 is an antineoplastic drug. It is the active metabolite of irinotecan (an analog of camptothecin - a topoisomerase I inhibitor) but is 1000 times more active than irinotecan. So far, two ADCs bearing SN38 as payload, both developed by Immunomedics, have entered clinical trial stage. CBL has extensive experience with SN38-related payload-linker synthesis. The successful cases include the synthesis of Mc-vc-PAB-SN38, a relatively simple synthesis involving 3 steps, and that of CL2A-SN38 (proprietary for Immunomedics), a much more complex construct that requires 10-step synthesis.
SN38 ADCs in clinical trials SN38 (irinotecan prodrug) Sacituzumab govitecan
IgG1 TROP2
CL2A-SN38
TNBC
Immunomedics
(also known as IMMU-132)
NCT02574455
(licensed to Seattle Genetics) (phase III) Metastatic CRC NCT01915472
Immunomedics
IgG1 CEACAM5 CL2A-SN38
Labetuzumab govitecan
(phase II)
(also known as IMMU-130)
Case 1: Mc-vc-PAB-SN38 OH O
O
O
NH O
Mc
NH 2
Di-pep�de vc
2.
Self-elimina�on PAB
Compound specifics: 1. 2. 3.
3.
Chemical formula: C51H58N8O13 MW: 991.05 Elemental analysis: C-61.82; H-5.86; N-11.31; O-21.01
496.2
Reactions for each step were carried out in optimized conditions with suitable solvents and catalysts, if necessary. The yields of each step were recorded and reported, and the products from each step were characterized by LC-MS to ensure the correct MW. The final product: Mc-vc-PAB-SN38 was characterized by both LC-MS and 1HNMR to assess purity and validate structure. Synthesis lead time was 3 weeks.
1.
O
N O
Ret. Time: 1.68
ES-API Positive
N
4.
17500 15000 10000 7500 5000 2500
130.2 186.2
0
991.0
200
400
600
800
1000
1200
1400
m/z
2. Structure confirmation: compound structure validated by 1HNMR
Synthesis route design: Based on the structure of Mc-vc-PAB-SN38, a 3-step synthesis route was designed.
+
Molecular peak [M+H]
12500
4.193 3.450 3.376 3.361 3.347 3.104 3.089 2.635 2.500 2.423 2.362 2.182 2.167 2.153 2.140 2.126 1.491 1.476 1.458 1.311 1.297 1.281 1.195 1.180 1.165 0.908 0.894 0.879 0.852 0.839 0.821 0.808 -0.001
N H
Synthesis of Mc-vc-PAB-SN38:
5.504 5.320 5.310 5.099 5.071
O
O
8.089 8.075 8.066 8.048 7.811 7.793 7.606 7.589 7.435 7.417 7.412 7.307 7.290 6.996 6.944
H N
N
O
10.008
N H
O
O
10.342
O
Results
OH O O
Mc-vc-PAB-SN38 was successfully synthesized in 3 weeks. Final product is characterized as:
N H
O
O
H N
N
O O
N H
O
O N O
O
N NH
O
O
NH2
Chemical Formula: C51H58N8O13 Exact Mass: 990.41
1. Purity: > 95% by LC-MS
Proton peaks are labeled and integrated for structure identification
*ADC1 A, ELSD
0.76 1.77 1.68 1.57 1.59 2.10
0.83 0.71
Purity calculated based on peak areas
5.23 1.72
0.72
4.25
2.02
4.61
5.06
5.94
6.16
8.56
1.675
mAu 780 0
0.5
1
1.5
2
2.5
min* 10
8
6
2
4
0
PPM
Case 2: CL2A-SN38 OH H2N N
O
N H
O
O
O N
O
O
N N N
HN
O
N H
n
O
O
MMTNH
H2N
O O
O
O
FmocNH
OH
Fmoc NH
1
OH
N3
Chemical formula: C73H97N11O21 MW: 1479.7 Elemental analysis: C-59.88; H-6.63; O-22.97; N-10.52
O
On
OH
Cathepsin B cleavable peptide when AA=Phe
N N
[
O
]7
H N O
O
H [AA]-Lys-N
O
O N H
hRS7
N O
O
N
HN
N N N
O
O
6
O O
OH
O
O
O
H N
n
N
O NH HN
O
O
m
250 00
741. 0
100 150 00 00 50 000
537. 2
40 0
751. 8
60 0
80 0
100 0
120 0
140 0
160 0
180 0
m/ z
2. Structure confirmation: compound structure validated by 1HNMR
N
O O
O
2.5
O O
TM n=7 OH
O
Cl
N
HN O
O pH-dependent cleavage site
AA = Phe AA=None
Synthesis of CL2A-SN38:
N
O
O O
OH
SN-38
O O O
O
O O
HN
OH Cl
Cl
Cl
HN
O
O
O
O
O
O
O
O
1. 2.
3. 4.
Reactions for each step were carried out in optimized conditions with suitable solvents and catalysts, if necessary. The yields of each step were recorded and reported, and the products from each step were characterized by LC-MS to ensure the correct MW. The final product: CL2A-SN38 was characterized by both LC-MS and 1HNMR to assess purity and validate structure. Synthesis lead time was 10 weeks.
Conclusions 1. Using the "DrugLnk" custom synthesis service, Creative Biolabs successfully synthesized two SN38-linker complexes. 2. "DrugLnk" custom synthesis service is well-suited for simple synthesis tasks with only a few steps and complex organic synthesis with multiple steps (in this case, a 10-step synthesis was successfully demonstrated). 3. The compounds prepared by the "DrugLnk" custom synthesis service are correct in structure and show excellent purity.
O
2
494. 4
O
O
CL2-SN-38: CL2A-SN-38:
O
On
O
H N
N
Purity calculated based on peak areas
1.5
H 2N
CH2O
O
O
N3
O
O
1
200 00
N
O
NH HN
0.5
O
N
N
N
O
O
steps
O
7
{
{
Short PEG for solubility
N O
7
62%
H2O
O
HO
O O
MMTNH
H2N
CL2A-SN38 structure breakdown: 10
Cl
5
O
O
0
ES-API Positive
OH
4
OTBS
N
NH HN
O
H N
HN
OTBS O
O
70%
H2N
OH
3
0
O
88%
Fmoc NH HN
2
MMTNH
Compound specifics:
O
O
91%
mAU
MMTNH
MMTNH
NH2
O
52%
n=7
O
1. 2. 3.
*DAD1 A, Sig=214,4 Ref=off
OH
N
O
CL2A-SN38 was successfully synthesized in 10 weeks. Final product is characterized as: 1.437
HN
Results
Based on the structure of CL2A-SN38, a 10-step synthesis route was designed.
8.099 8.077 8.039 8.020 7.978 7.794 7.537 7.513 7.494 7.473 7.457 7.284 7.263 7.239 7.219 7.151 6.685 5.974 5.724 5.682 5.372 5.330 5.301 5.292 5.261 5.177 4.933 4.903 4.556 4.533 4.516 4.501 4.488 4.130 4.099 4.056 3.882 3.870 3.813 3.622 3.617 3.609 3.582 3.514 3.496 3.479 3.461 3.367 3.333 3.316 3.071 3.063 3.053 2.992 2.193 2.174 2.133 2.115 2.098 2.080 1.856 1.824 1.734 1.704 1.699 1.671 1.642 1.485 1.425 1.400 1.354 1.335 1.316 1.290
O
Synthesis route design:
O
O
N N N
HN O O
N
O
35.41
9.04 Proton peaks are6.88 labeled and integrated for structure identification 5.19 4.25 0.86
10 NMRB003, CDCl3, F1: 400.132 EX: zg30
F2: 1.000
1.83 1.13 1.13
2.03 1.84
2.00
2.28 0.98 2.35 1.961.00
8 SW1: 8224 PW: 13.8 usec
6
PD: 1.0 sec
2.02
2.412.19 2.25
4 OF1: 2467.4 NA: 8
3.16
8.23 3.35
2
LB: 0.0
0
PPM
USER: nmrB601 -- DATE: Fri Jan 13 02:21:33 2017 PTS1d: 32768 Nuts - $pdata
References 1. Ramesh, M., Ahlawat, P., Srinivas, N.R. Biomed Chromatogr. 2010, 24: 104-123. 2. Cardillo, T.M., Govindan, S.V., Sharkey, R.M., et al. Bioconjug Chem. 2015, 26: 919-931. 3. Sharkey, R.M., Govindan, S.V., Cardillo, T.M., et al. Mol Cancer Ther. 2012,11: 224-234. 4. Jain, N., Smith, S.W., Ghone, S., et al. Pharm Res. 2015, 32: 3526-3540. Address: 45-1 Ramsey Road, Shirley, NY 11967, USA Tel: 1-631-357-2254 Fax: 1-631-207-8356 Email: info@creative-biolabs.com Web:www.creative-biolabs.com/adc