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.https://www.creative-biolabs.com/adc

1. ADC Case Study
Custom Synthesis of ADC Linker-payload SET
Introduction
SN38: a novel payload for ADC development
Conclusions References
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 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 Immunomed-
ics),a much more complex construct that requires 10-step synthesis.
SN38 ADCs in clinical trials
Chemical formula: C51H58N8O13
MW: 991.05
Elemental analysis: C-61.82; H-5.86; N-11.31; O-21.01
Mc-vc-PAB-SN38 was successfully synthesized in 3 weeks.
Final product is characterized as:
Proton peaks are labeled and integrated for structure identification
Proton peaks are labeled and integrated for structure identification
CL2A-SN38 was successfully synthesized in 10 weeks. Final product is
characterized as:
2. Structure confirmation: compound structure validated by 1HNMR
Based on the structure of CL2A-SN38, a 10-step synthesis
route was designed.
1. Purity: > 95% by LC-MS
2. Structure confirmation: compound structure validated by 1HNMR
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.
2.
3.
1.
2.
3.
4.
Reactions for each step were carried out in optimized
conditionswithsuitablesolventsandcatalysts,ifnecessary.
The yields of each step were recorded and reported, and
theproductsfromeachstepwerecharacterizedbyLC-MS
toensurethecorrectMW.
The final product: CL2A-SN38 was characterized by both
LC-MSand1HNMRtoassesspurityandvalidatestructure.
Synthesisleadtimewas10weeks.
1.
2.
3.
4.
Using the "DrugLnk" custom synthesis service, Creative Biolabs successfully synthesized two
SN38-linker complexes.
"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).
The compounds prepared by the "DrugLnk" custom synthesis service are correct in structure
and show excellent purity.
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.
1.
2.
3.
Compound specifics:
Chemical formula: C73H97N11O21
MW: 1479.7
Elemental analysis: C-59.88; H-6.63; O-22.97; N-10.52
1.
2.
3.
Compound specifics:
Results
Results
Case 1: Mc-vc-PAB-SN38
Case 2: CL2A-SN38
Synthesis route design:
CL2A-SN38 structure breakdown:
Synthesis route design:
Synthesis of Mc-vc-PAB-SN38:
Synthesis of CL2A-SN38:
Based on the structure of Mc-vc-PAB-SN38, a 3-step synthesis
route was designed.
Purity calculated
based on peak areas
min*0 0.5 1 1.5 2 2.5
mAu
780
*ADC1 A, ELSD
1.675
Ret. Time: 1.68
m/z200 400 600 800 1000 1200 1400
0
2500
5000
7500
10000
12500
15000
17500
ES-API Positive
186.2 991.0130.2
496.2
Molecular peak [M+H]
+
ES-API
Positive
494.
4
250
00
200
00
150
00
741.
0
100
00
50
00
537.
2
751.
80
40
0
60
0
80
0
100
0
120
0
140
0
160
0
180
0
m/
z
m0 0.5 1 1.5 2 2.5
mAU
0
*DAD1 A, Sig=214,4 Ref=off
1.437
Purity calculated
based on peak areas
Mc Di-pep�de vc Self-elimina�on PAB
N
N
O
O
O
O
H
N
NH
NH2O
O
N
H
O
O
N
H
N
O
O
O
O
OH
10.342
10.008
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
5.504
5.320
5.310
5.099
5.071
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
2.101.59
1.77
1.57
0.76
1.680.710.83 0.72
5.23
1.72 2.02
8.56
6.16
5.945.064.614.25
10 8 6 4 2 0 PPM
N
N
O
O
O
O
H
N
NH
NH2O
O
N
H
O
O
N
H
N
O
O
O
O
OH
Chemical Formula: C51H58N8O13
Exact Mass: 990.41
NH
O
N
O
O O
N
NN
O
N
N
O
O
OO
O
O
OH
N
H
O
NH
O
O
NH2
N
H
O
n
n=7
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
0.86 1.13 1.84 2.00 0.98 2.35 1.00
5.19 4.25
2.031.131.83 1.96 2.02 2.252.19
35.41
2.41
6.88
2.28 3.35
8.239.04
3.16
10 8 6 4 2 0 PPM
N
N
O
O
OO
O
O
O H
H N
O
H N
O
O
H 2N
H N
O
O
O
O
O
O
O
O
O
N
N
N
H N
O
N
O
O
C l
C l
O H
O
C l
C l
O H
O
USER: nmrB601 -- DATE: Fri Jan 13 02:21:33 2017
Nuts - $pdata
NMRB003, CDCl3,
86723:d1STP4.7642:1FO4228:1WS000.1:2F231.004:1F
0.0:BL8:ANces0.1:DPcesu8.31:WP03gz:XE
SN38 (irinotecan prodrug)
Sacituzumab govitecan
(also known as IMMU-132)
IgG1 TROP2 CL2A-SN38 TNBC
(phase III)
NCT02574455
NCT01915472Metastatic CRC
(phase II)
Immunomedics
(licensed to Seattle Genetics)
ImmunomedicsCL2A-SN38IgG1 CEACAM5Labetuzumab govitecan
(also known as IMMU-130)
H2N
OH
O
OH
O
NHFmocFmocNH
MMTNH
HN
O
NHFmoc
MMTNH
NH2
OH
OH
1 2 3
HN
O
H2N
MMTNH
OH
4
52% 91% 88%
HN
O
NH
MMTNH
OH
O
O
O
H
N
O
O
N3 n
HN
O
NH
MMTNH
O
O
O
O
H
N
O
ON3 n
N
N
O
OTBS
O
O
O
Cl
O
N
N
O
OTBS
O
O
O
O
H2O
5
6
7
70%
62%
N
N NN
O
HN
O
O
HN
O
NH
H2N
O
O
O
O
H
N
O
O
n
N
N
O
OH
O
O
O
O
TM n=7
steps
HO
N
N
N
N
N
N
O
O
O
OO
O
OO
10 7
7
O
O
O
hRS7
OO
Short PEG
for solubility
Cathepsin B
cleavable peptide
when AA=Phe
pH-dependent
cleavage site
CL2-SN-38: AA = Phe
CL2A-SN-38: AA=None
SN-38
CH2
O
H
N
H
N
N
H
[AA]-Lys-[ ]
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Address: 45-1 Ramsey Road, Shirley, NY 11967, USA
Tel: 1-631-357-2254
Email: info@creative-biolabs.com
Fax: 1-631-207-8356
Web:www.creative-biolabs.com/adc