ADC Case StudyCustom 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: C51H58N8O13MW: 991.05Elemental 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 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.

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: C73H97N11O21MW: 1479.7Elemental 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

1.67

5

Ret. Time: 1.68

m/z200 400 600 800 1000 1200 1400

0250050007500

10000125001500017500

ES-API Positive

186.2 991.0130.2

496.2

Molecular peak [M+H] +

ES-APIPositive

494.4

25000

20000

15000

741.0

10000

5000

537.2 751.

80

400

600

800

1000

1200

1400

1600

1800

m/z

m0 0.5 1 1.5 2 2.5

mAU

0

*DAD1 A, Sig=214,4 Ref=off

1.43

7 Purity calculated based on peak areas

Mc Di-pep�de vc Self-elimina�on PAB

N

N

OO

OOH

N

NH

NH2O

O

NH

O

ONH

N

O

O

O

O

OH

10.

342

10.

008

8.0

89 8

.075

8.0

66 8

.048

7.8

11 7

.793

7.6

06 7

.589

7.4

35 7

.417

7.4

12 7

.307

7.2

90 6

.996

6.9

44

5.5

04 5

.320

5.3

10 5

.099

5.0

71

4.1

93 3

.450

3.3

76 3

.361

3.3

47 3

.104

3.0

89 2

.635

2.5

00 2

.423

2.3

62 2

.182

2.1

67 2

.153

2.1

40 2

.126

1.4

91 1

.476

1.4

58 1

.311

1.2

97 1

.281

1.1

95 1

.180

1.1

65 0

.908

0.8

94 0

.879

0.8

52 0

.839

0.8

21 0

.808

-0.0

01

2.10 1.59 1.77

1.57 0.76

1.68 0.71 0.83 0.72 5.23

1.72 2.02 8.56 6.16

5.94 5.06 4.61 4.25

10 8 6 4 2 0 PPM

N

N

OO

OOH

N

NH

NH2O

O

NH

O

ONH

N

O

O

O

O

OH

Chemical Formula: C51H58N8O13Exact Mass: 990.41

NH

O

NO

O ONNN

O

N

N

OO

OO

OO

OH

NH

O

NH

O

O

NH2

NH

O

n

n=7

8.0

99 8

.077

8.0

39 8

.020

7.9

78 7

.794

7.5

37 7

.513

7.4

94 7

.473

7.4

57 7

.284

7.2

63 7

.239

7.2

19 7

.151

6.6

85 5

.974

5.7

24 5

.682

5.3

72 5

.330

5.3

01 5

.292

5.2

61 5

.177

4.9

33 4

.903

4.5

56 4

.533

4.5

16 4

.501

4.4

88 4

.130

4.0

99 4

.056

3.8

82 3

.870

3.8

13 3

.622

3.6

17 3

.609

3.5

82 3

.514

3.4

96 3

.479

3.4

61 3

.367

3.3

33 3

.316

3.0

71 3

.063

3.0

53 2

.992

2.1

93 2

.174

2.1

33 2

.115

2.0

98 2

.080

1.8

56 1

.824

1.7

34 1

.704

1.6

99 1

.671

1.6

42 1

.485

1.4

25 1

.400

1.3

54 1

.335

1.3

16 1

.290

0.86 1.13 1.84 2.00 0.98 2.35 1.00 5.19 4.25 2.03 1.13 1.83 1.96 2.02 2.25 2.19

35.41

2.41 6.88

2.28 3.35 8.23 9.04

3.16

10 8 6 4 2 0 PPM

N

N

OO

OO

O

O

O H

H N

O

H N

O

O

H 2N

H NO

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 :d1STP 4.7642 :1FO 4228 :1WS 000.1 :2F231.004 :1F

0.0 :BL 8 :ANces 0.1 :DPcesu 8.31 :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

OH1 2 3

HN

O

H2N

MMTNH

OH4

52% 91% 88%

HN

O

NH

MMTNH

OH

O

O

O

HN

OO

N3 n HN

O

NH

MMTNH

O

O

O

O

HNOON3 n

N

N

O

OTBS

OOO

ClO

N

N

O

OTBS

OOO

O

H2O

5

6

7

70%

62%

N

N NN

O

HNO

O

HN

O

NH

H2N

O

O

O

O

HN

OO

n

N

N

O

OH

OO

O

O

TM n = 7

steps

HO

N

NNN

N

N

O

O

O

OOO

OO

10 7

7

OO

O

hRS7

OO

Short PEGfor solubility

Cathepsin Bcleavable peptide

when AA=Phe

pH-dependentcleavage site

CL2-SN-38: AA = PheCL2A-SN-38: AA=None

SN-38

CH2OHN

HN

NH

[AA]-Lys-[ ]

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