Design, synthesis and biological evaluation of phenol-linked uncialamycin antibody-drug conjugates

Journal Pre-proofs Design, Synthesis and Biological Evaluation of Phenol-linked Uncialamycin Antibody-drug Conjugates Yam B. Poudel, Chetana Rao, Srikanth Kotapati, Madhura Deshpande, Lourdes Thevanayagam, Chin Pan, Josephine Cardarelli, Naidu Chowdari, Mahammed Kaspady, Ramesh Samikannu, Prakasam Kuppusamy, Pon Saravanakumar, Pirama N. Arunachalam, Shrikant Deshpande, Vangipuram Rangan, Richard Rampulla, Arvind Mathur, Gregory D. Vite, Sanjeev Gangwar PII: DOI: Reference:

S0960-894X(19)30747-4 https://doi.org/10.1016/j.bmcl.2019.126782 BMCL 126782

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

7 September 2019 18 October 2019 24 October 2019

Please cite this article as: Poudel, Y.B., Rao, C., Kotapati, S., Deshpande, M., Thevanayagam, L., Pan, C., Cardarelli, J., Chowdari, N., Kaspady, M., Samikannu, R., Kuppusamy, P., Saravanakumar, P., Arunachalam, P.N., Deshpande, S., Rangan, V., Rampulla, R., Mathur, A., Vite, G.D., Gangwar, S., Design, Synthesis and Biological Evaluation of Phenol-linked Uncialamycin Antibody-drug Conjugates, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126782

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Graphical Abstract

Design, Synthesis and Biological Evaluation Leave this area blank for abstract info. of Phenol-linked Uncialamycin Antibodydrug Conjugates Yam B. Poudel,*a Chetana Rao,a Srikanth Kotapati,a Madhura Deshpande,a Lourdes Thevanayagam,a Chin Pan,a Josephine Cardarelli,a Naidu Chowdari,a Mahammed Kaspady,b Ramesh Samikannu,b Prakasam Kuppusamy,b Pon Saravankumar,b Pirama N. Arunachalam,b Shrikant Deshpande, a, Vangipuram Rangan,a Richard Rampulla,c Arvind Mathur,c Gregory D. Vite,c Sanjeev Gangwara Me

N H 2N

H N

S

N

NH O

O N H Uncialamycin ADC

HN

O

O

H N

S O

O

O

H N O

O N H

O

OH OH

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com

Design, Synthesis and Biological Evaluation of Phenol-linked Uncialamycin Antibody-drug Conjugates Yam B. Poudel,*a Chetana Rao,a Srikanth Kotapati,a Madhura Deshpande,a Lourdes Thevanayagam,a Chin Pan,a Josephine Cardarelli,a Naidu Chowdari,a Mahammed Kaspady,b Ramesh Samikannu,b Prakasam Kuppusamy,b Pon Saravanakumar,b Pirama N. Arunachalam,b Shrikant Deshpande,a Vangipuram Rangan,a Richard Rampulla,c Arvind Mathur,c Gregory D. Vite,c Sanjeev Gangwara a

Bristol-Myers Squibb Research & Development, 700 Bay Road, Redwood City, California 94063, United States

b

Biocon-Bristol-Myers Squibb R &D Center (BBRC), Biocon Park, Jigani Link Rd, Banglore 560099, India

c Bristol-Myers

Squibb Research & Development, Princeton, New Jersey, 08543, United States

ARTICLE INFO

ABSTRACT

Article history: Received Revised Accepted Available online

Uncialamycin is one of the structurally simpler and newer members of enediyne family of natural products. It exhibits highly potent activity against several types of bacteria and cancer cells. Described herein is a strategy for the targeted delivery of this cytotoxic agent to tumors using an antibody-drug conjugate (ADC) approach. Central to the design of ADC were the generation of potent and chemically stable uncialamycin analogues and attachment of protease cleavable linkers to newly realized phenolic handles to prepare linker-payloads. Conjugation of the linker-payloads to tumor targeting antibody, in vitro activity and in vivo evaluation are presented.

Keywords: Enediyne Uncialamycin Antibody-drug conjugate (ADC) Cancer Linker

Enediynes are among the most structurally intriguing and biologically active families of natural products isolated from bacterial source. This class of compounds exhibits extremely potent activity against various bacteria and tumor cells. Mechanistic studies have shown that the activity of these natural products stems from their ability to form highly reactive biradical species upon activation in biological system.1 This leads to the cleavage of single or double strands of DNA in living cells, ultimately causing cell death. A number of enediyne natural products have been pursued as cancer therapeutic agents. However, because of their extreme potency, systemic delivery leads to indiscriminate cell killing rendering these compounds unsuitable for use as anticancer agents. Despite this, neocarzinostatin polymer drug conjugate has been approved in Japan for the treatment of several forms of cancer.2 In addition, cancer cell targeted delivery of calicheamicin using a CD33antibody-drug conjugate was developed by Pfizer and has been approved for the treatment of acute myeloid leukemia.3 Recently, a new enediyne natural product called uncialamycin was isolated in extremely small quantity from streptomycete isolated from lichen found in British Columbia.4 The initially assigned

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structure was confirmed by total synthesis. Further mechanistic studies verified that this agent too forms highly reactive diradical species causing DNA cleavage similar to other enediyne natural products.5 Despite being much simpler in structure, uncialamycin showed potent cytotoxic activity against a panel cancer cell lines.

HO H3CS CH3

O

I

S

O H3C HO H3CO

H3C

OCH3

O

O

OH

E

1 3

7

A

B

8

DO

C 13

O

OH

1 Uncialamycin

B

DO

17

O

O

Me

O

H 3C N H Neocarzinostatin

CH3

O

R

A

H N

OH

Since then, a number of highly potent synthetic analogues have been prepared as potential payloads for antibody-drug conjugates.6 In this paper, we wish to report the synthesis of potent and chemically more stable uncialamycin analogues, attachment of protease cleavable linkers to them, preparation of corresponding antibody-drug conjugates (ADC) and initial in vitro and in vivo activity of the ADCs. We have previously reported the synthesis of several uncialamycin analogues that were over 100-fold more potent than the natural product.7 One of the structural features these highly potent analogues share is the presence of a primary or secondary benzylic amine moiety on the A-ring of uncialamycin (examples 2 and 3, table 1). Although the exact origin of such potent activity is unknown, it was thought that a relatively facile Bergman cyclization was responsible for such high potency. However, a more facile Bergman cyclization was accompanied by the observation that the more potent analogues tended to be chemically less stable. As expected, the products of Bergman cyclization were found to be biologically inactive. Thus preparation, purification and further manipulation including attachment of linker to prepare ADCs from analogues such as 2 proved to be quite challenging. During the course of our structure-activity relationship studies, we discovered that the instability issue could be resolved if the amine is simply converted into the corresponding benzamide (example 4). It was found that these amides were several-fold less potent than the parent amines. However, potency can be brought back to some degree with the introduction of a terminal arylamine (such as in examples 5-8). Although slightly less potent, these analogues were chemically stable for synthesis and purification under mild acidic or basic conditions. Thus, the amide formation approach allowed us to generate potent and stable analogues for attachment of linker and finally for the preparation of antibody-drug conjugates.

B

8

OH

E

DO

26

17

OH OH

C

2: 3: 4: 5: 6: 7: 8:

R=H R = Me R = PhCO R = p-NH2PhCO R = Z (X = H) R = Z (X = Me) R = Z (X = CF3)

N H 2N

X

S Z

O

13

O

OH

Figure 1: Structure of uncialamycin and other clinical enediyne natural products

HN 3

7

OH OH OH

2-a

O

O

HO

Bergman H N cyclization 2

1

O

dmso

OH

O

OH

O HN

2

OCH3

H 3C

Me

OH OH

13

O

OH OH

26

C

8

O O

26

17

A

H 2N

O

HN 3

7

H3C

Me HN

O

O

N O

Calicheamicin Mylotarg® payload

Me E

1

O

O O

HO

H3C

O

H3C

CH2CH3

OH

N H

S

HN

OCH3

O

O OCH 3

S

OH

In Vitro Cytotoxicity of Analogues (IC50 in pm)a compound

1 2 3 4 a

H226b

ADRc

compound

H226

ADR

1770 28 10 3164

388 20 15 5191

5 6 7 8

706 189 214 284

300 336 344 805

For detailed biological assay, see SI, b Lung and c Multidrug resistant cancer cell line

Table 1: Structure and cytotoxicity of some analogues With the preparation of potent and chemically stable analogues, we focused our attention towards attaching protease cleavable linkers to the payload.8 To this end, chemistry was developed to alkylate C-13 phenolic group of fully a protected uncialamycin analogue using valine-alanine benzyl iodide 10. This selective alkylation of the phenol provided the desired arylbenzyl ether 11 in good yield. It was necessary to use fully protected starting material because direct linker attachment on unprotected analogue such as 6 was not successful due to poor alkylation regiochemistry. Removal of TES protecting group under mild conditions provided the corresponding alcohol. Phthalamide protecting group was then removed under standard conditions to provide benzylamine which was surprisingly stable for isolation and purification purposes. We discovered that as long as the amine at N1 position was protected, these compounds were chemically stable. The free amine was then directly coupled with 2-aminothiazole-5-carbozylic acid directly to provide amide 12. Removal of both alloc groups simultaneously using palladium tetrakis(triphenylphosphine)palladium(0) provided bis-amine 13. It is noteworthy that removal of N1 alloc group was accompanied by the development of intense purple color, characteristic of uncialamycin chemotype that remained throughout the subsequent steps. Chemoselective amide formation on the more reactive aliphatic amine using maleamide caprioicNHS ester 14 provided the desired product 15 with a maleamide handle for conjugation to the antibody. Once the linker-payload 15 was prepared, the serum stability and the protease cleavability of the dipeptide linker was evaluated. While stability studies of full ADCs were done later, that of linkerpayload provided initial assessment of potential metabolic pathways and helped to screen for linker-payloads with desired stability

alloc N O O

Me

O

9

OH

Me

10 N

1. K2CO3 2. 3HF.Et3N 31% (2 steps)

N O

N H

O

OTES OH

O

I

O

H N

allocHN

H 2N

O

N

Me

alloc N O

O

N O H N

allocHN

11

H 2N

H N

S

H 2N

HN

CO2H

Me

O

O N H

O

O

O

O

OH OH

cathepsin B or sera

N H

O 15-NAC

OH OH

Me

O

O

Pd(PPh3)4

O

N

12

N H

O

O

morpholine

O

H N

allocHN

HN

O

H N

H N

S

O H N

S O

S

Me O

L-N-acetyl cysteine

N H

O

N

OH

N

alloc N O N

H 2N

N H

O

O O

2. EDC, HOBT N

O

O

O

15

1. Me NH2

O

O

OH OH

O

O

OH OH

O

O

N H

HN

H N

S O

O

O

H 2N

HN

OH OH

O

H N

S

self immolation OH

O

O

H 2N

O

Me

H 2N

HN

O H N

O

O

Me

N

HN

O

OH OH

Me O

S

O

O H N

H 2N

HN

OH OH

O

H N

S O

O

OH

6

Figure 3: Stability studies of linker-payload

H N O

O

6-PABE

N

O

N H

O

H 2N

N

13

N H

H 2N

O

Hunig's base 10% (4 steps)

O

O

N

14

O

O

H 2N

O

N

H N

S

O

O

OH OH

O

O

O N H

O

15

Figure 2: Synthesis and stability linker-payload N-acetyl cysteine (NAC) derivative to avoid the side reactions arising from free maleamide and to mimic the antibody amino acid residue (Figure 3). It is worthwhile to note that uncialamycin has been shown to be activated by free thiols in the biological system leading to Bergman cyclization. However, treatment of 15 with NAC at pH 7 aqueous buffer did not provide any Bergman cyclization product. Digestion of NAC derivative with cathepsin-B showed that the linker was cleaved by enzyme at the amide position, followed by selfimmolation of para-aminobenzyl alcohol to release free payload as desired. We believe that extended  conjugation present in uncialamycin leads to such a facile self-immolation. When the NAC derivative was incubated with PBS buffer and various sera, it was found that the linker-payload was stable in PBS and human serum but was less stable in mouse (100% payload release in 2h) and rat serum, releasing the payload.9 The instability in mouse and rat serum was later addressed by preparing the ADCs which reduced such payload cleavage to some degree.

The maleamide N87 Proliferation group on 15 was then used to 120000 CD70-16 conjugate the Meso-16 linker-payload to 90000 two different tumor targeting 60000 antibodies, antiCD70 and anti30000 mesothelin (meso) using maleamide0 cysteine chemistry 10 -4 10 -2 10 0 10 2 10 4 Drug Concentration (nM) to generate antibody-drug conjugates (Figure 4). These antibodies were chosen based on their higher expression on certain types of tumors. For example, CD70 is highly expressed on the surface of 780-0 renal cancer cells whereas mesothelin is overexpressed in N87 (gastric cancer) and H226 (lung cancer) cells. For conjugation, most accessible lysines present on the surface of antibody were first reacted with excess (35-fold) Traut’s reagent (2-iminothiolane) to generate nucleophilic thiols.10 Use of this reagent maintains positive charges similar to that of native lysines. Conjugate addition of newly generated thiols to maleamide group of the linker-payload 15 generated antibody-drug conjugates in 71% yield. The unreacted thiols were finally capped with N-ethyl maleamide. This protocol provided randomly conjugated ADCs with drug to antibody ratio (DAR) of 2.3 which was determined using the extinction coefficient of the linker-payload. Presence of intense purple color of the uncialamycin -max at 515 nm) independent of antibody was used in monitoring the conjugation reaction and to measure the extinction coefficient. This protocol 3H Thymidine Incorporation (cpm)

O N

3H Thymidine Incorporation (cpm)

generated high quality conjugates with <1% aggregation and <1.0 EU/mg endotoxin. The ADCs were completely stable in human serum and found to be much more stable (25% payload release in 24h) in mouse serum, than parent linker-payload. S 15

NH

H N

NH2

SH

786-0 Proliferation 250000

Meso-16 CD70-16

200000 150000 100000 50000 0 10 -4

10 -2

NH Me

N H 2N

S

S

O

N

NH

N H

O

16

HN

O

O

O

OH OH

H N O

O

H N

O

H N

O N H

O

Figure 4: Generation of antibody-drug conjugates

3H Thymidine Incorporation (cpm)

The two ADCs were then taken for in vitro study in three different tumor cell lines, N87-gastric, 786-0-renal and H226-lung cell lines (Figure 5). Both H226 and N87 have overexpression of mesothelin protein on their surfaces and respond to anti-mesothelin conjugates, whereas anti-CD70 ADC does not bind to them and served as a control. In contrast, 786-0 cell line is sensitive to antiCD70 conjugate and thus anti-mesothelin conjugates were used as non-binding controls. It was observed that for H226 cell line, mesoconjugate was quite potent (EC50 = 0.2 nM) whereas the control H226 Proliferation

10 2

10 4

for the anti-Meso ADC. Mice were administrated with single dose of either Meso- or control ADC at 0.1 M uncialamycin payload/kg dosed intraperitoneally and were observed for 6 weeks. Mice treated with anti–Meso-ADC showed complete tumor growth inhibition for 6 weeks after a single dose. On the other hand, the non-binding control anti-CD70 ADC had partial tumor growth inhibition for only two weeks, and tumors started to grow afterwards. This experiment further verified the target-mediated tumor growth inhibition as observed in in vitro experiments. Thus, a single dose of anti-Meso-uncialamycin ADC showed specific durable tumor growth inhibition of mesothelin-positive H226 tumors at a tolerated dose. Tumor Volume (LWH/2 mm3 )

antibody-thiol

antbody

10 0

Drug Concentration (nM)

40000

1000

Vehicle

800 600 400

CD70-16 0.1

200

Meso-16 0.1

0 0

20

40

Days Post Initial Dosing

CD70-16 Meso-16

30000 20000 10000 0 10 -2

10 0

10 2

10 4

Figure 6: In vivo efficacy of uncialamycin ADCs In summary, we have designed highly potent and chemically stable uncialamcyin analogues that were appended with a protease cleavable linker using newly developed phenol alkylation chemistry. Conjugation of resulting payload with appropriate antibodies provided ADCs which showed antigen-specific antitumor activity both in vitro and in vivo.

Drug Concentration (nM)

References and notes

cell line

CD70-1-16a

Meso-16a

specificityb

H226

6.3

0.2

31.5

1.

N87

21

2.0

10.5

2.

786-0

0.2

20.8

104

a

IC50 in nM, b specificity in fold

Figure5: In vitro cytotoxicity and selectivity of ADCs CD70 conjugate was 32 fold less potent (EC50 = 6.3 nM). On the other hand N87 cell line was less sensitive to both conjugates with lower selectivity. However, 786-0 cell line that overexpresses CD70 responded well to anti CD70 conjugate (EC50 = 0.2 nM) and displayed 104-fold selectivity over the non-binding anti-Meso conjugates. These data suggest that the difference in antiproliferative activity of the conjugates was mainly due to target mediated delivery of the drug to the respective cells. Thus, both antimesothelin and CD70 uncialamycin ADCs inhibited cell proliferation in antigen-specific manner. Both anti-CD70 and Meso-ADCs were further tested in vivo using H226 human lung carcinoma xenograft model in mice. In this model, anti-CD70.1 conjugate served as non-binding control

3.

4. 5. 6.

7.

Smith, A. L.; Nicolaou, K. C. The Enediyne Antibiotics, J. Med. Chem. 1996, 39, 2103−2117. Maeda H., Konno T. Metamorphosis of Neocarzinostatin to SMANCS: Chemistry, Biology, Pharmacology, and Clinical Effect of the First Prototype Anticancer Polymer Therapeutic. In: Maeda H., Edo K., Ishida N. (eds) Neocarzinostatin. Springer, Tokyo, 1997, 227−268. Perez, H.L.; Cardarelli, P. M.; Deshpande S.; Gangwar, S.; Schroeder, G. M.; Vite, G. D.; Borzilleri; R. M. Antibody-drug conjugates: current status and future directions, Drug Discov. Today. 2014, 19, 869−881. Davies, J.; Wang, H.; Taylor, T.; Warabi, K.; Huang, X.−H.; Andersen, R. Uncialamycin, A New Enediyne Antibiotics, J. Org. Lett., 2005, 7, 5233−5236. Nicolaou, K. C.; Chen, J. S.; Zhang, H.; Montero, A. Asymmetric synthesis and biological properties of uncialamycin and 26-epiuncialamycin Angew. Chem., Int. Ed. 2008, 47, 185−189. (a) Chowdari, N. S.; Gangwar, S.; Sufi, B. Enediyne Compounds, Conjugates Thereof, and Uses and Methods Thereof. US8709431 B2, April 29, 2014. (b) Nicolaou, K. C.; Lu, M.; Mandal, D.; Gangwar, S.; Chowdari, N. S.; Poudel, Y. B. Derivatives of Uncialamycin, Methods of Synthesis and Their Use as Antitumor Agents. WO2015023879 A1, February 19, 2015. Nicolaou, K. C.; Wang, Y.; Lu, M.; Mandal, D.; Pattanayak, M. R.; Yu, R.; Shah, A. A.; Chen, J. S.; Zhang, H.; Crawford, J. J.;

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9.

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Traut, R. R.; Bollen, A; Sun, T. T.; Hershey, J. W.; Sundberg, J; Pierce, L. R. Methyl 4-mercaptobutyrimidate as a cleavable crosslinking reagent and its application to the Escherichia coli 30S ribosome. Biochemistry. 1973, 12, 3266–3273.

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