Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents

• US 6,441,163 B1
• Filed: 05/31/2001
• Issued: 08/27/2002
• Est. Priority Date: 05/31/2001
• Status: Expired due to Term

• Current Assignee: ImmunoGen Incorporated
• Original Assignee: ImmunoGen Incorporated
• Inventors: Widdison, Wayne C., Chari, Ravi V. J.
• Primary Examiner(s): Kifle, Bruck
• Application Number:
  US09/867,598
• Time in Patent Office: 453 Days
• Field of Search: 540/458
• US Class Current: 540/458
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07D 498/18 Bridged systems

Abstract

The present invention discloses a one-step process for the production of cytotoxic conjugates of maytansinoids and cell binding agents. Maytansinoids having a disulfide linker that bears a reactive moiety are linked to cell binding agents, such as antibodies, without prior modification of the cell binding agent. These conjugates are useful as therapeutic agents which are delivered specifically to target cells and are cytotoxic.

Claims

1. A method for producing a maytansinoid having a disulfide moiety that bears a reactive ester comprising reacting the carboxyl group of N^2′

-deacetyl-N^2′

-[3-(carboxyalkyldithio)-1-oxopropyl]-maytansine with a hydroxy compound to yield a maytansinoid having a disulfide moiety that bears a reactive ester.

2. The method according to claim 1, wherein the N^2′

-deacetyl- N^2′

-[3-(carboxyalkyldithio)-1-oxopropyl]-maytansine is N^2′

- deacetyl-N^2′

-[3-(3-carboxy-1- methyl-propyldithio)-1-oxopropyl]-maytansine (6);

3. The method according to claim 1, further comprising a step of isolating the maytansinoid ester product of the reaction.

4. The method according to claim 1, wherein said hydroxy compound is selected from the group consisting of N-hydroxysuccinimide, N-hydroxysulfosuccinimide, N-hydroxyphthalimide, N-hydroxysulfophthalimide, 2-nitrophenol, 4-nitrophenol, 2,4-dinitrophenol, 3-sulfonyl-4-nitrophenol, and 3-carboxy-4-nitrophenol.

5. The method according to claim 1, wherein said hydroxy compound is N-hydroxysuccinimide.

6. The method according to claim 1, wherein said hydroxy compound is N-hydroxysulfosuccinimide.

7. The method according to claim 1, wherein said hydroxy compound is N-hydroxysuccinimide and said maytansinoid having a disulfide moiety that bears a reactive ester is the N-succinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (2);

8. The method according to claim 1, wherein said hydroxy compound is N-hydroxysulfosuccinimide and said maytansinoid having a disulfide moiety that bears a reactive ester is the N-sulfosuccinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (3a);

26. The method according to claim 1, wherein said hydroxy compound is N-hydroxysulfosuccinimide and said maytansinoid having a disulfide moiety that bears a reactive ester is a N-sulfosuccinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine.

9. A method for producing a maytansinoid having a disulfide moiety that bears a reactive ester comprising performing a disulfide exchange reaction between a thiol-containing maytansinoid and the reactive disulfide moiety of a compound containing a reactive carboxylic acid ester.

10. The method according to claim 9, further comprising the step of isolating the maytansinoid having a disulfide moiety that bears a reactive ester.

11. The method according to claim 9, wherein the reactive carboxylic acid ester containing a reactive disulfide is N-succinimidyl 4-(2-pyridyldithio)-pentanoate (7):

12. The method according to claim 9, wherein said maytansinoid having a disulfide moiety that bears a reactive ester is the N-succinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (2);

13. A method for producing a maytansinoid having a disulfide moiety that bears a reactive ester comprising performing a disulfide exchange reaction between a thiol-containing maytansinoid and the reactive disulfide moiety of a compound containing a water soluble reactive carboxylic acid ester.

14. The method according to claim 13, further comprising the step of isolating the maytansinoid having a disulfide moiety that bears a reactive ester.

15. The method according to claim 13, wherein the water soluble reactive carboxylic acid ester containing a reactive disulfide is sulfosuccinimidyl 4-(2-pyridyldithio)-pentanoate (8a):

16. The method according to claim 13, wherein said maytansinoid having a disulfide moiety that bears a reactive ester is the N-sulfosuccinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (3a);

27. The method according to claim 13, wherein said maytansinoid having a disulfide moiety that bears a reactive ester is a N-sulfosuccinimidyl ester of N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine.

17. A method for producing N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (6);

comprising reacting a thiol-containing maytansinoid with 4-(2-pyridyldithio)-pentanoic acid (5);

18. The method according to claim 17, further comprising the step of isolating the N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)]-1-oxopropyl]-maytansine (6);

19. A method for producing N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (6)

comprising the steps of;

(a) combining N-methyl-N-(3-mercaptopropanoyl)-L-alanine and trimethylsilylethyl 4-mercaptopentanoate (9)

to form a mixed disulfide compound,

(b) coupling maytansinol to the product of (a) to produce a maytansinoid ester (10),

and

(c) deprotecting the maytansinoid ester (10).

20. The method according to claim 19, further comprising the step of isolating the N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine (6);

21. A maytansinoid having a disulfide moiety that bears a reactive ester comprising the following formula 11:

22. The compound according to claim 21, where R₁ is H, R₂ is CH₃, n is 2, and X is N-succinimidyl.

23. The compound according to claim 21, where R₁ is H, R₂ is CH₃, n is 2, and X is N-sulfosuccinimidyl.

24. N^2′

-deacetyl-N^2′

-[3-(3-carboxy-1-methyl-propyldithio)-1-oxopropyl]-maytansine comprising the following formula;

25. The maytansinoid ester compound (10) comprising the following formula: