Process for the preparation and purification of thiol-containing maytansinoids

1. A process for preparing a thiol-containing maytansinoid wherein the thiol group is part of the ester moiety at C-3, comprising the steps of:

(1) conducting reductive hydrolysis of a maytansinoid C-3 ester with a reducing agent selected from the group consisting of lithium trimethoxyaluminum hydride (LiAl(OMe) 3 H), lithium triethoxyaluminum hydride (LiAl(OEt) 3 H) and lithium tripropoxyaluminum hydride (LiAl(OPr) 3 H), to yield a maytansinol;

(2) purifying the maytansinol to remove side products when present;

(3) esterifying the purified maytansinol with a carboxylic acid to yield a reaction mixture of an L- and a D-aminoacyl ester of maytansinol;

(4) separating the L-aminoacyl ester of maytansinol from the reaction mixture in (3);

(5) reducing the L-aminoacyl ester of maytansinol to yield a thiol-containing maytansinoid; and

(6) purifying the thiol-containing maytansinoid.

2. The process of claim 1 , wherein the reducing agent in (1) is lithium trimethoxyaluminum hydride.

3. The process of claim 1 , wherein the reducing agent in (1) is used in a concentration of from about 5 to 100 equivalents per mole of the maytansinoid C-3 ester.

4. The process of claim 1 , wherein the reducing agent in (1) is used in a concentration of from about 7.5 to 30 equivalents per mole of the maytansinoid C-3 ester.

5. The process of claim 1 , wherein the reducing agent in (1) is used in a concentration of from about 10 to 20 equivalents per mole of the maytansinoid C-3 ester.

6. The process of claim 1 , wherein the reductive hydrolysis in (1) is conducted at a temperature of from about −80° C. to 0° C.

7. The process of claim 1 , wherein the reductive hydrolysis in (1) is conducted at a temperature of from about −45° C. to −27.5° C.

8. The process of claim 1 , wherein the reductive hydrolysis in (1) is conducted at a temperature of from about −35° C. to −30° C.

9. The process of claim 1 , wherein the reducing agent in (1) is added over a period of from about 5 to 40 minutes.

10. The process of claim 1 , wherein the reducing agent in (1) is added over a period of from about 7 to 20 minutes.

11. The process of claim 1 , wherein the reducing agent in (1) is added over a period of from about 8 to 12 minutes.

12. The process of claim 1 , wherein the maytansinol is purified in (2) by chromatography.

13. The process of claim 12 , wherein the chromatography is silica gel column chromatography, preparative thin-layer chromatography on silica gel or cyano-bonded silica HPLC column chromatography.

14. The process of claim 12 , wherein the chromatography is silica gel column chromatography.

15. The process of claim 12 , wherein the purification is performed at ambient temperature.

16. The process of claim 12 , wherein the maytansinol is purified to a purity of about 95%.

17. The process of claim 1 , wherein the carboxylic acid in (3) is selected from the group consisting of N-methyl-N-methyldithioacetyl-L-alanine, N-methyl-N-(3-methyldithio-propanoyl)-L-alanine, N-methyl-N-(3-methyldithio-butanoyl)-L-alanine, N-methyl-N-(4-methyldithio-butanoyl)-L-alanine, N-methyl-N-(5-methyldithio-pentanoyl)-L-alanine, N-methyl-N-(3-phenyldithio-propanoyl)-L-alanine, N-methyl-N-(3-(4-nitrophenyldithio)propanoyl)L-alanine, N-acetyl-N-methylmethyldithiocysteine and N-acetyl-N-methyl-methyldithiohomocysteine.

18. The process of claim 1 , wherein the carboxylic acid in (3) is N-methyl-N-(3-methyldithio-propanoyl)-L-alanine.

19. The process of claim 1 , wherein the esterification in (3) is conducted at ambient temperature.

20. The process of claim 1 , wherein the esterification in (3) further comprises the use of dicyclohexylcarbodiimide and zinc chloride.

21. The process of claim 1 , wherein the separating in (4) is carried out by passing the reaction mixture over a cyano-bonded silica HPLC column.

22. The process of claim 1 , wherein the separating in (4) is carried out at about 25° C.

23. The process of claim 1 , wherein the reduction in (5) uses dithiothreitol as the reducing agent.

24. The process of claim 1 , wherein the reduction in (5) is carried out in a mixture of ethyl acetate-methanol-aqueous buffer which is capable of keeping buffer salts, dithiothreitol, unreduced maytansinoids and reduced maytansinoids in solution.

25. The process of claim 24 , wherein the mixture of ethyl acetate-methanol-aqueous buffer is 1:1.5:1, v/v/v, ethyl acetate:methanol:aqueous buffer.

26. The process of claim 24 , wherein the concentration of the thiol-containing maytansinoid is such that the thiol-containing maytansinoid remains soluble in ethyl acetate-methanol-aqueous buffer.

27. The process of claim 26 , wherein the concentration of the thiol-containing maytansinoid is about 4 g/L.

28. The process of claim 1 , wherein the reduction in (5) is carried out in an oxygen-free atmosphere.

29. The process of claim 1 , wherein the reduction in (5) is carried out at about 25° C.

30. The process of claim 1 , wherein the purifying of the thiol-containing maytansinoid in (6) is by chromatography.

31. The process of claim 30 , wherein the purifying of the thiol-containing maytansinoid in (6) is by a cyano-bonded HPLC column chromatography.

32. The process of claim 31 , wherein the chromatography is by a cyano-bonded HPLC column equilibrated and run in an organic solvent.

33. The process of claim 32 , wherein the organic solvent is a mixture of hexanes:2-propanol:ethyl acetate.

34. The process of claim 33 , wherein the organic solvent is a 78.0:5.5:16.5, v/v/v, mixture of hexanes:2-propanol:ethyl acetate.

35. A process for isolating maytansinol from a mixture containing unreduced and over- reduced maytansinoids by separating the maytansinol by normal - phase high performance liquid chromatography ( HPLC ) on a chemically modified silica stationary phase.

36. The process of claim 35 , wherein the chemically modified silica is cyano- bonded silica.

37. The process of claim 35 , further comprising converting the maytansinol prepared by the process into a cell- binding agent maytansinoid complex.

38. A process for isolating maytansinol from a mixture containing unreduced and over- reduced maytansinoids by separating the maytansinol by large - scale preparative normal - phase high performance liquid chromatography ( HPLC ) on a chemically modified silica stationary phase.

39. The process of claim 38 wherein the chemically modified silica is cyano- bonded silica.

40. The process of claim 38 further comprising converting the maytansinol prepared by the process into a cell- binding agent maytansinoid complex.