Intermediates and methods for synthesizing calicheamicin derivatives

Abstract

The present invention relates to intermediates of Formula I ##STR00001##
and to methods of synthesizing and purifying calicheamicin derivatives.

Claims

1. A method for synthesizing a compound of Formula I ##STR00072## wherein R.sup.12 is selected from straight and branched-chain C.sub.1-C.sub.8 alkyl; each R.sup.10 is independently selected from hydrogen, R.sup.12 and OR.sup.12; R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, or SR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-C.sub.5 alkyl; r is an integer 0 or 1; G is oxygen or sulfur; Z.sup.1 is H or straight or branched-chain C.sub.1-C.sub.5 alkyl; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two or three groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two, three, or four groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14; wherein each R.sup.14 is independently selected from (C.sub.1-C.sub.5)alkyl and each R.sup.14 is independently optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; W is selected from O, S, C(O)NH, NHC(O), and NR.sup.15, wherein R.sup.15 is a (C.sub.1-C.sub.5)alkyl and R.sup.15 is optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; and Y is a straight or branched-chain (C.sub.1-C.sub.6)alkylene group or a straight or branched-chain (C.sub.2-C.sub.6)alkenylene group; which method comprises reacting a compound of Formula II ##STR00073## with a compound of Formula III ##STR00074##

2. A method according to claim 1, wherein r is 0, G is oxygen, Z.sup.1 is methyl, Ar is 1,4-phenylene, W is O, and Y is (CH.sub.2).sub.3-.

3. A method according to claim 1, wherein R.sup.8 and R.sup.9 are methyl.

4. A method according to claim 1, wherein each R.sup.10 is hydrogen.

5. A method according to claim 4, wherein R.sup.12 is methyl.

6. A method for synthesizing a compound of Formula IV ##STR00075## which method comprises treating a compound of Formula I ##STR00076## wherein R.sup.12 is selected from straight and branched-chain C.sub.1-C.sub.8 alkyl; each R.sup.10 is independently selected from hydrogen, R.sup.12 and OR.sup.12; R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, or SR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-C.sub.5 alkyl; r is an integer 0 or 1; G is oxygen or sulfur; Z.sup.1 is H or straight or branched-chain C.sub.1-C.sub.5 alkyl; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two or three groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two, three, or four groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14; wherein each R.sup.14 is independently selected from (C.sub.1-C.sub.5)alkyl and each R.sup.14 is independently optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; W is selected from O, S, C(O)NH, NHC(O), and NR.sup.15, wherein R.sup.15 is a (C.sub.1-C.sub.5)alkyl and R.sup.15 is optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; and Y is a straight or branched-chain (C.sub.1-C.sub.6)alkylene group or a straight or branched-chain (C.sub.2-C.sub.6)alkenylene group, with a strong acid to form a mixture comprising the compound of Formula IV.

7. A method according to claim 6, wherein the strong acid is trifluoroacetic acid or sulfuric acid.

8. A method according to claim 6, wherein R.sup.8 and R.sup.9 are methyl, r is 0, G is oxygen, Z.sup.1 is methyl, Ar is 1,4-phenylene, W is O, and Y is (CH.sub.2).sub.3.

9. A method according to claim 6, wherein each R.sup.10 is hydrogen.

10. A method according to claim 9, wherein R.sup.12 is methyl.

11. A method for synthesizing a linker intermediate of Formula V ##STR00077## wherein R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, orSR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-C.sub.5 alkyl; r is an integer selected from 0 and 1; G is oxygen or sulfur; Z.sup.1 is H or straight or branched-chain C.sub.1-C.sub.5 alkyl; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two or three groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two, three, or four groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14; wherein each R.sup.14 is independently selected from (C.sub.1-C.sub.5)alkyl and each R.sup.14 is independently optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; W is selected from O, S, C(O)NH, NHC(O), and NR.sup.15-, wherein R.sup.15 is a (C.sub.1-C.sub.5)alkyl and R.sup.15 is optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; Y is a straight or branched-chain (C.sub.1-C.sub.6)alkylene group or a straight or branched-chain (C.sub.2-C.sub.6)alkylene group; and Z is selected from the group consisting of ##STR00078## which method comprises a) treating a compound of Formula I ##STR00079## wherein R.sup.12 is selected from straight and branched-chain C.sub.1-C.sub.8 alkyl, and each R.sup.10 is independently selected from hydrogen, R.sup.12 and OR.sup.12; with a strong acid to form a mixture comprising a compound of Formula IV ##STR00080## and b) reacting the compound of Formula IV with a compound ZH; thereby synthesizing the linker intermediate of Formula V.

12. A method according to claim 11, wherein the strong acid is trifluoroacetic acid or sulfuric acid.

13. A method according to claim 11, wherein ZH is ##STR00081##

14. A method according to claim 11, wherein the linker intermediate is of the structure ##STR00082##

15. A method according to claim 11, wherein the compound of Formula I is obtained by reacting a compound of Formula II ##STR00083## with a compound of Formula III ##STR00084##

16. A method of synthesizing a calicheamicin derivative of Formula VI ##STR00085## wherein J is ##STR00086## R.sub.1 is ##STR00087## or CH .sub.3; R.sub.2 is ##STR00088## or H; R.sub.3 is ##STR00089## or H; R.sub.4 is ##STR00090## or H; R.sup.5 is CH.sub.3, C.sub.2H.sub.5, or CH(CH.sub.3).sub.2; X is an iodine or bromine atom; R.sup.5 is a hydrogen or the group RCO, wherein R is hydrogen, branched or unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbon atoms, aryl of 6 to 11 carbon atoms, a (C.sub.6-C.sub.11) aryl-alkyl (C.sub.1-C.sub.5) group, or a heteroaryl or heteroaryl-alkyl (C.sub.1-C.sub.5) group wherein heteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or 5-(N-methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, (C.sub.1-C.sub.3) alkoxy, or thioalkoxy of 1 to 5 carbon atoms; R.sub.6 and R.sub.7 are each independently selected from H and ##STR00091## R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, or SR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-C.sub.5 alkyl; r is an integer 0 or 1; G is oxygen or sulfur; Z.sup.1 is H or straight or branched-chain C.sub.1-C.sub.5 alkyl; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two or three groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two, three, or four groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14; wherein each R.sup.14 is independently selected from (C.sub.1-C.sub.5)alkyl and each R.sup.14 is independently optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; W is selected from O, S, C(O)NH, NHC(O), and NR.sup.15-, wherein R.sup.15 is a (C.sub.1-C.sub.5)alkyl and R.sup.15 is optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; Y is a straight or branched-chain (C.sub.1-C.sub.6)alkylene group or a straight or branched-chain (C.sub.1-C.sub.6)alkenylene group; and Z is selected from the group consisting of ##STR00092## which method comprises a) treating a compound of Formula I ##STR00093## wherein R.sup.12 is selected from straight and branched-chain C.sub.1-C.sub.8 alkyl, and each R.sup.10 is independently selected from hydrogen, R.sup.12 and OR.sup.12; with a strong acid to form a mixture comprising a compound of Formula IV ##STR00094## b) reacting the compound of Formula IV with a compound ZH; to form a linker intermediate of Formula V ##STR00095## and c) reacting the linker intermediate of Formula V resulting from step (b) with a methyltrisulfide compound CH.sub.3SSS-J; thereby synthesizing a calicheamicin derivative of Formula VI.

17. A method according to claim 16, wherein the compound of Formula I is obtained by reacting a compound of Formula II ##STR00096## with a compound of Formula III ##STR00097##

18. A method according to claim 16, wherein one of R.sub.6 and R.sub.7 is hydrogen and the other of R.sub.6 and R.sub.7 is ##STR00098##

19. A method according to claim 16, further comprising d) conjugating the calicheamicin derivative of Formula VI resulting from step (c) to a monoclonal antibody.

20. A method according to claim 19, wherein the monoclonal antibody is inotuzumab or gemtuzumab.

21. A method according to claim 16, wherein the methyltrisulfide compound CH.sub.3SSS-J is ozogamicin.

22. A method according to claim 21, further comprising d) conjugating the calicheamicin derivative of Formula VI resulting from step (c) to a monoclonal antibody.

23. A method according to claim 22, wherein the monoclonal antibody is inotuzumab.

24. A method according to claim 22, wherein the monoclonal antibody is gemtuzumab.

25. A method of synthesizing a calicheamicin derivative of Formula VI ##STR00099## wherein J is ##STR00100## R.sub.1 is ##STR00101## or CH.sub.3; R.sub.2 is ##STR00102## or H; R.sub.3 is ##STR00103## or H; R.sub.4 is ##STR00104## or H; R.sup.5 is CH.sub.3, C.sub.2H.sub.5, or CH(CH.sub.3).sub.2; X is an iodine or bromine atom; R.sup.5 is a hydrogen or the group RCO, wherein R is hydrogen, branched or unbranched alkyl of 1 to 10 carbon atoms, alkylene of 2 to 10 carbon atoms, aryl of 6 to 11 carbon atoms, a (C.sub.6-C.sub.11) aryl-alkyl (C.sub.1-.sub.5) group, or a heteroaryl or heteroaryl-alkyl (C.sub.1-C.sub.5) group wherein heteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3-, or 4-pyridinyl, 2-, 4-, or 5-(N -methylimidazolyl), 2-, 4-, or 5-oxazolyl, 2-, 3-, 5-, or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolyl, or 1-, 3-, 4-, 5-, 6-, 7-, or 8-isoquinolyl, all aryl and heteroaryl groups optionally substituted by one or more hydroxy, amino, carboxy, halo, nitro, (C.sub.1-C.sub.3) alkoxy, or thioalkoxy of 1 to 5 carbon atoms; R.sub.6 and R.sub.7 are each independently selected from H and ##STR00105## R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, or SR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-.sub.5 alkyl; r is an integer 0 or 1; G is oxygen or sulfur; Z.sup.1 is H or straight or branched-chain C.sub.1-C.sub.5 alkyl; Ar is 1,2-, 1,3-, or 1,4-phenylene optionally substituted with one, two or three groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14, or Ar is a 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6-, or 2,7-naphthylidene optionally substituted with one, two, three, or four groups independently selected from straight or branched-chain C.sub.1-C.sub.6 alkyl, OR.sup.14, SR.sup.14, halogen, nitro, COOR.sup.14, C(O)NHR.sup.14, O(CH.sub.2).sub.nCOOR.sup.14, S(CH.sub.2).sub.nCOOR.sup.14, O(CH.sub.2).sub.nC(O)NHR.sup.14, and S(CH.sub.2).sub.nC(O)NHR.sup.14; wherein each R.sup.14 is independently selected from (C.sub.1-C.sub.5)alkyl and each R.sup.14 is independently optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; each n is an integer independently selected from 0, 1, 2, 3, 4, and 5; W is selected from O, S, C(O)NH, NHC(O), and NR.sup.15-, wherein R.sup.15 is a (C.sub.1-C.sub.5)alkyl and R.sup.15 is optionally substituted with one or two groups selected from OH, (C.sub.1-C.sub.4)alkyl, and S(C.sub.1-C.sub.4)alkyl; Y is a straight or branched-chain (C.sub.1-C.sub.6)alkylene group or a straight or branched-chain (C.sub.2-C.sub.6)alkenylene group; and Z is selected from the group consisting of ##STR00106## which method comprises reacting a linker intermediate of Formula V ##STR00107## with a methyltrisulfide compound CH.sub.3SSS-J, in the presence of a carbodiimide; thereby synthesizing a calicheamicin derivative of Formula VI.

26. A method according to claim 25, wherein one of R.sub.6 and R.sub.7 is hydrogen and the other of R.sub.6 and R.sub.7 is ##STR00108##

27. A method according to claim 25, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

28. A method according to claim 25, further comprising subjecting the calicheamicin derivative of Formula VI to reversed phase purification protocol.

29. The method of claim 28, wherein the calicheamicin derivative of Formula VI resulting from the reversed phase purification protocol is subsequently subjected to a solid phase extraction protocol.

30. A method according to claim 25, wherein the methyltrisulfide compound has an initial concentration in said reaction of greater than about 3 g/L of the reaction mixture.

31. A method according to claim 30, wherein the methyltrisulfide compound has an initial concentration in said reaction of between about 10 g/L and 110 g/L of the reaction mixture.

32. A method according to claim 25, further comprising conjugating the resulting calicheamicin derivative of Formula VI to a monoclonal antibody.

33. A method according to claim 32, wherein the monoclonal antibody is inotuzumab or gemtuzumab.

34. A method according to claim 25, wherein the methyltrisulfide compound CH.sub.3SSS-J is ozogamicin.

35. A method according to claim 34, further comprising conjugating the resulting calicheamicin derivative of Formula VI to a monoclonal antibody.

36. A method according to claim 35, wherein the monoclonal antibody is inotuzumab.

37. A method according to claim 35, wherein the monoclonal antibody is gemtuzumab.

38. A method of synthesizing a compound of Formula II ##STR00109## wherein R.sup.12 is selected from straight and branched-chain C.sub.1-C.sub.8 alkyl; each R.sup.10 is independently selected from hydrogen, R.sup.12 and OR.sup.12; R.sup.8 and R.sup.9 are each independently selected from hydrogen and straight and branched-chain C.sub.1-C.sub.8 alkyl, wherein each said alkyl for R.sup.8 and R.sup.9 is independently optionally substituted by NH.sub.2, NHR.sup.11, NR.sup.11R.sup.13, OR.sup.11, OH, or SR.sup.11, wherein each R.sup.11 and each R.sup.13 are independently selected from straight and branched-chain C.sub.1-C.sub.5 alkyl; r is an integer selected from 0 and 1; and G is oxygen or sulfur; which method comprises treating a compound of Formula VII ##STR00110## wherein R.sup.16 is C(O)OH or C(V)SH, wherein V is oxygen or sulfur, or R.sup.16 is NH.sub.2; with an azole activating agent of Formula IX ##STR00111## wherein V is oxygen or sulfur; and wherein E is ##STR00112## wherein m is an integer 0, 1, 2, or 3; q is an integer 0, 1 or 2; and p is an integer 0, 1, 2, 3, or 4; and wherein each R.sup.17 attached to E is independently selected from straight and branched-chain (C.sub.1-C.sub.6)alkyl groups; in an organic solvent to form a compound of Formula VIII ##STR00113## wherein when R.sup.16 is C(O)OH, r is 0 and G is oxygen; when R.sup.16 is C(V)SH, r is 0 and G is V; and when R.sup.16 is NH.sub.2, r is 1 and G is V; followed by combining the compound of Formula VIII with hydrazine, thereby forming a compound of Formula II.

39. The method of claim 38, wherein the azole activating agent is selected from carbonyl diimidazole; thiocarbonyl diimidazole; carbonyl bis-pyrazole wherein each pyrazole optionally substituted with from one to three (C.sub.1-C.sub.6) alkyl groups; carbonyl bis-1,2,3-triazole; carbonyl bis-benzotriazole, and carbonyl bis-1,2,4-triazole.

40. The method of claim 39, wherein the azole activating agent is carbonyl diimidazole.

41. The method of claim 38, wherein r is 0 and G is oxygen.

42. The method of claim 38, wherein R.sup.16 in the compound of Formula VII is C(O)OH.

43. The method of claim 42, wherein the azole activating agent is carbonyl diimidazole.

44. The method of claim 38, wherein the hydrazine is anhydrous hydrazine.

45. The method of claim 38, wherein the hydrazine is hydrazine monohydrate.

46. The method of claim 38, wherein the hydrazine is an aqueous solution of hydrazine.

47. The method of claim 38, wherein the hydrazine is a tetrahydrofuran solution of hydrazine.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The method of the present invention for synthesizing a linker intermediate of Formula V is described in the following reaction Schemes I-IV. In the chemical formulae in Schemes I-IV, R.sup.8, R.sup.9, R.sup.10, R.sup.12, Q, Ar, W, Y, Z.sup.1, and Z are as defined above herein.

(2) ##STR00063##

(3) Referring to Scheme I, a (4-alkoxyphenyl)methanethiol 1, wherein R.sup.12 is as described herein, is reacted with methyl senecioate 2 to yield the carboxylic acid intermediate compound 3. Intermediate 3 is then charged with a suitable organic solvent such as tetrahydrofuran and an azole activating agent of the Formula IX as described herein, such as carbonyl diimidazole (CDI). Intermediate 3.5 is obtained. This reaction is followed by combining 3.5 with a hydrazine. The hydrazine source may be anhydrous hydrazine as is described in WO 2008/147765; however, preferably, the hydrazine source is aqueous hydrazine, such as hydrazine monohydrate. This reaction yields the intermediate compound 4. The compound p-methoxybenzylthioether hydrazide, which is an intermediate compound 4 wherein R.sup.12 is methyl and each R.sup.10 is hydrogen, is described in WO 2008/147765, the entire contents of which are incorporated herein by reference.

(4) ##STR00064##

(5) In Scheme II, intermediate 4 is reacted with the compound 5 wherein Z.sup.1 is as described herein, for example 4-(4-acyl-phenoxy)butanoic acid, in an inert (in other words, non-reactive) solvent, optionally with an acidic catalyst, to provide the intermediate 6. Examples of inert solvents that may be used in this reaction include, but are not limited to, alcohols (for example, methanol), ethers and esters such as ethyl acetate. A person of ordinary skill in the art can determine a suitable inert solvent for this reaction. Acidic catalysts can also be determined by a person of ordinary skill in the art; examples of acidic catalysts include, but are not limited to acetic acid.

(6) ##STR00065##

(7) In Scheme III, intermediate compound 6 is deprotected to form compound 7. In this reaction, compound 6 is charged with methoxybenzene and a strong acid optionally under heat, for example trifluoroacetic acid under heat, yielding intermediate compound 7. Other strong acids may be used instead of trifluoroacetic acid, for example sulfuric acid.

(8) ##STR00066##

(9) In Scheme IV, intermediate 7 is converted to a compound 8, which is an embodiment of the linker intermediate of Formula V described herein. Intermediate 7 can be converted to a linker intermediate such as 8 as is described in the art, such as in U.S. Pat. No. 8,273,862, which is incorporated herein by reference in its entirety. Preferably, however, as depicted in Scheme IV, intermediate 7 is reacted with a tertiary amine base such as triethylamine (TEA) and with trimethylacetyl chloride (PivCl) in the presence of an inert solvent such as tetrahydrofuran. Subsequently, a compound of Formula ZH, for example N-hydroxysuccinimide, is introduced to provide the linker intermediate 8.

(10) After synthesis of the linker intermediate of Formula V (such as a compound 8), a calicheamicin derivative of Formula VI is then synthesized using the linker intermediate from the reaction of Scheme IV and methods known in the art, for example as is described in U.S. Pat. No. 8,273,862. For example, the linker intermediate of Formula V can be reacted first with an alkali methyl carbonate, which includes but is not limited to sodium carbonate, forming the sodium salt of the linker intermediate in acetonitrile by heating at gentle reflux. Further reaction of the sodium salt of the linker intermediate with the methyltrisulfide CH.sub.3SSS-J at about 15 C., in an inert organic solvent, preferably acetonitrile gives the calicheamicin derivative of Formula VI. Preferred is the reaction in acetonitrile at about 0 C. Optionally an organic base may replace the alkali metal carbonate which preferably includes triethylamine, in acetonitrile at about 0 C.

(11) Alternatively, a calicheamicin derivative of Formula VI can be synthesized from the linker intermediate of Formula V by reaction with a molecule of formula CH.sub.3SSS-J by using the method described herein comprising a carbodiimide.

(12) The calicheamicin derivative of Formula VI can be further conjugated to a biomacromolecule, such as a monoclonal antibody, to form an antibody-drug conjugate, using techniques described in the art, for example the methods described in U.S. Pat. No. 5,053,394, and in U.S. Pat. No. 5,770,701, both of which are incorporated by reference herein in their entireties.

(13) The following examples are presented to illustrate certain embodiments of the present invention, but should not be construed as limiting the scope of this invention. Those skilled in the art will readily understand that known variations of the specific conditions of the following examples can be used.

EXAMPLE 1

3-(4-methoxybenzylthio)-3-methylbutanoic Acid

(14) ##STR00067##

(15) 85 g of 1 and 255 mL of 2-Methyltetrahydrofuran were added to a reactor at 20-25 C. 125.05 g of 2 was added to the reactor and the reactor was degassed by bubbling a nitrogen stream into the stirred solution for 15-20 min. Tetrabutylammonium fluoride (1M in THF, 0.05 equiv., 36.1 mL) was added to the reactor and the reaction was maintained at 20-30 C. for 2 hours.

(16) A solution of calcium chloride dihydrate (0.35 equiv., 28.060 g) in 255 mL of water was added. After stirring for 20 minutes the lower aqueous phase was removed. To the upper organic phase was added 252 mL of methanol and 3 equiv. of NaOH in 252 mL of water. The reaction mixture was stirred until complete consumption of the intermediate ester (3) is observed.

(17) The reaction was cooled to 15 C. and 2-methyltetrahydrofuran was added (252 mL) followed by 252 mL of water. Concentrated HCl was added (3.1 equiv., 184 mL) slowly, maintaining the reaction in the range 15-30 C. The lower aqueous phase was removed. The organic layer was washed with 1M HCl (252 mL) and heptanes was added (1940 mL). The solution was distilled to remove 2-methyltetrahydrofuran. The resulting slurry was stirred for an hour, then filtered and dried. The mother liquor was concentrated to give a second crop of solids.

(18) Total yield of the title compound acid 4 was 162.59 g (88.5%). .sup.1H NMR (CDCl.sub.3): (ppm) 1.5 (s, 6H), 2.3 (s, 2H), 3.6 (m, 5H), 6.7 (d, 2H), 7.6 (d, 2H).

EXAMPLE 2

3-(4-methoxybenzylthio)-3-methylbutanehydrazide

(19) ##STR00068##

(20) Acid 4 (81.67 g, 321 mmol) was added to 375 mL of THF (tetrahydrofuran). CDI (1.05 eq., 54.7 g) was charged in three portions and the reaction was stirred at 20 C. for 2.5 hours. In a separate reactor a solution of hydrazine monohydrate (2.5 equiv., 40.19 g) in 200 mL of THF was prepared. The solution of intermediate 5 was added to the hydrazine hydrate solution keeping the internal temperature at 20 C. After the addition was complete the reaction was stirred for 18 hours, then concentrated to 100 mL. 850 mL of EtOAc (ethyl acetate) was added, and the solution was washed 3 times with 500 mL of water then 200 mL of brine. The organic layer was dried with Na.sub.2SO.sub.4, filtered through diatomaceous earth and concentrated on a rotary evaporator to a white slurry. 300 mL of heptane was added and 200 mL was removed on a rotary evaporator. Another 200 mL of heptane was added and stripped to a thick white slurry. The mixture was filtered, washed with heptanes and dried. 83.15 g (96.5%) of the title compound 6 was obtained from 4. MS 269 (M+1), 121, 120.

EXAMPLE 3

4-(4-(1-(2-(3-(4-methoxybenzylthio)-3-methylbutanoyl)hydrazono)-ethyl)phenoxy)butanoic Acid

(21) ##STR00069##

(22) 68 g of 6 and 57.43 g of 7 were added to 680 mL of methanol (MeOH). 68 mL of acetic acid (HOAc) was added and the mixture was heated at 45 C. for 3 hours, cooled to 20 C., and held for 16 hours. The slurry was filtered, washed with methanol and dried. 112.60 g of the title compound 8 as a mixture of E and Z isomers was obtained. .sup.1H NMR (DMSO-d.sub.6): (ppm) 1.5 (m, 6H), 2.0 (m, 2H), 2.2 (m, 3H), 2.4 (m, 2H), 2.7 (s, 1.1H), 3.0 (s, 0.9H), 3.7 (m, 3H), 3.8 (m, 2H), 4.0 (m, 2H), 6.8 (m 2H), 6.9 (m, 2H), 7.3 (m, 2H), 7.7 (m, 2H), 10.2&10.3 (s, 1H), 12.1 (s, 1H). LC-MS m/z 473 [M+H].sup.+.

EXAMPLE 4

4-(4-(1-(2-(3-mercapto-3-methylbutanoyl)hydrazono)ethyl)-phenoxy)butanoic Acid

(23) ##STR00070##

(24) 290.5 g, (614.7 mmol) of 8 and 1162 mL of anisole were charged to a reactor at 20-25 C. Trifluoroacetic acid (1162 mL) was added over 2 minutes, and the reaction was heated to 65-70 C. for 2.5 hours. The reaction was cooled to 40 C. The TFA (trifluoroacetic acid) was vacuum distilled and replaced with 2-methyltetrahydrofuran (2905 mL). The distillation was continued until a thick slurry was observed (final volume of the slurry was approximately 2 L). The slurry was cooled to 15 C. and filtered. The crude product was reslurried in methanol (1836.6 mL), heated to 55 C. and then cooled to 20 C. overnight. The slurry was filtered, washed with methanol and dried. 171.80 g (93.54%) of the title compound 9 as an E and Z mixture of isomers was obtained from 8. .sup.1H NMR (DMSO-d.sub.6): (ppm) 1.5 (m, 6H), 2.0 (m, 2H), 2.2 (m, 3H), 2.4 (m, 2H), 2.7 (s, 1.1H), 3.0 (s, 0.9H), 3.3 (s 1H), 4.0 (m, 2H), 6.9 (m, 2H), 7.7 (m, 2H), 10.2&10.3 (s, 1H), 12.1 (s, 1H).

EXAMPLE 5

2,5-dioxopyrrolidin-1-yl 4-(4-(1-(2-(3-mercapto-3-methylbutanoyl)-hydrazono)ethyl)phenoxy)butanoate

(25) ##STR00071##

(26) Reactor setup: 2-L jacketed reactor, Tr probe, nitrogen inlet.

(27) 60 g (170.3 mmol) was added to 2400 mL of THF and cooled to 10 C. Triethylamine (2 equiv., 34.46 g) was added, then trimethylacetyl chloride (1.1 equiv., 22.81 g) was added slowly over 10 minutes, maintaining the temperature in the range 10-20 C. The mixture was stirred at 10-20 C. for 30 minutes. N-hydroxysuccinimide (1.1 equiv., 21.99 g) was added to the reactor and stirred at 20-25 C. for 30 minutes.

(28) The slurry was filtered to remove the TEA-HCl salts and concentrated under vacuum to a volume of approximately 800 mL. Hexane (780 mL) was slowly added to crystallize the product. The slurry was stirred for 1.5 hours, then filtered, washed with heptanes and dried. 70.4 g (92% yield) of the title compound linker intermediate 10 was obtained from 9.

(29) The crude product was recrystallized by adding 144 g of 10 to 2100 mL of THF and heated to 60 C. The mixture was filtered through diatomaceous eart, and 2100 mL of hexane slowly added and cooled to 20 C. over 1.5 hours. The slurry was filtered, washed with cold THF/hexane (1:1), then hexane and dried. 126 g (87.5% recovery) of the title compound linker intermediate 10 was obtained from 9. .sup.1H NMR (DMSO-d.sub.6): (ppm) 1.5 (m, 6H), 2.1 (m, 2H), 2.2 (m, 3H), 2.7 (s, 1.1H), 2.9 (M, 5H) 3.0 (s, 1.9H), 3.3 (s 1H), 4.3 (m, 2H), 7.0 (m, 2H), 7.5 (m, 2H), 10.2&10.3 (s, 1H).

EXAMPLE 6

Preparation of Butanoic Acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio--D-ribo-hexopyranosyl]oxy]amino]--D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-, 2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxyl]phenyl]ethylidene]hydrazide

(30) To a solution of N-acetyl-calicheamicin (50 mg, 0.035 mmol) in acetonitrile (1.0 mL) at room temperature was added linker intermediate butanoic acid, 3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)-oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide (31.9 mg, 0.07 mmol) in one portion followed by 1,(3-dimethylaminopropyl)-3-ethylcarbodiimide (6.7 mg, 0.035 mmol) and then triethylamine (5.3 mg, 7.3 L, 0.053 mmol). The reaction mixture (a slurry) was stirred for 1 hour at room temperature at which point it became a yellow solution. The yield was determined by area percent relative to a derivative standard (60% yield).

(31) Mass Spec:

(32) (M+Na)=1801.4578

(33) .sup.1H NMR:

(34) .sup.1H NMR (CDCl.sub.3+5% CD.sub.3OD, 400 MHz): 8.69 (s, 1H, 18CNHN.sub.), 7.75 (d, J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H, 14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd, J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz, 1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d, J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B), 4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A), 3.91 (m, 1H, 15), 3.89 (s, 3H, 2COCH.sub.3), 3.84 (s, 3H, 3COCH.sub.3), 3.8 (m, 1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs, 3H, 10-NHCOOCH.sub.3), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H, 3D-OCH.sub.3), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH.sub.3), 3.30 (m, 2H, 4E-NCH.sub.2CH.sub.3), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H, 27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4 (m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H, 19), 2.08 (s, 3H, 4E-NCOCH.sub.3), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50 (s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H, 2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19 (t, J=7.2 Hz, 3H, 4E-NCH.sub.2CH.sub.3).

EXAMPLE 7

Preparation of Butanoic Acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio--D-ribo-hexopyranosyl]oxy]amino]--D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-, 2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide, Without Using EDC

(35) As a comparison to the method of the present invention illustrated in Example 6, the following reaction was conducted: To a solution of N-acetyl-calicheamicin (50 mg, 0.035 mmol) in acetonitrile (1.0 mL) at room temperature was added linker intermediate butanoic acid, 3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]-ethylidene]-hydrazide (31.9 mg, 0.07 mmol) in one portion followed by triethylamine (5.3 mg, 7.3 uL, 0.053 mmol). The reaction mixture (a slurry) was stirred for 1 hour at room temperature at which point it became a yellow solution. The yield was determined by area percent relative to a derivative standard (35% yield).

(36) Mass Spec:

(37) (M+Na)=1801.4578

(38) .sup.1H NMR:

(39) .sup.1H NMR (CDCl.sub.3+5% CD.sub.3OD, 400 MHz): 8.69 (s, 1H, 18CNHN.sub.), 7.75 (d, J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H, 14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd, J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz, 1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d, J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B), 4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A), 3.91 (m, 1H, 15), 3.89 (s, 3H, 2COCH.sub.3), 3.84 (s, 3H, 3COCH.sub.3), 3.8 (m, 1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs, 3H, 10-NHCOOCH.sub.3), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H, 3D-OCH.sub.3), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH.sub.3), 3.30 (m, 2H, 4E-NCH.sub.2CH.sub.3), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H, 27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4 (m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H, 19), 2.08 (s, 3H, 4E-NCOCH.sub.3), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50 (s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H, 2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19 (t, J=7.2 Hz, 3H, 4E-NCH.sub.2CH.sub.3)

EXAMPLE 8

Large Scale Preparation of Butanoic Acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3-O-methyl-a-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl-a-L-mannopyranosyl)oxy]-3-iodo-5,6-dimethoxy-2-methylbenzoyl]-4-thio--D-ribo-hexopyranosyl]oxy]amino]--D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxobicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-, 2-[(1E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide, Followed by Purification

(40) To a solution of N-acetyl-calicheamicin (60.2 g, 42.7 mmol) in acetonitrile (900 mL) at 4 C. was added linker intermediate butanoic acid, 3-mercapto-3-methyl-,2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide (38.4 g, 85.4 mmol) in one portion followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (8.2 g, 42.7 mmol). The bottles containing linker and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were rinsed with acetonitrile (300 mL) and added to reaction mixture. Triethylamine (6.5 g, 8.9 mL, 64.1 mmol) was then added to reaction mixture. The reaction mixture (a slurry) was stirred for 1 hour at 4 C. at which point it becomes a yellow solution. The reaction mixture was further diluted with acetonitrile (600 mL) and 20 mM sodium acetate buffer, pH 5.0 (1200 mL) and purified by a reversed phase HPLC column using a gradient of mobile phases A and B. (Mobile phase A: 55:45 (v/v) 20 mM NaOAc buffer pH 5.0 (pH 4.5-5.5): acetonitrile: Mobile phase B: acetonitrile.) The fractions of desired purity were pooled and then subjected to solid phase extraction (SPE). In SPE the purified fractions were loaded onto the column and then washed with a water/acetonitrile mixture and then eluted with acetonitrile to yield concentrated fractions containing product. The resulting fractions were concentrated in vacuo, taken up in ethyl acetate and precipitated by adding hexane. The solids were filtered and dried to provide the title compound as a white solid of 96.9% purity by HPLC. (45.2 g, 60% yield).

(41) Mass Spec:

(42) (M+Na)=1801.4578

(43) .sup.1H NMR:

(44) .sup.1H NMR (CDCl.sub.3+5% CD.sub.3OD, 400 MHz): 8.69 (s, 1H, 18CNHN.sub.), 7.75 (d, J=8.7 Hz, 2H, 22), 6.95 (d, J=8.7 Hz, 2H, 23), 6.38 (br t, J=7 Hz, 1H, 14), 6.23 (d, J=1.5 Hz, 1H, 8), 5.90 (d, J=9.5 Hz, 1H, 4), 5.80 (dd, J=9.5, 1.5 Hz, 1H, 5), 5.72 (d, J=1.5 Hz, 1H, 1D), 5.63 (br d, J=2.2 Hz, 1H, 1E), 5.03 (dd, J=11.5, 1.6 Hz, 1H, 1B), 4.70 (m, 1H, 5E), 4.61 (d, J=7.8 Hz, 1H, 1A), 4.6 (m, 1H, 3E), 4.49 (m, 1H, 2D), 4.31 (m, 1H, 3B), 4.20 (m, 1H, 5D), 4.10 (m, 2H, 25), 4.07 (m, 1H, 5B), 4.03 (m, 1H, 3A), 3.91 (m, 1H, 15), 3.89 (s, 3H, 2COCH.sub.3), 3.84 (s, 3H, 3COCH.sub.3), 3.8 (m, 1H, 3D), 3.76 (m, 1H, 15), 3.75 (m, 1H, 4B), 3.65 (m, 1H, 5A), 3.63 (bs, 3H, 10-NHCOOCH.sub.3), 3.62 (m, 1H, 2A), 3.6 (m, 1H, 4D), 3.57 (s, 3H, 3D-OCH.sub.3), 3.4 (m, 1H, 5E), 3.37 (s, 3H, 3E-OCH.sub.3), 3.30 (m, 2H, 4E-NCH.sub.2CH.sub.3), 3.12 (d, J=17.6 Hz, 1H, 12), 3.0 (m, 1H, 4E), 2.85 (m, 2H, 27), 2.85 (bs, 4, 30), 2.72 (d, J=17.6 Hz, 1H, 12), 2.5 (m, 2H, 17), 2.4 (m, 1H, 2E equatorial), 2.33 (m, 1H, 4A), 2.25 (m, 2H, 26), 2.18 (s, 3H, 19), 2.08 (s, 3H, 4E-NCOCH.sub.3), 2.0 (m, 1H, 2B), 1.8 (m, 1H, 2B), 1.50 (s, 3H, 16a), 1.44 (s, 3H, 16b), 1.42 (d, J=6.2 Hz, 3H, 6B), 1.4 (m, 1H, 2E axial), 1.31 (d, J=6.1 Hz, 3H, 6A), 1.31 (d, J=6.1 Hz, 3H, 6D), 1.19 (t, J=7.2 Hz, 3H, 4E-NCH.sub.2CH.sub.3)