Intermediates and methods for synthesizing calicheamicin derivatives
The present invention relates to intermediates of Formula I and to methods of synthesizing and purifying calicheamicin derivatives.
1. A method for synthesizing a compound of Formula I
wherein R 12 is selected from straight and branched-chain C 1 -C 8 alkyl;
each R 10 is independently selected from hydrogen, R 12 and —OR 12 ;
R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or —SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 -C 5 alkyl;
r is an integer 0 or 1;
G is oxygen or sulfur;
Z 1 is H or straight or branched-chain C 1 -C 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 14 ;
wherein each R 14 is independently selected from (C 1 -C 5 )alkyl and each R 14 is independently optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 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 15 —, wherein R 15 is a (C 1 -C 5 )alkyl and R 15 is optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 4 )alkyl; and Y is a straight or branched-chain (C 1 -C 6 )alkylene group or a straight or branched-chain (C 2 -C 6 )alkenylene group; which method comprises reacting a compound of Formula II
with a compound of Formula III
2. A method according to claim 1 , wherein r is 0, G is oxygen, Z 1 is methyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH 2 ) 3 -.
3. A method according to claim 1 , wherein R 8 and R 9 are methyl.
4. A method according to claim 1 , wherein each R 10 is hydrogen.
5. A method according to claim 4 , wherein R 12 is methyl.
6. A method for synthesizing a compound of Formula IV
which method comprises treating a compound of Formula I
wherein R 12 is selected from straight and branched-chain C 1 -C 8 alkyl;
each R 10 is independently selected from hydrogen, R 12 and —OR 12 ;
R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or —SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 -C 5 alkyl;
r is an integer 0 or 1;
G is oxygen or sulfur;
Z 1 is H or straight or branched-chain C 1 -C 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 14 ;
wherein each R 14 is independently selected from (C 1 -C 5 )alkyl and each R 14 is independently optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 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 15 —, wherein R 15 is a (C 1 -C 5 )alkyl and R 15 is optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 4 )alkyl; and
Y is a straight or branched-chain (C 1 -C 6 )alkylene group or a straight or branched-chain (C 2 -C 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 8 and R 9 are methyl, r is 0, G is oxygen, Z 1 is methyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH 2 ) 3 —.
9. A method according to claim 6 , wherein each R 10 is hydrogen.
10. A method according to claim 9 , wherein R 12 is methyl.
11. A method for synthesizing a linker intermediate of Formula V
wherein R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or—SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 -C 5 alkyl;
r is an integer selected from 0 and 1;
G is oxygen or sulfur;
Z 1 is H or straight or branched-chain C 1 -C 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 14 ;
wherein each R 14 is independently selected from (C 1 -C 5 )alkyl and each R 14 is independently optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 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 15 -, wherein R 15 is a (C 1 -C 5 )alkyl and R 15 is optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 4 )alkyl;
Y is a straight or branched-chain (C 1 -C 6 )alkylene group or a straight or branched-chain (C 2 -C 6 )alkylene group; and
Z is selected from the group consisting of
which method comprises
a) treating a compound of Formula I
wherein R 12 is selected from straight and branched-chain C 1 -C 8 alkyl, and each R 10 is independently selected from hydrogen, R 12 and —OR 12 ;
with a strong acid to form a mixture comprising a compound of Formula IV
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
14. A method according to claim 11 , wherein the linker intermediate is of the structure
15. A method according to claim 11 , wherein the compound of Formula I is obtained by reacting a compound of Formula II
with a compound of Formula III
16. A method of synthesizing a calicheamicin derivative of Formula VI
wherein J is
R 1 is
or CH 3 ; R 2 is
or H;
R 3 is
or H; R 4 is
or H;
R 5 is —CH 3 , —C 2 H 5 , or —CH(CH 3 ) 2 ;
X is an iodine or bromine atom;
R 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 6 -C 11 ) aryl-alkyl (C 1 -C 5 ) group, or a heteroaryl or heteroaryl-alkyl (C 1 -C 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 1 -C 3 ) alkoxy, or thioalkoxy of 1 to 5 carbon atoms;
R 6 and R 7 are each independently selected from H and
R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or —SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 -C 5 alkyl;
r is an integer 0 or 1;
G is oxygen or sulfur;
Z 1 is H or straight or branched-chain C 1 -C 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 14 ;
wherein each R 14 is independently selected from (C 1 -C 5 )alkyl and each R 14 is independently optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 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 15 -, wherein R 15 is a (C 1 -C 5 )alkyl and R 15 is optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 4 )alkyl;
Y is a straight or branched-chain (C 1 -C 6 )alkylene group or a straight or branched-chain (C 1 -C 6 )alkenylene group; and
Z is selected from the group consisting of
which method comprises
a) treating a compound of Formula I
wherein R 12 is selected from straight and branched-chain C 1 -C 8 alkyl, and each R 10 is independently selected from hydrogen, R 12 and —OR 12 ; with a strong acid to form a mixture comprising a compound of Formula IV
b) reacting the compound of Formula IV with a compound ZH;
to form a linker intermediate of Formula V
and
c) reacting the linker intermediate of Formula V resulting from step (b) with a methyltrisulfide compound CH 3 —S—S—S-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
with a compound of Formula III
18. A method according to claim 16 , wherein one of R 6 and R 7 is hydrogen and the other of R 6 and R 7 is
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 3 —S—S—S-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
wherein J is
R 1 is
or CH 3 ; R 2 is
or H;
R 3 is
or H; R 4 is
or H;
R 5 is —CH 3 , —C 2 H 5 , or —CH(CH 3 ) 2 ;
X is an iodine or bromine atom;
R 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 6 -C 11 ) aryl-alkyl (C 1 - 5 ) group, or a heteroaryl or heteroaryl-alkyl (C 1 -C 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 1 -C 3 ) alkoxy, or thioalkoxy of 1 to 5 carbon atoms;
R 6 and R 7 are each independently selected from H and
R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or —SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 - 5 alkyl;
r is an integer 0 or 1;
G is oxygen or sulfur;
Z 1 is H or straight or branched-chain C 1 -C 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 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 1 -C 6 alkyl, —OR 14 , —SR 14 , halogen, nitro, —COOR 14 , —C(═O)NHR 14 , —O(CH 2 ) n COOR 14 , —S(CH 2 ) n COOR 14 , —O(CH 2 ) n C(═O)NHR 14 , and —S(CH 2 ) n C(═O)NHR 14 ;
wherein each R 14 is independently selected from (C 1 -C 5 )alkyl and each R 14 is independently optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 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 15 -, wherein R 15 is a (C 1 -C 5 )alkyl and R 15 is optionally substituted with one or two groups selected from —OH, —(C 1 -C 4 )alkyl, and —S(C 1 -C 4 )alkyl;
Y is a straight or branched-chain (C 1 -C 6 )alkylene group or a straight or branched-chain (C 2 -C 6 )alkenylene group; and
Z is selected from the group consisting of
which method comprises reacting a linker intermediate of Formula V
with a methyltrisulfide compound CH 3 —S—S—S-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 6 and R 7 is hydrogen and the other of R 6 and R 7 is
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 3 —S—S—S-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
wherein R 12 is selected from straight and branched-chain C 1 -C 8 alkyl;
each R 10 is independently selected from hydrogen, R 12 and —OR 12 ;
R 8 and R 9 are each independently selected from hydrogen and straight and branched-chain C 1 -C 8 alkyl, wherein each said alkyl for R 8 and R 9 is independently optionally substituted by —NH 2 , —NHR 11 , —NR 11 R 13 , —OR 11 , —OH, or —SR 11 , wherein each R 11 and each R 13 are independently selected from straight and branched-chain C 1 -C 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
wherein R 16 is —C(═O)OH or —C(═V)SH, wherein V is oxygen or sulfur, or R 16 is —NH 2 ;
with an azole activating agent of Formula IX
wherein V′ is oxygen or sulfur; and
wherein E is
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 17 attached to E is independently selected from straight and branched-chain (C 1 -C 6 )alkyl groups;
in an organic solvent to form a compound of Formula VIII
wherein when R 16 is —C(═O)OH, r is 0 and G is oxygen; when R 16 is —C(═V)SH, r is 0 and G is V; and when R 16 is —NH 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 1 -C 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 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.