Enzymatic transamination of cyclopamine analogs

1. A process for preparing a compound of formula (II):

or a salt thereof;

from a compound of formula (I):

or a salt thereof;

wherein:

R 1 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, —OR 16 , —C(O)R 16 , —CO 2 R 16 , —SO 2 R 16 , —C(O)N(R 17 )(R 17 ), —[C(R 16 ) 2 ] q —R 16 , —[(W)—N(R 17 )C(O)] q R 16 , —[(W)—C(O)] q R 16 , —[(W)—C(O)O] q R 16 , —[(W)—CO(O)] q R 16 , —[(W)—SO 2 ] q R 16 , —[(W)—N(R 17 )SO 2 ] q R 16 , —[(W)—C(O)N(R 17 )] q R 17 , —[(W)—O] q R 16 , —[(W)—N(R 17 )] q R 16 , or —[(W)—S] q R 16 ; wherein W is a diradical and q is 1, 2, 3, 4, 5, or 6;

each R 2 and R 3 is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, haloalkyl, halo, —OR 16 , —OR 16 , —N(R 17 ) 2 , or —SR 16 , or R 2 and R 3 taken together form a double bond or form a group

wherein Z is NR 17 , O, or C(R 18 ) 2 ;

R 4 is independently H, halo, —OR 16 , —N(R 17 ) 2 , or —SR 16 ;

each R 5 and R 6 , is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR 16 , —N(R 17 ) 2 , or —SR 16 ; or R 5 and R 6 taken together with the carbon to which they are bonded form C═O, C═S, C═N—OR 17 , C═N—R 17 , C═N—N(R 17 ) 2 , or form an optionally substituted 3-8 membered ring;

each R 7 , R 8 , R 9 , R 10 , R 11 , R 12 and R 13 is, independently, H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —OR 16 , —N(R 17 ) 2 , or —SR 16 ;

or R 10 and R 11 taken together, or R 11 and R 12 taken together, form a double bond or form a group

wherein Z is NR 17 , O, or C(R 18 ) 2 ;

each R 14 and R 15 is, independently, H, halo, —OR 16 , —N(R 17 ) 2 , or —SR 16 ; or R 14 and R 15 taken together with the carbon to which they are bonded form C═O or C═S;

X is a bond or the group —C(R 19 ) 2 —; wherein each R 19 is, independently, H, alkyl, aralkyl, halo, —CN, —OR 16 , or —N(R 17 ) 2 ;

R 16 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl or —[C(R 20 ) 2 ] p —R 21 wherein p is 0-6; or any two occurrences of R 16 on the same substituent are taken together to form a 4-8 membered optionally substituted ring;

R 17 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, —C(═O)R 20 , —C(═O)OR 20 , —SO 2 R 20 , —C(═O)N(R 20 ) 2 , or —[C(R 20 ) 2 ] p —R 21 wherein p is 0-6; or any two occurrences of R 17 on the same substituent are taken together to form a 4-8 membered optionally substituted ring;

R 18 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl, halo, —CN, —OR 20 , —OSi(R 20 ) 3 , —C(═O)R 20 , —C(═O)OR 20 , —SO 2 R 20 or —C(═O)N(R 20 ) 2 ;

R 20 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, or heteroaralkyl; or any two occurrences of R 20 on the same substituent are taken together to form a 4-8 membered optionally substituted ring;

R 21 is —OR 22 , —N(R 22 )C(═O)R 22 , —N(R 22 )C(═O)OR 22 , —N(R 22 )SO 2 (R 22 ), —C(═O)R 22 N(R 22 ) 2 , —OC(═O)R 22 N(R 22 )(R 22 ), —SO 2 N(R 22 )(R 22 ), —N(R 22 )(R 22 ), —C(═O)OR 22 , —C(═O)N(OH)(R 22 ), —OS(O) 2 OR 22 , —S(O) 2 OR 22 , —OP(═O)(OR 22 )(OR 22 ), —N(R 22 )P(O)(OR 22 )(OR 22 ), or —P(═O)(OR 22 )(OR 22 ); and

R 22 is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl; or any two occurrences of R 22 on the same substituent are taken together to form a 4-8 membered optionally substituted ring;

said process comprising contacting a compound of formula (I) or a salt thereof, an amino donor molecule, an amine transaminase enzyme and a cofactor in a solution to provide a compound of formula (II) or a salt thereof.

2. The process according to claim 1 , wherein the co-factor is pyridoxal phosphate (PLP).

3. The process according to claim 1 , wherein the co-factor is a co-enzyme.

4. The process according to claim 3 , wherein the co-enzyme is selected from L-alanine dehydrogenase (LADH), lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NAD), formate dehydrogenase (FDH), and glucose dehydrogenase (GDH).

5. The process according to claim 1 , wherein the process further comprises adding a pyruvate reductase mix to the solution.

6. The process according to claim 1 , wherein the enzyme preferentially generates a compound of formula (II) or a salt thereof wherein the newly-formed amino group has (R) stereochemistry or (S) stereochemistry.

7. The process according to claim 1 , wherein the enzyme preferentially generates an enantiomerically pure compound of formula (II) or salt thereof.

8. The process according to claim 1 , wherein the enzyme is an omega amine transaminase, a broad-range transaminase, a glutamate-pyruvate transaminase or a glutamate-oxaloacetic transaminase.

9. The process according to claim 8 , wherein the enzyme is an omega amine transaminase.

10. The process according to claim 9 , wherein the omega amine transaminase is selected from the group consisting of ATA-101, ATA-102, ATA-103, ATA-104, ATA-105, ATA-106, ATA-107, ATA-108, ATA-109, ATA-110, ATA-113, ATA-114, ATA-115, ATA-116, ATA-117, ATA-124, an omega amine transaminase from Chromobacterium violaceum , an omega amine transaminase from Alcaligenes denitrificans , an omega amine transaminase from Arthrobactercitreus , an omega amine transaminase from Klebsiella pneumoniae , an omega amine transaminase from Bacillus thuringiensis , an omega amine transaminase from Bacillus cereus , and an omega amine transaminase from Vibrio fluvialis.

11. The process according to claim 1 , wherein the amino donor molecule is an amine or salt thereof.

12. The process according to claim 1 , wherein the amino donor molecule is an amino acid, a polypeptide and/or a salt thereof.

13. The process according to claim 1 , wherein the amino donor molecule is selected from pyridoxamine, methylbenzylamine, 2-aminobutane, propyl amine, isopropyl amine, 1,1,1-trifluoropropan-2-amine, 1,1,1,3,3,3-hexafluoropropan-2-amine, benzyl amine, 2-amino-1-butanol, 1-amino-1-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane, 1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane, 1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane, 1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane, 2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane, 1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane, 1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-1-(2-naphthyl)ethane, cis-2-methylcyclopentanamine, trans-2-methylcyclopentanamine, cis-3-methylcyclopentanamine, trans-3-methylcyclopentanamine, cis-2-ethylcyclopentanamine, trans-2-ethylcyclopentanamine, cis-3-ethylcyclopentanamine, trans-3-ethylcyclopentanamine, cis-2-methylcyclohexanamine, trans-2-methylcyclohexanamine, cis-3-methylcyclohexanamine, trans-3-methylcyclohexanamine, cis-2-ethylcyclohexanamine, trans-2-ethylcyclohexanamine, cis-3-ethylcyclohexanamine, trans-3-ethylcyclohexanamine, 1-aminotetralin, 2-aminotetralin, 2-amino-5-methoxytetralin, 1-aminoindane, 2-aminoindane, 2-amino-1-propanol, cis-1-amino-2-indanol, trans-1-amino-2-indanol, 1-amino-6-hydroxyindanamine, taurine, and salts thereof.

14. The process according to claim 1 , wherein the amino donor molecule is selected from glycine, alanine, aspartic acid, phenylalanine, 2-aminopentanedioic acid, 3-aminobutyrate, γ-aminobutyrate, β-alanine, asparagine, cysteine, glutamic acid, glutamine, proline, selenocysteine, serine, tyrosine, arginine, histidine, ornithine, isoleucine, leucine, lysine, methionine, threonine, tryptophan, valine, and polypeptides thereof and/or salts thereof.

15. The process according to claim 1 , wherein the amino donor molecule is a chiral amino donor molecule.

16. The process according to claim 1 , wherein the solution is a buffered solution.

17. The process according to claim 1 , wherein the pH of the solution is between about 5 and about 9.

18. The process according to claim 1 , wherein R 1 is H, aralkyl or —CO 2 R 16 .

19. The process according to claim 1 , wherein R 2 and R 3 taken together form a double bond or form a group:

wherein Z is NR 17 , O, or C(R 18 ) 2 .

20. The process according to claim 1 , wherein R 2 and R 3 taken together form a double bond.

21. The process according to claim 1 , wherein X is a bond.

22. The process according to claim 1 , wherein X is the group —C(R 19 ) 2 —.

23. The process according to claim 1 , wherein R 4 is H.

24. The process according to claim 1 , wherein each of R 5 and R 6 is independently H, or R 5 and R 6 taken together with the carbon to which they are bonded form C═O.

25. The process according to claim 1 , wherein R 7 and R 8 are each H.

26. The process according to claim 1 , wherein R 9 and R 10 are each H.

27. The process according to claim 1 , wherein R 11 is a H.

28. The process according to claim 1 , wherein R 12 and R 13 are each H.

29. The process according to claim 1 , wherein R 9 is H and R 10 and R 11 taken together form a double bond.

30. The process according to claim 1 , wherein R 13 is H, and R 11 and R 12 taken together form a double bond.

31. The process according to claim 1 , wherein each of R 9 , R 10 , R 11 , R 12 and R 13 is H.

32. The process according to claim 1 , wherein R 14 and R 15 are each H.

33. The process according to claim 1 , wherein the compound of formula (I) or a salt thereof and a compound of formula (II) or a salt thereof are selected from the compounds provided in Tables 1, 2, 3, 4 or 5.

34. The process according to claim 1 , wherein the compound of formula (I) is a compound of formula (I-a):

or salt thereof,

and the compound of formula (II) is a compound of formula (R)-(II-a):

or a salt thereof.

35. The process according to claim 1 , further comprising contacting a compound of formula (II) or a salt thereof with a sulfonylating agent to provide a compound of formula (III):

or a salt thereof,

wherein R 23 is alkyl or aryl.

36. The process according to claim 35 , wherein the sulfonylating agent is selected from benzenesulfonyl chloride, benzenesulfonyl anhydride, p-toluenesulfonyl chloride, p-toluenesulfonyl anhydride, methanesulfonyl chloride, and methanesulfonyl anhydride.

37. The process according to claim 36 , wherein the sulfonylating agent is methanesulfonyl chloride or methanesulfonyl anhydride, and R 23 is —CH 3 .