Cytotoxic and anti-mitotic compounds, and methods of using the same

1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

R 1 is selected from: amino-C 1 -C 6 alkyl, amino-aryl, amino-C 3 -C 7 cycloalkyl, amino-heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from aryl, aryl-C 1 -C 6 alkyl, C 1 -C 6 alkyl, C 1 -C 6 alkylthio, carboxyl, carboxamide, C 3 -C 7 cycloalkyl, C 3 -C 7 cycloalkyl-C 1 -C 6 alkyl, guanidino, halo, C 1 -C 6 haloalkyl, heterocyclyl, heterocyclyl-C 1 -C 6 alkyl, hydroxyl, and thio; or

R 1 is R a R b NCH(R c )—;

R a is selected from: H and C 1 -C 6 alkyl;

R b is C 1 -C 6 alkyl; and

R c is R d —C(CH 3 ) 2 —; and

R d is selected from: H, aryl, C 3 -C 7 cycloalkyl, and heteroaryl, each of which is optionally substituted with one or more substituents selected from: C 1 -C 4 acylthio, C 2 -C 4 alkenyl, C 1 -C 4 alkyl, C 1 -C 4 alkylamino, C 1 -C 4 alkyloxy, amino, amino-C 1 -C 4 alkyl, halo, C 1 -C 4 haloalkyl, hydroxyl, hydroxy-C 1 -C 4 alkyl, and thio, wherein C 2 -C 4 alkenyl, C 1 -C 4 alkylamino and C 1 -C 4 alkyloxy are further optionally substituted with one substituent selected from C 1 -C 4 alkylaryl, hydroxyl, and thio; or

R b and R c taken together with the atoms to which they are each bonded form a heterocyclyldiyl;

R 2 is aryl or aryl-C 1 -C 6 alkyl each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl; and

X is —C(O)NHCH(CH 2 R 3 )—; and

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl.

2. The compound according to claim 1 , wherein R 1 is selected from: amino-C 1 -C 6 alkyl, amino-aryl, amino-C 3 -C 7 cycloalkyl, amino-heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from C 1 -C 6 alkyl and halo.

3. The compound according to claim 1 , wherein R 1 is R a R b NCH(R c )—.

4. The compound according to claim 1 having Formula Ie:

or a pharmaceutically acceptable salt thereof,

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl;

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl; and

R 4 and R 5 are each independently selected from: H and C 1 -C 6 alkyl.

5. The compound according to claim 4 , wherein R 4 and R 5 are each C 1 -C 6 alkyl.

6. The compound according to claim 4 , wherein R 4 is H, and R 5 is C 1 -C 6 alkyl.

7. The compound according to claim 1 having Formula Ih:

or Formula Ik:

or a pharmaceutically acceptable salt thereof,

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl; and

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl.

8. The compound according to claim 7 , wherein R 3 is aryl optionally substituted with one substituent selected from amino and hydroxyl.

9. The compound according to claim 1 selected from the following, or a pharmaceutically acceptable salt thereof:

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4- aminophenylsulfonamido)-1-oxo-3- phenylpropan-2-ylamino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 11); and

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4- aminophenylmethylsulfonamido)-1- oxo-3-phenylpropan-2-ylamino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 14).

10. A pharmaceutical composition comprising the compound according to claim 1 , or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

11. A method of inhibiting cancer in a mammal, relieving cancer in a mammal, or relieving the symptoms of cancer in a mammal comprising administering to a mammal in need thereof an effective amount of the compound according to claim 1 .

12. A method of inhibiting tumor growth in a mammal comprising administering to a mammal in need thereof an effective amount of the compound according to claim 1 .

13. A composition of Formula II:

(T)-(L)-(D)  II

wherein (T) is a targeting moiety, (L) is a linker, and (D) is the compound according to claim 1 .

14. The composition according to claim 13 , wherein R 1 is selected from: amino-C 1 -C 6 alkyl, amino-aryl, amino-C 3 -C 7 cycloalkyl, amino-heterocyclyl, and heterocyclyl, each optionally substituted with one or more substituents selected from C 1 -C 6 alkyl and halo.

15. The composition according to claim 13 , wherein R 1 is R a R b NCH(R c )—.

16. The composition according to claim 13 , wherein (D) is a compound having Formula Ie:

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl;

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl; and

R 4 and R 5 are each independently selected from: H and C 1 -C 6 alkyl.

17. The composition according to claim 16 , wherein R 4 and R 5 are each C 1 -C 6 alkyl.

18. The composition according to claim 16 , wherein R 4 is H, and R 5 is C 1 -C 6 alkyl.

19. The composition according to claim 13 , wherein (D) is a compound having Formula Ih:

or Formula Ik:

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl; and

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl.

20. The composition according to claim 19 , wherein R 3 is aryl optionally substituted with one substituent selected from amino and hydroxyl.

21. The composition according to claim 13 , wherein (D) is a compound selected from the following:

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4- aminophenylsulfonamido)-1-oxo-3- phenylpropan-2-ylamino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 11); and

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4- aminophenylmethylsulfonamido)-1- oxo-3-phenylpropan-2-ylamino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 14).

22. The composition according to claim 13 , wherein (L)-(D) has the following structure:

23. The composition according to claim 13 , wherein (T) is a monoclonal antibody or antibody fragment.

24. The composition according to claim 23 , wherein the monoclonal antibody or antibody fragment is a bispecific antibody or antibody fragment.

25. The composition according to claim 23 , wherein the monoclonal antibody or antibody fragment specifically binds to a cancer cell antigen.

26. A pharmaceutical composition comprising the composition according to claim 13 and a pharmaceutically acceptable carrier, diluent or excipient.

27. A method of inhibiting cancer in a mammal, relieving cancer in a mammal, or relieving the symptoms of cancer in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 13 .

28. A method of inhibiting tumor growth in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 13 .

29. A compound selected from the following, or a pharmaceutically acceptable salt thereof:

(S)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N-((3R,4S,5S)- 3-methoxy-1-((S)-2-((1R,2R)-1- mcthoxy-2-methyl-3-oxo-3-((S)-1- oxo-3-phenyl-1-(4-(2,2,2- trifluoroacetamido)phenyl- sulfonamido)propan-2- ylamino)propyl)pyrrolidin-1-yl)-5- methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide (Compound 10); and

(S)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N-((3R,4S,5S)- 3-methoxy-1-((S)-2-((1R,2R)-1- methoxy-2-methyl-3-oxo-3-((S)-1- oxo-3-phenyl-1-(4-(2,2,2- trifluoroacetamido) phenylmethylsulfonamido)propan- 2-ylamino)propyl)pyrrolidin-1-yl)- 5-methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide (Compound 13).

30. The composition according to claim 13 , wherein (L)-(D) is formed by reacting (D) with a compound:

HO-AA 1 -AA 2 -NH-Anchor

wherein AA 1 and AA 2 are each independently an amino acid, and Anchor comprises a functional group that forms a covalent bond with (T).

31. The composition according to claim 30 , wherein Anchor further comprises a stretcher.

32. The composition according to claim 13 , wherein (L) does not include a self-immolative group.

33. The composition according to claim 21 , wherein (L)-(D) is formed by reacting (D) with a compound:

HO-AA 1 -AA 2 -NH-Anchor

wherein AA 1 and AA 2 are each independently an amino acid, and Anchor comprises a functional group that forms a covalent bond with (T).

34. The composition according to claim 33 , wherein Anchor further comprises a stretcher.

35. The composition according to claim 21 , wherein (L) does not include a self-immolative group.

36. The composition according to claim 21 , wherein (T) is a monoclonal antibody or antibody fragment.

37. The composition according to claim 36 , wherein the monoclonal antibody or antibody fragment is a bispecific antibody or antibody fragment.

38. The composition according to claim 36 , wherein the monoclonal antibody or antibody fragment specifically binds to a cancer cell antigen.

39. A pharmaceutical composition comprising the composition according to claim 21 and a pharmaceutically acceptable carrier, diluent or excipient.

40. A method of inhibiting cancer in a mammal, relieving cancer in a mammal, or relieving the symptoms of cancer in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 21 .

41. A method of inhibiting tumor growth in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 21 .

42. The composition according to claim 22 , wherein (T) is a monoclonal antibody or antibody fragment.

43. The composition according to claim 42 , wherein the monoclonal antibody or antibody fragment is a bispecific antibody or antibody fragment.

44. The composition according to claim 42 , wherein the monoclonal antibody or antibody fragment specifically binds to a cancer cell antigen.

45. A pharmaceutical composition comprising the composition according to claim 22 and a pharmaceutically acceptable carrier, diluent or excipient.

46. A method of inhibiting cancer in a mammal, relieving cancer in a mammal, or relieving the symptoms of cancer in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 22 .

47. A method of inhibiting tumor growth in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 22 .

48. The compound according to claim 1 having Formula Ib:

or a pharmaceutically acceptable salt thereof,

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl;

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl; and

R 4 and R 5 are each independently selected from: H and C 1 -C 6 alkyl.

49. The composition according to claim 13 , wherein (D) is a compound having Formula Ib:

or a pharmaceutically acceptable salt thereof,

wherein:

R 2 is aryl or aryl-C 1 -C 6 alkyl, each substituted with one or more substituents selected from: amino, amino-C 1 -C 6 alkyl and amino-C 3 -C 7 cycloalkyl;

R 3 is selected from: aryl, heteroaryl, and C 3 -C 7 cycloalkyl, each optionally substituted with one substituent selected from amino and hydroxyl; and

R 4 and R 5 are each independently selected from: H and C 1 -C 6 alkyl.

50. The compound according to claim 1 selected from the following, or a pharmaceutically acceptable salt thereof:

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((S)-1-((4- (aminomethyl)phenyl)sulfonamido)- 1-oxo-3-phenylpropan-2-yl)amino)- 1-methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 24);

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((S)-1-(((4- (aminomethyl)phenyl)methyl) sulfonamido)-1-oxo-3- phenylpropan-2-yl)amino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 28); and

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4-(1- aminocyclopropyl)phenylsulfonamido)- 1-oxo-3-phenylpropan-2- ylamino)-1-methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 32).

51. The composition according to claim 13 , wherein (D) is a compound selected from the following:

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((S)-1-((4- (aminomethyl)phenyl)sulfonamido)- 1-oxo-3-phenylpropan-2-yl)amino)- 1-methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 24);

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((S)-1-(((4- (aminomethyl)phenyl)methyl) sulfonamido)-1-oxo-3- phenylpropan-2-yl)amino)-1- methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 28); and

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-((S)-1-(4-(1- aminocyclopropyl)phenylsulfonamido)- 1-oxo-3-phenylpropan-2- ylamino)-1-methoxy-2-methyl-3- oxopropyl)pyrrolidin-1-yl)-3- methoxy-5-methyl-1-oxoheptan-4- yl)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N,3- dimethylbutanamide (Compound 32).

52. The composition according to claim 51 , wherein (L)-(D) is formed by reacting (D) with a compound:

HO-AA 1 -AA 2 -NH-Anchor

wherein AA 1 and AA 2 are each independently an amino acid, and Anchor comprises a functional group that forms a covalent bond with (T).

53. The composition according to claim 52 , wherein Anchor further comprises a stretcher.

54. The composition according to claim 51 , wherein (L) does not include a self-immolative group.

55. The composition according to claim 51 , wherein (T) is a monoclonal antibody or antibody fragment.

56. The composition according to claim 55 , wherein the monoclonal antibody or antibody fragment is a bispecific antibody or antibody fragment.

57. The composition according to claim 55 , wherein the monoclonal antibody or antibody fragment specifically binds to a cancer cell antigen.

58. A pharmaceutical composition comprising the composition according to claim 51 and a pharmaceutically acceptable carrier, diluent or excipient.

59. A method of inhibiting cancer in a mammal, relieving cancer in a mammal, or relieving the symptoms of cancer in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 51 .

60. A method of inhibiting tumor growth in a mammal comprising administering to a mammal in need thereof an effective amount of the composition according to claim 51 .

61. A compound selected from the following, or a pharmaceutically acceptable salt thereof:

(S)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N-((3R,4S,5S)- 3-methoxy-1-((S)-2-((1R,2R)-1- methoxy-2-methyl-3-oxo-3-(((S)-1- oxo-3-phenyl-1-((4-((2,2,2- trifluoroacetamido)methyl)phenyl) sulfonamido)propan-2- yl)amino)propyl)pyrrolidin-1-yl)-5- methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide (Compound 23);

(S)-2-((S)-2-(dinielhylamino)-3- methylbutanamido)-N-((3R,4S,5S)- 3-methoxy-1-((S)-2-((1R,2R)-1- methoxy-2-methyl-3-oxo-3-(((S)-1- oxo-3-phenyl-1-(((4-((2,2,2- trifluoroacetamido)methyl)phenyl) methyl)sulfonamido)propan-2- yl)amino)propyl)pyrrolidin-1-yl)-5- methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide (Compound 27); and

(S)-2-((S)-2-(dimethylamino)-3- methylbutanamido)-N-((3R,4S,5S)- 3-methoxy-1-((S)-2-((1R,2R)-1- methoxy-2-methyl-3-oxo-3-((S)-1- oxo-3-phenyl-1-(4-(1-(2,2,2- trifluoroacetamido)cyclo- propyl)phenylsulfonamido)propan-2- ylamino)propyl)pyrrolidin-1-yl)-5- methyl-1-oxoheptan-4-yl)-N,3- dimethylbutanamide (Compound 31).