Tubulysin compounds, methods of making and use

1. A compound having a structure represented by formula (I)

wherein

R 1 is H, unsubstituted or substituted C 1 -C 10 alkyl, unsubstituted or substituted C 2 -C 10 alkenyl, unsubstituted or substituted C 2 -C 10 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted (CH 2 ) 1-2 O(C 1 -C 10 alkyl), unsubstituted or substituted (CH 2 ) 1-2 O(C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2 ) 1-2 O(C 2 -C 10 alkynyl), (CH 2 ) 1-2 OC(═O)(C 1 -C 10 alkyl), unsubstituted or substituted (CH 2 ) 1-2 OC(═O)(C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2 ) 1-2 OC(═O)(C 2 -C 10 alkynyl), unsubstituted or substituted C(═O)(C 1 -C 10 alkyl), unsubstituted or substituted C(═O)(C 2 -C 10 alkenyl), unsubstituted or substituted C(═O)(C 2 -C 10 alkynyl), unsubstituted or substituted cycloaliphatic, unsubstituted or substituted heterocycloaliphatic, unsubstituted or substituted arylalkyl, or unsubstituted or substituted alkylaryl;

R 2 is H, unsubstituted or substituted C 1 -C 10 alkyl, unsubstituted or substituted C 2 -C 10 alkenyl, unsubstituted or substituted C 2 -C 10 alkynyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, unsubstituted or substituted (CH 2 ) 1-2 O(C 1 -C 10 alkyl), unsubstituted or substituted (CH 2 ) 1-2 O(C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2 ) 1-2 O(C 2 -C 10 alkynyl), (CH 2 ) 1-2 OC(═O)(C 1 -C 10 alkyl), unsubstituted or substituted (CH 2 ) 1-2 OC(═O)(C 2 -C 10 alkenyl), unsubstituted or substituted (CH 2 ) 1-2 OC(═O)(C 2 -C 10 alkynyl), unsubstituted or substituted C(═O)(C 1 -C 10 alkyl), unsubstituted or substituted C(═O)(C 2 -C 10 alkenyl), unsubstituted or substituted C(═O)(C 2 -C 10 alkynyl), unsubstituted or substituted cycloaliphatic, unsubstituted or substituted heterocycloaliphatic, unsubstituted or substituted arylalkyl, unsubstituted or substituted alkylaryl, or

wherein each R 2a is independently H, NH 2 , NHMe, Cl, F, Me, Et, or CN;

R 3a and R 3b are independently H, C 1 -C 5 alkyl, CH 2 (C 5 -C 6 cycloalkyl), CH 2 C 6 H 5 , C 6 H5, or CH 2 CH 2 OH;

R 4 is

wherein R 4a is H or C 1 -C 3 alkyl; and Y is H, OH, Cl, F, CN, Me, Et, NO 2 , or NH 2 ;

R 5 is H, C 1 -C 5 alkyl, C 2 -C 5 alkenyl, C 2 -C 5 alkynyl, CO(C 1 -C 5 alkyl), CO(C 2 -C 5 alkenyl), or CO(C 2 -C 5 alkynyl);

W is O or S; and

n is 0, 1, or 2;

or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 , having a structure represented by formula (Ia)

wherein Y is H or NO 2 ; R 4a is H, Me, or Et; and R 3a and R 3b are independently H, Me, or Et.

3. A compound according to claim 1 , having a structure represented by formula (Ib):

where R 4a is H, Me, or Et; R 3a and R 3b are independently H, Me, and Et; and R 6 is C 1 -C 5 alkyl, CH 2 OC(═O)C 1 -C 5 alkyl, or (CH 2 ) 1-2 C 6 H 5 .

4. A compound according to claim 3 , wherein one of R 3a and R 3b is H and the other is Me.

5. A compound according to claim 3 , having a structure represented by formula (Ib′):

where R 4a is H, Me, or Et and R 6 is Me or n-Pr.

6. A conjugate comprising a compound according to claim 1 covalently linked to a targeting moiety that specifically or preferentially binds to a tumor associated antigen.

7. A conjugate according to claim 6 , having a structure represented by formula (II-1′):

where R 6 is Me or n-Pr and Ab is an antibody.

8. A conjugate according to claim 7 , wherein the antibody is an anti-CD70, anti-mesothelin, or anti-glypican 3 antibody.

9. A conjugate according to claim 6 , having a structure represented by formula (II)

[D(X D ) a C(X Z ) b ] m Z  (II)

wherein

Z is a targeting moiety;

X D is a first spacer moiety;

X Z is a second spacer moiety;

C is a cleavable group;

subscripts a and b are independently 0 or 1;

subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

D is according to formula (D-a)

or formula (D-b)

wherein Y is H or NO 2 ; R 4a is H, Me, or Et; R 3a and R 3b are independently H, Me, or Et; and R 6 is C 1 -C 5 alkyl, CH 2 OC(═O)C 1 -C 5 alkyl, or (CH 2 ) 1-2 C 6 H 5 ;

or a pharmaceutically acceptable salt thereof.

10. A conjugate according to claim 9 , wherein Z is an antibody.

11. A drug-linker compound having a structure according to formula (III)

D-(X D ) a C(X Z ) b —R 31   (III)

wherein

R 31 is a reactive functional group;

X D is a first spacer moiety;

X Z is a second spacer moiety;

C is a cleavable group;

subscripts a and b are independently 0 or 1;

subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and

D is according to formula (D-a)

or formula (D-b)

wherein Y is H or NO 2 ; R 4a is H, Me, or Et; and R 3a and R 3b are independently H, Me, or Et; and R 6 is C 1 -C 5 alkyl, CH 2 OC(═O)C 1 -C 5 alkyl, or (CH 2 ) 1-2 C 6 H 5 ;

or a pharmaceutically acceptable salt thereof.

12. A drug-linker compound according to claim 11 , wherein R 31 is —NH 2 , —OH, —CO 2 H, —SH, maleimido, cyclooctyne, azido, hydroxylamino, or N-hydroxysuccinimido.

13. A drug-linker compound according to claim 11 , having a structure represented by formula (III-a):

where

R 3a and R 3b are independently H, Me, or Et;

R 6 is Me, Et, or n-Pr;

AA a and each AA b are independently selected from the group consisting of alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;

p is 1, 2, 3, or 4;

q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

r is 1, 2, 3, 4, or 5;

s is 0 or 1; and

R 31 is selected from the group consisting of

14. A drug-linker compound according to claim 11 , having a structure represented by formula (III-b):

where

R 6 is Me or n-Pr;

q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

r is 1, 2, 3, 4, or 5;

s is 0 or 1; and

R 31 is selected from the group consisting of

15. A pharmaceutical composition comprising a compound according to claim 1 , or a conjugate thereof with a targeting moiety, and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition according to claim 15 , wherein the compound according to claim 1 is conjugated to a targeting moiety that is an antibody.

17. A compound according to claim 1 , having a structure represented by formula (I- 9 ):

18. A conjugate according to claim 7 , wherein the antibody is an anti-mesothelin antibody and R 6 is n-Pr.

19. A drug-linker compound according to claim 11 , having a structure represented by formula (III- 8 ):