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1. (WO2017004025) CONJUGATES OF CYSTEINE ENGINEERED ANTIBODIES
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An antibody-cytotoxic agent conjugate represented by the following formula:


or a pharmaceutically acceptable salt thereof, wherein:

Ab is an antibody having a cysteine residue at the EU/OU numbering position 442 of a heavy chain of the antibody, and is covalently linked to a linking moiety JCB ' through the thiol group S442 of the cysteine residue;

D is a cytotoxic agent covalently linked to a linker L that is covalently linked

w is 1 or 2.

The conjugate of claim 1, wherein the cysteine residue at position 442 is

recombinantly introduced into said Ab.

The conjugate of claim 1 or 2, wherein:


si is the site covalently linked to the cysteine residue, and s2 is the site covalently linked to the group L; and,

Ra, Rb, Rc, and Re, for each occurrence, are independently H or an optionally substituted alkyl.

The conjugate of claim 1 or 2, wherein:


si is the site covalently linked to the cysteine residue, and s2 is the site covalently linked to the group L; and,

Ra, Rb, Rc, and Re, for each occurrence, are independently H or an optionally substituted alkyl.

The conjugate of claim 3 or 4, wherein Ra, Rb, Rc, and Re are each H.

The conjugate of claim 1, wherein JCB' is

The conjugate of any one of claims 1-6, wherein -L- is represented by the following structural formula:


wherein:

s3 is the site covalently linked to JCB', and s4 is the site covalently linked to D; E is -(CRioRn)q-, cycloalkyl, or cycloalkylalkyl;

Z is absent, -S02NR9-, -NR9S02-, -C(=0)-NR9-, -NR9-C(=0)-, -C(=0)-0-, -0-C(=0)-, -C(=0)-NR9-(CH2CH20)P-, -NR9-C(=0)-(CH2CH20)P-,

-(OCH2CH2)p-C(=0)NR9-, or -(OCH2CH2)p-NR9-C(=0)-;

p is an integer from 1 to 24;

Q is H, a charged substituent, or an ionizable group;

R9, Rio, Rn, R12, and R13, for each occurrence, are independently H or an optionally substituted alkyl; and,

q and r, for each occurrence, are independently an integer between 0 and 10.

8. The conjugate of claim 7, wherein E is -(CRioRn)q-.

9. The conjugate of claim 7, wherein E is

10. The conjugate of any one of claims 7-9, wherein Z is

11. The conjugate of claim 10, wherein R9 is -H.

12. The conjugate of any one of claims 7-11, wherein Q is:

i) H;

ii) -SO3H, -Z'-S03H, -OP03H2, -Z'-OP03H2, -P03H2, -Z'-P03H2, -C02H, -Z'- C02H, -NR11R12, or -Z'-NRi4Ri5, or a pharmaceutically acceptable salt thereof; or,

iii) -N+Ri4Ri5Ri6X" or -Z'-N+Ri4Ri5Ri6X";

Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene, or an optionally substituted phenylene;

Ri4, Ri5 and Ri6 are each independently an optionally substituted alkyl; and, X" is a pharmaceutically acceptable anion.

13. The conjugate of claim 12, wherein Z' is an optionally substituted alkylene.

14. The conjugate of any one of claims 7-11, wherein Q is H or -S03M, wherein M is H+, Na+ or K+.

15. The conjugate of any one of claims 7-14, wherein R9, Rio, Rn, R12, and Ri3 are all H; and q and r are each independently an integer between 1 and 6.

16. The conjugate of claim 1 or 2, wherein -JCB'-L- is represented by any one of the

following structural formulae:


wherein M is H+ or a pharmaceutically acceptable cation.

17. The conjugate of any one of claims 1-6, wherein -L- is represented by the following structural formula:


wherein:

s3 is the site covalently linked to JCB', and s4 is the site covalently linked to D; Ri9 to P22, for each occurrence, are independently H or an optionally substituted alkyl;

m and n are each independently an integer between 0 and 10;

Rh is H or an optionally substituted alkyl;

P is an optionally substituted alkylene, -(CH2-CH2-0)j- (wherein the oxygen atom is connected to the -(C=0)- group connected to P), an amino acid residue or a peptide containing 2 to 20 amino acid residues; and

j is an integer from 1 to 24.

18. The conjugate of claim 17, wherein R19, R20, R21 and R22 are each H; and m and n are each independently an integer between 1 and 6.

19. The conjugate of claim 17 or 18, wherein P is an amino acid residue or a peptide

containing 2 to 10 amino acid residues.

20. The conjugate of claim 19, wherein P is a peptide containing 2 to 5 amino acid

residues.

21. The conjugate of any one of claims 17-20, wherein each amino acid residue is the residue of an amino acid independently selected from: a naturally occurring amino acid, a synthetic amino acid, an amino acid analog, and an amino acid mimetic that functions in a manner similar to the naturally occurring amino acids.

22. The conjugate of claim 21, wherein each amino acid residue is the residue of an

amino acid independently selected from the group consisting of: Histidine, Alanine, Isoleucine, Arginine, Leucine, Asparagine, Lysine, Aspartic acid, Methionine, Cysteine, Phenylalanine, Glutamic acid, Threonine, Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine, N-methyl-Histidine, N-methyl- Alanine, N-methyl- Isoleucine, N-methyl-Arginine, N-methyl-Leucine, N-methyl- Asparagine, N-methyl- Lysine, N-methyl- Aspartic acid, N-methyl-Methionine, N-methyl-Cysteine, N- methyl-Phenylalanine, N-methyl-Glutamic acid, N-methyl-Threonine, N-methyl- Glutamine, N-methyl- Tryptophan, N-methyl-Glycine, N-methyl- Valine, N-methyl- Proline, N-methyl-Serine, N-methyl-Tyrosine, hydroxyproline, γ-carboxyglutamate, selinocysteine, O-phospho serine, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, citrulline, Ornithine, cysteine sulfonic acid, cysteine sulfinic acid, 3-aminoalanine, 3-dimethylaminoalanine, 2-amino-4- (dimethylamino)butanoic acid, 2,4-diaminobutanoic acid, 2-amino-6- (dimethylamino)hexanoic acid, 2-amino-5-(dimethylamino)pentanoic acid, and β- alanine, each independently as an L or D isomer.

23. The conjugate of claim 22, wherein each amino acid residue is the residue of an

independently selected glycine or alanine.

24. The conjugate of any one of claims 17-23, wherein P is a peptide cleavable by a

protease.

25. The conjugate of claim 17, wherein P is selected from the group consisting of: Val- Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, He- Ala-Leu, Val- Ala- Val, Ala-Leu- Ala-Leu (SEQ ID NO: 14), β- Ala- Leu-Ala-Leu (SEQ ID NO: 15), Gly-Phe-Leu-Gly (SEQ ID NO: 16), Val-Arg, Arg- Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D- Val-Lys, D- Val-Arg, D-Val-D-Cit, D- Val-D-Lys, D- Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D- Ala-Ala, and D-Ala-D-Ala., Gly-Gly-Gly, Ala- Ala- Ala, D- Ala- Ala- Ala, Ala-D- Ala- Ala, Ala-Ala-D-Ala, Ala-Val-Cit, Ala- Val- Ala, and β-Ala-Gly-Gly-Gly.

26. The conjugate of claim 25, wherein P is Gly-Gly-Gly, Ala- Ala- Ala, D-Ala-Ala-Ala, Ala-D- Ala- Ala, Ala- Val- Ala, or β-Ala-Gly-Gly-Gly.

The conjugate of claim 17, wherein -JCB'-L- is represented by the following structural formula


The conjugate of any one of claims 1-6, wherein -L- is represented by the following structural formula:


wherein:

s3 is the site covalently linked to JCB', and s4 is the site covalently linked to D; R23 and R24, for each occurrence, are independently H or an optionally substituted alkyl;

m' is an integer between 0 and 10; and

Rh' is H or an optionally substituted alkyl.

29. The conjugate of claim 28, wherein R23 and R24 are both H; and m' is an integer

between 1 and 6.

30. The conjugate of claim 28 or 29, wherein Rh' is H.

31. The conjugate of claim 28, wherein -JCB'-L- is represented by the following structural formula:


The conjugate of any one of claims 1-31, wherein D is a benzodiazepine compound.

The conjugate of claim 32, wherein D is a indolinobenzodiazepine compound.

The conjugate of any one of claims 1-27, wherein D is represented by the following structural formula:


or a pharmaceutically acceptable salt thereof, wherein:

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is selected from -H, or an amine protecting group; and Y is selected from -OR, -OCOR', -SR, -NR'R", -S03M, -S02M or -OS03M, wherein M is -H or a cation;

R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group -(CH2CH20)n-Rc, wherein n is an integer from 1 to 24, and Rc is a linear or branched alkyl having 1 to 4 carbon atoms;

R' and R" are each independently selected from -H, -OH, -OR, -NRRg , -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted aryl having from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P, a PEG group -(CH2CH20)n-Rc, and Rg is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group -(CH2CH20)n-Rc;

X' is selected from the group consisting of -H, -OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and an amine-protecting group;

Y' is selected from the group consisting of -H, an oxo group, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

A and A' are selected from -O- and -S-;

W is absent, or selected from -0-, -N(Re)-, -N(Re)-C(=0)-, -N(C(=0)Re)-, -S-or -CH2-S-, -CH2NRe-;

Rx is absent or selected from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms;

Re is -H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or -(CH2-CH2-0)n-Rk, wherein Rk is a -H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g. , -NHR101) or tertiary amino (-NR101R102) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R101 and R102 are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

G is selected from -CH- or -N-; and

R6 is -H, -R, -OR, -SR, -NR'R", -N02, or halogen.

35. The conjugate of claim 34, wherein D is represented by the following structural

formul


36. The conjugate of claim 34 or 35, wherein:

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H, and Y is -OH or -SO3M;

M is -H or a pharmaceutically acceptable cation;

X' and Y' are both -H;

A and A' are both -0-;

R6 is -OMe; and

Rx is a linear or branched alkyl having 1 to 6 carbon atoms.

37. The conjugate of claim 34, wherein D is represented by the following structural

formula:


or a pharmaceutically acceptable salt thereof.

The conjugate of any one of claims 34-37, wherein Y is -SO3M, and M is H+, Na+ or K+.

The conjugate of any one of claims 1-6 and 28-31, wherein D is represented by the followin structural formula:


or a pharmaceutically acceptable salt thereof, wherein:

one of L', L", and L" ' is represented by the following formula:


and the other two are each independently selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, halogen, guanidinium

[-NH(C=NH)NH2], -OR, -NR'R", -N02, -NR'COR", -SR, -SOR', -S02R', -S03H, -OS03H, -S02NR'R", cyano, an azido, -COR', -OCOR', and -OCONR'R" ;

one of the Zi and Z2 is -C(=0)-, and the other is -NR5-;

Pi is an amino acid residue or a peptide containing between 2 to 20 amino acid residues;

Rxi is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

Re is -H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or -(CH2-CH2-0)n-Rk, wherein Rk is a -H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g. , -NHR101) or tertiary amino (-NR101R102) group or a 5- or 6-membered nitrogen containing

heterocycle, wherein R and R are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond Xi is absent and Yi is -H, or a linear or branched alkyl having 1 to 4 carbon atoms, and when it is a single bond, Xi is -H or an amine protecting moiety; and Yi is a leaving group selected from -OR, -OCOR', -OCOOR', -OCONR'R", -NR'R", -NR'COR", -NR'NR'R", an optionally substituted 5- or 6-membered nitrogen-containing heterocycle, a guanidinium represented by -NR'(C=NH)NR'R", an amino acid, or a peptide represented by -NRCOP', -SR, -SOR', halogen, cyano, azido, -OS03H (or a salt thereof), sulfite (-S03H or -SO2H or a salt thereof), metabisulfite (H2S205 or a salt thereof), mono-, di-, tri-, and tetra- thiophosphate (P03SH3, P02S2H2, POS3H2, PS4H2 or a salt thereof), thio phosphate ester (R'O^PSCOR1), R'S-, R'SO, ^802, R'SOS, thiosulfate (HS203 or a salt thereof), dithionite (HS204 or salt thereof), phosphorodithioate

(P(=S)(ORk')(S)(OH) or a salt thereof), hydroxamic acid (Rk'C(=0)NOH or a salt thereof), and formaldehyde sulfoxylate (HOCH2SO2 or a salt thereof) or a mixture thereof, wherein R1 is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N(RJ)2, -CO2H, -S03H, and -P03H; R1 can be further optionally substituted with a substituent for an alkyl described herein; R' is a linear or branched alkyl having 1 to 6 carbon atoms; Rk is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl;

P' is an amino acid residue or a peptide containing between 2 to 20 amino acid residues,

R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(Cf^Cf O R0, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;

R' and R" are each independently selected from -H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, and an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P;

Rc is -H or an optionally substituted linear or branched alkyl having 1 to 4 carbon atoms;

n is an integer from 1 to 24;

Xi' is selected from -H, an amine-protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;

Yi' is selected from -H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted 6- to 18-membered aryl, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms;

Ri, R2, R3, R4, Ri', R2', R3' and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, halogen, guanidinium [-NH(C=NH)NH2], -OR, -NR'R", -N02, -NCO, -NR'COR", -SR, -SOR', -S02R', -SO3 H, -OSO3H, -S02NR'R", cyano, an azido, -COR', -OCOR', and -OCONR'R" ;

R6 is -H, -R, -OR, -SR, -NR'R", -N02, or halogen;

G is -CH- or -N-;

A and A' are the same or different, and are independently selected from -0-, oxo (-C(=0)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR5 and -CRR'N(R5)-; and

R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms.

The conjugate of claim 39, wherein D is represented by the following structural formula:


or a pharmaceutically acceptable salt thereof.

41. The conjugate of claim 39 or 40, wherein one of L', L" and L'" is represented by formula (Α') or (D'), and the others are -H, an linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (Ci-C6)alkoxy, or -N02.

42. The conjugate of claim 41, wherein L' is represented by formula (A'); and L" and L'" are both -H.

43. The conjugate of claim 39 or 40, L' is represented by formula (D'); and L" and L'" are both -H.

44. The conjugate of any one of claims 39-43, wherein Rxi is a linear, branched or cyclic alkyl having 1 to 6 carbon atoms optionally substituted with halogen, -OH, -SO3H, (Ci-C3)alkyl, (Ci-C3)alkoxy, halo(Ci-C3)alkyl, or a charged substituent or an ionizable group Q.

45. The conjugate of any one of claims 42-44, wherein L' is represented by the following formula:

-NR5-Pi-C(=0)-(CRaRb)s-C(=0)- (B l');

-NR5-Pi-C(=0)-Cy-(CRaRb)sr-C(=0)- (B2');

-C(=0)-Pi-NR5-(CRaRb)s-C(=0)- (CI'), or

-C(=0)-Pi-NR5-Cy-(CRaRb)sr-C(=0)- (C2') wherein:

Ra and Rb, for each occurrence, are each independently -H, (Ci-C3)alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

si' is 0 or an integer from 1 to 6; and

Cy is a cyclic alkyl having 5 or 6 ring carbon atoms optionally substituted with halogen, -OH, (Ci-C3)alkyl, (Ci-C3)alkoxy, or halo(Ci-C3)alkyl.

46. The conjugate of claim 45, wherein Ra and R are both H; Cy in formulas (Β2') and (C2') is cyclohexane; and R5 is H or Me.

47. The conjugate of claim 45 or 46, wherein si ' is 0 or 1.

48. The conjugate of any one of claims 39-47, wherein the charged substituent or an

ionizable group Q is i) -S03H, -Z'-S03H, -OP03H2, -Z'-OP03H2, -P03H2, -Z'-P03H2, -C02H, -Z'-C02H, -NRiiRi2, or -Z'-NRnRi2, or a pharmaceutically acceptable salt thereof; or, ii) -N+Ri4Ri5Ri6X" or -Z'-N+Ri4RisRi6X ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted phenylene; Ri4 to Ri6 are each independently an optionally substituted alkyl; and X" is a pharmaceutically acceptable anion.

49. The conjugate of claim 48, wherein Q is -S03H or a pharmaceutically acceptable salt thereof.

50. The conjugate of any one of claims 39-49, wherein Pi is a peptide containing 2 to 10 amino acid residues.

51. The conjugate of claim 50, wherein Pi is a peptide containing 2 to 5 amino acid

residues.

52. The conjugate of claim 51, wherein Pi is Gly-Gly-Gly, Ala- Val, Val- Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, He- Ala-Leu, Val- Ala- Val, Ala-Leu- Ala-Leu (SEQ ID NO: 14), β- Ala- Leu-Ala-Leu (SEQ ID NO: 15), Gly-Phe-Leu-Gly (SEQ ID NO: 16), Val-Arg, Arg- Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D- Val-Cit, D- Val-Lys, D- Val-Arg, D-Val-D-Cit, D- Val-D-Lys, D- Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D- Ala-Ala, D-Ala-D-Ala, Ala-Met, or Met- Ala.

53. The conjugate of claim 52, wherein Pi is Gly-Gly-Gly, Ala- Val, Ala- Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

54. The conjugate of any one of claims 39-53, wherein the double line ~ between N and C represents a double bond.

55. The conjugate of any one of claims 39-53, wherein the double line ~ between N and C represents a single bond, Xi is -H or an amine protecting group; and Yi is selected from -OR, -OCOR', -SR, -NR'R," an optionally substituted 5- or 6-membered

nitrogen-containing heterocycle, -SO3M, -SO2M and -OSO3M, wherein M is H+ or a pharmaceutically acceptable cation.

56. The conjugate of claim 55, wherein Yi is selected from -SO3M, -OH, -OMe, -OEt or -NHOH.

57. The conjugate of claim 56, wherein Yi is -SO3M or -OH, wherein M is H+, Na+ or K+.

58. The conjugate of any one of claims 39-57, wherein Xi' is selected from the group consisting of -H, -OH, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, and phenyl.

59. The conjugate of claim 58, wherein Xi ' is -H, -OH, (Ci-C3)alkyl, halo(Ci-C3)alkyl, or phenyl.

60. The conjugate of claim 59, wherein Xi ' is -H, -OH or -Me.

61. The conjugate of claim 59, wherein Xi ' is -H.

62. The conjugate of any one of claims 39-61, wherein Yi' is selected from the group consisting of -H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms.

63. The conjugate of claim 62, wherein Yi ' is -H, an oxo group, (Ci-C3)alkyl or halo(Ci- C3)alkyl.

64. The conjugate of claim 62, wherein Yi ' is -H or oxo.

65. The conjugate of claim 62, wherein Yi ' is -H.

66. The conjugate of any one of claims 39-65, wherein A and A' are the same or

different, and are selected from -0-, -S-, -NR5-, and oxo -(C=0)-.

67. The conjugate of claim 66, wherein A and A' are the same or different, and are

selected from -O- and -S-.

68. The conjugate of claim 67, wherein A and A' are -0-.

69. The conjugate of any one of claims 39-68, wherein R6 is -OMe.

70. The conjugate of any one of claims 39-69, wherein Ri, R2, R3, R4, Ri', R2', R3' and R4' are independently -H, halogen, -N02, -OH, (Ci-C3)alkyl, halo(Ci-C3)alkyl or (Q- C3)alkoxy.

71. The conjugate of claim 70, wherein Ri, R2, R3, R4, Ri', R2', R3' and R4' are all -H.

72. The conjugate of any one of claims 39-71, wherein R, R', R" and R5 are each

independently -H or (Ci-C3)alkyl.

73. The conjugate of any one of claims 39-53, wherein:

the double line ~ between N and C represents a single bond or double bond, provided that when it is a double bond, Xi is absent and Yi is -H; and when it is a single bond, Xi is -H, and Yi is -OH or -S03M;

Ri, R2, R3, R4, Ri', R2', R3' and R4' are all -H;

R6 is -OMe;

Xi' and Yi' are both -H;

A and A' are -0-; and

M is H+, Na+ or K+.

The conjugate of claim 39, wherein D is represented by the following structural formula:

wherein M is H+ or a pharmaceutically acceptable cation.

75. The conjugate of claim 1, wherein the conjugate is represented by the following structural formula:

- 139 -

- 140 -

- 141 -

or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody; and M is H+ or a pharmaceutically acceptable cation.

76. The conjugate of any one of claims 1-75, wherein M, when present, is H+, Na+ or K+.

77. The conjugate of any one of claims 1-76, wherein w is 2.

78. A compound represented by the following structural formula:

D-L-JcB,

or a pharmaceutically acceptable salt thereof, wherein:

D is represented by the following structural formula:


or a pharmaceutically acceptable salt thereof, wherein:

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is selected from -H, or an amine protecting group; and Y is selected from -OR, -OCOR', -SR, -NR'R", -S03M, -S02M or -OS03M, wherein M is -H or a cation;

R is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group -(CH2CH20)n-Rc, wherein n is an integer from 1 to 24, and Rc is a linear or branched alkyl having 1 to 4 carbon atoms;

R' and R" are each independently selected from -H, -OH, -OR, -NRRg , -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted aryl having from 6 to 18 carbon atoms, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms selected from O, S, N and P, a PEG group -(CH2CH20)n-Rc, and Rg is -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms or a PEG group -(CH2CH20)n-Rc;

X' is selected from the group consisting of -H, -OH, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, phenyl, and an amine-protecting group;

Y' is selected from the group consisting of -H, an oxo group, a substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

A and A' are selected from -O- and -S-;

W is absent, or selected from -0-, -N(Re)-, -N(Re)-C(=0)-, -N(C(=0)Re)-, -S-

Rx is absent or selected from a linear, branched or cyclic alkyl having 1 to 10 carbon atoms;

Re is -H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or -(CH2-CH2-0)n-Rk, wherein Rk is a -H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g. , -NHR101) or tertiary amino (-NR101R102) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R101 and R102 are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

G is selected from -CH- or -N-;

R6 is -H, -R, -OR, -SR, -NR'R", -N02, or halogen;

-L- is represented b the following structural formula:


wherein:

s3 is the site covalently linked to JCB, and s4 is the site covalently linked to D;

E is -(CRioRn)q-, cycloalkyl or cycloalkylalkyl;

Z is absent, -S02NR9-, -NR9S02-, -C(=0)-NR9-, -NR9-C(=0)-, -C(=0)-0-, -0-C(=0)-, -C(=0)-NR9-(CH2CH20)p-, -NR9- C(=0)-(CH2CH20)p-, -(OCH2CH2)p-C(=0)NR9-, or -(OCH2CH2)p-NR9- C(=0)-;

p is an integer from 1 to 1000;

Q is H, a charged substituent or an ionizable group;

R9, Rio, R11, R12, and R13, for each occurrence, are independently H or an optionally substituted alkyl;

q and r, for each occurrence, are independently an integer between 0 and 10; and


79. The compound of claim 78, wherein JCB is
X'-CRbRc


80. The compound of claim 78 or 79, wherein D is represented by the following structural formul


or a pharmaceutically acceptable salt thereof.

81. The compound of claim 78, 79 or 80, wherein Ra, Rb, Rc, and Re are each H.

82. The compound of claim 78 or 79, wherein JCB is

83. The compound of any one of claims 78-82, wherein:

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond X is absent and Y is -H, and when it is a single bond, X is -H; Y is -OH or -S03M;

M is -H or a pharmaceutically acceptable cation (e.g. , Na+);

X' and Y' are both -H;

A and A' are both -0-;

R6 is -OMe; and

Rx is a linear or branched alkyl having 1 to 6 carbon atoms.

84. The compound of claim 78 or 79, wherein D is represented by the following structural formula:


or a pharmaceutically acceptable salt thereof.

85. The compound of any one of claims 78-84, wherein Y is -SO3M; and M is H+, Na+ or K+.

86. The compound of any one of claims 78-85, wherein E is -(CRioRn)q-.

The compound of claim 86, wherein E

88. The compound of any one of claims 78-87, wherein Z is
or -NR9- C(=0)-.

89. The compound of claim 88, wherein R9 is -H.

90. The compound of any one of claims 78-89 wherein Q is:

i) H;

ii) -SO3H, -Z'-S03H, -OPO3H2, -Z'-OP03H2, -P03H2, -Z'-P03H2, -C02H, -Z'- C02H, -NR11R12, or -Z'-NRi4Ri5, or a pharmaceutically acceptable salt thereof; or,

iii) -N+Ri4Ri5Ri6X" or -Z'-N+Ri4Ri5Ri6X";

Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene, or an optionally substituted phenylene;

Ri4, Ri5 and Ri6 are each independently an optionally substituted alkyl; and,

X" is a pharmaceutically acceptable anion.

91. The compound of claim 90, wherein Z' is an optionally substituted alkylene.

92. The compound of any one of claims 78-89, Q is H, or -SO3M, wherein M is H+, Na+ or K+.

The compound of any one of claims 78-92, wherein R9, Rio, Rn, R12, and R13 are H; and q and r are each independently an integer between 1 and 6.

The compound of claim 78-85, wherein -L-JCB is represented by the following structural formula:


wherein M is H+ or a pharmaceutically acceptable cation.

95. The compound of claim 78, wherein the compound is represented by the following structural formula

D-L-JcB, harmaceutically acceptable salt thereof, wherein:

D is represented by the following structural formula:


or a pharmaceutically acceptable salt thereof, wherein:

one of L', L", and L" ' is represented by the following formula:


and the other two are each independently selected from -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, halogen, guanidinium [- NH(C=NH)NH2], -OR, -NR'R", -N02, -NR'COR", -SR, -SOR', -S02R', -S03H, -OS03H, -S02NR'R", cyano, an azido, -COR', -OCOR', and -OCONR'R" ;

one of the Zi and Z2 is -C(=0)-, and the other is -NR5-;

Pi is an amino acid residue or a peptide containing between 2 to 20 amino acid residues;

Rxi is an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms;

Re is -H, a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms or -(CH2-CH2-0)n-Rk, wherein Rk is a -H, a linear, branched cyclic alkyl having 1 to 6 carbon atoms, optionally bearing a secondary amino (e.g. , -NHR101) or tertiary amino (-NR101R102) group or a 5- or 6-membered nitrogen containing heterocycle, wherein R101 and R102 are each independently a linear, branched, or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms;

the double line ~ between N and C represents a single bond or a double bond, provided that when it is a double bond Xi is absent and Yi is -H, or a linear or branched alkyl having 1 to 4 carbon atoms, and when it is a single bond, Xi is -H or an amine protecting moiety; and Yi is a leaving group selected from -OR, -OCOR', - OCOOR', -OCONR'R", -NR'R", -NR'COR", -NR'NR'R", an optionally substituted 5- or 6-membered nitrogen-containing heterocycle, a guanidinium represented by -NR'(C=NH)NR'R", an amino acid, or a peptide represented by - NRCOP', -SR, -SOR', halogen, cyano, azido, -OS03H (or a salt thereof), sulfite (- SO3H or -SO2H or a salt thereof), metabisulfite (H2S205 or a salt thereof), mono-, di-, tri-, and tetra- thiophosphate (PO3SH3, P02S2H2, POS3H2, PS4H2 or a salt thereof), thio phosphate ester (RiO)2PS(ORi), R;S-, R'SO, ^802, R'SOS, thiosulfate (HS203 or a salt thereof), dithionite (HS204 or salt thereof), phosphorodithioate

(P(=S)(ORk')(S)(OH) or a salt thereof), hydroxamic acid (Rk C(=0)NOH or a salt thereof), and formaldehyde sulfoxylate (HOCH2SO2 or a salt thereof) or a mixture thereof, wherein R1 is a linear or branched alkyl having 1 to 10 carbon atoms and is substituted with at least one substituent selected from -N(RJ)2, -CO2H, -SO3H, and -PO3H; R1 can be further optionally substituted with a substituent for an alkyl described herein; R' is a linear or branched alkyl having 1 to 6 carbon atoms; Rk is a linear, branched or cyclic alkyl, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl;

P' is an amino acid residue or a peptide containing between 2 to 20 amino acid residues,

R, for each occurrence, is independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(Cf^Cf O R0, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, or an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;

R' and R" are each independently selected from -H, -OH, -OR, -NHR, -NR2, -COR, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(Cf^Cf O R0, and an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms independently selected from O, S, N and P;

Rc is -H or an optionally substituted linear or branched alkyl having 1 to 4 carbon atoms;

n is an integer from 1 to 24;

Xi' is selected from -H, an amine-protecting group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(Cf^Cf O R0, an optionally substituted aryl having 6 to 18 carbon atoms, an optionally substituted 5- to 18-membered heteroaryl ring

containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, and an optionally substituted 3- to 18-membered heterocyclic ring containing 1 to 6 heteroatoms independently selected from O, S, N and P;

Yi' is selected from -H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, an optionally substituted 6- to 18-membered aryl, an optionally substituted 5- to 18-membered heteroaryl ring containing one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur, an optionally substituted 3- to 18-membered heterocyclic ring having 1 to 6 heteroatoms;

Ri, R2, P3, P4, Ri', R2', R3' and R4' are each independently selected from the group consisting of -H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, a polyethylene glycol unit -(CH2CH20)n-Rc, halogen, guanidinium [-NH(C=NH)NH2], -OR, -NR'R", -N02, -NCO, -NR'COR", -SR, -SOR', -S02R', -S03"H, -OS03H, -S02NR'R", cyano, an azido, -COR', -OCOR', and -OCONR'R" ;

R6 is -H, -R, -OR, -SR, -NR'R", -N02, or halogen;

G is -CH- or -N-;

A and A' are the same or different, and are independently selected from -0-, oxo (-C(=0)-), -CRR'O-, -CRR'-, -S-, -CRR'S-, -NR5 and -CRR'N(R5)-; and

R5 for each occurrence is independently -H or an optionally substituted linear or branched alkyl having 1 to 10 carbon atoms;

-L- is represented by the followin structural formula:


wherein:

s3 is the site covalently linked to JCB', and s4 is the site covalently linked to D; R23 and R24, for each occurrence, are independently H or an optionally substituted alkyl;

m' is an integer between 0 and 10;

Rh' is H or an optionally substituted alkyl; and

97.


The compound of claim 96 or 97, wherein D is represented by the following structural formula:


harmaceutically acceptable salt thereof.

99. The compound of claim 96, 97 or 98, wherein Ra, Rb, Rc, and Re are each H.

100. The compound of any one of claims 96-99, wherein JCB is

101. The compound of any one of claims 96-100, wherein one of L', L" and L'" is represented by formula (Α') or (D'), and the others are -H, an linear or branched alkyl having from 1 to 6 carbon atoms, halogen, -OH, (Ci-C6)alkoxy, or -N02.

102. The compound of claim 101, wherein L' is represented by formula (A'); and L" and L'" are both -H.

103. The compound of any one of claims 96-100, wherein L' is represented by formula (D'); and L" and L'" are both -H.

104. The compound of any one of claims 96-103, wherein Rxi is a linear, branched or

cyclic alkyl having 1 to 6 carbon atoms optionally substituted with halogen, -OH, - SO3H, (Ci-C3)alkyl, (Ci-C3)alkoxy, halo(Ci-C3)alkyl, or a charged substituent or an ionizable group Q.

105. The compound of any one of claims 96-104, wherein L' is represented by the

following formula:

-NR5-Pi-C(=0)-(CRaRb)s-C(=0)- (B l');

-NR5-Pi-C(=0)-Cy-(CRaRb)sr-C(=0)- (B2');

-C(=0)-Pi-NR5-(CRaRb)s-C(=0)- (CI'), or

-C(=0)-Pi-NR5-Cy-(CRaRb)sr-C(=0)- (C2') wherein:

Ra and Rb, for each occurrence, are each independently -H, (Ci-C3)alkyl or a charged substituent or an ionizable group Q;

s is an integer from 1 to 6;

si' is 0 or an integer from 1 to 6; and

Cy is a cyclic alkyl having 5 or 6 ring carbon atoms optionally substituted with halogen, -OH, (Ci-C3)alkyl, (Ci-C3)alkoxy, or halo(Ci-C3)alkyl.

106. The compound of claim 105, wherein Ra and Rb are both H; Cy for formulas (Β2') and (C2') is cyclohexane; and R5 is H or Me.

107. The compound of claim 105 or 106, wherein si ' is 0 or 1.

108. The compound of any one of claims 96-107, wherein the charged substituent or an ionizable group Q is i) -SO3H, -Z'-S03H, -OP03H2, -Z'-OP03H2, -P03H2, -Z'-P03H2, -C02H, -Z'-C02H, -NRnRi2, or -Z'-NRnRi2, or a pharmaceutically acceptable salt thereof; or, ii) -N+Ri4RisRi6X" or -Z'-N+Ri4RisRi6X ; Z' is an optionally substituted alkylene, an optionally substituted cycloalkylene or an optionally substituted

phenylene; R14 to Ri6 are each independently an optionally substituted alkyl; and X" is a pharmaceutically acceptable anion.

109. The compound of claim 108, wherein Q is SO3H or a pharmaceutically acceptable salt thereof.

110. The compound of any one of claims 96-109, wherein Pi is a peptide containing 2 to 10 amino acid residues.

111. The compound of claim 110, wherein Pi is a peptide containing 2 to 5 amino acid residues.

112. The compound of claim 111, wherein Pi is selected from Gly-Gly-Gly, Ala-Val, Val- Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe-N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly- Phe-Lys, Leu- Ala-Leu, He- Ala-Leu, Val- Ala- Val, Ala-Leu-Ala-Leu (SEQ ID NO: 14), β-Ala-Leu- Ala-Leu (SEQ ID NO: 15), Gly-Phe-Leu-Gly (SEQ ID NO: 16), Val- Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D- Val-Cit, D- Val-Lys, D- Val-Arg, D- Val-D-Cit, D- Val-D-Lys, D- Val-D-Arg, D-Arg-D-Arg, Ala- Ala, Ala-D- Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala.

113. The compound of claim 112, wherein Pi is Gly-Gly-Gly, Ala-Val, Ala- Ala, Ala-D- Ala, D-Ala-Ala, and D-Ala-D-Ala.

114. The compound of any one of claims 96-113, wherein the double line ~ between N and C represents a double bond.

115. The compound of any one of claims 96-113, wherein the double line ~ between N and C represents a single bond, Xi is -H or an amine protecting group; and Yi is selected from -OR, -OCOR', -SR, -NR'R," an optionally substituted 5- or 6- membered nitrogen-

containing heterocycle, -SO3M, -SO2M and -OSO3M, wherein M is H+ or a pharmaceutically acceptable cation.

116. The compound of claim 115, wherein Yi is selected from -SO3M, -OH, -OMe, -OEt or -NHOH.

117. The compound of claim 116, wherein Yi is -SO3M or -OH, and M is H+, Na+ or K+.

118. The compound of any one of claims 96-117, wherein Xi' is selected from the group consisting of -H, -OH, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms, and phenyl.

119. The compound of claim 118, wherein Xj' is -H, -OH, (Ci-C3)alkyl, halo(Ci-C3)alkyl, or phenyl.

120. The compound of claim 119, wherein Xi' is -H, -OH or -Me.

121. The compound of claim 120, wherein Xi' is -H.

122. The compound of any one of claims 96-121, wherein Yi' is selected from the group consisting of -H, an oxo group, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl having from 1 to 10 carbon atoms.

123. The compound of claim 122, wherein Yi' is -H, an oxo group, (Ci-C3)alkyl or

halo(Ci-C3)alkyl.

124. The compound of claim 123, wherein Yi' is -H or oxo.

125. The compound of claim 123, wherein Yi' is -H.

126. The compound of any one of claims 96-125, wherein A and A' are the same or

different, and are selected from -0-, -S-, -NR5-, and oxo -(C=0)-.

127. The compound of claim 126, wherein A and A' are the same or different, and are selected from -O- and -S-.

128. The compound of claim 126, wherein A and A' are -0-.

129. The compound of any one of claims 96-128, wherein R6 is -OMe.

130. The compound of any one of claims 96-129, wherein Ri, R2, R3, R4, Ri', R2', R3' and R4' are independently -H, halogen, -N02, -OH, (Ci-C3)alkyl, halo(Ci-C3)alkyl or (Q- C3)alkoxy.

131. The compound of claim 130, wherein Ri, R2, R3, R4, Ri', R2', R3' and R4' are all -H.

132. The compound of any one of claims 96-131, wherein R, R', R" and R5 are each

independently -H or (Ci-C3)alkyl.

133. The compound of any one of claims 96-113, wherein:

the double line ~ between N and C represents a single bond or double bond, provided that when it is a double bond Xi is absent and Yi is -H, and when it is a single bond, Xi is -H, Y is -OH or -S03M;

Ri, R2, R3, R4, Ri', R2', R3' and R4' are all -H;

R6 is -OMe;

Xi' and Yi' are both -H;

A and A' are -0-; and

M is H+, Na+ or K+.

The compound of claim 96, wherein D is represented by the following structural formula:

or a pharmaceutically acceptable salt thereof.

135. The compound of any one of claims 96-134, wherein M is H+, Na+ or K+.

136. The compound of any one of claims 96-135, wherein R23 and R24 are both H; and m' is an integer between 1 and 6.

137. The compound of claims 136, wherein Rh' is H.

138. The compound of claim 96-137, wherein -L-JCB is represented by the following structural formula:

The compound of claim 96, wherein the compound is represented by the following structural formula:


wherein M is H+ or a pharmaceutically acceptable cation.

140. The compound of claim 139, wherein M is H+, Na+ or K+.

141. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 77 and a pharmaceutically acceptable carrier.

142. A method of inhibiting abnormal cell growth or treating a proliferative disorder in a mammal, comprising administering to said mammal a therapeutically effective amount of the conjugate of any one of claims 1 to 77, and, optionally, a second therapeutic agent.

143. The method of claim 142, wherein the proliferative disorder is cancer.

144. The method of claim 143, wherein the cancer is a hematological cancer or a solid tumor.

145. The method of claim 144, wherein the hematological cancer is leukemia (e.g. , acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) such as acute B lymphoblastic leukemia (B-ALL), chronic myelogenous leukemia (CML), or chronic lymphocytic leukemia (CLL)) or lymphoma.

146. The method of claim 144, wherein the solid tumor is melanoma, lung cancer (e.g. , no n- small cell lung cancer), ovarian cancer, endometrial cancer, peritoneal cancer, pancreatic cancer, breast cancer, prostate cancer, or cervical cancer.