Pyrrolobenzodiazepines and targeted conjugates

1. A conjugate of formula I:

L-(LU-D) p ,   (I)

or a pharmaceutically acceptable salt thereof, wherein

L is a Ligand Unit selected from the group consisting of an antibody, an antigen-binding fragment of an antibody and an Fc fusion protein;

LU is a Linker Unit of formula 1a:

-A 1 -L 1 -,   (1a)

wherein:

L 1 is an amino acid sequence comprising a dipeptide of formula —NH—X 1 —X 2 —CO—, wherein —NH— is the amino group of X 1 , and CO is the carbonyl group of X 2 , and wherein the peptide is cleavable by the action of an enzyme to release D as a free drug, wherein the enzyme is a cathepsin;

A 1 is a Stretcher Unit, wherein A 1 comprises the functionality —CO— connected directly to the amino group of X 1 , thereby forming an amide link with —X 1 —, and wherein A 1 is further comprised of the structure of:

wherein

S is a sulfur atom derived from the Ligand Unit;

the wavy line indicates the point of attachment to the Ligand Unit; and the asterisk indicates the point of attachment to the remaining portion of A 1 ;

subscript p is from 1 to 20; and

D is a Drug Unit, wherein the Drug Unit is a PBD dimer of formula II:

wherein:

—R 2 is of formula III:

wherein A is a C 5-7 aryl group and X is selected from the group consisting of:

wherein R N is selected from the group consisting of H and C 1-4 alkyl;

the asterisk indicates the point of attachment to Q 2 , and the wavy line indicates the point of attachment to LU; and either:

(i) Q 1 is a single bond and Q 2 is a single bond or —Z—(CH 2 ) n —, wherein Z is selected from the group consisting of a single bond, O, S and NH; and subscript n is from 1 to 3, or

(ii) Q 1 is —CH═CH— and Q 2 is a single bond; and

R 12 is a C 5-10 aryl group, substituted by a group selected from the group consisting of —OH, —CO 2 H, and —CO 2 R ∘ , wherein R ∘ is C 1-4 alkyl;

R 6 and R 9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo;

R 7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo,

wherein R and R′ are independently selected from the group consisting of optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups; and

either:

(a) R 10 is H, and R 11 is OH or OR A , wherein R A is C 1-4 alkyl, or

(b) R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or

(c) R 10 is H and R 11 is SO z M, where subscript z is 2 or 3 and M is a pharmaceutically acceptable cation;

and

R″ is a C 3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms selected from the group consisting of O, S, and NR N2 ,wherein R N2 is H or C 1-4 alkyl, and/or by and aromatic ring;

Y and Y′ are independently selected from the group consisting of O, S, and NH;

R 6′ , R 7′ , R 9′ are selected from the same groups as R 6 , R 7 and R 9 , respectively, and R 10′ and R 11′ are the same as R 10 and R 11 , wherein if R 11 and R 11′ are each SO z M, then each M is an independently selected pharmaceutically acceptable monovalent cation or both M together is a pharmaceutically acceptable divalent cation; and

wherein LU is connected to D via the X substituent of R 2 ; and

wherein C 3-20 heterocyclyl is a monovalent moiety derived by removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S.

2. The conjugate of claim 1 , wherein the dipeptide of formula —NH—X 1 —X 2 —CO— is selected from the group consisting of -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-, wherein Cit is citrulline.

3. The conjugate of claim 2 , wherein R 7 is selected from the group consisting of H, OH and OR.

4. The conjugate of claim 2 , wherein R 7 is a C 1-4 alkyloxy group.

5. The conjugate of claim 3 , wherein each of Y and Y′ is O.

6. The conjugate of claim 5 , wherein R″ is C 3-7 alkylene.

7. The conjugate of claim 6 , wherein R 9 is H.

8. The conjugate of claim 7 , wherein R 6 is selected from the group consisting of H and halo.

9. The conjugate of claim 1 , wherein A is phenyl, X is selected from the group consisting of —O—, —S—, and —NH—, and Q 1 is a single bond.

10. The conjugate of claim 9 , wherein X is NH.

11. The conjugate of claim 1 , wherein R 10 and R 11 form a nitrogen-carbon double bond.

12. The conjugate of claim 11 , wherein R 10′ and R 11′ form a nitrogen-carbon double bond.

13. The conjugate of claim 1 , wherein R 6′ , R 7′ , R 9′ , and Y′ are the same as R 6 , R 7 , R 9 , and Y, respectively.

14. The conjugate of claim 1 , wherein D has the formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein the wavy line indicates the point of attachment to LU.

15. The conjugate of claim 12 , wherein L 1 is a dipeptide selected from the group consisting of valine-alanine, valine-citrulline and phenylalanine-lysine.

16. The conjugate of claim 15 , wherein D has the formula:

or

a pharmaceutically acceptable salt thereof, wherein the wavy line indicates the point of attachment to LU.

17. The conjugate of claim 15 , wherein D has the formula:

or

a pharmaceutically acceptable salt thereof, wherein the wavy line indicates the point of attachment to LU.

18. The conjugate of claim 1 , wherein the Ligand Unit is an antibody or antigen binding fragment thereof and wherein the sulfur atom bonding the antibody Ligand Unit to A 1 of LU is from a reactive thiol group of a cysteine residue of the antibody or antigen binding fragment thereof.

19. The conjugate of claim 18 , wherein the cysteine residue is an introduced cysteine residue in the heavy chain or light chain of the antibody or antigen binding fragment thereof.

20. The conjugate of claim 19 , wherein the introduced cysteine is at amino acid heavy chain position 239 of the antibody, according to the EU numbering system.

21. The conjugate of claim 18 , wherein the antibody is a humanized 1F6 antibody.

22. The conjugate of claim 20 , wherein the antibody is a humanized 1F6 antibody.

23. A drug Linker compound having the formula of:

G 1 -L 1 -D,

or a salt thereof;

wherein L 1 is an amino acid sequence comprising a dipeptide of formula —NH—X 1 —X 2 —CO—, wherein —NH— is the amino group of X 1 , and CO is the carbonyl group of X 2 , and wherein the peptide is cleavable by the action of an enzyme to release D as a fee drug, wherein the enzyme is a cathepsin;

G 1 is a Stretcher Unit for connection to a Ligand Unit, wherein G 1 is comprised of a maleimide group for reaction with a reactive thiol functional group provided by the Ligand Unit for said connection, and wherein G 1 further comprises the functionality —CO— connected directly to the amino terminus of X 1 , thereby forming an amide link with —X 1 —;

D is a Drug Unit, wherein the Drug Unit is a PBD dimer of formula II:

wherein:

—R 2 is of formula III:

wherein A is a C 5-7 aryl group and X is selected from the group consisting of:

wherein R N is selected from the group consisting of H and C 1-4 alkyl;

the asterisk indicates the point of attachment to Q 2 and the wavy line indicates the point of attachment to L 1 ; and either:

(i) Q 1 is a single bond and Q 2 is a single bond or —Z—(CH 2 ) n —, wherein Z is selected from the group consisting of a single bond, O, S and NH; and subscript n is from 1 to 3, or

(ii) Q 1 is —CH═CH— and Q 2 is a single bond; and

R 12 is a C 5-10 aryl group, substituted by a group selected from the group consisting of —OH, —CO 2 H, and —CO 2 R ∘ , where R ∘ is C 1-4 alkyl;

R 6 and R 9 are independently selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo;

R 7 is selected from the group consisting of H, R, OH, OR, SH, SR, NH 2 , NHR, NRR′, nitro, Me 3 Sn and halo,

wherein R and R′ are independently selected from the group consisting of optionally substituted C 1-12 alkyl, C 3-20 heterocyclyl and C 5-20 aryl groups; and

either:

(a) R 10 is H, and R 11 is OH or OR A , wherein R A is C 1-4 alkyl, or

(b) R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound, or

(c) R 10 is H and R 11 is SO z M, wherein subscript z is 2 or 3 and M is a cation;

R″ is a C 3-12 alkylene group, which chain is optionally interrupted by one or more heteroatoms selected from the group consisting of O, S, and NR N2 , wherein R N2 is H or C 1-4 alkyl, and/or by and aromatic ring;

Y and Y′ are independently selected from the group consisting of O, S, and NH;

R 6′ , R 7′ , R 9′ are selected from the same groups as R 6 , R 7 and R 9 , respectively, and R 10′ and R 11′ are the same as R 10 and R 11 , wherein if R 11 and R 11′ are each SO z M, then each M is an independently selected monovalent cation or both M together is a divalent cation; and

wherein L 1 is connected to D via the X substituent of R 2 ; and

wherein C 3-20 heterocyclyl is a monovalent moiety derived from removing a hydrogen atom of a heterocyclic compound which has 3 to 20 ring atoms, of which 1 to 10 are heteroatoms selected from the group consisting of N, O and S.

24. The drug linker compound of claim 23 , wherein the dipeptide of formula —NH—X 1 —X 2 —CO— is selected from the group consisting of -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, -Val-Cit-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, and -Trp-Cit-, wherein Cit is citrulline.

25. The drug linker compound of claim 24 , wherein R 7 is selected from the group consisting of H, OH and OR.

26. The drug linker compound of claim 24 , wherein R 7 is a C 1-4 alkyloxy group.

27. The drug linker compound of claim 25 , wherein each of Y and Y′ is O.

28. The drug linker compound of claim 27 , wherein R″ is C 3-7 alkylene.

29. The drug linker compound of claim 28 , wherein R 9 is H.

30. The drug linker compound of claim 29 , wherein R 6 is selected from the group consisting of H and halo.

31. The drug linker compound of claim 23 , wherein A is phenyl, X is selected from the group consisting of —O—, —S—, and —NH—, and Q 1 is a single bond.

32. The drug linker compound of claim 31 , wherein X is NH.

33. The drug linker compound of claim 32 , wherein Q 1 is a single bond and Q 2 is a single bond.

34. The drug linker compound of claim 23 , wherein R 10 and R 11 form a nitrogen-carbon double bond.

35. The drug linker compound of claim 34 , wherein R 10′ and R 11′ form a nitrogen-carbon double bond.

36. The drug linker compound of claim 23 , wherein R 6′ , R 7′ , R 9′ , and Y′ are the same as R 6 , R 7 , R 9 , and Y, respectively.

37. The drug linker compound of claim 23 , wherein D has the formula selected from the group consisting of:

and salts thereof, wherein the wavy line indicates the point of attachment to L 1 .

38. The drug linker compound of claim 34 , wherein L 1 is a dipeptide selected from the group consisting of valine-alanine, valine-citrulline and phenylalanine-lysine.

39. The drug linker compound of claim 38 , wherein D has the formula:

or a salt thereof, wherein the wavy line indicates the point of attachment to L 1 .

40. The drug linker compound of claim 38 , wherein D has the formula:

or a salt thereof, wherein the wavy line indicates the point of attachment to L 1 .

41. A method of preparing an antibody drug conjugate of claim 1 in which L is an antibody Ligand Unit,

the method comprising the step of contacting an antibody having a reactive cysteine thiol functional group with a Drug Linker compound of claim 22 so that the sulfur atom of the reactive thiol functional group reacts with the maleimide group of G 1 of the Drug Linker compound to form the Stretcher Unit (A 1 ) of the antibody drug conjugate, wherein the reactive thiol functional group is obtained by disulfide reduction of the antibody or by introduction of a cysteine residue in the heavy chain of the antibody.