Cytotoxic agents and conjugates thereof

1. A compound of the general formula I or a salt thereof:

in which R represents a group —Y—OH, —Y—O—R x , —Y—SH, —Y—S—R x , —Y—S(O) 2 NH—R x , —Y—NHS(O) 2 —R x , —Y—C(O)H, —Y—CO 2 H, —Y—C(O)—R x , —Y—C(O)NH—R x , —Y—NHC(O)—R x , —Y—NHR y , —Y—NR x R y , —Y—NR y —NHR z , —Y—CR y ═NOH, —Y—C(NH 2 )═NOH, —Y—C(O)NH 2 , —Y—C(O)NH—NH 2 , or —Y—S(O) 2 NH 2 , in which either Y is not present or Y represents a C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene or C 1-6 alkyleneoxy group which may be interrupted by an oxygen atom and/or which may optionally be substituted by —OH or —OC 1-4 alkyl, or Y represents a phenylene or C 5-10 heteroarylene group;

R x represents a C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, phenyl, C 5-10 heteroaryl or benzyl group which is substituted by —OH, —SH, —NHR y , or —CO 2 H;

each of R y and R z independently represents a hydrogen atom, a C 1-4 alkyl group, phenyl, C 5-10 heteroaryl or a benzyl group;

X represents OH, OC 1-4 alkyl, SH, S 1-4 alkyl, or CN;

Ra represents a hydrogen atom or a C 1-4 alkyl group;

Rb represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—;

Rc represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—;

Rd represents a C 1-4 alkyl group;

each Re independently represents a halogen atom, an optionally substituted C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl or C 1-6 alkoxy group each of which may be optionally interrupted by an oxygen atom, an optionally substituted phenyl or C 5-10 heteroaryl group, —OH, —CO 2 R v , —C(O)NR v R w , —NR y C(O)R w , NR v R w , —S(O)—R v , S(O) 2 —R v , —S(O) 2 NR v R w , a —CN group, or a —NO 2 group; R v and R w are each independently selected from the group consisting of hydrogen, phenyl, benzyl, and an optionally substituted C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl group each of which may be optionally interrupted by an oxygen atom; and n is 0, 1, 2, 3 or 4;

Rf represents a hydrogen atom or a C 1-4 alkyl group; and

Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

2. The compound as claimed in claim 1 , wherein R is an —OH, —NH 2 , —SH, —CONH 2 , —SO 2 NH 2 , —CO 2 H, CH 2 OH, or —NHC(O)—C 1-6 alkylene-SH group.

3. The compound as claimed in claim 1 , wherein R is a —NHC(O)—C 1-6 alkyl which alkyl is substituted by —SH.

4. The compound as claimed in claim 1 , wherein any Re group present is selected from the group consisting of a halogen atom, a methoxy group, a —CN group or a —NO 2 group; and/or wherein Rg represents C 1-4 alkyl which is unsubstituted or substituted by N(R i )(R ii ); R i represents a C 1-4 alkyl group; and R ii represents a —C(O)—C 1-6 alkyl group.

5. The compound as claimed in claim 1 , wherein R represents a group —Y—OH, —Y—O—R x , —Y—SH, —Y—CO 2 H, —Y—NHR y , —Y—NR y —NHR z , or —Y—CR y ═NOH, in which either Y is not present or Y represents a C 1-6 alkylene or C 1-6 alkyleneoxy group either of which may be interrupted by an oxygen atom, R x represents a C 1-4 alkyl group substituted by —OH, —SH, —NHR y , or —CO 2 H, and each of R y and R z independently represents a hydrogen atom or a C 1-4 alkyl group; X represents OH, OC 1-4 alkyl, SH, S 1-4 alkyl, or CN; Ra represents a hydrogen atom or a C 1-4 alkyl group; Rb represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—; Rc represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—; Rd represents a C 1-4 alkyl group; each Re independently represents a halogen atom, a CF 3 group, or a C 1-4 alkyl or C 1-4 alkoxy group, and n is 0, 1, 2, 3 or 4; Rf represents a hydrogen atom or a C 1-4 alkyl group; and Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

6. The compound as claimed in claim 1 , in which:

i) Y is not present, or in which Y represents a C 1-4 alkylene or C 1-4 alkyleneoxy group, which may be interrupted by an oxygen atom; and/or

ii) R is an —OH, —NH 2 , —CONH 2 or —CO 2 H group, or a C 1-4 alkylene group substituted by an —OH, —NH 2 , —CONH 2 or —CO 2 H group; and/or R is in the 3- or 4-position of the phenyl ring; and/or

iii) any Re group present is a halogen atom or a methyl or methoxy group; and/or

iv) n is 0, 1 or 2; and/or

v) X represents OH; and/or

vi) Ra represents C 1-4 alkyl; Rb represents hydrogen; Rc represents hydrogen or methoxy; Rd represents C 1-4 alkyl; Re represents chlorine or hydrogen; Rf represents C 1-4 alkyl; and Rg represents C 1-4 alkyl.

7. The compound as claimed in claim 1 , which is a compound of the general formula Ia or a salt thereof:

8. The compound as claimed in claim 1 , which is a compound of the general formula Ib or a salt thereof:

9. A conjugate comprising the compound as claimed in claim 1 linked to a binding protein via a linker, said linker being connected to said compound via the group R of the general formula I;

wherein:

i) the linker includes the group —S—C 1-6 alkylene-; and/or

ii) the binding protein is a full length antibody or an antibody fragment comprising an antigen-binding region of the full length antibody.

10. The conjugate as claimed in claim 9 , wherein the binding protein is IgG1 or IgG4 or a fragment of IgG1 or IgG4.

11. The conjugate as claimed in claim 9 , which includes a portion:

in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, each of A and B independently represents a C 1-5 alkylene or alkenylene chain, and Pr represents said binding protein bonded to A and B via nucleophiles Nu; or includes a portion:

˜W′—(CH═CH) p —(CH 2 ) 2 -Nu-Pr

in which W′ represents an electron withdrawing group or a group obtained by reduction of an electron withdrawing group, p is 0 or an integer from 1 to 4, and Pr represents said binding protein bonded to the rest of the molecule via a nucleophile Nu.

12. The conjugate as claimed in claim 9 , which includes a portion:

or which includes a portion:

˜NH—CO—Ar′—CO—(CH 2 ) 2 -Nu-Pr

in which Ar′ represents an optionally substituted aryl group.

13. The conjugate as claimed in claim 9 , in which each Nu represents a sulfur atom present in a cysteine residue in the binding protein Pr; or in which each Nu represents an imidazole group present in a polyhistidine tag attached to the binding protein.

14. The conjugate as claimed in claim 9 , in which:

i) the linker includes a pendant polyethylene glycol chain which has a terminal end group of formula —CH 2 CH 2 OR r in which R r represents a hydrogen atom, an alkyl group, or an optionally substituted aryl group; or

ii) at least two ˜(CH 2 —CH 2 —O—)˜ units within a ring; and/or

iii) the linker includes a peptidyl group comprising at least two naturally-occurring alpha amino acids.

15. The conjugate as claimed in claim 9 , wherein the linker includes the sequence Val-Cit-PAB or Val-Ala.

16. A conjugating reagent comprising the compound as claimed in claim 1 , attached via a linker to at least one functional group capable of reacting with a binding protein, said linker being connected to said compound via the group R of the general formula I; and wherein:

i) the conjugating reagent includes a portion

or a salt thereof; or

ii) the conjugating reagent includes a portion:

or

iii) the functional group of the conjugating reagent has the formula:

in which W represents an electron-withdrawing group; each of A and B independently represents a C 1-5 alkylene or alkenylene chain; and either each L independently represents a leaving group, or both Ls together represent a leaving group; or

in which W and A have the meanings given above, L represents a leaving group, and m is 0 to 4; or

˜W—(CH═CH) p —(CH 2 ) 2 -L or ˜W—(CH═CH) p —CH═CH 2

in which W represents an electron withdrawing group, p represents 0 or an integer of from 1 to 4, and L represents a leaving group.

17. The conjugating reagent as claimed in claim 16 , in which said functional group has the formula:

in which Ar′ represents an optionally substituted aryl group.

18. The conjugating reagent as claimed in claim 16 , in which:

i) the or each leaving group includes a portion —(CH 2 CH 2 O) q — in which q is a number of six or more; or

ii) the linker

a) includes a pendant polyethylene glycol chain which has a terminal end group of formula —CH 2 CH 2 OR r in which R r represents a hydrogen atom, an alkyl group, or an optionally substituted aryl group; or

b) has at least two ˜(CH 2 —CH 2 —O—)˜ units within a ring; and/or

c) includes a peptidyl group comprising at least two naturally-occurring alpha amino acids.

19. A pharmaceutical composition comprising the compound as claimed in claim 1 , or a conjugate comprising the compound of claim 1 , together with a pharmaceutically acceptable carrier, optionally together with an additional therapeutic agent.

20. A process for the preparation of a compound of the general formula I or a salt thereof as claimed in claim 1 , which comprises reacting a compound of the general formula:

with an aryl-organometallic reagent in which the aryl moiety is a phenyl group substituted by (Re) n and by R or a protected version of R, the reaction being carried out in the presence of a transition metal catalyst;

wherein, optionally, the aryl-organometallic reagent is an aryl-boronic acid or aryl-boronate ester, and in which the reaction is carried out in the presence of a palladium catalyst in the presence of water and in the absence or substantial absence of oxygen.

21. The process as claimed in claim 20 , wherein R represents a group —Y—OH, —Y—O—R x , —Y—SH, —Y—S—R x , —Y—CO 2 H, —Y—CO—R x , —Y—NHR y , —Y—NR y —NHR z , or —Y—CR y ═NOH, in which either Y is not present or Y represents a C 1-6 alkylene or C 1-6 alkyleneoxy group either of which may be interrupted by an oxygen atom, R x represents a C 1-4 alkyl group substituted by —OH, —SH, —NHR y , or —CO 2 H, and each of R y and R z independently represents a hydrogen atom or a C 1-4 alkyl group; X represents OH, OC 1-4 alkyl, SH, S 1-4 alkyl, or CN; Ra represents a hydrogen atom or a C 1-4 alkyl group; Rb represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—; Rc represents hydrogen, OH, C 1-4 alkoxy or C 1-4 alkylC(O)O—; Rd represents a C 1-4 alkyl group; each Re independently represents a halogen atom, a CF 3 group, or a C 1-4 alkyl or C 1-4 alkoxy group, and n is 0, 1, 2, 3 or 4; Rf represents a hydrogen atom or a C 1-4 alkyl group; and Rg represents a hydrogen atom or an optionally substituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or heteroaryl group.

22. The process as claimed in claim 20 , in which the aryl-organometallic reagent is a boronic acid of the general formula:

or a protected version thereof and the reaction is carried out in the presence of a palladium catalyst.

23. An intermediate useful for preparing a compound of the general formula I or a salt thereof as claimed in claim 1 , which has the general formula

and R prot is the group R of the general formula I carrying a protecting group; or a salt thereof;

wherein optionally, R includes an —OH or —SH group and the protecting group is a silyl group, an acyl group, or an arylmethyl group; R includes a —CO 2 H group and the protecting group is methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, or diphenylmethyl; or R includes an —NHR′, —NHR″ or —NHR′″ group and the protecting group is t-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, or t-butyldimethylsilyl.

24. A compound as claimed in claim 1 , wherein R is selected from —Y—OH, —Y—SH, —Y—S(O) 2 NH—R x , —Y—NHS(O) 2 —R x , —Y—CO 2 H, —Y—C(O)NH—R x , —Y—NHC(O)—R x , —Y—NHR y or —Y—S(O) 2 NH 2 , in which either Y is not present or Y represents a C 1-6 alkylene, and wherein R x represents a C 1-6 alkyl which is substituted by —OH, —SH, —NH 2 or —CO 2 H; and R y represents a hydrogen atom or a C 1-4 alkyl group.

25. A compound as claimed in claim 1 , wherein R represents a group selected from —Y—SH, —Y—S(O) 2 NH—R x , —Y—NHS(O) 2 —R x , —Y—C(O)NH—R x , —Y—NHC(O)—R x , or —Y—S(O) 2 NH 2 , wherein R x represents a C 1-6 alkyl which is substituted by —OH, —SH, —NH 2 or —CO 2 H.

26. A compound as claimed in claim 1 , wherein R represents —Y—NHC(O)—R x , wherein Y is absent and R x represents a C 1-6 alkyl which is substituted by —OH, —SH, —NH 2 or —CO 2 H.

27. A compound as claimed in claim 1 , wherein Rd is methyl.

28. A compound as claimed in claim 1 , wherein X represents OH.

29. A compound as claimed in claim 1 , wherein Ra represents a C 1-4 alkyl group and Rb represents hydrogen.

30. A compound as claimed in claim 1 , wherein Rc represents hydrogen or methoxy.

31. A compound as claimed in claim 1 , wherein each Re independently represents a halogen atom, a CF 3 group, a C 1-4 alkyl group, a C 1-4 alkoxy group, a —CN group, or a NO 2 group, and wherein n is 0, 1 or 2.

32. A compound as claimed in claim 1 , wherein Rg represents C 1-4 alkyl which is unsubstituted or substituted by N(R i )(R ii ), and wherein R i represents a C 1-4 alkyl group, and R ii represents a —C(O)—C 1-6 alkyl group.