AZETIDOBENZODIAZEPINE DIMERS AND CONJUGATES COMPRISING THEM FOR USE IN THE TREATMENT OF CANCER

Abstract

A compound of formula IV: as well as drug-linkers and conjugates comprising this compound, and the use of the conjugates in treating cancer.

##STR00001##

Claims

1.-23. (canceled)

24. A compound of formula IV: ##STR00079## and salts and solvates thereof, wherein: R.sup.2 and R.sup.2′ are H; R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups; either (a) R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; or (b) R.sup.7 and R.sup.7′ together form a group which is: (i) —O—(CH.sub.2).sub.n—O—, where n is from 7 to 16; or (ii) —O—(CH.sub.2CH.sub.2O).sub.m—, where m is 2 to 5; R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R.sup.6 and R.sup.9′ are selected from the same groups as R.sup.6, and R.sup.9 respectively; either (i-a) R.sup.10 and R.sup.11 together form a double bond between the N and C atoms to which they are bound; or (i-b) R.sup.10 is H and R.sup.11 is selected from OH and OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or (i-c) R.sup.10 and R.sup.11 are both H; either (ii-a) R.sup.20 and R.sup.21 together form a double bond between the N and C atoms to which they are bound; or (ii-b) R.sup.20 is H and R.sup.21 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl; or (ii-c) R.sup.20 and R.sup.21 are both H.

25. A compound according to claim 24, wherein: a) both Y and Y′ are O; and/or b) R″ is C.sub.3-7 alkylene; or c) R″ is a group of formula: ##STR00080## where r is 1 or 2; and/or d) R.sup.6′ is the same group as R.sup.6, R.sup.7′ is the same group as R.sup.7, R.sup.9′ is the same group as R.sup.9 and Y is the same group as Y.

26. A compound according to claim 24, wherein R.sup.9 is H, R.sup.6 is H, R.sup.7 and R.sup.7′ are independently a C.sub.1-4 alkyloxy group.

27. A compound according to claim 24, which is of formulae IVa, IVb or IVc: ##STR00081## where R.sup.1a is selected from methyl and benzyl.

28. A compound of formula I: ##STR00082## and salts and solvates thereof, wherein: Y, Y′, R″, R.sup.2, R.sup.2′, R.sup.6, R.sup.6′, R.sup.7, R.sup.7′, R.sup.9 and R.sup.9′ are as defined in claim 1; R.sup.11b is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; and R.sup.L is a linker for connection to a cell binding agent, which is selected from: (iiia): ##STR00083## wherein Q is: ##STR00084## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is: ##STR00085## where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; G.sup.L is a linker for connecting to a Ligand Unit; and (iiib): ##STR00086## where R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or 1; either: (a) R.sup.30 and R.sup.31 together form a double bond between the N and C atoms to which they are bound; or (b) R.sup.30 is H and R.sup.31 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl. (c) R.sup.30 and R.sup.31 are both H; or (d) R.sup.31 is OH or OR.sup.B, where R.sup.B is C.sub.1-4 alkyl and R.sup.30 is selected from: ##STR00087## where R.sup.Z is selected from: (z-i) ##STR00088## (z-ii) OC(═O)CH.sub.3; (z-iii) NO.sub.2; (z-iv) OMe; (z-v) glucoronide; (z-vi) NH—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—C(═O)—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.

29. A compound according to claim 28, wherein R.sup.30 and R.sup.31 together form a double bond between the N and C atoms to which they are bound.

30. A compound according to claim 28, which is of formula Ia, Ib or Ic: ##STR00089## where R.sup.1a is selected from methyl and benzyl.

31. A compound according to claim 28, wherein R.sup.L is of formula IIIa, and Q is a dipeptide residue selected from: .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Ala-.sup.NH, .sup.CO-Val-Lys-.sup.NH, .sup.CO-Ala-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH, .sup.CO-Phe-Cit-.sup.NH, .sup.CO-Leu-Cit-.sup.NH, .sup.CO-Ile-Cit-.sup.NH, .sup.CO-Phe-Arg-.sup.NH, and .sup.CO-Trp-Cit-.sup.NH.

32. A compound according to claim 28, wherein R.sup.L is of formula IIIa and, a is 0, c is 1 and d is 2, and b is from 0 to 8.

33. A compound according to claim 32, wherein b is 0, 4 or 8.

34. A compound according to claim 28, wherein R.sup.L is of formula IIIa and G.sup.L is selected from: ##STR00090## ##STR00091## where Ar represents a C.sub.5-6 arylene group.

35. A compound according to claim 34, wherein G.sup.L is G.sup.L1-1.

36. A compound according to claim 28, wherein the compound is of formula Id: ##STR00092## where Q is selected from: (a) —CH.sub.2—; (b) —C.sub.3H.sub.6—; and (c) ##STR00093##

37. A conjugate of formula II:
L-(D.sup.L).sub.p  (II) wherein L is a Ligand unit (i.e., a targeting agent), D.sup.L is a Drug Linker unit of formula I′: ##STR00094## wherein: R.sup.2 and R.sup.2′ are H; R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups; either (a) R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; R.sup.7′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; or (b) R.sup.7 and R.sup.7′ together form a group which is: (i) —O—(CH.sub.2).sub.n—O—, where n is from 7 to 16; or (ii) —O—(CH.sub.2CH.sub.2O).sub.m—, where m is 2 to 5; R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine; Y and Y′ are selected from O, S, or NH; R.sup.6′ and R.sup.9′ are selected from the same groups as R.sup.6, and R.sup.9 respectively; R.sup.11b, R.sup.30 and R.sup.31 are as defined in claim 28; R.sup.LL is a linker for connection to a cell binding agent, which is selected from: (iiia): ##STR00095## where Q and X are as defined in claim 8 and G.sup.LL is a linker connected to a Ligand Unit; and (iiib): ##STR00096## where R.sup.L1 and R.sup.L2 are as defined in claim 28; wherein p is an integer of from 1 to 20.

38. A conjugate according to claim 37, wherein G.sup.LL is selected from: ##STR00097## ##STR00098## where Ar represents a C.sub.5-6 arylene group.

39. A conjugate according to claim 37, wherein D.sup.L is of formula (Id′): ##STR00099## where Q is selected from: (a) —CH.sub.2—; (b) —C.sub.3H.sub.6—; and (c) ##STR00100##

40. A composition comprising a mixture of conjugates according to claim 37, wherein the average p in the mixture of conjugates is about 1 to about 8.

41. A pharmaceutical composition comprising the conjugate of claim 37, and a pharmaceutically acceptable diluent, carrier or excipient.

42. A method of treating cancer in a subject comprising administering to a patient the pharmaceutical composition of claim 41.

43. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to claim 37.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0406] FIG. 1 shows the effect on the growth of a tumour cell line when treated by a control or by a conjugate of the present invention.

FURTHER PREFERENCES

[0407] The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

[0408] R.sup.6′ and R.sup.9′ are selected from the same groups as R.sup.6 and R.sup.9 respectively. In some embodiments, R.sup.6′, R.sup.7′, R.sup.9′, and Y′ are the same as R.sup.6, R.sup.7, R.sup.9, and Y respectively.

Dimer Link

[0409] In some embodiments, Y and Y′ are both O.

[0410] In some embodiments, R″ is a C.sub.3-7 alkylene group with no substituents. In some of these embodiments, R″ is a C.sub.3, C.sub.5 or C.sub.7 alkylene. In particular, R″ may be a C.sub.3 or C.sub.5 alkylene.

[0411] In other embodiments, R″ is a group of formula:

##STR00032##

[0412] where r is 1 or 2.

[0413] The phenylene group may be replaced by a pyridylene group.

[0414] R.sup.6 to R.sup.9

[0415] In some embodiments, R.sup.9 is H.

[0416] In some embodiments, R.sup.6 is selected from H, OH, OR, SH, NH.sub.2, nitro and halo, and may be selected from H or halo. In some of these embodiments R.sup.6 is H.

[0417] In some embodiments, R.sup.7 is selected from H, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, and halo. In some of these embodiments R.sup.7 is selected from H, OH and OR, where R is selected from optionally substituted C.sub.1-7 alkyl, C.sub.3-10 heterocyclyl and C.sub.5-10 aryl groups. R may be more preferably a C.sub.1-4 alkyl group, which may or may not be substituted. A substituent of interest is a C.sub.5-6 aryl group (e.g. phenyl). Particularly preferred substituents at the 7-positions are OMe and OCH.sub.2Ph. Other substituents of particular interest are dimethylamino (i.e. —NMe.sub.2); —(OC.sub.2H.sub.4).sub.qOMe, where q is from 0 to 2; nitrogen-containing C heterocyclyls, including morpholino, piperidinyl and N-methyl-piperazinyl.

[0418] These embodiments and preferences apply to R.sup.9′, R.sup.6′ and R.sup.7′ respectively.

[0419] In other embodiments, R.sup.7 and R.sup.7′ together form a group which is —O—(CH.sub.2).sub.n—O—, where n is from 7 to 16. n may be at least 7, 8, 9, 10 or 11. N may be at most 16, 15, 14 or 13.

[0420] In other embodiments, R.sup.7 and R.sup.7′ together form a group which is —O—(CH.sub.2CH.sub.2O).sub.m—, where m is 2 to 5. m may be at least 2, 3 or 4. m may be at most 5, 4 or 3.

R.sup.10, R.sup.11, R.sup.20, R.sup.21 (Formula IV)

[0421] In some embodiments, R.sup.10 and R.sup.11 together form a double bond between the N and C atoms to which they are bound. In some of these embodiments, R.sup.20 and R.sup.21 together form a double bond between the N and C atoms to which they are bound. In other of these embodiments, R.sup.20 and R.sup.21 are both H.

[0422] In some embodiments, R.sup.10 is H and R.sup.11 is selected from OH and OR.sup.A, where R.sup.A is C.sub.1-4 alkyl. In some of these embodiments, R.sup.20 is H and R.sup.21 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl. In other of these embodiments, R.sup.20 and R.sup.21 are both H.

[0423] In some embodiments, R.sup.10 and R.sup.11 are both H. In some of these embodiments, R.sup.20 and R.sup.21 together form a double bond between the N and C atoms to which they are bound. In other of these embodiments, R.sup.20 is H and R.sup.21 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl.

[0424] In some embodiments, R.sup.A is methyl. In some embodiments, R.sup.B is methyl.

[0425] In some embodiments, only one of the pairs of R.sup.10 and R.sup.11 and R.sup.20 and R.sup.21 are both H. In other embodiments, neither of the pairs of R.sup.10 and R.sup.11 and R.sup.20 and R.sup.21 are both H.

[0426] In some embodiments, R.sup.10, R.sup.11, R.sup.20 and R.sup.21 are all H.

N10′-C11′ (Formulae I and I*)

[0427] In some embodiments, R.sup.30 and R.sup.31 together form a double bond between the N and C atoms to which they are bound.

[0428] In some embodiments, R.sup.30 is H and R.sup.31 is selected from OH and OR.sup.B, where R.sup.B is O.sub.1_4 alkyl. In some of these embodiments, R.sup.B is methyl.

[0429] In some embodiments, R.sup.30 is H and R.sup.31 is H.

[0430] In some embodiments, R.sup.31 is OH or OR.sup.B, where R.sup.B is C.sub.1-4 alkyl and R.sup.30 is selected from:

##STR00033## ##STR00034##

[0431] —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH— represent a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids. The dipeptide may be the site of action for cathepsin-mediated cleavage.

[0432] In one embodiment, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0433] -Phe-Lys-, [0434] -Val-Ala-, [0435] -Val-Lys-, [0436] -Ala-Lys-, [0437] -Val-Cit-, [0438] -Phe-Cit-, [0439] -Leu-Cit-, [0440] -Ile-Cit-, [0441] -Phe-Arg-, [0442] -Trp-Cit-
where Cit is citrulline.

[0443] Preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: [0444] -Phe-Lys-, [0445] -Val-Ala-, [0446] -Val-Lys-, [0447] -Ala-Lys-, [0448] -Val-Cit-.

[0449] Most preferably, the dipeptide, —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is -Phe-Lys- or -Val-Ala-.

[0450] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.

[0451] In one embodiment, the amino acid side chain is derivatised, where appropriate. For example, an amino group or carboxy group of an amino acid side chain may be derivatised.

[0452] In one embodiment, an amino group NH.sub.2 of a side chain amino acid, such as lysine, is a derivatised form selected from the group consisting of NHR and NRR′.

[0453] In one embodiment, a carboxy group COOH of a side chain amino acid, such as aspartic acid, is a derivatised form selected from the group consisting of COOR, CONH.sub.2, CONHR and CONRR′.

[0454] In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. The present inventors have established that protected amino acid sequences are cleavable by enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

[0455] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective Groups in Organic Synthesis, Greene and Wuts.

[0456] Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality: [0457] Arg: Z, Mtr, Tos; [0458] Asn: Trt, Xan; [0459] Asp: Bzl, t-Bu; [0460] Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt; [0461] Glu: Bzl, t-Bu; [0462] Gln: Trt, Xan; [0463] His: Boc, Dnp, Tos, Trt; [0464] Lys: Boc, Z—Cl, Fmoc, Z, Alloc; [0465] Ser: Bzl, TBDMS, TBDPS; [0466] Thr: Bz; [0467] Trp: Boc; [0468] Tyr: Bzl, Z, Z—Br.

[0469] In one embodiment, the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present. Thus, the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group.

[0470] In other embodiments of the invention, the amino acids selected are those having no reactive side chain functionality. For example, the amino acids may be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro, and Val.

[0471] It is particularly preferred in the present invention, that if Q comprises a dipeptide, then —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH— is the same dipeptide. An example of a preferred group is:

##STR00035##

[0472] Other preferred R.sup.30 groups include:

##STR00036##

R.SUP.11b .(Formulae I and I*)

[0473] In some embodiments, R.sup.11b is OH.

[0474] In some embodiments, R.sup.11b is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl. In some of these embodiments, R.sup.A is methyl.

Further Formulae

[0475] In some embodiments of the first aspect of the present invention are of formulae Va, IVb or IVc:

##STR00037##

[0476] where R.sup.1a is selected from methyl and benzyl;

[0477] R.sup.10, R.sup.11, R.sup.20 and R.sup.21 are as defined above.

[0478] In some embodiments of the second aspect of the present invention are of formulae Ia, Ib or Ic:

##STR00038##

[0479] where R.sup.1a is selected from methyl and benzyl;

[0480] R.sup.30, R.sup.31, R.sup.L an R.sup.11b are as defined above.

[0481] These embodiments and preferences also apply to the third aspect of the invention.

Linker (R.SUP.L.)

[0482] In some embodiments, R.sup.L is a formula IIIa.

[0483] In some embodiments, R.sup.LL is a formula IIIa′.

G.SUP.L

[0484] G.sup.L may be selected from

##STR00039## ##STR00040##

where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.

[0485] In some embodiments, G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2. In some of these embodiments, G.sup.L is G.sup.L1-1.

G.SUP.LL

[0486] G.sup.LL ma be selected from:

##STR00041## ##STR00042##

where Ar represents a C.sub.5-6 arylene group, e.g. phenylene.

[0487] In some embodiments, G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2. In some of these embodiments, G.sup.LL is G.sup.LL1-1.

X

[0488] X is:

##STR00043##

[0489] where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5.

[0490] a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.

[0491] b may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0 to 12. In some of these embodiments, b is 0 to 8, and may be 0, 2, 4 or 8.

[0492] c may be 0 or 1.

[0493] d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2.

[0494] In some embodiments of X, a is 0, c is 1 and d is 2, and b may be from 0 to 8. In some of these embodiments, b is 0, 4 or 8.

Q

[0495] In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid.

[0496] In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0497] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

[0498] In one embodiment, Q is selected from: [0499] .sup.CO-Phe-Lys-.sup.NH, [0500] .sup.CO-Val-Ala-.sup.NH, [0501] .sup.CO-Val-Lys-.sup.NH, [0502] .sup.CO-Ala-Lys-.sup.NH, [0503] .sup.CO-Val-Cit-.sup.NH, [0504] .sup.CO-Phe-Cit-.sup.NH, [0505] .sup.CO-Leu-Cit-.sup.NH, [0506] .sup.CO-Ile-Cit-.sup.NH, [0507] .sup.CO-Phe-Arg-.sup.NH, and [0508] .sup.CO-Trp-Cit-.sup.NH;

[0509] where Cit is citrulline.

[0510] Preferably, Q is selected from: [0511] .sup.CO-Phe-Lys-.sup.NH, [0512] .sup.CO-Val-Ala-.sup.NH, [0513] .sup.CO-Val-Lys-.sup.NH, [0514] .sup.CO-Ala-Lys-.sup.NH, [0515] .sup.CO-Val-Cit-.sup.NH.

[0516] Most preferably, Q is selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.

[0517] Other dipeptide combinations of interest include: [0518] .sup.CO-Gly-Gly-.sup.NH, [0519] .sup.CO-Pro-Pro-.sup.NH, and [0520] .sup.CO-Val-Glu-.sup.NH.

[0521] Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which is incorporated herein by reference.

[0522] In some embodiments, Q.sup.X is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin. Tripeptide linkers of particular interest are: [0523] .sup.CO-Glu-Val-Ala-.sup.NH [0524] .sup.CO-Glu-Val-Cit-.sup.NH [0525] .sup.CO-αGlu-Val-Ala-.sup.NH [0526] .sup.CO-αGlu-Val-Cit-.sup.NH

[0527] In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

[0528] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.

[0529] In some embodiments, R.sup.L is of formula IIIb.

[0530] In some embodiments, R.sup.LL is of formula IIIb′.

[0531] R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group.

[0532] In some embodiments, both R.sup.L1 and R.sup.L2 are H.

[0533] In some embodiments, R.sup.L1 is H and R.sup.L2 is methyl.

[0534] In some embodiments, both R.sup.L1 and R.sup.L2 are methyl.

[0535] In some embodiments, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.

[0536] In some embodiments, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

[0537] In the group IIIb, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be in any available position of the ring. In some of these embodiments, it is in the ortho position. In others of these embodiments, it is in the para position.

[0538] In one particular embodiment, the second aspect of the invention comprises a compound of formula Id:

##STR00044##

[0539] where Q is selected from:

[0540] (a) —CH.sub.2—;

[0541] (b) —C.sub.3H.sub.6—; and

##STR00045##

[0542] In one particular embodiment, the third aspect of the invention, the Drug linker (D.sup.L) is of formula (Id′):

##STR00046##

[0543] where Q is selected from:

[0544] (a) —CH.sub.2—;

[0545] (b) —C.sub.3H.sub.6—; and

##STR00047##

[0546] In some embodiments of the present invention, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:

##STR00048##

[0547] In other embodiments, the C11 substituent may be in the following stereochemical arrangement relative to neighbouring groups:

##STR00049##

EXAMPLES

General Information

[0548] Manual flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. Extraction and chromatography solvents were bought and used without further purification from Fisher Scientific, U.K. All chemicals were purchased from Aldrich, Lancaster or BDH. Automated flash chromatography was performed using a Biotage Isolera 1™ using gradient elution starting from either 88% hexane/EtOAc or 99.9% DCM/MeOH until all UV active components (detection at 214 and 254 nm) eluted from the column. The gradient was manually held whenever substantial elution of UV active material was observed. Fractions were checked for purity using thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. All fine chemicals were purchased from Sigma-Aldrich or TCI Europe unless otherwise stated. Pegylated reagents were obtained from Quanta biodesign US via Stratech UK.

The LC/MS Conditions were as Follow:

[0549] Positive mode electrospray mass spectrometry was performed using a Waters Aquity H-class. Mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid).

[0550] LCMS 3 min: initial composition was 5% B held over 0.25 min, then increase from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutes and hold there for 0.05 min. Total gradient run time equals 3 min. Flow rate 0.8 mL/min. Detection was at 254 nm. Columns: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

[0551] LCMS 15 min: initial composition 5% B held over 1 min, then increase from 5% B to 100% B over a 9 min period. The composition was held for 2 min at 100% B, then returned to 5% B in 0.10 minutes and hold there for 3 min. Total gradient run time equals 15 min. Flow rate 0.6 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50° C. Column: ACE Excel 2 C18-AR, 2μ, 3.0×100 mm.

Preparative HPLC:

[0552] Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a Shimazdzu Prominence® machine using a Phenomenex® Gemini NX 5μ C18 column (at 50° C.) dimensions: 150×21.2 mm. Eluents used were solvent A (H.sub.2O with 0.1% formic acid) and solvent B (CH.sub.3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: Initial composition 13% B increased to 60% B over a 15 minute period then increased to 100% B over 2 minutes. The composition was held for 1 minute at 100% B, then returned to 13% B in 0.1 minute and held there for 1.9 minutes. The total duration of the gradient run was 20.0 minutes. Flow rate was 20.0 mL/minute and detection was at 254 and 280 nm.

Example 1

[0553] ##STR00050## ##STR00051##

a) 1-((benzyloxy)carbonyl)azetidine-2-carboxylic Acid (2)

[0554] (2S)-Azetidine-2-carboxylic acid 1 (3 g, 29.674 mmol) and sodium bicarbonate (6.3 g, 75 mmol) were solubilised in H.sub.2O (25 mL, 1387.75 mmol) and N-(benzyloxycarbonyl)succinimide (8.5 g, 34 mmol) in THF (25 mL, 307 mmol, 100 mass %) were added dropwise. After stirring at room temperature for 12 h, the two phases were allowed to separate. The aqueous phase was washed with diethyl ether (50 mL), cooled in an ice bath, and then acidified to pH=2 with conc. HCl. The aqueous layer was extracted with ethyl acetate (2×50 mL) and the combined organic extracts were dried (MgSO.sub.4) and the excess solvent evaporated in vacuo to give crude product as a clear oil. The crude material was used without purification in the next step. LCMS 3 min: ES.sup.+=1.34 min, m/z 258.2 [M+Na].sup.+.

b) (1-benzyl 2-methyl (S)-azetidine-1,2-dicarboxylate (3)

[0555] In a dry round bottom flask, (2S)-1-benzyloxycarbonylazetidine-2-carboxylic acid 2 (6.98 g, 29.7 mmol) was solubilised in MeOH (65 mL) and sulfuric acid (3 mL) was added. The mixture was heated to reflux and left to stir overnight. The mixture was left to cool down to r.t. and quenched with Net.sub.3 (to pH=7) before being stirred for 1 h. Methanol was removed in vacuo. The residue was taken up in EtOAc, washed with H.sub.2O and brine before being dried with MgSO.sub.4 and filtered. The organics were removed in vacuo to give crude product 3 (8.004 g, 32.11 mmol) as a clear oil. LCMS 3 min: ES.sup.+=1.53 min, m/z no ionisation

c) Benzyl (S)-2-(hydroxymethyl)azetidine-1-carboxylate (4)

[0556] O1-benzyl O2-methyl (2S)-azetidine-1,2-dicarboxylate 3 (7.6 g, 30 mMol) was solubilised in THF (75 mL, 922 mmol), cooled to 0° C. and LiBH.sub.4 (1 g, 45 mMol,) was added. The mixture was allowed to warm to r.t. and stirred for a further hour at which point the reaction is complete. The reaction mixture was cooled to 0° C. before being quench with H.sub.2O and 1M HCl. The volatiles were removed in vacuo. The residue was taken up in EtOAc and washed with brine (2×50 mL), dried with MgSO.sub.4, filtered and the solvent removed by rotary evaporation under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:2) afforded product a 4 as a clear oil (4.076 g, 60% yield over 3 steps). LCMS 3 min: ES.sup.+=1.36 min, m/z 222.3 [M+H].sup.+.

[0557] d) Benzyl (S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carboxylate (5) Benzyl (2S)-2-(hydroxymethyl)azetidine-1-carboxylate 4 (4.0766 g, 18.425 mmol) was solubilised in dry CH.sub.2Cl.sub.2 (20 mL, 312.0 mmol) and the mixture was cooled to 0° C. before adding imidazole (2.508 g, 36.84 mmol) and TBS-Cl (4.16 g, 27.6 mmol). The mixture was allowed to warm to room temperature and left to stir. LCMS shows reaction was complete within 5 min. The organics were washed with sat. NH.sub.4Cl, water, brine, dried with MgSO.sub.4, filtered and the volatiles removed in vacuo. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 9:1) afforded product a 5 (6.90 g, not completely dry, quantitative). LCMS 3 min: ES.sup.+=2.15 min, m/z 336.9 [M+H].sup..+.

e) (S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine (6)

[0558] Palladium on carbon (10%) (100 mg, 0.93 mMol) was treated with EtOAc (5 mL) dropwise and the resulting slurry added to a suspension of 5 (6.9027 g, 20.57 mmol) in EtOH (100 mL) at room temperature in a Parr hydrogenation bottle. The reaction mixture was subjected to H.sub.2 gas at 20 psi then the bottle was evacuated under vacuum (repeated 3 times). The bottle was then topped up to 38 psi H.sub.2 and shaken for 1 hour. The pressure dropped to ˜30 psi during this time and the bottle was topped up again to 40 psi and shaken for a further hour. No further decreases in pressure were observed and the reaction was deemed complete. This was confirmed by LC-MS. The mixture was filtered through celite and the filtrate evaporated in vacuo to provide the crude product 6 as a brown oil (3.761 g, 90% yield). LCMS 3 min: ES.sup.+=1.70 min, m/z no ionisation.

f) ((S)-2-(((Tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)(4-(6-(4-((2R)-2-(((tert-butyldimethylsilyl)oxy)methyl)cyclobutane-1-carbonyl)-2-methoxy-5-nitrophenoxy)hexyl)-5-methoxy-2-nitrophenyl)methanone (8)

[0559] DCC (3.8 g, 18 mmol) was added to a solution of 7 (3.9 g, 7.9 mMol) and HOBt (2.3 g, 17 mMol) in CH.sub.2Cl.sub.2 (200 mL) at 0° C. The cold bath was removed and the reaction was allowed to proceed for 30 min at room temperature, at which time a solution of 6 (3.65 g, 18 mMol) and triethylamine (3.2 mL, 23 mmol) in CH.sub.2Cl.sub.2 (200 mL) was added rapidly at −10° C. under argon The reaction mixture was allowed to stir at room temperature and monitored by LC/MS. After 2 min, the reaction was complete. The solids were removed by filtration over celite and the organic phase was washed with cold aqueous 0.1 M HCl until the pH was measured at 2. The organic phase was then washed with water, followed by saturated aqueous sodium bicarbonate, brine, dried with MgSO.sub.4, filtered and vacced down under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc/CH.sub.2Cl.sub.2, 100% to 1:2:1) afforded product 8 (5.9 g, 87% yield). The product is contaminated with some mono-coupled product (impurity does not separate upon chromatography). LCMS 3 min: ES.sup.+=2.35 min, m/z 862.2 [M+H].sup..+.

g) ((Pentane-1,5-diylbis(oxy))bis(2-amino-5-methoxy-4,1-phenylene))bis(((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)methanone) (9)

[0560] Zinc (4.65 g, 71.1 mmol) was slowly added to a solution of 8 (2.45 g, 2.85 mmol) in a mixture of MeOH/H.sub.2O/formic acid 90:5:5 (66 mL). The resulting exotherm was controlled using an ice bath to maintain the temperature of the reaction mixture below 40° C. Upon completion, the solids were removed by filtration over celite and the organic phase was washed with water and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Crude material 9 (2.28 g, quantitative) was used as such in the next step. LCMS 3 min: ES.sup.+=2.32 min, m/z 802.3 [M+H].sup..+.

h) Diallyl ((pentane-1,5-diylbis(oxy))bis(6-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxy-3,1-phenylene))dicarbamate (10)

[0561] Compound 9 (2.23 g, 2.78 mmol) was solubilised in CH.sub.2Cl.sub.2 (50 mL) under an argon atmosphere. The mixture was cooled to −78° C. before pyridine (0.99 mL, 12.3 mMol) and allyl chloroformate (0.738 mL, 2.49 mmol) were added. The reaction was left to stir at −78° C. for 10 min before being allowed to warm up to room temp. After 15 min the reaction was complete. The organics were washed with sat. CuSO.sub.4, H.sub.2O, brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Crude product 10 (1.47 g, 1.52 mMol, quantitative) was used as such in the next step. LCMS 3 min: ES.sup.+=2.53 min, m/z 970.3 [M+H].sup..+.

i) Diallyl ((pentane-1,5-diylbis(oxy))bis(6-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-3,1-phenylene))dicarbamate (11)

[0562] Compound 10 (1.47 g, 1.52 mMol) was solubilised in a 3:1:1 mixture of H.sub.2O/THF/acetic acid (16 mL) and the reaction was left to stir over the weekend. The mixture was extracted with CH.sub.2Cl.sub.2 and washed with sat. NaHCO.sub.3, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) afforded product 11 (859 mg, 76.5% Yield) as a clear oil. LCMS 3 min: ES.sup.+=1.75 min, m/z 742.0 [M+H].sup..+.

j) Diallyl 7,7′-(pentane-1,5-diylbis(oxy))(10aS,10a′S)-bis(10-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (12)

[0563] Compound 11 (850 mg, 1.14 mMol) was solubilised in CH.sub.2Cl.sub.2 (60 mL). 1-hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.7 mL, 1140 mmol, 0.2 mMl/L) and tetrakisacetonitrile copper(I) triflate (55 mg, 0.145 mMol) were subsequently added ant the mixture stirred at 35° C. with 2 balloon of air pressing in. The reaction was left to stir overnight before being vacuumed to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 95:5) afforded product 12 (346 g, 0.47 mMol, 41% yield). LCMS 3 min: ES.sup.+=1.48 min, m/z 737.9 [M+H].sup..+.

k) (10aS,10a′S)-7,7′-(pentane-1,5-diylbis(oxy))bis(6-methoxy-1,10a-dihydroazeto[1,2-a]benzo[e][1,4]diazepin-4(2H)-one) (Ex1)

[0564] Compound 12 (335 mg, 0.45 mmol) was solubilised in CH.sub.2Cl.sub.2 (20 mL) in a flask under Argon. Pyrrolidine (650 μL, 7.8 mMol) and Pd(PPh.sub.3).sub.4 (50 mg, 0.004 mMol) were subsequently added and the mixture left to stir at r.t until complete. The organics were washed with sat. NH.sub.4Cl, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by isolera chromatography (CH.sub.2Cl.sub.2/(CH.sub.2Cl.sub.2+10% MeOH) 92:7 to 10:90. Two fractions containing the product were isolate but with insufficient purity. The fractions were combined and repurified by manual chromatography and pure product Ex1 was isolated (146 mg, 0.27 mMol, 24% yield).

[0565] LCMS 3 min: ES.sup.+=1.32 min, m/z 533.8 [M+H].sup..+. LCMS 15 min: ES.sup.+=4.83 min, m/z 533.9 [M+H].sup..+.

Example 2

[0566] ##STR00052##

a) ((S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (13)

[0567] DCC (4.021 g, 19.49 mmol) was added to a solution of 5-methoxy-2-nitro-4-triisopropylsilyloxy-benzoic acid 13 (6 g, 16.24 mmol), and HOPO (1.984 g, 17.86 mMol) in CH.sub.2Cl.sub.2 (100 mL) at 0° C. The cold bath was removed and the reaction was allowed to proceed for 30 min at room temperature, at which time a solution of [(2S)-azetidin-2-yl]methoxy-tert-butyl-dimethyl-silane 6 (3.761 g, 18.68 mmol) and triethylamine (3.39 mL, 33.5 mmol) in CH.sub.2Cl.sub.2 (100 mL) was added rapidly at −10° C. under argon The reaction mixture was allowed to stir at room temperature and monitored by LC/MS. After 2 min, the reaction was complete. The solids were removed by filtration over celite and the organic phase was washed with cold aqueous 0.1 M HCl until the pH was measured at 2. The organic phase was then washed with water, followed by saturated aqueous sodium bicarbonate, brine, dried with MgSO.sub.4, filtered and vacced down under reduced pressure.

[0568] Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) afforded product 14 (8.6737 g, 96.63% yield). LCMS 3 min: ES.sup.+=2.44 min, m/z 554.2 [M+H].sup..+.

b) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidin-1-yl)methanone (15)

[0569] Zinc (10 g, 152.9 mMol) was slowly added to a solution of 14 (8.6737 g, 15.69 mMol) in a mixture of MeOH/H.sub.2O/formic acid 90:5:5 (200 mL). The resulting exotherm was controlled using an ice bath to maintain the temperature of the reaction mixture below 40° C. Upon completion, the solids were removed by filtration over celite and the organic phase was washed with water and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Crude material 15 (7.6343 g, 14.6 mMol, 93.05% yield) was used as such in the next step. LCMS 3 min: ES.sup.+=2.42 min, m/z 524.4 [M+H].sup..+.

c) allyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (16)

[0570] Compound 15 (7.6343 g, 14.60 mMol) was solubilised in CH.sub.2Cl.sub.2 (100 mL) under an argon atmosphere. The mixture was cooled to −78° C. before pyridine (2.6 mL, 32 mMol) and allyl chloroformate (1.7 mL, 16 mMol) were added. The reaction was left to stir at −78° C. for 10 min before being allowed to warm up to room temp. After 15 min the reaction was complete. The organics were washed with sat. CuSO.sub.4, H.sub.2O, brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Crude product 16 (8.9129 g, 14.69 mMol, quantitative) was used as such in the next step. LCMS 3 min: ES.sup.+=2.53 min, m/z 608.2 [M+H].sup.+.

d) Allyl (S)-(2-(2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (17)

[0571] Compound 16 (8.9129 g, 14.69 mMol) was solubilised in a 3:1:1 mixture of H.sub.2O/THF/acetic acid (80 mL) and the reaction was left to stir over the weekend. The mixture was extracted with CH.sub.2Cl.sub.2 and washed with sat. NaHCO.sub.3, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (Hex/EtOAc, 100% to 1:1) afforded product 17 (5.5572 g, 76.80% yield) as a clear oil. LCMS 3 min: ES.sup.+=1.97 min. m/z 494.0 [M+H].sup.+.

e) Allyl (10aS)-10-hydroxy-6-methoxy-4-oxo-7-((triisopropylsilyl)oxy)-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (18)

[0572] Compound 17 (5.5572 g, 11.28 mMol) was solubilised in CH.sub.2Cl.sub.2 (40 mL). 1-hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (6 mL, 1 mMol) and tetrakisacetonitrile copper(I) triflate (425 mg, 1.1279 mMol) were subsequently added ant the mixture stirred at 35° C. with 2 balloon of air pressing in. The reaction was left to stir overnight before being vacuumed to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 97:3) afforded product 18 (5.3835 g, 10.97 mMol, 97.27% yield) as a light orange foam. LCMS 3 min: ES.sup.+=2.00 min, m/z 491.8 [M+H].sup..+.

f) Allyl (10aS)-10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-7-((triisopropylsilyl)oxy)-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (19)

[0573] Compound 18 (5.3835 g, 10.97 mMol) was solubilised in CH.sub.2Cl.sub.2 (50 mL) and the mixture was cooled to −78° C. 2,6-Lutidine (2.55 mL, 21.9 mMol) and TBS-OTf (3.78 mL, 16.4 mMol) were subsequently added. The mixture was left for 10 min before removing the cooling bath and allowing to warm to r.t. The organics were washed with, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 95:5) afforded product 19 (6.8532 g, quantitative). LCMS 3 min: ES.sup.+=2.47 min, m/z 606.0 [M+H].sup..+.

g) Allyl (10aS)-10-((tert-butyldimethylsilyl)oxy)-7-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (20)

[0574] Compound 19 (6.8 g, 14 mMol) was solubilised in DMF (10 mL). LiOAc.2H.sub.2O (1.4 g, 14 mmMl) and H.sub.2O (3 mL or as much as possible) were added. When the solution becomes clear again, add a few drops of water. Keep repeating the process until the reaction is complete. The organics were diluted with CHCl.sub.3 and washed with a citric acid solution (pH=3), H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 95:5) afforded product 20 (5.2885 g, 11.79 mMol, 85% yield) as a yellow oil. LCMS 3 min: ES.sup.+=1.86 min, m/z 449.8 [M+H].sup..+.

h) Diallyl 7,7′-(propane-1,3-diylbis(oxy))(10aS,10a′S)-bis(10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (21)

[0575] 1,3-Dibromopropane (204.9 mg, 1.015 mMol) and compound 20 (1 g, 2.030 mMol) were solubilised in CH.sub.2Cl.sub.2 (50 mL) under an argon atmosphere. K.sub.2CO.sub.3 (280 mg, 2.026 mMol) and TBAI (149 mg, 0.2 mMol) were subsequently added and the mixture was allowed to stir at 40° C. until complete. The mixture was left to stir overnight but the reaction does not go to completion instead an impurity formed. The organics were washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 97:3) afforded product 21 (482 mg, 0.471 mMol, 46.50% yield), contaminated with an inseparable impurity (r.t=9.95 min on LCMS 15 min). LCMS 15 min: ES.sup.+=9.86 min, m/z 938.3 [M+H].sup..+.

i) (10aS,10a′S)-7,7′-(propane-1,3-diylbis(oxy))bis(6-methoxy-1,10a-dihydroazeto[1,2-a]benzo[e][1,4]diazepin-4(2H)-one) (Ex2A)

[0576] Compound 21 (482 mg, 0.5143 mMol) was solubilised in CH.sub.2Cl.sub.2 (20 mL) in a flask under argon. Pyrrolidine (786 μL, 9.44 mMol) and Pd(PPh.sub.3).sub.4 (54 mg, 0.046 mMol) were subsequently added and the mixture left to stir at r.t until complete. The organics were washed with sat. NH.sub.4Cl, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by isolera chromatography (CH.sub.2Cl.sub.2/(CH.sub.2Cl.sub.2+10% MeOH) 98:2 to 30:70. Two fractions containing the product were isolated but with insufficient purity. The fractions were combined and repurified by isolera chromatography (same solvent system) and pure product Ex2A was isolated (35.1 mg, 0.135 mMol, 13.5% yield). LCMS 3 min: ES.sup.+=1.23 min, m/z 505.8 [M+H].sup..+.

j) Diallyl 7,7′-((1,3-phenylenebis(methylene))bis(oxy))(10aS,10a′S)-bis(10-((tert-butyldimethylsilyl)oxy)-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (22)

[0577] 1,3-Bis(bromomethyl)benzene (267.9 mg, 1.011 mMol) and compound 20 (1 g, 2.030 mMol) were solubilised in DMF (5 mL) under an argon atmosphere. K.sub.2CO.sub.3 (280 mg, 2.026 mMol) and TBAI (749 mg, 2.027 mMol) were subsequently added and the mixture was allowed to stir at 40° C. until complete. The mixture was left to stir overnight but the reaction did not go to completion and an impurity formed. The mixture was diluted with CH.sub.2Cl.sub.2 and washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 97:3) afforded product 22 (467 mg, 0.43 mMol, 42.47% yield)+398 mg of mixed fractions. LCMS 3 min: ES.sup.+=2.30 min, m/z 1000.5 [M+H].sup..+.

k) (10aS,10a′S)-7,7′-((1,3-phenylenebis(methylene))bis(oxy))bis(6-methoxy-1,10a-dihydroazeto[1,2-a]benzo[e][1,4]diazepin-4(2H)-one) (Ex2B)

[0578] Compound 22 (455 mg, 0.419 mMol) was solubilised in CH.sub.2Cl.sub.2 (20 mL) in a flask under Argon. Pyrrolidine (600 μL, 7.2 mMol) and Pd(PPh.sub.3).sub.4 (48 mg, 0.041 mMol) were subsequently added and the mixture left to stir at r.t until complete. The organics were washed with sat. NH.sub.4Cl, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by isolera chromatography (CH.sub.2Cl.sub.2/(CH.sub.2Cl.sub.2+10% MeOH) 98:2 to 30:70. Two fractions containing the product were isolate but with insufficient purity. The fractions were combined and repurified by isolera chromatography (same solvent system) and pure product Ex2B was isolated (214.5 mg, 0.378 mMol, 90.5% yield) as a white solid. LCMS 3 min: ES.sup.+=1.38 min, m/z 567.8 [M+H].sup..+.

Example 3

[0579] ##STR00053## ##STR00054##

a) Allyl (5-((5-(5-amino-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamate (23)

[0580] Compound 9 (1.192 g, 1.488 mMol) was solubilised in CH.sub.2Cl.sub.2 (250 mL) under an argon atmosphere. The mixture was cooled to −78° C. before pyridine (0.241 mL, 2.98 mMol) and allyl chloroformate (0.158 mL, 1.484 mMol) were added. The reaction was left to stir at −78° C. for 10 min before being allowed to warm up to room temp. After 15 min the reaction was complete. The organics were washed with sat. CuSO.sub.4, H.sub.2O, brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH) afforded a mixture of mono and bis-alloc which was purified further with a second column (Hex/EtOAc) to give pure product 23 (499.2 g, 37.9% yield out of 50% possible). LCMS 3 min: ES.sup.+=2.41 min, m/z 886.6 [M+H].sup..+.

b) Allyl (5-((5-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamate (24)

[0581] Triphosgene (68.8 mg, 0.232 mMol) was added in one portion to a mixture of 23 (620 mg, 0.7 mMol) and TEA (203 μL, 1.46 mMol) in CH.sub.2Cl.sub.2 (50 mL) at 0° C. The ice bath was removed, and after 15 min, Alloc-Val-Ala-PAB-OH (275 mg, 0.728 mMol) was added in one portion as a fine powder, followed by more TEA (73 μL, 0.524 mMol,) and Dibutyltin dilaurate (39.6 μL, 0.07 mMol). The reaction mixture was allowed to stir at 37° C. for 4 h, followed by stirring at room temperature overnight. The organics were washed with H.sub.2O, sat. NH.sub.4Cl and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH) to give pure product 24 (414 g, 45.9% yield). LCMS 3 min: ES.sup.+=2.43 min, m/z 1289.5 [M+H].sup..+.

c) Allyl (5-((5-(5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-2-methoxyphenoxy)pentyl)oxy)-2-((S)-2-(hydroxymethyl)azetidine-1-carbonyl)-4-methoxyphenyl)carbamate (25)

[0582] Compound 24 (414 mg, 0.32 mMol) was solubilised in a 3:1:1 mixture of H.sub.2O/THF/acetic acid (10 mL) and the reaction was left to stir over the weekend. The mixture was extracted with CH.sub.2Cl.sub.2 and washed with sat. NaHCO.sub.3, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 94:6) afforded product 25 (326 mg, 95.7% Yield). LCMS 3 min: ES.sup.+=1.80 min, m/z 1060.1 [M+H].sup..+.

d) Allyl (10aS)-7-((5-(((10S,10aS)-9-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-10-hydroxy-6-methoxy-4-oxo-1,2,4,9,10,10a-hexahydroazeto[1,2-a]benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy)-10-hydroxy-6-methoxy-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (26)

[0583] Compound 25 (202.4 mg, 0.3 mMol) was solubilised in CH.sub.2Cl.sub.2 (20 mL). 1-hydroxy-2,2,6,6-tetramethyl-piperidine; 1-methylimidazole; 2-(2-pyridyl)pyridine (0.4 mL, 0.03 mMol) and tetrakisacetonitrile copper(I) triflate (11 mg, 0.03 mMol) were subsequently added ant the mixture stirred at 35° C. with 2 balloon of air pressing in. The reaction was left to stir overnight before being vacuumed to dryness in a rotary evaporator. Purification by silica gel column chromatography (CHCl.sub.3/MeOH, 100% to 97:3) afforded product 26 (313 mg, 0.19 mMol, 64.5% yield). LCMS 3 min: ES.sup.+=1.59 min, m/z 1057.1 [M+H].sup..+.

e) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (10S,10aS)-10-hydroxy-6-methoxy-7-((5-(((S)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy)-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate) (27)

[0584] Compound 26 (195 mg, 0.184 mMol) was solubilised in CH.sub.2Cl.sub.2 (10 mL) in a flask under Argon. Pyrrolidine (262 μL, 3.15 mMol) and Pd(PPh.sub.3).sub.4 (21 mg, 0.018 mMol) were subsequently added and the mixture left to stir at r.t until complete. The organics were washed with sat. NH.sub.4Cl, H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by isolera chromatography (CH.sub.2Cl.sub.2/(CH.sub.2Cl.sub.2+10% MeOH) 98:2 to 30:70 gave product 27 (141 mg, 0.16 mMol, 87.7% yield). LCMS 3 min: ES.sup.+=1.23 min, m/z 870.9 [M+H].sup..+.

f) 4-((2S,5S)-37-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (10S,10aS)-10-hydroxy-6-methoxy-7-((5-(((S)-6-methoxy-4-oxo-1,2,4,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepin-7-yl)oxy)pentyl)oxy)-4-oxo-1,2,10,10a-tetrahydroazeto[1,2-a]benzo[e][1,4]diazepine-9(4H)-carboxylate (Ex3)

[0585] The reaction was conducted in a glovebox. Compound 27 (70 mg, 0.080 mMol) was solubilised in CH.sub.2Cl.sub.2 (10 mL) in a flask under argon at room temperature. Mal-dPEG.sub.8-OH (50 mg, 0.084 mMol) and EDCI.HCl (15.4 mg, 0.080 mMol) were added and the mixture was stirred until completion. The organics were washed with H.sub.2O and brine before being dried with MgSO.sub.4, filtered and the volatiles removed under reduced pressure. Purification by isolera chromatography (CH.sub.2Cl.sub.2/(CH.sub.2Cl.sub.2+10% MeOH) 98:2 to 30:70 gave an impure product. Further purification by reverse phase isolera gave pure Ex3 (4 mg, 0.027 mMol, 3.4% yield) plus some unclean fractions (22 mg). LCMS 3 min: ES.sup.+=1.51 min, m/z 1445.6 [M+H].sup..+.

Example 4

ConjA (Her2-Ex3)

[0586] A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (50 molar equivalent/antibody, 7.6 micromoles, 762.7 μL) to a 20.8 mL solution of tratuzumab (22.9 mg, 153 nanomoles) in reduction buffer containing 30 mM histidine/histidine HCl, 30 mM arginine, pH 6.8 and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 1.1 mg/mL. The reduction mixture was allowed to react at 37° C. for 2 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody solution was buffer exchanged (to remove all the excess reducing agent), via spin filter centrifugation, into a conjugation buffer containing 30 mM histidine/histidine HCl, 30 mM arginine and 1 mM EDTA for a final antibody concentration of 1.1 mg/mL. Ex3 was added as a DMSO solution (12.5 molar equivalent/antibody, 1.9 micromoles, in 2.1 mL DMSO) to 18.6 mL of this reduced antibody solution (20.5 mg, 136 nanomoles) for a 10% (v/v) final DMSO concentration. The solution was mixed for 17 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (8.5 micromoles, 68 μL at 100 mM), then purified via spin filter centrifugation using a 15 mL Amicon Ultracell 30 KDa MWCO spin filter, sterile-filtered and analysed.

[0587] UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjA at 214 nm and 330 nm (SG3931 specific) shows a mixture of unconjugated light chains, light chains attached to a single molecule of SG3931, unconjugated heavy chains and heavy chains attached to up to three molecules of SG3931, consistent with a drug-per-antibody ratio (DAR) of 7.32 molecules of SG3931 per antibody.

[0588] UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minute sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomer purity of 94.2%. UHPLC SEC analysis gives a concentration of final ConjA at 1.29 mg/mL in 5.8 mL, obtained mass of ConjA is 7.5 mg (37% yield).

Example 5—Cytoxicity Assay

[0589] The potency of the molecules were measured via in vitro cytotox assays in the carcinoma cell line NCI-N87.

[0590] Solid material was dissolved in DMSO to a 2 mM stock solution, from which eight serial dilutions were made at a 1:10 ratio in DMSO and stored at −20° C. until use.

[0591] Adherent NCI-N87 cells were washed with D-PBS and detached with Trypsin-EDTA, cell density and viability were then determined in duplicate by Trypan blue exclusion assay using an automated cell counter (LUNA-II™). Cell suspension was diluted to 1×10.sup.5 cells/ml in growth media (RPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum) and vortexed before dispensing 2 mL per well into sterile 3 mL polypropylene plates. Warhead dilutions were then dispensed into the appropriate wells at 10 μl/well and mixed by repeat pipetting. For control wells 10 μl of DMSO was dispensed onto 2 mL cell suspension, and thoroughly mixed. 100 μl of each sample was then aliquoted into 2 replicate wells of a sterile flat 96-well microplate and incubated in a 37° C. CO.sub.2-gassed (5%) incubator. At the end of the incubation period time (7 days), cell viability was measured by CellTiter 96 Aqueous One (MTS) assay, which was dispensed at 20 μl/well and incubated for 4 hours at 37° C., 5% CO.sub.2. Plates were then read on an EnVision Multi-label Plate Reader (Perkin Elmer) using absorbance at 490 nm.

[0592] Cell survival percentage was calculated from the mean absorbance of the 2 replicate wells for each sample, compared to the mean absorbance in the two control wells treated with DMSO only (100%). The IC.sub.50 was determined by fitting each data set to sigmoidal dose-response curves with a variable slope using the non-linear curve fit algorithm on the GraphPad Prism software (San Diego, Calif.).

[0593] All the experiments in this report were carried out and tested in three independent experiments. Data are reported as the mean of the three independent replicates.

TABLE-US-00001 IC.sub.50 (nM) Ex2A 70.11 Ex1 2.202 Ex2B 4.035

Example 6—ADC Cytoxicity Assay

[0594] The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2×10.sup.5/ml, dispensed (50 μl per well) into 96-well flat-bottom plates.

[0595] A stock solution (1 ml) of the test antibody drug conjugate (ADC) (20 μg/ml) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 8×10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 μl into 900 μl of cell culture medium. ADC dilution was dispensed (50 μl per well) into 4 replicate wells of the 96-well plate, containing 50 μl cell suspension seeded the previously. Control wells received 50 μl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37C in a CO.sub.2-gassed incubator for the exposure time.

[0596] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 μl per well) into each well and incubated for 4 hours at 37° C. in the CO.sub.2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC.sub.50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.

[0597] ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87. MDA-MB-468 and NCI-N87 were cultured in RPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum.

[0598] The EC.sub.50 values were determined by fitting data to a sigmoidal dose-response curve with variable slope using GraphPad Prism software v6.05 (GraphPad, San Diego, Calif.).

TABLE-US-00002 EC.sub.50 (μg/ml) NCI-N87 MDA-MB-468 ConjA 0.002285 15.71

Example 7—Xenograft Testing

NCI-N87 Xenografted Mice

[0599] Female severe combined immune-deficient mice (Fox Chase SCID®, C.B-17/lcr-Prkdcscid, Charles River) were eight weeks old with a body weight (BW) range of 16.5 to 21.6 grams on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm Cl), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fibre. The mice were housed on irradiated Enricho'cobs™ Laboratory Animal Bedding in static micro-isolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at CR Discovery Services is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals.

Tumour Cell Culture

[0600] Human NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate and 25 μg/mL gentamicin. The cells were grown in tissue culture flasks in a humidified incubator at 37° C., in an atmosphere of 5% CO.sub.2 and 95% air.

In Vivo Implantation and Tumour Growth

[0601] The NCI-N87 cells used for implantation were harvested during log phase growth and Re-suspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumour implant, each test mouse was injected subcutaneously in the right flank with 1×10.sup.7 cells (0.1 mL cell suspension), and tumour growth was monitored as the average size approached the target range of 100 to 150 mm.sup.3. Fourteen days later, designated as Day 1 of the study, mice were sorted according to calculated tumour size into groups each consisting of ten animals with individual tumour volumes ranging from 108 to 144 mm.sup.3 and group mean tumour volumes of 115 mm.sup.3.

[0602] Tumours were measured in two dimensions using calipers, and volume was calculated using the formula:

[00001]$\mathrm{Tumour}\mathrm{Volume}\left({\mathrm{mm}}^3\right)=\frac{w^2\times l}{2}$

where w=width and l=length, in mm, of the tumour. Tumour weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumour volume.

Treatment

[0603] Treatment began on Day 1 in groups of 10 mice (n=10) with established subcutaneous NCI-N87 tumours (108-144 mm.sup.3). ConjA (4 mg/kg) was administered intravenously once on Day 1 (qd×1). A vehicle-treated group served as the control group for efficacy analysis. Tumours were measured twice per week until the study was ended on Day 79. Each mouse was euthanized when its tumour reached the endpoint volume of 800 mm.sup.3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse.

[0604] The results are illustrated in FIG. 1 which shows the change in normalised tumour growth (.square-solid.—control; .diamond-solid.—ConjA).

Endpoint and Tumor Growth Delay (TGD) Analysis

[0605] Tumors were measured using calipers twice per week, and each animal was euthanized when its tumor reached the endpoint volume of 800 mm.sup.3 or at the end of the study (Day 79), whichever came first. Animals that exited the study for tumor volume endpoint were documented as euthanized for tumor progression (TP), with the date of euthanasia. The time to endpoint (TTE) for analysis was calculated for each mouse by the following equation:

[00002]$\mathrm{TTE}=\frac{{\log }_{10}\left(\mathrm{endpoint}\mathrm{volume}\right)-b}{m}$

where TTE is expressed in days, endpoint volume is expressed in mm.sup.3, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. The data set consisted of the first observation that exceeded the endpoint volume used in analysis and the three consecutive observations that immediately preceded the attainment of this endpoint volume. The calculated TTE is usually less than the TP date, the day on which the animal was euthanized for tumor size. Animals with tumors that did not reach the endpoint volume were assigned a TTE value equal to the last day of the study (Day 79). In instances in which the log-transformed calculated TTE preceded the day prior to reaching endpoint or exceeded the day of reaching tumor volume endpoint, a linear interpolation was performed to approximate the TTE. Any animal classified as having died from NTR (non-treatment-related) causes due to accident (NTRa) or due to unknown etiology (NTRu) were excluded from TTE calculations (and all further analyses). Animals classified as TR (treatment-related) deaths or NTRm (non-treatment-related death due to metastasis) were assigned a TTE value equal to the day of death. Treatment outcome was evaluated from tumor growth delay (TGD), which is defined as the increase in the median time to endpoint (TTE) in a treatment group compared to the control group:

TGD=T−C,

expressed in days, or as a percentage of the median TTE of the control group:

[00003]$\%\mathrm{TGD}=\frac{T-C}{C}\times 100$

where:
T=median TTE for a treatment group, and
C=median TTE for the designated control group.

Tumour Growth Inhibition

[0606] Tumor growth inhibition (TGI) analysis evaluates the difference in median tumor volumes (MTVs) of treated and control mice. For this study, the endpoint for determining TGI was Day 19, which was the last day that all evaluable control mice remained in the study. The MTV (n), the median tumor volume for the number of animals, n, on the day of TGI analysis, was determined for each group. Percent tumor growth inhibition (% TGI) was defined as the difference between the MTV of the designated control group and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:

[00004]$\%\mathrm{TGI}=\left(\frac{{\mathrm{MTV}}_{\mathrm{control}}-{\mathrm{MTV}}_{\mathrm{drug}-\mathrm{treated}}}{{\mathrm{MTV}}_{\mathrm{control}}}\right)\times 100=\left[1-\left({\mathrm{MTV}}_{\mathrm{drug}-\mathrm{treated}}/{\mathrm{MTV}}_{\mathrm{control}}\right)\right]\times 100$

[0607] The data set for TGI analysis included all animals in a group, except those that died due to treatment-related (TR) or non-treatment-related (NTR) causes prior to the day of TGI analysis.

MTV and Criteria for Regression Responses

[0608] Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume. Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm.sup.3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm.sup.3 for three consecutive measurements during the course of the study. Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.

Toxicity

[0609] Animals were weighed daily on Days 1-5, then twice per week until the completion of the study. The mice were observed frequently for overt signs of any adverse, treatment-related (TR) side effects, and clinical signs were recorded when observed. Individual body weight was monitored as per protocol, and any animal with weight loss exceeding 30% for one measurement or exceeding 25% for three consecutive measurements was euthanized as a TR death. Group mean body weight loss was also monitored according to CR Discovery Services protocol. Acceptable toxicity was defined as a group mean body weight (BW) loss of less than 20% during the study and no more than 10% TR deaths. Dosing was suspended in any group where mean weight loss exceeded acceptable limits. If group mean body weight recovered to acceptable levels, then dosing was modified to lower levels and/or reduced frequency then resumed. Deaths were classified as TR if it was attributable to treatment side effects as evidenced by clinical signs and/or necropsy. A TR classification was also assigned to deaths by unknown causes during the dosing period or within 14 days of the last dose. A death was classified as non-treatment-related (NTR) if there was no evidence that death was related to treatment side effects. NTR deaths are further categorized as follows: NTRa describes deaths due to accidents or human error; NTRm is assigned to deaths thought to result from tumor dissemination by invasion and/or metastasis based on necropsy results; NTRu describes deaths of unknown causes that lack available evidence of death related to metastasis, tumor progression, accident or human error. It should be noted that treatment side effects cannot be excluded from deaths classified as NTRu.

Statistical and Graphical Analyses

[0610] GraphPad Prism 8.0 for Windows was used for all statistical analysis and graphical presentations. Study groups experiencing toxicity beyond acceptable limits (>20% group mean body weight loss or greater than 10% treatment-related deaths) or having fewer than five evaluable observations, were not included in the statistical analysis. The logrank test was employed to assess the significance of the difference between the overall survival experiences of two groups. The logrank test analyzes the individual TTEs for all animals in a group, except those lost to the study due to NTR death. Statistical analyses of the differences between Day 19 median tumor volumes (MTVs) of control and treated groups were accomplished using the Mann-Whitney U-test. For statistical analyses, two-tailed tests were conducted at significance level P=0.05. Prism summarizes test results as not significant (ns) at P>0.05, significant (symbolized by “*”) at 0.01<P≤0.05, very significant (“**”) at 0.001<P≤0.01, and extremely significant (“***”) at P s 0.001. Because tests of statistical significance do not provide an estimate of the magnitude of the difference between groups, all levels of significance were described as either significant or not significant within the text of this report.

TABLE-US-00003 Median MTV (n), n TTE T-C % TGD Day 79 Vehicle — 10 24.8 — — 466 (10) ConjA 4 mg/kg 10 79.0 54.2 219 32 (9) PR CR TFS BW Nadir TR NTRm NTR Vehicle 0 0 0 −2.0 (2) 0 0 0 ConjA 6 4 0 −1.9 (2) 0 0 0

[0611] The Day 19 MTV(10) for animals treated with ConjA was 32 mm.sup.3, or a significant 93% TGI (P<0.001, Mann-Whitney). Nine animals survived the study and the assigned median TTE was 79.0 days; this represents the maximally possible, significant 219% TGD (P<0.001, logrank). The MTV(9) on Day 79 was 320 mm.sup.3 and there were six PRs and four CRs.

[0612] All documents and other references mentioned above are herein incorporated by reference.

Statements of Invention

[0613] 1. A compound of formula IV:

##STR00055##

[0614] and salts and solvates thereof, wherein:

[0615] R.sup.2 and R.sup.2′ are H;

[0616] R.sup.6 and R.sup.9 are independently selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo;

[0617] where R and R′ are independently selected from optionally substituted C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.5-20 aryl groups;

[0618] either

[0619] (a) R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; R.sup.7′ is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NRR′, nitro, Me.sub.3Sn and halo; or

[0620] (b) R.sup.7 and R.sup.7′ together form a group which is: (i) —O—(CH.sub.2).sub.n—O—, where n is from 7 to 16; or

[0621] (ii) —O—(CH.sub.2CH.sub.2O).sub.m—, where m is 2 to 5;

[0622] R″ is a C.sub.3-12 alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, NR.sup.N2 (where R.sup.N2 is H or C.sub.1-4 alkyl), and/or aromatic rings, e.g. benzene or pyridine;

[0623] Y and Y′ are selected from O, S, or NH;

[0624] R.sup.6′ and R.sup.9′ are selected from the same groups as R.sup.6, and R.sup.9 respectively; either

[0625] (i-a) R.sup.10 and R.sup.11 together form a double bond between the N and C atoms to which they are bound; or

[0626] (i-b) R.sup.10 is H and R.sup.11 is selected from OH and OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; or

[0627] (i-c) R.sup.10 and R.sup.11 are both H; either

[0628] (ii-a) R.sup.20 and R.sup.21 together form a double bond between the N and C atoms to which they are bound; or

[0629] (ii-b) R.sup.20 is H and R.sup.21 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl; or

[0630] (ii-c) R.sup.20 and R.sup.21 are both H.

[0631] 2. A compound according to statement 1, wherein both Y and Y′ are O.

[0632] 3. A compound according to either statement 1 or statement 2, wherein R″ is C.sub.3-7 alkylene.

[0633] 4. A compound according to either statement 1 or statement 2, wherein R″ is a group of formula:

##STR00056##

[0634] where r is 1 or 2.

[0635] 5. A compound according to any one of statements 1 to 4, wherein R.sup.9 is H.

[0636] 6. A compound according to any one of statements 1 to 5, wherein R.sup.6 is H.

[0637] 7. A compound according to any one of statements 1 to 6, wherein R.sup.7 is selected from H, OH and OR and R.sup.7′ is selected from H, OH and OR

[0638] 8. A compound according to statement 7, wherein R.sup.7 is a C.sub.1-4 alkyloxy group and R.sup.7′ is a C.sub.1-4 alkyloxy group.

[0639] 9. A compound according to any one of statements 1 to 8, wherein R.sup.2′ is the same as R.sup.2, R.sup.6′ is the same group as R.sup.6, R.sup.7′ is the same group as R.sup.7, R.sup.9′ is the same group as R.sup.9 and Y′ is the same group as Y.

[0640] 10. A compound according to any one of statements 1 to 9, wherein R.sup.10 and R″ together form a double bond between the N and C atoms to which they are bound.

[0641] 11. A compound according to any one of statements 1 to 9, wherein R.sup.10 is H and R″ is selected from OH and OR.sup.A.

[0642] 12. A compound according to statement 11, wherein R.sup.A is methyl.

[0643] 13. A compound according to any one of statements 1 to 9, wherein R.sup.10 and R.sup.11 are both H.

[0644] 14. A compound according to any one of statements 1 to 13, wherein R.sup.20 and R.sup.21 together form a double bond between the N and C atoms to which they are bound.

[0645] 15. A compound according to any one of statements 1 to 13, wherein R.sup.20 is H and R.sup.21 is selected from OH and OR.sup.B.

[0646] 16. A compound according to statement 14, wherein R.sup.B is methyl.

[0647] 17. A compound according to any one of statements 1 to 13, wherein R.sup.20 and R.sup.21 are both H.

[0648] 18. A compound according to statement 1 which is of formulae IVa, IVb or IVc:

##STR00057##

[0649] where R.sup.1a is selected from methyl and benzyl.

[0650] 19. A compound of formula I:

##STR00058##

[0651] and salts and solvates thereof, wherein:

[0652] Y, Y′, R″, R.sup.2, R.sup.2′, R.sup.6, R.sup.6′, R.sup.7, R.sup.7′, R.sup.9 and R.sup.9′ are as defined in any one of statements 1 to 18;

[0653] R.sup.11b is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; and

[0654] R.sup.L is a linker for connection to a cell binding agent, which is selected from:

[0655] (iiia):

##STR00059##

[0656] wherein

[0657] Q is:

##STR00060##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue;

[0658] X is:

##STR00061##

[0659] where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;

[0660] G.sup.L is a linker for connecting to a Ligand Unit; and

[0661] (iiib):

##STR00062##

[0662] where R.sup.L1 and R.sup.L2 are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group;

[0663] and e is 0 or 1;

[0664] either: [0665] (a) R.sup.30 and R.sup.31 together form a double bond between the N and C atoms to which they are bound; or [0666] (b) R.sup.30 is H and R.sup.31 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl; or [0667] (c) R.sup.30 and R.sup.31 are both H; or [0668] (d) R.sup.31 is OH or OR.sup.B, where R.sup.B is C.sub.1-4 alkyl and R.sup.30 is selected from:

##STR00063##

where R.sup.Z is selected from:

##STR00064## [0669] (z-i); [0670] (z-ii) OC(═O)CH.sub.3; [0671] (z-iii) NO.sub.2; [0672] (z-iv) OMe; [0673] (z-v) glucoronide; [0674] (z-vi) NH—C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—C(═O)—R.sup.ZC, where —C(═O)—X.sub.1—NH— and —C(═O)—X.sub.2—NH— represent natural amino acid residues and R.sup.ZC is selected from Me, OMe, CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.

[0675] 20. A compound according to statement 19, wherein R.sup.30 and R.sup.31 together form a double bond between the N and C atoms to which they are bound.

[0676] 21. A compound according to statement 19, wherein R.sup.30 is H and R.sup.31 is selected from OH and OR.sup.B, where R.sup.B is C.sub.1-4 alkyl.

[0677] 22. A compound according to statement 19, wherein R.sup.30 and R.sup.31 are both H.

[0678] 23. The compound according to statement 19, wherein R.sup.31 is OH or OR.sup.A and R.sup.30 is selected from:

##STR00065## ##STR00066##

[0679] 24. The compound according to statement 23, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Ala-Lys-, and -Val-Cit-.

[0680] 25. The compound according to statement 23, wherein —C(═O)—X.sub.1—NHC(═O)X.sub.2—NH—, is selected from: -Phe-Lys-, and -Val-Ala-.

[0681] 26. The compound according to any one of statements 23 to 25 wherein R.sup.ZC is selected from CH.sub.2CH.sub.2OMe, and (CH.sub.2CH.sub.2O).sub.2Me.

[0682] 27. The compound according to statement 26 wherein R.sup.ZC is (CH.sub.2CH.sub.2O).sub.2Me.

[0683] 28. A compound according to statement 19, which is of formulae Ia, Ib or Ic:

##STR00067##

[0684] where R.sup.1a is selected from methyl and benzyl.

[0685] 29. A compound according to any one of statements 19 to 28, wherein R.sup.11b is OH.

[0686] 30. A compound according to any one of statements 19 to 29, wherein R.sup.11b is OR.sup.A, where R.sup.A is C.sub.1-4 alkyl.

[0687] 31. A compound according to statement 30, wherein R.sup.A is methyl.

[0688] 32. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIa, and Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0689] 33. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIa, and Q is a dipeptide residue selected from: [0690] .sup.CO-Phe-Lys-.sup.NH, [0691] .sup.CO-Val-Ala-.sup.NH, [0692] .sup.CO-Val-Lys-.sup.NH, [0693] .sup.CO-Ala-Lys-.sup.NH, [0694] .sup.CO-Val-Cit-.sup.NH, [0695] .sup.CO-Phe-Cit-.sup.NH, [0696] .sup.CO-Leu-Cit-.sup.NH, [0697] .sup.CO-Ile-Cit-.sup.NH, [0698] .sup.CO-Phe-Arg-.sup.NH, and [0699] .sup.CO-Trp-Cit-.sup.NH.

[0700] 34. A compound according to statement 33, wherein Q is selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Ala-.sup.NH.

[0701] 35. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIa, and Q is a tripeptide residue selected from: [0702] .sup.CO-Glu-Val-Ala-.sup.NH, [0703] .sup.CO-Glu-Val-Cit-.sup.NH, [0704] .sup.CO-αGlu-Val-Ala-.sup.NH, and [0705] .sup.CO-αGlu-Val-Cit-.sup.NH.

[0706] 36. A compound according to any one of statements 19 to 35, wherein R.sup.L is of formula IIIa and a is 0 to 3.

[0707] 37. A compound according to statement 36, wherein a is 0.

[0708] 38. A compound according to any one of statements 19 to 37, wherein R.sup.L is of formula IIIa and b is 0 to 12.

[0709] 39. A compound according to statement 38, wherein b is 0 to 8.

[0710] 40. A compound according to any one of statements 19 to 39, wherein R.sup.L is of formula IIIa and d is 0 to 3.

[0711] 41. A compound according to statement 38, wherein d is 2.

[0712] 42. A compound according to any one of statements 19 to 35, wherein R.sup.L is of formula IIIa and, a is 0, c is 1 and d is 2, and b is from 0 to 8.

[0713] 43. A compound according to statement 42, wherein b is 0, 4 or 8.

[0714] 44. A compound according to any one of statements 19 to 43 wherein R.sup.L is of formula IIIa and G.sup.L is selected from:

##STR00068## ##STR00069##

[0715] where Ar represents a C.sub.5-6 arylene group.

[0716] 45. A compound according to statement 44, wherein Ar is a phenylene group.

[0717] 46. A compound according to either statement 44 or statement 45, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

[0718] 47. A compound according to statement 46, wherein G.sup.L is G.sup.L1-1.

[0719] 48. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIb, and both R.sup.L1 and R.sup.L2 are H.

[0720] 49. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIb, R.sup.L1 is H and R.sup.L2 is methyl.

[0721] 50. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIb, and both R.sup.L1 and R.sup.L2 are methyl.

[0722] 51. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIb, and, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.

[0723] 52. A compound according to any one of statements 19 to 31, wherein R.sup.L is of formula IIIb, and, R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

[0724] 53. A compound according to any one of statements 19 to 31 and 48 to 52, wherein R.sup.L is of formula IIIb, and e is 0.

[0725] 54. A compound according to any one of statements 19 to 31 and 48 to 52, wherein R.sup.L is of formula IIIb, and e is 1.

[0726] 55. A compound according to statement 54, wherein the nitro group is in the para position.

[0727] 56. A compound according to statement 19, wherein the compound is of formula Id:

##STR00070##

[0728] where Q is selected from:

[0729] (a) —CH.sub.2—;

[0730] (b) —C.sub.3H.sub.6—; and

##STR00071##

[0731] 57. A conjugate of formula II:

L-(D.sup.L).sub.p  (II)

[0732] wherein L is a Ligand unit (i.e., a targeting agent), D.sup.L is a Drug Linker unit of formula I′:

##STR00072##

[0733] wherein:

[0734] Y, Y′, R″, R.sup.2, R.sup.2′, R.sup.6, R.sup.6′, R.sup.7′, R.sup.7′, R.sup.9 and R.sup.9′ are as defined in any one of statements 1 to 18;

[0735] R.sup.11b, R.sup.30 and R.sup.31 are as defined in any one of statements 19 to 27 and 29 to 31;

[0736] R.sup.LL is a linker for connection to a cell binding agent, which is selected from:

[0737] (iiia):

##STR00073##

[0738] where Q and X are as defined in any one of statements 19 and 32 to 43 and G.sup.LL is a linker connected to a Ligand Unit; and

[0739] (iiib):

##STR00074##

[0740] where R.sup.L1 and R.sup.L2 are as defined in any one of statements 19 and 48 to 52;

[0741] wherein p is an integer of from 1 to 20.

[0742] 58. A conjugate according to statement 57, wherein G.sup.LL is selected from:

##STR00075## ##STR00076##

[0743] where r represents a C.sub.5-6 arylene group.

[0744] 59. A conjugate according to statement 58, wherein Ar is a phenylene group.

[0745] 60 A conjugate according to either statement 58 or statement 59, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

[0746] 61. A conjugate according to statement 60, wherein G.sup.LL is G.sup.LL-11.

[0747] 62. A conjugate according to statement 57, wherein D.sup.L is of formula (Id′):

##STR00077##

[0748] where Q is selected from:

[0749] (a) —CH.sub.2—;

[0750] (b) —C.sub.3H.sub.6—; and

[0751] (c)

##STR00078##

[0752] 63. A conjugate according to any one of statements 57 to 62, wherein the Ligand Unit is an antibody or an active fragment thereof.

[0753] 64. The conjugate according to statement 63, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.

[0754] 65. The conjugate according to statement 64, wherein the antibody or antibody fragment is an antibody which binds to one or more tumor-associated antigens or cell-surface receptors selected from (1)-(88):

[0755] (1) BMPR1B;

[0756] (2) E16;

[0757] (3) STEAP1;

[0758] (4) 0772P;

[0759] (5) MPF;

[0760] (6) Napi3b;

[0761] (7) Sema 5b;

[0762] (8) PSCA hlg;

[0763] (9) ETBR;

[0764] (10) MSG783;

[0765] (11) STEAP2;

[0766] (12) TrpM4;

[0767] (13) CRIPTO;

[0768] (14) CD21;

[0769] (15) CD79b;

[0770] (16) FcRH2;

[0771] (17) HER2;

[0772] (18) NCA;

[0773] (19) MDP;

[0774] (20) IL20R-alpha;

[0775] (21) Brevican;

[0776] (22) EphB2R;

[0777] (23) ASLG659;

[0778] (24) PSCA;

[0779] (25) GEDA;

[0780] (26) BAFF-R;

[0781] (27) CD22;

[0782] (28) CD79a;

[0783] (29) CXCR5;

[0784] (30) HLA-DOB;

[0785] (31) P2X5;

[0786] (32) CD72;

[0787] (33) LY64;

[0788] (34) FcRH1;

[0789] (35) IRTA2;

[0790] (36) TENB2;

[0791] (37) PSMA-FOLH1;

[0792] (38) SST;

[0793] (38.1) SSTR2;

[0794] (38.2) SSTR5;

[0795] (38.3) SSTR1;

[0796] (38.4)SSTR3;

[0797] (38.5) SSTR4;

[0798] (39) ITGAV;

[0799] (40) ITGB6;

[0800] (41) CEACAM5;

[0801] (42) MET;

[0802] (43) MUC1;

[0803] (44) CA9;

[0804] (45) EGFRvIII;

[0805] (46) CD33;

[0806] (47) CD19;

[0807] (48) IL2RA;

[0808] (49) AXL;

[0809] (50) CD30-TNFRSF8;

[0810] (51) BCMA-TNFRSF17;

[0811] (52) CT Ags-CTA;

[0812] (53) CD174 (Lewis Y)-FUT3;

[0813] (54) CLEC14A;

[0814] (55) GRP78-HSPA5;

[0815] (56) CD70;

[0816] (57) Stem Cell specific antigens;

[0817] (58) ASG-5;

[0818] (59) ENPP3;

[0819] (60) PRR4;

[0820] (61) GCC-GUCY2C;

[0821] (62) Liv-1-SLC39A6;

[0822] (63) 5T4;

[0823] (64) CD56-NCMA1;

[0824] (65) CanAg;

[0825] (66) FOLR1;

[0826] (67) GPNMB;

[0827] (68) TIM-1-HAVCR1;

[0828] (69) RG-1/Prostate tumor target Mindin-Mindin/RG-1;

[0829] (70) B7-H4-VTCN1;

[0830] (71) PTK7;

[0831] (72) CD37;

[0832] (73) CD138-SDC1;

[0833] (74) CD74;

[0834] (75) Claudins-CLs;

[0835] (76) EGFR;

[0836] (77) Her3;

[0837] (78) RON-MST1R;

[0838] (79) EPHA2;

[0839] (80) CD20-MS4A1;

[0840] (81) Tenascin C-TNC;

[0841] (82) FAP;

[0842] (83) DKK-1;

[0843] (84) CD52;

[0844] (85) CS1-SLAMF7;

[0845] (86) Endoglin-ENG;

[0846] (87) Annexin A1-ANXA1;

[0847] (88) V-CAM (CD106)-VCAM1;

[0848] (89) ASCT2 (SLC1A5).

[0849] 66. The conjugate of any one of statements 63 to 65 wherein the antibody or antibody fragment is a cysteine-engineered antibody.

[0850] 67. The conjugate according to any one of statements 57 to 66 wherein p is an integer from 1 to 8.

[0851] 68. The conjugate according to statement 67, wherein p is 1, 2, 3, or 4.

[0852] 69. A composition comprising a mixture of conjugates according to any one of statements 57 to 68, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.

[0853] 70. The conjugate according to any one of statements 57 to 68, for use in therapy.

[0854] 71. A pharmaceutical composition comprising the conjugate of any one of statements 57 to 68, and a pharmaceutically acceptable diluent, carrier or excipient.

[0855] 72. The conjugate according to any one of statements 57 to 68 or the pharmaceutical composition according to statement 71, for use in the treatment of a proliferative disease in a subject.

[0856] 73. The conjugate for use according to statement 72, wherein the disease treated is cancer.

[0857] 74. Use of a conjugate according to any one of statements 57 to 68 or the pharmaceutical composition according to statement 71 in a method of medical treatment.

[0858] 75. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 71.

[0859] 76. The method of statement 75 wherein the method of medical treatment is for treating cancer.

[0860] 77. The method of statement 76, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.

[0861] 78. Use of a conjugate according to any one of statements 57 to 68 in a method of manufacture of a medicament for the treatment of a proliferative disease.

[0862] 79. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of statements 57 to 68 or the pharmaceutical composition according to statement 71.