Pyrrolobenzodiazepines and conjugates thereof as antitumour agents

Abstract

A compound with the formula I: (I) and salts and solvates thereof, wherein: R″ is a group of formula II: (II) where each of n and m are independently selected from 1, 2 and 3. ##STR00001##

Claims

1. A compound of formula I: ##STR00182## or a pharmaceutically acceptable salt thereof, wherein: R″ is a group of formula II: ##STR00183## where each of n and m are independently selected from 1, 2 and 3; R.sup.O is selected from the group consisting of H, methyl, ethyl, iso-propyl and benzyl; Q.sup.C is selected from N and CH; Y and Y′ are selected from O, S, or NH; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: (ia) C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group, wherein said C.sub.6-10 carboaryl or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group, a C.sub.6-10 carboaryl group or a C.sub.5-10 heteroaryl group; (ib) C.sub.1-5 alkyl; (ic) C.sub.3-6 cycloalkyl; ##STR00184## wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; ##STR00185## wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00186## where R.sup.14 is selected from: H; C.sub.1-3 alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R.sup.2 is H or ##STR00187## where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of: (iia) C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group, wherein said C.sub.6-10carboaryl or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group, a C.sub.6-10 aryl group or a C.sub.5-10 heteroaryl group; (iib) C.sub.1-5 alkyl; (iic) C.sub.3-6 cycloalkyl; ##STR00188## wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2′ group is no more than 5; ##STR00189## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00190## where R.sup.24 is selected from: H; C.sub.1-3 alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2′ and C3′, R.sup.2′ is H or ##STR00191## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; 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 C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.6-20 aryl groups; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively; R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; and either (a) R.sup.30 is H, and R.sup.31 is OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R.sup.11a and R.sup.31 are SO.sub.zM, M may represent a divalent pharmaceutically acceptable cation R.sup.L is a linker for connection to an antibody Or an antigen-binding fragment of an antibody, which is selected from: ##STR00192## wherein Q is: ##STR00193## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is: ##STR00194## 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 an antibody or an antigen-binding fragment of an antibody; and ##STR00195## 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; wherein the heterocyclyl group in C.sub.3-7 heterocyclyl contains 1 to 4 ring heteroatoms selected from N, O and S; wherein the heterocyclyl group in C.sub.3-20 heterocyclyl contains 1 to 10 ring heteroatoms selected from N, O and S; and wherein the heteroaryl group in C.sub.5-10 heteroaryl contains 1 to 4 ring heteroatoms selected from N, O and S.

2. A compound according to claim 1, wherein: a) Y and Y′ are O, b) R.sup.O is H or methyl; c) R″ is of formula IIa: ##STR00196## where R.sup.O1 is selected from the group consisting of H and methyl; d) R.sup.6, R.sup.9, R.sup.6′ and R.sup.9′ are H, and R.sup.7 and R.sup.7′ are methoxy; e) R.sup.11a is OH; f) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

3. A compound according to claim 1 of: (i) formula Ia: ##STR00197## where R.sup.2a and R.sup.2a′ are the same and are selected from: ##STR00198## (ii) formula Ib: ##STR00199## (iii) formula Ic: ##STR00200## wherein R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.

4. A compound according to claim 2 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.

5. A compound according to claim 1, 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.

6. A compound according to claim 1, wherein R.sup.L is of formula IIIa and G.sup.L is selected from TABLE-US-00010 (G.sup.LL1-1) (G.sup.LL1-2) (G.sup.LL2) (G.sup.LL3-1) (G.sup.LL3-2) (G.sup.LL-4) (G.sup.LL5) (G.sup.LL6) (G.sup.LL7) (G.sup.LL8-1) (G.sup.LL8-2) (G.sup.LL9-1) (G.sup.LL9-2) G.sup.L10 G.sup.L11 G.sup.L12 G.sup.L13 where Ar represents phenylene, and X represents C.sub.1-4 alkyl.

7. A compound according to claim 1, wherein R.sup.L is of formula IIIb.

8. A conjugate comprising a compound of formula I according to claim 1, or a pharmaceutically acceptable salt thereof, linked to an antibody or an antigen-binding, fragment of an antibody.

9. A conjugate of formula IV:
L-(D.sup.L).sub.p  (IV) or a pharmaceutically acceptable salt thereof, wherein L is an antibody or an antigen-binding fragment of an antibody, D.sup.L is of formula III: ##STR00218## wherein: R″ is a group of formula II: ##STR00219## where each of n and m are independently selected from 1, 2 and 3; R.sup.O is selected from the group consisting of H, methyl, ethyl, iso-propyl and benzyl; Q.sup.C is selected from N and CH: Y and Y′ are selected from O, S, or NH; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: (ia) C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group, wherein said C.sub.6-10 carboaryl or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group or a C.sub.6-10 carboaryl or C.sub.5-10 heteroaryl group; (ib) C.sub.1-5 alkyl; (ic) C.sub.3-6 cycloalkyl; ##STR00220## wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; ##STR00221## wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00222## where R.sup.14 is selected from: H; C.sub.1-3 alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl: when there is a single bond present between C2 and C3, R.sup.2 is H or ##STR00223## where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of: (iia) C.sub.6-10 carboaryl group or a C.sub.5-10 heteroaryl group, wherein said C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group or a C.sub.6-10 carboaryl or a C.sub.5-10 heteroaryl group; (iib) C.sub.1-5 alkyl; (iic) C.sub.3-6 cycloalkyl; ##STR00224## wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2′ group is no more than 5; ##STR00225## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy: pyridyl; and thiophenyl; and ##STR00226## where R.sup.24 is selected from: H; C.sub.1-3 alkyl: C.sub.2-3 alkenyl: C.sub.2-3 alkynyl: cyclopropyl: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2′ and C3′, R.sup.2′ is H or ##STR00227## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; 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 C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.6-20 aryl groups; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively; R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; and either (a) R.sup.30 is H, and R.sup.31 is OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R.sup.11a and R.sup.31 are SO.sub.zM, M may represent a divalent pharmaceutically acceptable cation; R.sup.LL is a linker for connection to an antibody or an antigen-binding fragment of an antibody, which is selected from: ##STR00228## where Q is: ##STR00229## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; X is: ##STR00230## where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5; and G.sup.LL is a linker connected to an antibody or an antigen-binding fragment of an antibody; and ##STR00231## 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; wherein the heterocyclyl group in C.sub.3-7 heterocyclyl contains 1 to 4 ring heteroatoms selected from N, O and S; wherein the heterocyclyl group in C.sub.3-20 heterocyclyl contains 1 to 10 ring heteroatoms selected from N, O and S; and wherein the heteroaryl group in C.sub.5-10 heteroaryl contains 1 to 4 ring heteroatoms selected from N, O and S; wherein p is an integer of from 1 to 20.

10. A conjugate according to claim 9, wherein G.sup.LL is selected from: TABLE-US-00011 (G.sup.LL1-1) (G.sup.LL1-2) (G.sup.LL2) (G.sup.LL3-1) (G.sup.LL3-2) (G.sup.LL-4) (G.sup.LL5) (G.sup.LL6) (G.sup.LL7) (G.sup.LL8-1) (G.sup.LL8-2) (G.sup.LL9-1) (G.sup.LL9-2) G.sup.L10 G.sup.L11 G.sup.L12 G.sup.L13 where Ar represents phenylene and X represents C.sub.1-4 alkyl.

11. A conjugate according to claim 9, wherein D.sup.L is of: (i) formula IIIa: ##STR00249## where R.sup.2a and R.sup.2a′ are the same and are selected from: ##STR00250## (ii) formula IIIb: ##STR00251## (iii) formula IIIc: ##STR00252## where R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.

12. The conjugate according to claim 9, 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): (1) BMPR1B; (2) E16; (3) STEAP1; (4) 0772P; (5) MPF; (6) Napi3b; (7) Sema 5b; (8) PSCA hlg; (9) ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20R-alpha; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; (35) IRTA2; (36) TENB2; (37) PSMA—FOLH1; (38) SST; (38.1) SSTR2; (38.2) SSTR5; (38.3) SSTR1; (38.4) SSTR3; (38.5) SSTR4; (39) ITGAV; (40) ITGB6; (41) CEACAM5; (42) MET; (43) MUCI; (44) CA9; (45) EGFRvIII; (46) CD33; (47) CD19; (48) IL2RA; (49) AXL; (50) CD30—TNFRSF8; (51) BCMA—TNFRSF17; (52) CT Ags—CTA; (53) CD174 (Lewis Y)—FUT3; (54) CLEC14A; (55) GRP78—HSPA5; (56) CD70; (57) Stem Cell specific antigens; (58) ASG-5; (59) ENPP3; (60) PRR4; (61) GCC—GUCY2C; (62) Liv-1—SLC39A6; (63) 5T4; (64) CD56—NCMA1; (65) CanAg; (66) FOLR1; (67) GPNMB; (68) TIM-1—HAVCR1; (69) RG-1/Prostate tumor target Mindin—Mindin/RG-1; (70) B7-H4—VTCN1; (71) PTK7; (72) CD37; (73) CD138—SDC1; (74) CD74; (75) Claudins—CLs; (76) EGFR; (77) Her3; (78) RON—MST1R; (79) EPHA2; (80) CD20—MS4A1; (81) Tenascin C—TNC; (82) FAP; (83) DKK-1; (84) CD52; (85) CS1—SLAMF7; (86) Endoglin—ENG; (87) Annexin A1—ANXA1; (88) V-CAM (CD106)—VCAM1.

13. A composition comprising a mixture of conjugates according to claim 9, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.

14. A pharmaceutical composition comprising the conjugate of claim 8 and a pharmaceutically acceptable diluent, carrier or excipient.

15. A method of treating a mammal having gastric carcinoma, gastrointestinal cancer or leukaemia, comprising administering an effective amount of a conjugate of claim 8 or a pharmaceutical composition of claim 14.

16. A compound of formula REL: ##STR00253## or a pharmaceutically acceptable salt thereof, wherein: R″ is a group of formula II: ##STR00254## where each of n and m are independently selected from 1, 2 and 3; R.sup.O is selected from the group consisting of H, methyl, ethyl, iso-propyl and benzyl; Q.sup.C is selected from N and CH, Y and Y′ are selected from O, S, or NH; when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of: (ia) C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group, wherein said C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group or a C.sub.6-10 carboaryl or a C.sub.5-10 heteroaryl group; (ib) C.sub.1-5 alkyl; (ic) C.sub.3-6 cycloalkyl; ##STR00255## wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5; ##STR00256## wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00257## where R.sup.14 is selected from: H; C.sub.1-3 alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2 and C3, R.sup.2 is H or ##STR00258## where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of: (iia) C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group, wherein said C.sub.6-10 carboaryl group or C.sub.5-10 heteroaryl group is optionally substituted by one or more substituents selected from the group consisting of: halo, nitro, cyano, —OR, carboxy, —C(═O)OR, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene, wherein R is selected from a C.sub.1-7 alkyl group, a C.sub.3-20 heterocyclyl group or a C.sub.6-10 carboaryl or a C.sub.5-10 heteroaryl group; (iib) C.sub.1-5 alkyl; (iic) C.sub.3-6 cycloalkyl; ##STR00259## wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2′ group is no more than 5; ##STR00260## wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and ##STR00261## where R.sup.24 is selected from: H; C.sub.1-3 alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; when there is a single bond present between C2′ and C3′, R.sup.2′ is H or ##STR00262## where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester; 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 C.sub.1-12 alkyl, C.sub.3-20 heterocyclyl and C.sub.6-20 aryl groups; R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo; R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively; R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; and either (a) R.sup.30 is H, and R.sup.31 is OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl; (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R.sup.11a and R.sup.31 are SO.sub.zM, M may represent a divalent pharmaceutically acceptable cation; wherein the heterocyclyl group in C.sub.3-7 heterocyclyl contains 1 to 4 ring heteroatoms selected from N, O and S; wherein the heterocyclyl group in C.sub.3-20 heterocyclyl contains 1 to 10 ring heteroatoms selected from N, O and S; and wherein the heteroaryl group in C.sub.5-10 heteroaryl contains 1 to 4 ring heteroatoms selected from N, O and S.

Description

EXAMPLES

(1) General Information

(2) Flash chromatography was performed using a Biotage Isolera 1™ using gradient elution with hexane/EtOAc or CH.sub.2Cl.sub.2/MeOH mixtures as indicated until all UV active components 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 VWR.

(3) The analytical LC/MS conditions (for reaction monitoring and purity determination) were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H.sub.2O with 0.1% formic acid) and solvent B (CH.sub.3CN with 0.1% formic acid).

(4) LCMS-A: Gradient for 3-minute run: Initial composition 5% B held over 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 seconds' period. The composition was held for 50 seconds at 100% B, then returned to 5% B in 5 seconds and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes at a flow rate of 0.8 mL/min. Column: 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

(5) LCMS-B: Gradient for 15-minute run: Initial composition 5% B held over 1 minute, then increased from 5% B to 100% B over a 9 minute period. The composition was held for 2 minutes at 100% B, then returned to 5% B in 10 seconds and held there for 2 minutes 50 seconds. The total duration of the gradient run was 15.0 minutes at a flow rate of 0.6 mL/minute. Detection was monitored at 254 nm, 223 nm and 214 nm. ACE Excel 2 C18-AR, 2μ, 3.0×100 mm fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

(6) Intermediates

(7) TABLE-US-00003 Structure Reference I1 WO2011/130598; Compound 8 I2 WO2014/159981; Compound 1 I3 WO2013/053872; Compound 26 I4 WO2013/053872; Compound 32 I5 See below

Synthesis of tert-butyl(11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(I5)

(8) ##STR00097##

(9) I6 is Compound 152 of WO 2018/182341

(i) tert-Butyl (S)-(2-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (I7)

(10) I6 (400 g, 1 eq.), CH.sub.2Cl.sub.2 (4 L) and Boc.sub.2O (206.7 g, 1.3 eq.) were added to a reactor and stirred for 20 h at 70° C. The mixture was cooled to rt and concentrated in vacuo to afford 17 (450 g, 95%) which was used without further purification.

(ii) tert-Butyl (S)-(2-(2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (I8)

(11) I7 (210 g, 1.0 eq.) THE (76 mL), MeOH (76 mL) AcOH (533 mL) and H.sub.2O (151 mL) were added to a reactor and stirred for 20 h. The mixture was diluted with EtOAc (30 V) and H.sub.2O (20 V) and the mixture vigorously stirred. The organic phase was washed with sat. NaHCO.sub.3 solution (20 V), then brine (20 V) and dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Flash column chromatography (8:1-2:1 petroleum ether/EtOAc) afforded I8 as a white solid (150 g, 87%).

(iii) tert-Butyl (11S,11aS)-11-hydroxy-7-methoxy-2-methylene-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (I9)

(12) (COCl).sub.2 (28.5 g, 1.0 eq.) was added to CH.sub.2Cl.sub.2 (900 mL) in a reactor under N.sub.2 and the mixture was cooled to −80° C. A solution of 18 (120 g, 1.0 eq.) in CH.sub.2Cl.sub.2 (600 mL) was added dropwise over 30 min, then Et.sub.3N (113.5 g, 5.0 eq.) was added dropwise over 30 min. The mixture was warmed to rt and stirred for 5 h the quenched by the addition of 5% aqueous citric acid (10 V). The organic phase was washed successively with sat. NaHCO.sub.3 (5 V) and H.sub.2O (5 V), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude product was stirred in heptane (10 V) at 90° C., then cooled to 0-5° C. The resulting white precipitate was isolated by filtration and dried in a vacuum oven at 50° C. to afford 19 (105 g, 88%).

(iv) tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methylene-5-oxo-8-((triisopropylsilyl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (I10)

(13) I9 (150 g, 1.0 eq.) and CH.sub.2Cl.sub.2 (1.5 L, 10 V) were charged to a reactor and the mixture cooled to 0-5° C. Lutidine (120.6 g, 4.0 eq.), then TBSOTf (223.2 g, 3.0 eq.) were added and the mixture was stirred for 10 min at 0-10° C., then 4 h at rt. The reaction was quenched by the addition of 5% aqueous citric acid (5 V) and the organic phase was successively washed with sat. NaHCO.sub.3 (5 V), H.sub.2O (5 V) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford 5 (179 g, 98%) as an orange oil.

(v) tert-Butyl (11S,11aS)-11-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (I5)

(14) DMF (1.83 L, 1 V), H.sub.2O (37.4 mL, 0.2 V) and I10 (187 g, 1.0 eq.) were charged to a reactor. LiOAc (19.1 g, 1.0 eq.) was added and the mixture stirred for 20 h at 10-20° C. The reaction mixture was diluted with cold water (10 V) and EtOAc (20 V). The aqueous phase was washed with EtOAc (10 V), the organic phases combined, successively washed with 5% aqueous citric acid (10 V), 5% aqueous NaHCO.sub.3 (10 V) and H.sub.2O (10 V) and dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The resulting crude was dissolved in EtOAc (6 V) at 77° C., heptane (12 V) was added and the mixture cooled to 0-5° C., the white precipitate collected by filtration and dried at 50° C. in a vacuum oven to afford 6 (101 g, 71%).

Example 1: 3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (19)

(15) ##STR00098## ##STR00099##

(i) (((5-iodo-1,3-phenylene)bis(methylene))bis(oxy))bis(tert-butyldimethylsilane) (2)

(16) A round-bottomed flask was charged with a magnetic stirrer bar, DMF (100 mL), diol 1 (10.0 g, 37.87 mmol, 1.0 eq.), TBSCl (17.1 g, 113.6 mmol, 3.0 eq.) imidazole (15.5 g, 227.2 mmol, 6.0 eq.) and the mixture stirred for 20 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was filtered, diluted with EtOAc, washed with water, the organic phase dried over MgSO.sub.4 and concentrated in vacuo. Isolera (0-8% EtOAc in hexane) afforded the desired product as a colourless oil (17.9 g, 96%). LCMS-A: 2.29 min (ES+) no ionisation.

(ii) tert-butyl 3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)benzoate (3)

(17) An oven-dried 3-necked round-bottomed flask was charged with a magnetic stirrer bar anhydrous toluene (7.5 mL) and the flask purged with Ar and the flask cooled to −10° C. BuLi (5.6 mL of a 2.5 M solution in hexanes, 14.00 mmol, 2.0 eq.), then slowly BuMgCl (3.5 mL of a 2.0 M solution in THF, 7.000 mmol, 1.0 eq.) were added and stirred for 30 min. 2 (8.21 g, 16.67 mmol, 2.4 eq.) in anhydrous toluene (15 mL) was added slowly and stirred for 2 h at −10° C. Boc.sub.2O (4.55 g, 20.84 mmol, 3.0 eq.) in anhydrous toluene (7.5 mL) was added slowly at −10° C. and stirred for 1 h, then poured into cold 10% citric acid. The aqueous phase was extracted with EtOAc, the organics combined, dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (0-20% EtOAc in hexane afforded the product as a colourless oil (7.39 g, 95%). LCMS-A: 1.48 min (ES+) no ionisation.

(iii) tert-butyl 3,5-bis(hydroxymethyl)benzoate (4)

(18) A round-bottomed flask was charged with a magnetic stirrer, 3 (7.39 g, 15.83 mmol, 1.0 eq.) and THE (75 mL). TBAF (31.7 mL of a 1M solution, 31.70 mmol, 2.0 eq.) was added by syringe and stirred for 20 min whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with EtOAc, washed with H.sub.2O, dried over MgSO.sub.4 and concentrated in vacuo to afford the product as a white crystalline solid (3.29 g, 87%). LCMS-A: 1.24 min (ES+) no ionisation.

(iv) tert-butyl 3,5-bis(bromomethyl)benzoate (5)

(19) A round-bottomed flask was charged with a magnetic stirrer, 4 (3.29 g, 15.83 mmol, 1.0 eq.), CH.sub.2Cl.sub.2 (200 mL) and cooled to −78° C. PPh.sub.3 (17.4 g, 66.34 mmol, 4.2 eq.) was added, then CBr.sub.4 (23.1 g, 69.65 mmol, 4.4 eq.) was added portionwise and the reaction allowed to warm to rt. The reaction was filtered through SiO.sub.2 (CH.sub.2Cl.sub.2/hexane) and purified by isolera (0-20% EtOAc in hexane) to afford the product as a white solid (1.43 g, 26%). LCMS-A: 1.81 min (ES+) no ionisation.

(v) Methyl (S)-1-(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)benzoyl)-4-methylenepyrrolidine-2-carboxylate (8)

(20) A round-bottomed flask was charged with a magnetic stirrer, THE (15 mL), 6 (1.89 g, 5.118 mmol, 1.0 eq.), HOBt (761 mg, 5.630 mmol, 1.1 eq.), DIC (872 μL, 5.630 mmol, 1.1 eq.) and i-Pr.sub.2NEt (1.96 mL, 11.26 mmol, 2.2 eq.) and the mixture was stirred for 10 min. 7 (1.00 g, 5.630 mmol, 1.1 eq.) was added portionwise and the reaction mixture was stirred for 1 h. The reaction was diluted with CH.sub.2Cl.sub.2, washed with 0.2N HCl solution, the organics dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (20-30% EtOAc in hexane) afforded the desired product as a yellow oil (1.19 g, 47%). LCMS-A: 2.00 min (ES+) m/z 493 [M+H].sup.+, 515 [M+Na].sup.+

(vi) (S)-(2-(hydroxymethyl)-4-methylenepyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (9)

(21) A round-bottomed flask was charged with a magnetic stirrer, THE (15 mL), 8 (1.17 g, 2.383 mmol, 1.0 eq.) and the stirring mixture was cooled to 0° C. Cautiously, LiBH.sub.4 (156 mg, 7.149 mmol, 3.0 eq.) was added and the mixture was stirred for 1 h at 0° C. and a further 1.5 h at rt. The reaction was quenched by the addition of ice-cold H.sub.2O and the pH adjusted to ca. 6 with 1N HCl. The aqueous mixture was extracted with CH.sub.2Cl.sub.2, the organics combined, dried over MgSO.sub.4 and concentrated in vacuo to afford the desired product as a yellow foam (1.06 g, 96%) which was used without further purification. LCMS-A: 1.92 min (ES+) m/z 465 [M+H].sup.+, 487 [M+Na].sup.+

(vii) (S)-(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)(5-methoxy-2-nitro-4-((triisopropylsilyl)oxy)phenyl)methanone (10)

(22) A round-bottomed flask was charged with a magnetic stirrer, CH.sub.2Cl.sub.2 (10 mL), 9 (1.06 g, 2.281 mmol, 1.0 eq.), imidazole (467 mg, 6.858 mmol, 3.0 eq.) TBSCl (517 mg, 3.429 mmol, 1.5 eq.) and stirred for 1 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was filtered, the filtrate washed with H.sub.2O, brine, dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (20-30% EtOAc in hexane) afforded the desired product as a yellow oil (1.27 g, 96%). LCMS-A: 2.30 min (ES+) m/z 579 [M+H].sup.+, 601 [M+Na].sup.+

(viii) (S)-(2-amino-5-methoxy-4-((triisopropylsilyl)oxy)phenyl)(2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidin-1-yl)methanone (11)

(23) A three-necked round-bottomed flask was charged with a magnetic stirrer, 10 (10.0 g, 17.27 mmol, 1.0 eq.), EtOH (170 mL), Zn (23.6 g, 345.4 mmol, 20 eq.) and cooled to 0° C. 5% HCO.sub.2H in EtOH (340 mL) was added dropwise by dropping funnel and stirred for 30 min, whereupon, LCMS indicated reaction was complete. The reaction mixture was diluted with EtOAc (200 mL) and filtered through Celite. Sat. NaHCO.sub.3 solution was added to the filtrate until the pH of the aqueous phase was basic. The phases were separated, the aqueous phase was washed with EtOAc (2×100 mL), the organic phases combined, washed with brine, dried over MgSO.sub.4 and concentrated in vacuo to afford a yellow oil which was used without further purification (8.99 g, 95%). LCMS-A: 2.23 min (ES+) m/z 549 [M+H].sup.+, 571 [M+Na].sup.+

(ix) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-4-methoxy-5-((triisopropylsilyl)oxy)phenyl)carbamate (12)

(24) A round-bottomed flask was charged with a magnetic stirrer, 11 (8.99 g, 16.38 mmol, 1.0 eq.), THE (90 mL), Et.sub.3N (5.4 mL, 35.90 mmol, 2.2 eq.), the flask purged with Ar and the mixture cooled to −10° C. Triphosgene (1.75 g, 5.897 mmol, 0.36 eq.) was added in one portion over a flow of Ar. After 5 min, LCMS indicated isocyanate formation complete (by way of MeOH quench and observation of methyl carbamate). I1 (9.27 g, 24.57 mmol, 1.5 eq.) was added in one portion over a flow of Ar and the reaction mixture was warmed to room temperature and stirred for 24 h. The reaction mixture was filtered, concentrated onto SiO.sub.2 in vacuo and purified by flash-column chromatography (0-3% MeOH in CH.sub.2Cl.sub.2) to afford the product (10.9 g, 70%). LCMS-A: 2.20 min (ES+) m/z 952 [M+H].sup.+, 974 [M+Na].sup.+

(x) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (2-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-5-hydroxy-4-methoxyphenyl)carbamate (13)

(25) A round-bottomed flask was charged with a magnetic stirrer, silyl ether 12 (10.9 g, 11.47 mmol, 1.0 eq.) DMF (85 mL), H.sub.2O (1.7 mL) and LiOAc (1.17 g, 11.47 mmol, 1.0 eq.). The reaction mixture was stirred for 16 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with EtOAc (150 mL) and washed successively with H.sub.2O (50 mL) and brine (50 mL). The organic phase was dried over MgSO.sub.4 and purified by Isolera chromatography (0-4% MeOH in CH.sub.2Cl.sub.2) to afford the product as a yellow foam (6.65 g, 73%). LCMS-A: 1.78 min (ES+) m/z 796 [M+H].sup.+, 818 [M+Na].sup.+

(xi) tert-butyl 3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-5-(bromomethyl)benzoate (14)

(26) A round-bottomed flask was charged with a magnetic stirrer, phenol 13 (1.09 g, 1.364 mmol, 1 eq.), bis bromo 5 (1.43 g, 4.093 mmol, 3.0 eq.), DMF (10 mL) and K.sub.2CO.sub.3 (207 mg, 1.500 mmol, 1.1 eq.) and the reaction stirred for 2.5 h at 35° C., whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with H.sub.2O and washed with CH.sub.2Cl.sub.2, dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (10-50% EtOAc in hexane) afforded the product as an off-white foam (946 mg, 64%). LCMS-A: 2.06 min (ES+) m/z 1100.20 [M+Na].sup.+

(xii) (11S,11 aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-5-(tert-butoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (15)

(27) A round-bottomed flask was charged with a magnetic stirrer, 14 (946 mg, 0.8766 mmol, 1.0 eq.), 12 (444 mg, 0.9643 mmol, 1.1 eq.), DMF (12 mL) and K.sub.2CO.sub.3 (133 mg, 0.9643 mmol, 1.1 eq.) were stirred for 3.5 h at 35° C. whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with H.sub.2O, washed with EtOAc and the organic phase was dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (40-100% EtOAc in hexane afforded the product as a white foam (1.04 g, 82%). LCMS-A: 2.11 min (ES+) m/z 1458.20 [M+H].sup.+, 1480.35 [M+Na].sup.+

(xiii) tert-butyl (11S,11 aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-5-(tert-butoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (16)

(28) A round-bottomed flask was charged with a magnetic stirrer, 15 (1.04 g, 0.7129 mmol, 1.0 eq.), THE (40 mL) AcOH (82 μL, 1.430 mmol, 2.0 eq.) and TBAF (2.1 mL of a 1M solution, 2.100 mmol, 3.0 eq.) and the reaction stirred for 1 h whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CHCl.sub.3 and washed with sat. NaHCO.sub.3, the organic phase dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (0-4% MeOH in CH.sub.2Cl.sub.2) afforded the product as a white foam (961 mg, 100%). LCMS-A: 1.86 min (ES+) m/z 1344.70 [M+H].sup.+, 1366.85 [M+Na].sup.+

(xiv) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-((3-(tert-butoxycarbonyl)-5-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (17)

(29) A round-bottomed flask was charged with a magnetic stirrer, 16 (961 mg, 0.7150 mmol, 1.0 eq.) DMSO (7 mL) and IBX (489 mg, 0.7865, 1.1 eq.) and the reaction mixture stirred at 35° C. for 16 h. IBX (50 mg, 80.35 μmol, 0.11 eq.) was added and the reaction mixture stirred for 48 h, whereupon the reaction mixture was poured into water and washed with 10% MeOH in CH.sub.2Cl.sub.2, the organic layers combined, dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (0-4% MeOH in CH.sub.2Cl.sub.2) afforded the product as a white foam (492 mg, 51%). Mixed fractions were purified again by isolera (0-2.5% MeOH in CH.sub.2Cl.sub.2) affording the product as a white solid (302 mg, 31%). Both clean fractions were combined to afford the product as a white foam (794 mg, 83%). LCMS-A: 1.83 min (ES+) m/z 1364.55 [M+Na].sup.+

(xv) tert-butyl (11S,11aS)-8-((3-(tert-butoxycarbonyl)-5-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (18)

(30) Around-bottomed flask was charged with a magnetic stirrer, 17 (794 mg, 0.5914 mmol, 1.0 eq.), CH.sub.2Cl.sub.2 (50 mL), pyrrolidine (121 μL, 1.479 mmol, 2.5 eq.) and the flask purged with Ar. Pd(PPh).sub.4 (34 mg, 29.58 μmol, 0.050 eq.) was added over a flow of Ar and the reaction mixture stirred for 10 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was thoroughly concentrated in vacuo overnight to remove pyrrolidine traces. The resulting residue was dissolved in CH.sub.2Cl.sub.2 (10 mL) and maleimide propionic acid (78 mg, 0.4612, 1.1 eq.), EDCl.HCl (880 mg, 0.4589 mmol, 1.1 eq.) and i-Pr.sub.2NEt (218 μL, 1.252 mmol, 3.0 eq.) were added and the mixture stirred for 5 h, whereupon LCMS indicated the reaction was incomplete. A further 0.1 eq of EDCl.HCl, 0.1 eq. of maleimide propionic acid and 0.3 eq. of i-Pr.sub.2NEt were added and the mixture stirred for 16 h, whereupon LCMS indicated the reaction was incomplete. A further 0.2 eq of EDCl.HCl, 0.2 eq. of maleimide propionic acid and 0.6 eq. of i-Pr.sub.2NEt were added and the mixture stirred for 24 h, diluted with CH.sub.2Cl.sub.2 (20 mL), washed with H.sub.2O (10 mL) the organic phase dried over MgSO.sub.4 and concentrated in vacuo. Isolera chromatography (2-6% MeOH in CH.sub.2Cl.sub.2) afforded the desired product (205 mg, 35%). LCMS-A: 1.77 min (ES+) m/z 1410 [M+H].sup.+, 1431 [M+Na].sup.+

(xvi) 3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (19)

(31) A round-bottomed flask was charged with a magnetic stirrer, 18 (205 mg, 0.1454 mmol, 1.0 eq.) and cooled to ca. −5° C. 95:5 v/v TFA/H.sub.2O was cooled to ca. −20° C. and added to the reaction flask and stirred for 75 min, whereupon LCMS indicated the reaction was complete. The pH of the reaction mixture was adjusted to ca. 7 by the cautious addition of sat. NaHCO.sub.3, whereupon a precipitate formed. The aqueous mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Preparative HPLC (15-70% A in B) afforded the product as a white solid (52 mg, 31%, QC=94%). A=0.05% formic acid in acetonitrile. B=0.05% formic acid in water

(32) LCMS-B: 7.13 min (ES+) m/z 1151 [M+H].sup.+.

Example 2: 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (27)

(33) ##STR00100##

(i) Methyl 3,5-bis(((tert-butyldimethylsilyl)oxy)methyl)benzoate (20)

(34) An oven-dried 3-necked round-bottomed flask was charged with a magnetic stirrer bar anhydrous toluene (15 mL) and the flask purged with Ar and the flask cooled to −10° C. BuLi (9.9 mL of a 2.5 M solution in hexanes, 24.86 mmol, 2.0 eq.), then slowly BuMgCl (6.2 mL of a 2.0 M solution in THF, 12.40 mmol, 1.0 eq.) were added and stirred for 30 min. 2 (14.7 g, 29.84 mmol, 2.4 eq.) in anhydrous toluene (30 mL) was added slowly and stirred for 2 h at −10° C. ClCO.sub.2Me (2.9 mL, 37.29 mmol, 3.0 eq.) in anhydrous toluene (15 mL) was added slowly at −50° C. and stirred for 2 h, then poured into cold 10% citric acid. The aqueous phase was extracted with EtOAc, the organics combined, washed with NaHCO.sub.3, dried over MgSO.sub.4 and concentrated in vacuo. Isolera purification (0-20% EtOAc in hexane afforded the product as a pale-yellow oil (11.5 g, 91%). LCMS-A: 2.18 min (ES+) m/z 425 [M+H].sup.+

(ii) Methyl 3,5-bis(bromomethyl)benzoate (21)

(35) A round-bottomed flask was charged with a magnetic stirrer, AcOH (15 mL), 20 (1.00 g, 2.354 mmol, 1.0 eq.) and HBr (33% in AcOH) (10 mL) and the mixture was stirred for 2 h at 50° C. The reaction mixture was cooled to rt, poured into ice-cold water, the resulting precipitate collected by filtration, dissolved in CH.sub.2Cl.sub.2, dried over MgSO.sub.4 and concentrated in vacuo. Isolera (0-50% CH.sub.2Cl.sub.2 in hexane) afforded the desired product as an off-white solid (460 mg, 61%). LCMS-A: 1.59 min (ES+) No ionisation

(iii) tert-Butyl (11S,11aS)-8-((3-(bromomethyl)-5-(methoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (22)

(36) A round-bottomed flask was charged with a magnetic stirrer, DMF (20 mL), 12 (256 mg, 0.5559 mmol, 1.0 eq.) 21 (716 mg, 2.224 mmol, 4.0 eq.) and K.sub.2CO.sub.3 (84.5 mg, 0.6115 mmol, 1.1 eq.). The mixture was stirred for 1 h 45 min, whereupon LCMS indicated the reaction was complete. DMF was removed in vacuo, the resulting residue dissolved in CH.sub.2Cl.sub.2 and washed with sat. NH.sub.4Cl solution. The organic phase was collected, dried over MgSO.sub.4 and purified by isolera (10-60% EtOAc in heptane) to afford the desired product as a white solid (234 mg, 60%). LCMS-A: 1.77 min (ES+) m/z 701 [M+H].sup.+, 723 [M+Na].sup.+

(iv) tert-Butyl (11S,11aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (23)

(37) A round-bottomed flask was charged with a magnetic stirrer, DMF (2 mL), 22 (234 mg, 0.3335 mmol, 1.0 eq.), 13 (292 mg, 0.3669 mmol, 1.1 eq.) and K.sub.2CO.sub.3 (51 mg, 0.3690 mmol, 1.1 eq.). The mixture was stirred for 4 h whereupon LCMS indicated the reaction was complete. DMF was removed in vacuo, the resulting residue dissolved in CH.sub.2Cl.sub.2 and washed with sat. NH.sub.4Cl solution. The organic phase was collected, dried over MgSO.sub.4 and purified by isolera (0-3% MeOH in CH.sub.2Cl.sub.2) to afford the desired product as a white solid (450 mg, 95%). LCMS-A: 2.00 min (ES+) m/z 1416 [M+H].sup.+, 1438 [M+Na].sup.+

(v) tert-Butyl (11S,11aS)-8-((3-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (24)

(38) A round-bottomed flask was charged with a magnetic stirrer, THE (18 mL), 23 (450 mg, 0.3176 mmol, 1.0 eq.), AcOH (36.3 μL, 0.6352 mmol, 2.0 eq.) and TBAF (953 μL of 1M solution in THF, 0.9530 mmol, 3.0 eq.). The mixture was stirred for 7 h at rt, then a few drops of TBAF were added and stirred at rt for a further 3 h, whereupon LCMS indicated reaction was complete. The reaction mixture was diluted with EtOAc and washed with H.sub.2O, then brine, dried over MgSO.sub.4 and concentrated in vacuo. Isolera (0-4% MeOH in CH.sub.2Cl.sub.2) afforded the desired product with Bu.sub.4N+ salts as an inseparable mixture. The resulting material was used without further purification. LCMS-A: 1.73 min (ES+) m/z 1302 [M+H].sup.+, 1324 [M+Na].sup.+

(vi) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-((3-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (25)

(39) A round-bottomed flask was charged with a magnetic stirrer, DMSO (3 mL), 24 (ca. 0.3176 mmol, 1.0 eq.) and IBX (45% wt.) (237 mg, 0.3811 mmol, 1.2 eq.). The mixture was stirred at rt for 16 h, then a knifepoint of IBX was added and was stirred for 35° C. for 24 h. The reaction mixture was diluted with H.sub.2O and extracted with CHCl.sub.3. The organics were combined, dried over MgSO.sub.4 and concentrated in vacuo. Isolera (0-4% MeOH in CH.sub.2Cl.sub.2) afforded the desired product as a white solid (333 mg, 81% (2 steps)). LCMS-A: 1.71 min (ES+) m/z 1300 [M+H].sup.+, 1323 [M+Na].sup.+

(vii) tert-butyl (11S,11aS)-8-((3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (26)

(40) A round-bottomed flask was charged with a magnetic stirrer, CH.sub.2Cl.sub.2 (19 mL), 25 (333 mg, 0.2561 mmol, 1.0 eq.), pyrrolidine (42.1 μL, 0.5122 mmol, 2.0 eq.) and Pd(PPh).sub.4 (7.4 mg, 6.403 μmol, 0.025 eq.). The reaction mixture was stirred for 20 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was thoroughly concentrated in vacuo overnight to remove pyrrolidine traces. The resulting residue was dissolved in CH.sub.2Cl.sub.2 (3.5 mL) and maleimide propionic acid NHS ester (82 mg, 0.3080 mmol, 1.2 eq.) and i-Pr.sub.2NEt (53.5 μL, 0.3073 mmol, 1.2 eq.) were added and the mixture stirred for 2 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera (0-4% MeOH in CH.sub.2Cl.sub.2) to afford the desired product as a white solid (150 mg, 43%). LCMS-A: 1.65 min (ES+) m/z 1368 [M+H].sup.+, 1389 [M+Na].sup.+

(viii) 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (27)

(41) A round-bottomed flask was charged with a magnetic stirrer, 26 (150 mg, 0.1097 mmol, 1.0 eq.) and cooled to 0° C. 95% TFA in H.sub.2O was cooled to −20° C. and added to the reaction flask in one portion and the mixture stirred for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was poured into ice cold sat. NaHCO.sub.3 solution and solid NaHCO.sub.3 was added until the pH was approximately neutral. The aqueous mixture was extracted with 10% MeOH in CH.sub.2Cl.sub.2, the organics combined, dried over MgSO.sub.4 and concentrated in vacuo. Purification by isolera (0-5% MeOH in CH.sub.2Cl.sub.2) afforded the desired product as a white solid (52.8 mg, 41% yield, 98.1% QC). LCMS-A: 7.95 min (ES+) m/z 1165 [M+H].sup.+.

Example 3: 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(33) and 3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (34)

(42) ##STR00101##

(i) Allyl (11S,11aS)-8-((3-(bromomethyl)-5-(methoxycarbonyl)benzyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (28)

(43) A round-bottomed flask was charged with a magnetic stirrer, DMF (15 mL), 13 (184 mg, 0.3883 mmol, 1.0 eq.) 21 (500 mg, 1.553 mmol, 4.0 eq), K.sub.2CO.sub.3 (59 mg, 0.4271 mmol, 1.1 eq.) and TBAl (3.6 mg, 9.708 μmol, 0.025 eq.) and the mixture stirred at 65° C. for 16 h. DMF was removed in vacuo, the resulting residue dissolved in EtOAc and washed with 1N HCl solution. The organic phase was dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (0-30% EtOAc in hexane) afforded the desired product (183 mg, 69%). LCMS-A: 2.06 min (ES+) m/z 714 [M+H].sup.+, 737 [M+Na].sup.+

(ii) Allyl (11S,11aS)-8-((3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (29)

(44) A round-bottomed flask was charged with a magnetic stirrer, DMF (1 mL) 28 (128 mg, 0.1788 mmol, 1.0 eq.), 14 (353 mg, 0.4446 mmol, 2.5 eq.), K.sub.2CO.sub.3 (27 mg, 0.1967 mmol, 1.1 eq.) and TBAl (1.7 mg, 4.470 μmol, 0.025 eq.) and the mixture was stirred for 20 h at 65° C. DMF was removed in vacuo, the resulting residue dissolved in EtOAc and washed with H.sub.2O, then brine. The organic phase was dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (0-3% MeOH in CH.sub.2Cl.sub.2) afforded the desired product (117 mg, 46%). LCMSA: 2.20 min (ES+) m/z 1428 [M+H].sup.+, 1450 [M+Na].sup.+

(iii) 3-((((11S,11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (30)

(45) A microwave vial was charged with a magnetic stirrer, CHCl.sub.3 (1 mL) 29 (100 mg, 0.06999 mmol, 1.0 eq.) SnMe.sub.3OH (127 mg, 0.6999 mmol, 10 eq.) and irradiated at 100° C. for 10 h.

(46) The reaction mixture was filtered through Celite® and the filtrate concentrated in vacuo. The resulting residue was dissolved in THE (1 mL) and H.sub.2O (1 mL), K.sub.2CO.sub.3 (20 mg, 0.1400 mmol, 2.0 eq.) and allyl chloroformate (14 μL, 0.1400 mmol, 2.0 eq.) were added. The mixture was stirred for 2 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted (EtOAc), washed with 1N HCl solution, dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (70-100% EtOAc in hexane, 0.1% AcOH) afforded the desired product (67 mg, 68%). LCMS-A: 2.13 min (ES+) m/z 1414 [M+H].sup.+, 1436 [M+Na].sup.+

(iv-a) Allyl (11S,11aS)-8-((3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-11-hydroxy-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (31)

(47) A round-bottomed flask was charged with a magnetic stirrer, THE (10 mL), 29 (200 mg, 0.1400 mmol, 1.0 eq.), AcOH (32 μL, 0.5600 mmol, 4.0 eq.) and TBAF (560 μL of a 1M solution, 0.5600 mmol, 4.0 eq.) were added and the reaction was stirred for 16 h with gradual warming to rt, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with sat. NH.sub.4Cl, dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (3% MeOH in CHCl.sub.3) afforded the desired product (140 mg, 83%). LCMS-A: 1.62 min (ES+) m/z 1200 [M+H].sup.+, 1222 [M+Na].sup.+

(iv-b) 3-((((11S,11aS)-10-((allyloxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (32)

(48) A round-bottomed flask was charged with a magnetic stirrer, THE (10 mL), 30 (135 mg, 0.09542 mmol, 1.0 eq.), cooled to 0° C. and stirred for 10 minutes. AcOH (21.9 μL, 0.3817 mmol, 4.0 eq.) and TBAF (429 μL of a 1M solution, 0.4290 mmol, 4.0 eq.) were added and the reaction was stirred for 16 h with gradual warming to rt, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with sat. NH.sub.4Cl, dried over MgSO.sub.4 and concentrated in vacuo. Flash column chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) afforded the product contaminated with Bu.sub.4N salts. The resulting product was used without further purification and the yield was assumed to be 100%. LCMS-A: 1.63 min (ES+) m/z 1186 [M+H].sup.+, 1208 [M+Na].sup.+

(v-a) 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-((3-((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-(methoxycarbonyl)benzyl)oxy)-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (33)

(49) A round-bottomed flask was charged with a magnetic stirrer, CH.sub.2Cl.sub.2 (10 mL), 31 (120 mg, 99.98 μmol, 1.0 eq.), pyrrolidine (21 μL, 250 μmol, 2.5 eq.) and Pd(PPh).sub.4 (5.8 mg, 4.999 μmol, 0.050 eq.). The mixture was stirred under Ar for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was thoroughly concentrated in vacuo overnight to remove pyrrolidine traces. The resulting residue was dissolved in CH.sub.2Cl.sub.2 (3.5 mL) and maleimide propionic acid (14.9 mg, 88.31 μmol, 1.0 eq.) and EDCl.HCl (13.7 mg, 88.31 μmol, 1.0 eq.) were added and the mixture stirred for 2 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by flash column chromatography (3-5% MeOH in CH.sub.2Cl.sub.2) then preparative HPLC (15-90% A in B) to afford the desired product as a yellow solid (10 mg, 9.7% yield). A=0.05% formic acid in acetonitrile. B=0.05% formic acid in water. LCMS-B: 6.78 min (ES+) m/z 1165 [M+H].sup.+, 1187 [M+Na].sup.+

(v-b) 3-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-5-((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (34)

(50) A round-bottomed flask was charged with a magnetic stirrer, CH.sub.2Cl.sub.2 (8 mL) 32 (crude from previous step ca. 95.42 μmol, 1.0 eq.) pyrrolidine (19.6 μL, 238.6 μmol, 2.5 eq.), Pd(PPh).sub.4 (5.5 mg, 4.771 μmol, 0.050 eq.) and the mixture stirred for 30 min under Ar, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CHCl.sub.3, washed with sat. NH.sub.4Cl, the organics combined, dried over MgSO.sub.4 and concentrated in vacuo. The resulting residue was dissolved in THE (7 mL) and NaHCO.sub.3 (1 mL of a 0.1M solution) and maleimide propionic acid NHS ester (28 mg, 105 μmol, 1.1 eq.) were added. The mixture was stirred for 20 h, diluted with CHCl.sub.3, washed with sat. NH.sub.4C solution and concentrated in vacuo. Preparative HPLC (15-70% A in B) afforded the desired product as a yellow solid (6.4 mg, 5.8% yield, 84.6% QC). A=0.05% formic acid in acetonitrile. B=0.05% formic acid in water. LCMS-B: 6.52 min (ES+) m/z 1151 [M+H].sup.+.

Example 4: 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl(11S,11aS)-11-hydroxy-7-methoxy-8-((6-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate(44) and 2-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-6-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinic acid (45)

(51) ##STR00102##

(i) Methyl 2,6-bis(bromomethyl)isonicotinate (36)

(52) A round-bottomed flask was charged with a magnetic stirrer bar, acetonitrile (46 mL), 35 (924 mg, 4.686 mmol, 1.0 eq.) and the mixture cooled to 0° C. PPh.sub.3 (3.96 g, 14.06 mmol, 3.0 eq.) then slowly CBr.sub.4 (4.66 g, 14.06 mmol, 3.0 eq.) were added and the mixture stirred or 30 min. The reaction mixture was concentrated in vacuo and purified by isolera (0-100% EtOAc in hexane) to afford the desired product (1.01 g, 67%). LCMS-A: 1.46 min (ES+) m/z 321 [M+H].sup.+

(ii) Methyl 2-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-6-(bromomethyl)isonicotinate (37)

(53) A round-bottomed flask was charged with a magnetic stirrer bar, DMF (10 mL), 13 (814 mg, 1.022 mmol, 1.0 eq.), 36 (1.32 g, 4.087 mmol, 4.0 eq.) and K.sub.2CO.sub.3 (155 mg, 1.124 mmol, 1.1 eq.) and stirred for 40 min at 35° C., whereupon LCMS indicated the reaction was complete. DMF was removed in vacuo and the resulting residue was dissolved in EtOAc, washed with H.sub.2O, brine and dried over MgSO.sub.4. Isolera (50-100% EtOAc in hexane) afforded the desired product as a pale yellow foam (750 mg, 71%). LCMS-A: 1.90 min (ES+) m/z 1037 [M+H].sup.+

(iii) tert-Butyl (11S,11aS)-8-((6-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (38)

(54) A round-bottomed flask was charged with a magnetic stirrer bar, DMF (6 mL), 37 (750 mg, 0.7225 mmol, 1.0 eq.), 12 (333 mg, 0.7225 mmol, 1.0 eq.) and K.sub.2CO.sub.3 (100 mg, 0.7235 mmol, 1.0 eq.) and stirred for 80 min at 35° C., whereupon LCMS indicated the reaction was complete. DMF was removed in vacuo and the resulting residue was dissolved in EtOAc, washed with H.sub.2O and dried over MgSO.sub.4. In vacuo concentration afforded the desired product as a pale-yellow foam (979 mg, 96%). LCMS-A: 1.99 min (ES+) m/z 1417 [M+H].sup.+

(iv) tert-Butyl (11S,11aS)-8-((6-((5-((((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)amino)-4-((S)-2-(hydroxymethyl)-4-methylenepyrrolidine-1-carbonyl)-2-methoxyphenoxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (39)

(55) A round-bottomed flask was charged with a magnetic stirrer bar, THE (9 mL) 38 (879 mg, 0.6200 mmol, 1.0 eq.) TBAF (1.24 mL of a 1M solution in THF, 1.240 mmol, 2.0 eq.) and the mixture stirred for 80 min whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera (0-4% MeOH in CH.sub.2Cl.sub.2) to afford the desired product (746 mg, 83%). LCMS-A: 1.71 min (ES+) m/z 1303 [M+H].sup.+, 1325 [M+Na].sup.+

(v) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-8-((6-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (40)

(56) A round-bottomed flask was charged with a magnetic stirrer bar, acetonitrile (13 mL), 39 (64 mg, 0.4956 mmol, 1.0 eq.), Stahl aerobic oxidation TEMPO solution (248 μL of a 0.2M solution in acetonitrile, 0.04960 mmol, 0.10 eq.), Cu(MeCN).sub.4OTf (18.7 mg, 0.04956 mmol, 0.10 eq.) and the mixture stirred under a balloon of air for 3 d. The reaction mixture was concentrated in vacuo and purified by isolera (0-4% MeOH in CH.sub.2Cl.sub.2) to afford the desired product as a white solid (523 mg, 81%). LCMS-A: 1.69 min (ES+) m/z 1301 [M+H].sup.+, 1323 [M+Na].sup.+

(vi) 2-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-6-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinic acid (41)

(57) A microwave vial was charged with a magnetic stirrer bar, (CH.sub.2Cl).sub.2 (0.5 mL) 40 (100 mg, 64.62 μmol, 1.0 eq.), SnMe.sub.3OH (35.1 mg, 193.9 μmol, 3.0 eq.) and the vial sealed. The mixture was heated to 80° C. for 4 h, cooled to rt, poured into H.sub.2O, extracted with 10% MeOH in CH.sub.2Cl.sub.2, the organics combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Isolera (5-15% MeOH in CH.sub.2Cl.sub.2) afforded the desired product (53 mg, 64%). LCMS-A: min (ES+) m/z 1287 [M+H].sup.+, 1309 [M+Na].sup.+.

(vii-a) tert-Butyl (11S,11aS)-8-((6-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (42)

(58) A round-bottomed flask was charged with a magnetic stirrer bar, 40 (100 mg, 76.84 μmol, 1.0 eq.) CH.sub.2Cl.sub.2 (6 mL), pyrrolidine (12.6 μL, 153.7 μmol, 2.0 eq.) and Pd(PPh.sub.3).sub.4 (2.2 mg, 1.921 μmol, 0.025 eq.). The mixture was stirred under Ar for 25 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was thoroughly concentrated in vacuo overnight to remove pyrrolidine traces. The resulting residue was dissolved in CH.sub.2Cl.sub.2 (1 mL) and maleimide propionic acid NHS ester (24.5 mg, 92.21 μmol, 1.2 eq.) and i-Pr.sub.2NEt (16.1 μL, 92.21 μmol, 1.2 eq.) were added and the mixture stirred for 3.5 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera (0-4% MeOH in CH.sub.2Cl.sub.2) to afford the desired product as a white solid (89.5 mg, 85%). LCMS-A: 1.62 min (ES+) m/z 1368 [M+H].sup.+, 1390 [M+Na].sup.+

(vii-b) 2-((((11S,11aS)-10-(tert-butoxycarbonyl)-7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-6-((((11S,11 aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinic acid (43)

(59) A round-bottomed flask was charged with a magnetic stirrer bar, CH.sub.2Cl.sub.2 (3 mL), 41 (53 mg, 41.17 μmol, 1.0 eq.), pyrrolidine (10.1 μmol, 122.8 mmol, 3.0 eq.) Pd(PPh.sub.3).sub.4 (1.2 mg, 1.024 μmol, 0.025 eq.) and the mixture stirred under Ar for 30 min whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with NH.sub.4Cl, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The reaction mixture was thoroughly concentrated in vacuo overnight to remove pyrrolidine traces. The resulting residue was dissolved in THE (3 mL) and maleimide propionic acid NHS ester (13.1 mg, 49.13 μmol, 1.2 eq.) and NaHCO.sub.3 (0.43 mL of a 100 mM solution) were added and the mixture stirred for 5 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with EtOAc, washed with NH.sub.4Cl, dried over MgSO.sub.4 and concentrated in vacuo. Isolera (2-10% MeOH in CH.sub.2Cl.sub.2) afforded the desired product (39.9 mg, 72% yield). LCMS-A: 1.58 min (ES+) m/z 1354 [M+H].sup.+, 1376 [M+Na].sup.+

(viii-a) 4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl (11S,11aS)-11-hydroxy-7-methoxy-8-((6-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-4-(methoxycarbonyl)pyridin-2-yl)methoxy)-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate (44)

(60) A round-bottomed flask was charged with a magnetic stirrer bar, 42 (90 mg, 65.77 μmol, 1.0 eq.) and cooled to 0° C. 95% TFA in H.sub.2O (3.5 mL) was cooled to −20° C. and added in one portion. The reaction mixture was stirred for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was poured into H.sub.2O (10 mL), the pH adjusted to ca. 5 with NaHCO.sub.3 and the aqueous mixture extracted with 10% MeOH in CH.sub.2Cl.sub.2, the organics dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Isolera purification (0-8% MeOH in CH.sub.2Cl.sub.2) then preparative HPLC (15-70% A in B) afforded the desired product as a white solid (7 mg, 9.2% yield, 98.4% QC). A=0.05% formic acid in acetonitrile. B=0.05% formic acid in water. LCMS-B: 7.61 min (ES+) m/z 11.66 [M+H].sup.+

(viii-b) 2-((((11S,11aS)-10-(((4-((S)-2-((S)-2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3-methylbutanamido)propanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)-6-((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinic acid (45)

(61) A round-bottomed flask was charged with a magnetic stirrer bar, 43 (37 mg, 27.32 μmol, 1.0 eq.) and cooled to 0° C. 95% TFA in H.sub.2O (2 mL) was cooled to −20° C. and added in one portion. The reaction mixture was stirred for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was poured into H.sub.2O (10 mL) and pH adjusted to ca. 5 with NaHCO.sub.3. The solution was loaded onto an SPE cartridge (SCX-2, 10 g), washed with H.sub.2O (2×10 mL) then blown dry with air. The organics were eluted from the column with MeOH and concentrated in vacuo. Preparative HPLC (15-70% A in B) afforded the desired product as an off-white solid (5 mg, 16% yield, 92.5% QC). A=0.05% formic acid in acetonitrile. B=0.05% formic acid in water. LCMS-B: 6.93 min (ES+) m/z 1152.

Example 5—Conjugations

(62) Antibody-Drug Conjugate with Compound 19, ConjA

(63) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 12 micromoles, 240 μL) to a 7.5 mL solution of antibody Trastuzumab (45 mg, 300 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(64) The reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 15 molar equivalent/antibody, 4.5 micromoles, 90 μL) in DMSO was added and the reoxidation mixture was allowed to react for 2-3 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of 1.5 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 2.0 mg/mL. Compound 19 was added as a DMSO solution (10 molar equivalent/antibody, 0.9 micromole, in 1.0 mL DMSO) to 10.0 mL of this reoxidised antibody solution (13.5 mg, 90 nanomoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 2 hours at room temperature with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (4.5 micromoles, 45 μL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.

(65) 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 280 nm and 330 nm (Compound 19 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 19, consistent with a drug-per-antibody ratio (DAR) of 1.89 molecules of Compound 19 per antibody.

(66) 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 96%. UHPLC SEC analysis gives a concentration of final ConjA at 1.39 mg/mL in 8.0 mL, obtained mass of ConjA is 11.15 mg (74% yield).

(67) Antibody-Drug Conjugate with Compound 27, ConjB

(68) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 27 μmol, 536 μL) to a 25 mL solution of antibody Trastuzumab (100 mg, 0.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(69) The reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via Tangential Flow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filter with 115 cm.sup.2 surface area, into a reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent. The reduced antibody was centrifuged for 3 min at 4000 rpm and then filtered using 0.22 μM membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 12 micromoles, 240 μL) in DMSO was added and the reoxidation mixture was allowed to react for 2-3 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of ˜1.7 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 1.7 mg/mL. Compound 27 was added as a DMSO solution (12 molar equivalent/antibody, 1.2 micromole, in 1.5 mL DMSO) to 15.0 mL of this reoxidised antibody solution (15.3 mg, 0.1 μmoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 2 hours at room temperature with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (6.0 micromoles, 60 μL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.

(70) 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 ConjB at 280 nm and 330 nm (Compound 27 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 27, consistent with a drug-per-antibody ratio (DAR) of 1.69 molecules of Compound 27 per antibody.

(71) 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 ConjB at 280 nm shows a monomer purity of 99%. UHPLC SEC analysis gives a concentration of final ConjB at 2.31 mg/mL in 5.8 mL, obtained mass of ConjB is 13.4 mg (67% yield).

(72) Antibody-Drug Conjugate with Compound 33, ConjC

(73) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (125 molar equivalent/antibody, 25 micromoles, 500 μL) to a 7.5 mL solution of antibody (30 mg, 200 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(74) The reduction mixture was allowed to react at room temperature for 3-4 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged, via spin filter centrifugation, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 4 micromoles, 80 μL) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of 2.5 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 2.0 mg/mL. Compound 33 was added as a DMSO solution (10 molar equivalent/antibody, 1 micromole, in 0.75 mL DMSO) to 6.75 mL of this reoxidised antibody solution (15 mg, 100 nanomoles) for a 10% (v/v) final DMSO concentration. The solution was mixed for 4 hours at room temperature (0.5-1.0 mL propylene glycol added after 2 hours to aid solubility), then the conjugation was quenched by addition of N-acetyl cysteine (5 micromoles, 50 μL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.

(75) 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 ConjC at 280 nm and 330 nm (Compound 33 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 33, consistent with a drug-per-antibody ratio (DAR) of 1.63 molecules of Compound 33 per antibody.

(76) 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 ConjC at 280 nm shows a monomer purity of 99%. UHPLC SEC analysis gives a concentration of final ConjC at 1.89 mg/mL in 7.0 mL, obtained mass of ConjC is 13.2 mg (88% yield).

(77) Antibody-Drug Conjugate with Compound 34, ConjD

(78) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 28 micromoles, 560 μL) to a 26.25 mL solution of antibody Trastuzumab (105 mg, 700 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(79) The reduction mixture was allowed to react at room temperature for 20 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reduced antibody was buffer exchanged, via tangential flow filtration (TFF) using a Spectrum Labs KrosFlo Research IIi system with a 30 kDa MWCO, 115 cm.sup.2 surface area hollow fibre filter module at 50 mL/min in PBS with a transmembrane pressure (TMP) of 0.5-1.0 bar, into a reoxidation buffer containing PBS and 1 mM EDTA to remove all the excess reducing agent, recovery 97 mg (667 nanomoles). A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 12.9 micromoles, 259 μL) in DMSO was added and the reoxidation mixture was allowed to react for 17 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of 1.5 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 1.0 mg/mL. Compound 34 was added as a DMSO solution (15 molar equivalent/antibody, 1.4 micromoles, in 1.4 mL DMSO) to 12.6 mL of this reoxidised antibody solution (14 mg, 93 nanomoles) for a 10% (v/v) final DMSO concentration. The solution was mixed for 2 hours at room temperature, stored at +4° C. for 16 hours then the conjugation was quenched by addition of N-acetyl cysteine (3.7 micromoles, 37 μL at 100 mM). Reaction mixture was purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, further purified by TFF (800 mL permeated), sterile-filtered and analysed. UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjD at 280 nm and 330 nm (Compound 34 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 34, consistent with a drug-per-antibody ratio (DAR) of 1.81 molecules of Compound 34 per antibody.

(80) 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 ConjD at 280 nm shows a monomer purity of 97%. UHPLC SEC analysis gives a concentration of final ConjD at 0.49 mg/mL in 5.5 mL, obtained mass of ConjD is 2.59 mg (19% yield).

(81) Antibody-Drug Conjugate with Compound 44, ConjE

(82) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added Trastuzumab (40 molar equivalent/antibody, 27 μmol, 536 μL) to a 25 mL solution of antibody (100 mg, 0.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(83) The reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via Tangential Flow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filter with 115 cm.sup.2 surface area, into a reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent. The reduced antibody was centrifuged for 3 min at 4000 rpm and then filtered using 0.22 μM membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 12 micromoles, 240 μL) in DMSO was added and the reoxidation mixture was allowed to react for 2-3 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of ˜1.7 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 1.7 mg/mL. Compound 44 was added as a DMSO solution (10 molar equivalent/antibody, 1.0 micromole, in 1.5 mL DMSO) to 15.0 mL of this reoxidised antibody solution (15.3 mg, 0.1 μmoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 2 hours at room temperature with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (5.0 micromoles, 50 μL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.

(84) 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 ConjE at 280 nm and 330 nm (Compound 44 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 44, consistent with a drug-per-antibody ratio (DAR) of 1.85 molecules of Compound 44 per antibody.

(85) 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 ConjE at 280 nm shows a monomer purity of 99%. UHPLC SEC analysis gives a concentration of final ConjE at 2.06 mg/mL in 6.8 mL, obtained mass of ConjE is 14 mg (70% yield).

(86) Antibody-Drug Conjugate with Compound 45, ConjF

(87) A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 27 μmol, 536 μL) to a 25 mL solution of antibody Trastuzumab (100 mg, 0.67 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 4.0 mg/mL.

(88) The reduction mixture was allowed to react at room temperature for 17 hours (or until full reduction is observed by UHPLC) in an orbital shaker with gentle (60 rpm) shaking. After cooling down to room temperature, the reduced antibody was buffer exchanged, via Tangential Flow Filtration unit (TFF) using mPES, MidiKros® 30 kDa fiber filter with 115 cm.sup.2 surface area, into a reoxidation buffer containing PBS pH 7.4 and 1 mM EDTA to remove all the excess reducing agent. The reduced antibody was centrifuged for 3 min at 4000 rpm and then filtered using 0.22 μM membrane filter. A 50 mM solution of dehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 12 micromoles, 240 μL) in DMSO was added and the reoxidation mixture was allowed to react for 2-3 hours at room temperature with gentle (50 rpm) shaking at an antibody concentration of ˜1.7 mg/mL (or more DHAA added and reaction left for longer until full reoxidation of the cysteine thiols to reform the inter-chain cysteine disulfides is observed by UHPLC). The reoxidation mixture was then sterile-filtered and diluted in a conjugation buffer containing PBS and 1 mM EDTA for a final antibody concentration of 1.7 mg/mL. Compound 45 was added as a DMSO solution (10 molar equivalent/antibody, 1.0 micromole, in 1.5 mL DMSO) to 15.0 mL of this reoxidised antibody solution (15.3 mg, 0.1 μmoles) for a 10% (v/v) final DMSO concentration. The solution left to react at room temperature for 2 hours at room temperature with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (5.0 micromoles, 50 μL at 100 mM), then purified by spin filtration using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed.

(89) 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 ConjF at 280 nm and 330 nm (Compound 45 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 45, consistent with a drug-per-antibody ratio (DAR) of 1.72 molecules of Compound 45 per antibody.

(90) 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 ConjF at 280 nm shows a monomer purity of 99%. UHPLC SEC analysis gives a concentration of final ConjF at 2.02 mg/mL in 6.7 mL, obtained mass of ConjF is 13.5 mg (68% yield).

(91) Antibody-Drug Conjugate with Compound 19, ConjA*

(92) R347 antibody (30 mg) was loaded onto solid support and reduced, reoxidised, conjugated to Compound 19, purified, released from the resin and formulated onto 25 mM Histidine, 200 mM Sucrose, Tween-20 0.02%, pH 6.0 according to patent #US 2014/036111 A1.

(93) UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm mm column eluting with a gradient of water and acetonitrile on a reduced sample of Conjugate at 214 nm and 330 nm (Compound 19 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 19, consistent with a drug-per-antibody ratio (DAR) of 1.9 molecules of Compound 19 per antibody.

(94) 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 ADC at 280 nm shows a monomer purity of 97%. Nanodrop UV-Vis analysis gave a concentration of final ADC at 2.0 mg/mL in 107 mL, obtained mass of ADC is 214 mg (71% yield).

Example 6—In Vitro Cytotoxicity Assay

(95) Medium from sub-confluent (80-90% confluency) cell culture in a T75 flask was aspirated and the flask rinsed with PBS (about 20 ml) and emptied. Trypsin-EDTA (5 ml) was added, the flask returned to the 37 C gassed incubator for up to about 5 minutes, then rapped sharply to dislodge and dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube, diluted with growth medium to a final volume of 15 ml, then centrifuged (400 g for 5 min). The supernatant was aspirated and the pellet re-suspended in 10 ml culture medium. Repeated pipetting may be necessary to produce monodisperse cell suspensions. The cell concentration and viability are measured by trypan blue staining, and counting using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2×10.sup.5/ml, dispensed (50 μl/well) into 96-well flat-bottom plates and incubated overnight before use.

(96) A stock solution (1 ml) of 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/well) into 4 replicate wells of the 96-well plate, containing 50 μl cell suspension seeded the previous day. Control wells received 50 μl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37 C in a CO.sub.2-gassed incubator for the exposure time.

(97) 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, X is log(concentration). ADC incubation times were 4 and 7 days for MDA MB 468 and NCI N87 respectively. Cell growth medium for MDA MB 468 was RPMI 1640 with glutamax, 10% (v/v) HyClone™ Fetal Bovine Serum and for NCI N87 was RPMI 1640 with glutamax, 10% (v/v) HyClone™ Fetal Bovine Serum.

(98) TABLE-US-00004 EC.sub.50 (μg/ml) NCI-N87 MDA-MB-468 ConjA 0.00661 ~27500 ConjB 0.000670 10.4 ConjC 0.00135 0.829 ConjD 0.00783 ~1.35 ConjE 0.0124 ~1.69 ConjF 0.000968 2.62

Example 7—In Vivo Assay

(99) Mice

(100) Female severe combined immune-deficient mice (Fox Chase SCID, C.B-17/lcr-Prkdcscid, Charles River) were eight to twelve weeks old 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.

(101) Tumor Cell Culture

(102) 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, 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% C.sub.02 and 95% air.

(103) In Vivo Implantation and Tumor Growth

(104) The NCI-N87 cells used for implantation were harvested during log phase growth and resuspended in phosphate buffered saline (PBS) containing 50% Matrigel™ (BD Biosciences). On the day of tumor implant, each test mouse was injected subcutaneously in the right flank with 1×10.sup.7 cells (0.1 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 100 to 150 mm.sup.3. Eleven days later, designated as Day 1 of the study, mice were sorted according to calculated tumor size into groups each consisting of ten animals and equivalent group mean tumor volumes. Tumors were measured in two dimensions using calipers, and volume was calculated using the formula:
Tumour Volume (mm.sup.3)=0.5(w.sup.2×l)
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

(105) On Day 1 of the study, female SCID mice bearing established NCI-N87 xenografts were sorted into groups (n=10), and dosing was initiated as shown below. All agents were administered intravenously (i.v.) via tail vein injection once on Day 1 (qd×1). The dosing volume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaled to the body weight of each individual animal. A vehicle-treated group served as the control group for efficacy analysis. 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 85), whichever came first.

(106) Treatment outcome was determined from percent tumor growth delay (% TGD), defined as the percent increase in median TTE for treated versus control mice, with differences between groups deemed statistically significant at P≤0.05 using logrank survival analysis. 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.

(107) Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects. Treatment tolerability was assessed by body weight measurements and frequent observation for signs of treatment-related side effects.

(108) All regimens were acceptably tolerated. The median TTE for vehicle-treated controls was 33.5 days, establishing a maximum possible TGD of 51.5 days (154%) for the 85-day study.

(109) All ADC treatments produced TGD outcomes that were significantly different from the vehicle-treated controls (P<0.001).

(110) TABLE-US-00005 Dose % TGD PR CR TFS ConjA 1 mg/kg 154 0 0 0 ConjB 1 mg/kg 154 9 0 0 ConjC 1 mg/kg 154 7 1 1 ConjE 1 mg/kg 117 0 0 0 ConjF 1 mg/kg 154 8 0 0

Example 8—Synthesis of Drug Payloads

(111) ##STR00103##

(i) Diallyl 8,8′-(((5-(methoxycarbonyl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) (46)

(112) Phenol I3 (240 mg, 0.5 mmol, 2 eq.) and dibromo-m-xylene 21 (81 mg, 0.253 mmol, 1 eq.) were solubilised in dry DMF (20 mL) before TBAl (11 mg, 0.03 mmol, 0.1 eq.) and K.sub.2CO.sub.3 (70 mg, 0.5 mmol, 2 eq) were added. The mixture was heated to 60° C. for an hour at which point the reaction was completed. DMF was removed under vacuum and the crude product was purified by Biotage chromatography to give pure product 46 (273 mg, 97% yield). LCMS-A: r.t.=2.24 min, [M+H].sup.+=1109.

(ii) Methyl 3,5-bis((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoate (47)

(113) In a glovebox, ester 46 (60 mg, 0.054 mmol, 1 eq.) was solubilised in CH.sub.2Cl.sub.2 (5 mL) before PdP(Ph.sub.3).sub.4 (3.5 mg, 0.003 mmol, 0.05 eq.) and pyrrolidine (11 μL, 0.135 mmol, 2.5 eq.) were added and the mixture left to stir until complete. The organics were diluted with more CH.sub.2Cl.sub.2 and washed with sat NH.sub.4Cl.sub.(aq), water and brine before being dried with MgSO.sub.4, filtered and the volatiles removed in vacuo. The crude material was purified by Biotage chromatography to give pure product 47 (17.8 mg, 48% yield). LCMS-A: r.t.=1.42 min, [M+H].sup.+=677; LCMS.sup.2: r.t.=6.35 min, [M+H].sup.+=677.

(iii) 3,5-bis((((11S,11aS)-10-((allyloxy)carbonyl)-11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methyl-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (48)

(114) Ester 46 (185 mg, 0.17 mmol, 1 eq.) was solubilised in a 1:1:1 mixture of THF/H.sub.2O/MeOH (10 mL) and the mixture was stirred for 15h and after work up, product 48 (59 mg, 32% yield) was isolated. LCMS-A: r.t.=2.15 min, [M+H].sup.+=1095.

(iv) 3,5-bis((((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (49)

(115) In a glovebox, ester 48 (59 mg, 0.054 mmol, 1 eq.) was solubilised in CH.sub.2Cl.sub.2 (5 mL) before PdP(Ph).sub.4 (3.5 mg, 0.003 mmol, 0.05 eq.) and pyrrolidine (11 μL, 0.135 mmol, 2.5 eq.) were added and the mixture left to stir until complete. The organics were diluted with more CH.sub.2Cl.sub.2 and washed with sat NH.sub.4Cl.sub.(aq), water and brine before being dried with MgSO.sub.4, filtered and the volatiles removed in vacuo. The crude material was purified by Biotage chromatography to give pure product 49 (17.08 mg, 48% yield). LCMS-A: r.t.=1.35 min, [M+H].sup.+=663; LCMS-B: r.t.=5.70 min, [M+H].sup.+=663.

(116) ##STR00104##

(i) Di-tert-butyl 8,8′-(((5-(methoxycarbonyl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) (50)

(117) Phenol I2 (100 mg, 0.2171 mmol, 2.0 eq.), bromide 21 (35 mg, 0.1086 mmol, 1.0 eq.), TBAl (1 mg, 0.002715 mmol, 0.025 eq.) and K.sub.2CO.sub.3 (31.5 mg, 22.81 mmol, 2.1 eq.) were dissolved in DMF (1.5 mL) and the mixture stirred at 35° C. for 16 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (50-80% EtOAc in hexane) to afford 50 as an oil (98.3 mg, 84%).

(118) LCMS-A: r.t.=1.93, [M+H].sup.+=1103

(ii) Methyl 3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoate (51)

(119) 50 (86.6 mg, 80.09 μmol, 1.0 eq) was dissolved in 95% aq. TFA (0.5 mL) at 0° C. and the mixture stirred for 40 min, whereupon LCMS indicated the reaction was complete. Ice cold water was added to the reaction mixture and solid NaHCO.sub.3 was added until the pH was slightly basic. The aqueous was extracted with CHCl.sub.3, the combined organics dried over MgSO.sub.4 and concentrated in vacuo. HPLC (20-60% B in A*) afforded 51 as a yellow solid (6.2 mg, 11%). *A=0.1% HCO.sub.2H in H.sub.2O. B=0.1% HCO.sub.2H in MeCN. LCMS-B: r.t.=6.37 min, [M+H].sup.+=677.

(iii) Di-tert-butyl 8,8′-(((5-(tert-butoxycarbonyl)-1,3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) (52)

(120) Phenol I2 (100 mg, 0.2171 mmol, 2.0 eq.), bromide 6 (39.5 mg, 0.1086 mmol, 1.0 eq.), TBAl (1 mg, 0.002715 mmol, 0.025 eq.) and K.sub.2CO.sub.3 (31.5 mg, 22.81 mmol, 2.1 eq.) were dissolved in DMF (1.5 mL) and the mixture stirred at 35° C. for 16 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (50-80% EtOAc in hexane) to afford 52 as an oil (120 mg, 98%).

(121) LCMS-A: r.t.=2.03, [M+H].sup.+=1145

(iv) 3,5-bis((((S)-7-Methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)benzoic acid (53)

(122) 52 (106 mg, 94.36 μmol, 1.0 eq) was dissolved in 95% aq. TFA (0.5 mL) at 0° C. and the mixture stirred for 40 min. Ice cold water was added to the reaction mixture and solid NaHCO.sub.3 was added until the pH was neutral. The aqueous was extracted with CHCl.sub.3. The pH of the aqueous phase was adjusted to ca. pH 3 and extracted with CHCl.sub.3. The combined organics were dried over MgSO.sub.4 and concentrated in vacuo. HPLC (20-60% B in A*) afforded 53 as a yellow solid (3.8 mg, 6.1%). *A=0.1% HCO.sub.2H in H.sub.2O. B=0.1% HCO.sub.2H in MeCN. LCMS-B: r.t.=5.68, [M+H].sup.+=663

(123) ##STR00105##

(i) Di-tert-butyl 8,8′-(((4-(methoxycarbonyl)pyridine-2,6-diyl)bis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) (54)

(124) Phenol I5 (668 mg, 1.362 mmol, 2.2 eq.), bromide 36 (200 mg, 0.0.6192 mmol, 1.0 eq.) and K.sub.2CO.sub.3 (188 mg, 1.362 mmol, 2.2 eq.) were dissolved in DMF (3 mL) and the mixture stirred at rt for 3 h, then 2 h at 40° C., whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to afford 54 as a white solid (610 mg, 86%). LCMS-A: r.t.=2.17, [M+Na].sup.+=1164

(ii) Methyl 2,6-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinate (55)

(125) 54 (200 mg, 0.1751 mmol, 1.0 eq) was dissolved in 95% aq. TFA (14 mL) at 0° C. and the mixture stirred for 30 min. Ice cold water was added to the reaction mixture and solid NaHCO.sub.3 was added until the pH was neutral to slightly basic. The aqueous was extracted with CHCl.sub.3, dried over MgSO.sub.4 and concentrated. Isolera chromatography (0-4% MeOH in CH.sub.2Cl.sub.2) afforded 55 (61.4 mg, 52%). LCMS-A: r.t.=5.43 min, [M+H].sup.+=678

(iii) 2,6-Bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)methyl)isonicotinic acid (56)

(126) 55 (55 mg, 81.16 μmol, 1.0 eq.) and SnMe.sub.3OH (58.7 mg, 324.6 μmol, 4.0 eq.) were dissolved in (CH.sub.2C).sub.2 (0.6 mL) and the mixture warmed to 80° C. and stirred for 2.5 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CH.sub.2Cl.sub.2, filtered through celite and concentrated in vacuo. HPLC (5-70% B in A*) afforded 56 (20 mg, 37%). *A=0.1% HCO.sub.2H in H.sub.2O. B=0.1% HCO.sub.2H in MeCN.

(127) LCMS-B: r.t.=5.50 min, [M+H].sup.+=664

(128) ##STR00106##

(i) Di-tert-butyl 8,8′-((1,3-phenylenebis(methylene))bis(oxy))(11S,11aS,11′S,11a′S)-bis(11-((tert-butyldimethylsilyl)oxy)-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate) (57)

(129) Phenol I5 (400 mg, 0.8152 mmol, 2.1 eq.), bromide 56 (103 mg, 0.3902 mmol, 1.0 eq.) and K.sub.2CO.sub.3 (115 mg, 0.8321 mmol, 2.1 eq.) were dissolved in MEK (5 mL) and the mixture stirred at 40° C. for 2 h, then 2 h at 90° C., whereupon LCMS indicated the reaction was incomplete. KI (knifept.) was added and the mixture stirred at 90° C. for 2 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (10-25% EtOAc in isohexane) to afford 57 as an off-white foam (391 mg, 92%). LCMS-A: r.t.=2.47 min, [M+H].sup.+=1084

(ii) (11aS,11a′S)-8,8′-((1,3-phenylenebis(methylene))bis(oxy))bis(7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5-one) (58)

(130) 57 (98 mg, 0.09045 mmol, 1.0 eq) was dissolved in 95% aq. TFA (2 mL) at 0° C. and the mixture stirred for 30 min. Ice cold water was added to the reaction mixture and solid NaHCO.sub.3 was added until the pH was neutral to slightly basic. The aqueous was extracted with CHCl.sub.3, dried over MgSO.sub.4 and concentrated. Isolera chromatography (0-4% MeOH in CH.sub.2Cl.sub.2) afforded 58 (27.9 mg, 50%) as a pale-yellow solid. LCMS-B: r.t.=7.55 min, [M+H].sup.+=619.

(131) K562 Assay

(132) The potency of each of the PBD molecules were measured via in vitro cytotox assays in the carcinoma cell line K562.

(133) Solid PBD 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.

(134) Adherent K562 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 (4 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.

(135) 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.).

(136) 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.

(137) NCI-N87 Assay

(138) The potency of each of the PBD molecules were measured via in vitro cytotoxicity assays in the carcinoma cell line NCI-N87.

(139) Solid PBD 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.

(140) 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.

(141) Plates were then read on an EnVision® Multi-label Plate Reader (Perkin Elmer) using absorbance at 490 nm.

(142) 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.).

(143) 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.

(144) TABLE-US-00006 K562 (nM) NCI-N87 (nM) 47 0.162 49 1.84 51 0.06848 53 4.309 55 0.0168 56 20.7 58 0.0172

(145) These results show the acid payload to have a much lower activity in vitro than the ester, and that the addition of the ester group slightly reduces the activity of the payload.

Example 9—Toxicity of Conjugates

(146) A single dose nonGLP toxicity study was carried out on ConjA*.

(147) Male Sprague Dawley rats (Envigo, Inc) were dosed once by slow bolus intravenous injection via the tail vein with ADC. The vehicle for dilution used was 25 mM Histidine-HCl, 7% sucrose, 0.02% Polysorbate 80, pH 6.0. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, clinical pathology (clinical chemistry, hematology, and coagulation), and gross pathology findings. All animals were terminated on Study Day (SD) 29.

(148) ConjA*

(149) TABLE-US-00007 Group Dose (mg/kg) N 1 1.2 4 2 2 4

(150) Administration of either dose did not not result in any early mortalities. Test article-associated changes in the hematology data were indicative of myelotoxicity and included decreases in all cell lineages (red blood cells, reticulocytes, platelets, neutrophils, lymphocytes, monocytes and eosinophils in most dose groups. The values generally increased at subsequent intervals, indicating reversibility.

(151) In the coagulation data, increased fibrinogen on Day 29, in association with decreased albumin and A/G ratio, and increased globulin, was consistent with an acute systemic inflammatory response.

(152) In group 2, there was an increased mean ALT on Day 8 that correlated with increased liver weights and mild multifocal hepatocellular hypertrophy in that group. The activities of these enzymes were comparable to, or lower than, the control group at the subsequent collections on Days 15 and 29. ALP increases of a lesser magnitude were observed in group 1 on Day 8 only. Total cholesterol and triglycerides were also elevated in both groups on Days 15 and/or 29, consistent with altered lipid metabolism and likely associated with the liver enzymes and anatomic pathology findings. Microscopic changes included randomly oriented mild multifocal hepatocellular hypertrophy in all Group 2 animals. This hepatic change correlated to increased liver weights and increased liver enzymes.

(153) No significant changes in the bone marrow were seen in either group, which contrasts with the typical profile associated with pyrrolobenzodiazepine dimers.

Abbreviations

(154) Ac acetyl Acm acetamidomethyl Alloc allyloxycarbonyl Boc di-tert-butyldicarbonate t-Bu tert-butyl Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene Cbz or Z benzyloxy-carbonyl, where Z—Cl and Z—Br are chloro- and bromobenzyloxy carbonyl respectively DMF N,N-dimethylformamide Dnp dinitrophenyl DTT dithiothreitol Fmoc 9H-fluoren-9-ylmethoxycarbonyl imp N-10 imine protecting group: 3-(2-methoxyethoxy)propanoate-Val-Ala-PAB MC-OSu maleimidocaproyl-O—N-succinimide Moc methoxycarbonyl MP maleimidopropanamide Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl PAB para-aminobenzyloxycarbonyl PEG ethyleneoxy PNZ p-nitrobenzylcarbamate Psec 2-(phenylsulfonyl)ethoxycarbonyl TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl Teoc 2-(trimethylsilyl)ethoxycarbonyl Tos tosyl Troc 2,2,2-trichlorethoxycarbonylchloride Trt trityl Xan xanthyl

(155) Numbered Statements of the Invention

(156) 1. A compound with the formula I:

(157) ##STR00107##
and salts and solvates thereof, wherein:

(158) R″ is a group of formula II:

(159) ##STR00108##
where each of n and m are independently selected from 1, 2 and 3;

(160) R.sup.O is selected from the group consisting of H, methyl, ethyl, iso-propyl and benzyl;

(161) Q.sup.C is selected from N and CH;

(162) Y and Y′ are selected from O, S, or NH;

(163) when there is a double bond present between C2 and C3, R.sup.2 is selected from the group consisting of:

(164) (ia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;

(165) (ib) C.sub.1-5 saturated aliphatic alkyl;

(166) (ic) C.sub.3-6 saturated cycloalkyl;

(167) ##STR00109##
wherein each of R.sup.11, R.sup.12 and R.sup.13 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2 group is no more than 5;

(168) ##STR00110##
wherein one of R.sup.15a and R.sup.15b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

(169) ##STR00111##
where R.sup.14 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

(170) when there is a single bond present between C.sub.2 and C.sub.3,

(171) R.sup.2 is H or

(172) ##STR00112##
where R.sup.16a and R.sup.16b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.16a and R.sup.16b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;

(173) when there is a double bond present between C2′ and C3′, R.sup.2′ is selected from the group consisting of:

(174) (iia) C.sub.5-10 aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C.sub.1-7 alkyl, C.sub.3-7 heterocyclyl and bis-oxy-C.sub.1-3 alkylene;

(175) (iib) C.sub.1-5 saturated aliphatic alkyl;

(176) (iic) C.sub.3-6 saturated cycloalkyl;

(177) ##STR00113##
wherein each of R.sup.21, R.sup.22 and R.sup.23 are independently selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl, where the total number of carbon atoms in the R.sup.2′ group is no more than 5;

(178) ##STR00114##
wherein one of R.sup.25a and R.sup.25b is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and

(179) ##STR00115##
where R.sup.24 is selected from: H; C.sub.1-3 saturated alkyl; C.sub.2-3 alkenyl; C.sub.2-3 alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

(180) when there is a single bond present between C2′ and C3′,

(181) R.sup.2′ is H or

(182) ##STR00116##
where R.sup.26a and R.sup.26b are independently selected from H, F, C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C.sub.1-4 alkyl amido and C.sub.1-4 alkyl ester; or, when one of R.sup.26a and R.sup.26b is H, the other is selected from nitrile and a C.sub.1-4 alkyl ester;

(183) 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;

(184) 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;

(185) R.sup.7 is selected from H, R, OH, OR, SH, SR, NH.sub.2, NHR, NHRR′, nitro, Me.sub.3Sn and halo;

(186) R.sup.6′, R.sup.7′, R.sup.9′ are selected from the same groups as R.sup.6, R.sup.7 and R.sup.9 respectively;

(187) R.sup.11a is selected from OH, OR.sup.A, where R.sup.A is C.sub.1-4 alkyl, and SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

(188) and either

(189) (a) R.sup.30 is H, and R.sup.31 is OH, OR.sup.A, where R.sup.A is C.sub.1-4alkyl;

(190) (b) R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or

(191) (c) R.sup.30 is H and R.sup.31 is SO.sub.zM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation, wherein if R.sup.11a and R.sup.31 are SO.sub.zM, M may represent a divalent pharmaceutically acceptable cation

(192) R.sup.L is a linker for connection to a cell binding agent, which is selected from:

(193) ##STR00117##
wherein

(194) Q is:

(195) ##STR00118##
where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue;

(196) X is:

(197) ##STR00119##
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

(198) ##STR00120##
where R.sup.L 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.
2. A compound according to statement 1, wherein Y and Y′ are O.
3. A compound according to either statement 1 or statement 2, wherein n is 1.
4. A compound according to either statement 1 or statement 2, wherein n is 2.
5. A compound according to either statement 1 or statement 2, wherein n is 3.
6. A compound according to any one of statements 1 to 5, wherein m is 1.
7. A compound according to any one of statements 1 to 5, wherein m is 2.
8. A compound according to any one of statements 1 to 5, wherein m is 3.
9. A compound according to any one of statements 1 to 8, wherein Q.sup.C is N.
10. A compound according to any one of statements 1 to 8, wherein Q.sup.C is CH.
11. A compound according to any one of statements 1 to 10, wherein R.sup.O is H.
12. A compound according to any one of statements 1 to 10, wherein R.sup.O is methyl.
13. A compound according to any one of statements 1 to 10, wherein R.sup.O is ethyl.
14. A compound according to any one of statements 1 to 10, wherein R.sup.O is iso-propyl.
15. A compound according to any one of statements 1 to 10, wherein R.sup.O is benzyl.
16. A compound according to either statement 1 or statement 2, wherein R″ is of formula IIa:

(199) ##STR00121##
where R.sup.O1 is selected from the group consisting of H and methyl.
17. A compound according to any one of statements 1 to 16, wherein R.sup.9 and R.sup.9′ are H.
18. A compound according to any one of statements 1 to 17, wherein R.sup.6 and R.sup.6′ are independently selected from H and halo.
19. A compound according to statement 18, wherein R.sup.6 and R.sup.6′ are H.
20. A compound according to any one of statements 1 to 19, wherein R.sup.7 and R.sup.7′ are independently selected from H, OH and OR.
21. A compound according to statement 20, wherein R.sup.7 and R.sup.7′ are independently a C.sub.1-4 alkoxy group.
22. A compound according to statement 21, wherein R.sup.7 and R.sup.7′ are methoxy.
23. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.5-7 aryl group.
24. A compound according to statement 23, wherein R.sup.2 is phenyl.
25. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.8-10 aryl group.
26. A compound according to any one of statements 23 to 25, wherein R.sup.2 bears one to three substituent groups.
27. A compound according to any one of statements 23 to 26, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
28. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.1-5 saturated aliphatic alkyl group.
29. A compound according to statement 28, wherein R.sup.2 is methyl, ethyl or propyl.
30. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a C.sub.3-6 saturated cycloalkyl group.
31. A compound according to statement 30, wherein R.sup.2 is cyclopropyl.
32. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a group of formula:

(200) ##STR00122##
33. A compound according to statement 32, wherein the total number of carbon atoms in the R.sup.2 group is no more than 4.
34. A compound according to statement 32, wherein the total number of carbon atoms in the R.sup.2 group is no more than 3.
35. A compound according to any one of statements 32 to 34, wherein one of R.sup.11, R.sup.12 and R.sup.13 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
36. A compound according to any one of statements 32 to 34, wherein two of R.sup.11, R.sup.12 and R.sup.13 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
37. A compound according to any one of statements 1 to 22, wherein there is a double bond between C.sub.2 and C.sub.3, and R.sup.2 is a group of formula:

(201) ##STR00123##
38. A compound according to statement 37, wherein R.sup.2 is the group:

(202) ##STR00124##
39. A compound according to any one of statements 1 to 22, wherein there is a double bond between C2 and C3, and R.sup.2 is a group of formula:

(203) ##STR00125##
40. A compound according to statement 39, wherein R.sup.14 is selected from H, methyl, ethyl, ethenyl and ethynyl.
41. A compound according to statement 40, wherein R.sup.14 is selected from H and methyl.
42. A compound according to any one of statements 1 to 22, wherein there is a single bond between C2 and C3, R.sup.2 is H.
43. A compound according to any one of statements 1 to 22, wherein there is a single bond between C2 and C3, R.sup.2 is

(204) ##STR00126##
and R.sup.16a and R.sup.16b are both H.
44. A compound according to any one of statements 1 to 22, wherein there is a single bond between C2 and C3, R.sup.2 is

(205) ##STR00127##
and R.sup.16a and R.sup.16b are both methyl.
45. A compound according to any one of statements 1 to 22, wherein there is a single bond between C2 and C3, R.sup.2 is

(206) ##STR00128##
one of R.sup.16a and R.sup.16b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3alkenyl, which alkyl and alkenyl groups are optionally substituted.
46. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a C.sub.5-7 aryl group.
47. A compound according to statement 46, wherein R.sup.2′ is phenyl.
48. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a C.sub.8-10 aryl group.
49. A compound according to any one of statements 46 to 48, wherein R.sup.2′ bears one to three substituent groups.
50. A compound according to any one of statements 46 to 49, wherein the substituents are selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.
51. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a C.sub.1-5 saturated aliphatic alkyl group.
52. A compound according to statement 51, wherein R.sup.2′ is methyl, ethyl or propyl.
53. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a C.sub.3-6 saturated cycloalkyl group.
54. A compound according to statement 53, wherein R.sup.2′ is cyclopropyl.
55. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a group of formula:

(207) ##STR00129##
56. A compound according to statement 55, wherein the total number of carbon atoms in the R.sup.2′ group is no more than 4.
57. A compound according to statement 56, wherein the total number of carbon atoms in the R.sup.2′ group is no more than 3.
58. A compound according to any one of statements 55 to 57, wherein one of R.sup.21, R.sup.22 and R.sup.23 is H, with the other two groups being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
59. A compound according to any one of statements 55 to 57, wherein two of R.sup.21, R.sup.22 and R.sup.23 are H, with the other group being selected from H, C.sub.1-3 saturated alkyl, C.sub.2-3 alkenyl, C.sub.2-3 alkynyl and cyclopropyl.
60. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a group of formula:

(208) ##STR00130##
61. A compound according to statement 60, wherein R.sup.2′ is the group:

(209) ##STR00131##
62. A compound according to any one of statements 1 to 45, wherein there is a double bond between C2′ and C3′, and R.sup.2′ is a group of formula:

(210) ##STR00132##
63. A compound according to statement 62, wherein R.sup.24 is selected from H, methyl, ethyl, ethenyl and ethynyl.
64. A compound according to statement 63, wherein R.sup.24 is selected from H and methyl.
65. A compound according to any one of statements 1 to 45, wherein there is a single bond between C2′ and C3′, R.sup.2′ is H.
66. A compound according to any one of statements 1 to 45, wherein there is a single bond between C2′ and C3′, R.sup.2′ is

(211) ##STR00133##
and R.sup.26a and R.sup.26b are both H.
67. A compound according to any one of statements 1 to 45, wherein there is a single bond between C2′ and C3′, R.sup.2 is

(212) ##STR00134##
and R.sup.26a and R.sup.26b are both methyl.
68. A compound according to any one of statements 1 to 45, wherein there is a single bond between C2′ and C3′, R.sup.2′ is

(213) ##STR00135##
one of R.sup.26a and R.sup.26b is H, and the other is selected from C.sub.1-4 saturated alkyl, C.sub.2-3 alkenyl, which alkyl and alkenyl groups are optionally substituted.
69. A compound according to any one of statements 1 to 68, wherein R.sup.11a is OH.
70. A compound according to any one of statements 1 to 68, wherein R.sup.11a is OR.sup.A.
71. A compound according to statement 70, wherein R.sup.A is methyl.
72. A compound according to any one of statements 1 to 68, wherein R.sup.11a is SO.sub.zM.
73. A compound according to statement 72, wherein M is Na.sup.+ and z is 3.
74. A compound according to any one of statements 1 to 73, wherein R.sup.30 is H, and R.sup.31 is OH.
75. A compound according to any one of statements 1 to 73, wherein R.sup.30 is H, and R.sup.31 is OR.sup.A.
76. A compound according to statement 75, wherein R.sup.A is methyl.
77. A compound according to any one of statements 1 to 73, wherein R.sup.30 and R.sup.31 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
78. A compound according to any one of statements 1 to 73, wherein R.sup.30 is H and R.sup.31 is SO.sub.zM.
79. A compound according to statement 78, wherein M is Na.sup.+ and z is 3.
80. A compound according to statement 1 of formula Ia:

(214) ##STR00136##
where R.sup.2a and R.sup.2a′ are the same and are selected from:

(215) ##STR00137##
R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
81. A compound according to statement 1 of formula Ib:

(216) ##STR00138##
wherein R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
82. A compound according to statement 1 of formula Ic:

(217) ##STR00139##
wherein R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
83. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIa, and Q is an amino acid residue selected from Phe, Lys, Val, Ala, Cit, Leu, le, Arg, and Trp.
84. A compound according to any one of statements 1 to 82, 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;
85. A compound according to statement 84, wherein Q is selected from .sup.CO-Phe-Lys-.sup.NH, .sup.CO-Val-Cit-.sup.NH and .sup.CO-Val-Aa-.sup.NH.
86. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIa, and Q is a tripeptide residue.
87. A compound according to any one of statements 1 to 86, wherein R.sup.L is of formula IIIa and a is 0 to 3.
88. A compound according to statement 87, wherein a is 0.
89. A compound according to any one of statements 1 to 88, wherein R.sup.L is of formula IIIa and b is 0 to 12.
90. A compound according to statement 89, wherein b is 0 to 8.
91. A compound according to any one of statements 1 to 90, wherein R.sup.L is of formula IIIa and d is 0 to 3.
92. A compound according to statement 91, wherein d is 2.
93. A compound according to any one of statements 1 to 86, wherein R.sup.L is of formula IIIa and, a is 0, cis 1 and d is 2, and b is from 0 to 8.
94. A compound according to statement 93 wherein b is 0, 4 or 8.
95. A compound according to any one of statements 1 to 94, wherein R.sup.L is of formula IIIa and G.sup.L is selected from

(218) TABLE-US-00008 0 (G.sup.L1-1) (G.sup.L1-2) (G.sup.L2) (G.sup.L3-1) (G.sup.L3-2) (G.sup.L3-3) (G.sup.L3-4) (G.sup.L4) (G.sup.L5) (G.sup.L6) 0 (G.sup.L7) (G.sup.L8) (G.sup.L9) (G.sup.L10) (G.sup.L11) (G.sup.L12) (G.sup.L13)
where Ar represents a C.sub.5-6 arylene group, e.g. phenylene, and represents C.sub.1-4 alkyl.
96. A compound according to statement 95, wherein Ar is a phenylene group.
97. A compound according to either statement 95 or statement 96, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.
98. A compound according to statement 97, wherein G.sup.L is G.sup.L1-1.
99. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIb, and both R.sup.L and R.sup.L2 are H.
100. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIb, R.sup.L1 is H and R.sup.L2 is methyl.
101. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIb, and both R.sup.L and R.sup.L2 are methyl.
102. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIb, and, R.sup.L and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.
103. A compound according to any one of statements 1 to 82, wherein R.sup.L is of formula IIIb, and, R.sup.L and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.
104. A compound according to any one of statements 1 to 82 and 99 to 103, wherein R.sup.L is of formula IIIb, and e is 0.
105. A compound according to any one of statements 1 to 82 and 99 to 103, wherein R.sup.L is of formula IIIb, and e is 1.
106. A compound according to statement 105, wherein the nitro group is in the para position.
107. The use of a compound according to any one of statements 1 to 106 in the manufacture of a medicament for treating a proliferative disease.
108. A compound according to any one of statements 1 to 106 for use in the treatment of a proliferative disease.
109. A conjugate comprising a compound of formula I according to any one of statements 1 to 106, or a pharmaceutically acceptable salt or solvate thereof, linked to a Ligand unit.
110. A conjugate of formula IV:
L-(D.sup.L).sub.p  (IV)
or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit, D.sup.L is of formula III:

(219) ##STR00157##
wherein R.sup.2, R.sup.6, R.sup.7, R.sup.9, R.sup.11a, Y, R″, Y′, R.sup.2, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.30 and R.sup.31 are as defined in any one of statements 1 to 79;
R.sup.LL is a linker for connection to a cell binding agent, which is selected from:

(220) ##STR00158##
where Q and X are as defined in any one of statements 1 and 83 to 86 and G.sup.LL is a linker connected to a Ligand Unit; and

(221) ##STR00159##
where R.sup.L1 and R.sup.L2 are as defined in any one of statements 1 and 99 to 103;
wherein p is an integer of from 1 to 20.
111. A conjugate according to statement 110, wherein G.sup.LL is selected from:

(222) TABLE-US-00009 0 (G.sup.LL1-1) (G.sup.LL1-2) (G.sup.LL2) (G.sup.LL3-1) (G.sup.LL3-2) (G.sup.LL-4) (G.sup.LL5) (G.sup.LL6) (G.sup.LL7) (G.sup.LL8-1) 0 (G.sup.LL8-2) (G.sup.LL9-1) (G.sup.LL9-2) G.sup.L10 G.sup.L11 G.sup.L12 G.sup.L13
where Ar represents a C.sub.5-6 arylene group, e.g. phenylene and X represents C.sub.1-4 alkyl.
112. A conjugate according to statement 111, wherein Ar is a phenylene group.
113. A compound according to either statement 111 or statement 112, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.
114. A compound according to statement 112, wherein G.sup.LL is G.sup.LL1-1.
115. A conjugate according to statement 110, wherein D.sup.L is of formula IIIa:

(223) ##STR00177##
where R.sup.2a and R.sup.2a′ are the same and are selected from:

(224) ##STR00178##
R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
116. A conjugate according to statement 110, wherein D.sup.L is of formula IIIb:

(225) ##STR00179##
where R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
117. A conjugate according to statement 110, wherein D.sup.L is of formula IIIc:

(226) ##STR00180##
where R.sup.7a and R.sup.7a′ are the same and are selected from methoxy and benzyloxy.
118. A conjugate according to any one of statements 109 to 117, wherein the Ligand Unit is an antibody or an active fragment thereof.
119. The conjugate according to statement 118, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.
120. The conjugate according to statement 119, 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):
(1) BMPR1B;
(2) E16;
(3) STEAP1;
(4) 0772P;
(5) MPF;
(6) Napi3b;
(7) Sema 5b;
(8) PSCA hlg;
(9) ETBR;
(10) MSG783;
(11) STEAP2;
(12) TrpM4;
(13) CRIPTO;
(14) CD21;
(15) CD79b;
(16) FcRH2;
(17) HER2;
(18) NCA;
(19) MDP;
(20) IL20R-alpha;
(21) Brevican;
(22) EphB2R;
(23) ASLG659;
(24) PSCA;
(25) GEDA;
(26) BAFF-R;
(27) CD22;
(28) CD79a;
(29) CXCR5;
(30) HLA-DOB;
(31) P2X5;
(32) CD72;
(33) LY64;
(34) FcRH1;
(35) IRTA2;
(36) TENB2;
(37) PSMA—FOLH1;
(38) SST;
(38.1) SSTR2;
(38.2) SSTR5;
(38.3) SSTR1;
(38.4) SSTR3;
(38.5) SSTR4;
(39) ITGAV;
(40) ITGB6;
(41) CEACAM5;
(42) MET;
(43) MUC1;
(44) CA9;
(45) EGFRvIII;
(46) CD33;
(47) CD19;
(48) IL2RA;
(49) AXL;
(50) CD30—TNFRSF8;
(51) BCMA—TNFRSF17;
(52) CT Ags—CTA;
(53) CD174 (Lewis Y)—FUT3;
(54) CLEC14A;
(55) GRP78—HSPA5;
(56) CD70;
(57) Stem Cell specific antigens;
(58) ASG-5;
(59) ENPP3;
(60) PRR4;
(61) GCC—GUCY2C;
(62) Liv-1—SLC39A6;
(63) 5T4;
(64) CD56—NCMA1;
(65) CanAg;
(66) FOLR1;
(67) GPNMB;
(68) TIM-1—HAVCR1;
(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;
(70) B7-H4—VTCN1;
(71) PTK7;
(72) CD37;
(73) CD138—SDC1;
(74) CD74;
(75) Claudins—CLs;
(76) EGFR;
(77) Her3;
(78) RON—MST1R;
(79) EPHA2;
(80) CD20-MS4A1;
(81) Tenascin C—TNC;
(82) FAP;
(83) DKK-1;
(84) CD52;
(85) CS1—SLAMF7;
(86) Endoglin—ENG;
(87) Annexin A1—ANXA1;
(88) V-CAM (CD106)—VCAM1.
121. The conjugate of any one of statements 118 to 120 wherein the antibody or antibody fragment is a cysteine-engineered antibody.
122. The conjugate according to any one of statements 110 to 121 wherein p is an integer from 1 to 8.
123. The conjugate according to statement 122, wherein p is 1, 2, 3, or 4.
124. A composition comprising a mixture of conjugates according to any one of statements 110 to 123, wherein the average p in the mixture of conjugate compounds is about 1 to about 8.
125. The conjugate according to any one of statements 109 to 123, for use in therapy.
126. A pharmaceutical composition comprising the conjugate of any one of statements 109 to 123 and a pharmaceutically acceptable diluent, carrier or excipient.
127. The conjugate according to any one of statements 109 to 123 or the pharmaceutical composition according to statement 126, for use in the treatment of a proliferative disease in a subject.
128. The conjugate for use according to statement 127, wherein the disease treated is cancer.
129. Use of a conjugate according to any one of statements 109 to 123 or the pharmaceutical composition according to statement 126 in a method of medical treatment.
130. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 126.
131. The method of statement 130 wherein the method of medical treatment is for treating cancer.
132. The method of statement 131, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.
133. Use of a conjugate according to any one of statements 109 to 123 in a method of manufacture of a medicament for the treatment of a proliferative disease.
134. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate according to any one of statements 109 to
123 or the pharmaceutical composition according to statement 126.
135. A compound with the formula REL:

(227) ##STR00181##
and salts and solvates thereof, wherein R.sup.2, R, R.sup.7, R.sup.9, Y, R″, Y′, R.sup.2′, R.sup.6′, R.sup.7′, R.sup.9′, R.sup.30 and R.sup.31 are as defined in any one of statements 1 to 79.