COMPOUNDS AND CONJUGATES THEREOF

Abstract

A conjugate comprising the following topoisomerase inhibitor derivative (A*): where Y is H or F, with a single overall linker moiety connecting two topoisomerase inhibitor derivatives to a Ligand Unit, wherein the topoisomerase inhibitor derivatives are cleavable from the Ligand Unit. Also provided is A* with the linking unit attached, and intermediates for their synthesis.

##STR00001##

Claims

1. A compound with the formula I: ##STR00043## where X.sup.1 and X.sup.2 are independently selected from a group of formula Ia: ##STR00044## Q is: ##STR00045## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; a=0 to 5, b1=0 to 16, b2=0 to 16, wherein at least b1 or b2=0; Y is H or F; c1 is 0 to 5; c2 is 0 to 5; X.sup.3 is —CH.sub.2— or —C(═O)—; X.sup.4 is .sup.X3—(CH.sub.2).sub.d1—(C.sub.2H.sub.4O).sub.e—(CH.sub.2).sub.d2—.sup.GL, where d1 is 0 to 5, d2 is 0 to 5 and e is 0 to 16; and G.sup.L is a linker for connecting to a Ligand Unit.

2. The compound according to claim 1, wherein Q is: (a) an amino acid residue selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or (b) a dipeptide residue selected from: .sup.NH-Phe-Lys-.sup.C═O, .sup.NH-Val-Ala-.sup.C═O, .sup.NH-Val-Lys-.sup.C═O, .sup.NH-Ala-Lys-.sup.C═O, .sup.NH-Val-Cit-.sup.C═O, .sup.NH-Phe-Cit-.sup.C═O, .sup.NH-Leu-Cit-.sup.C═O, .sup.NH-Ile-Cit-.sup.C═O, .sup.NH-Phe-Arg-.sup.C═O, .sup.NH-Trp-Cit-.sup.C═O, and .sup.NH-Gly-Val-.sup.C═O; or (c) a tripeptide residue selected from: .sup.NH-Glu-Val-Ala-.sup.C═O, .sup.NH-Glu-Val-Cit-.sup.C═O, .sup.NH-αGlu-Val-Ala-.sup.C═O, and .sup.NH-αGlu-Val-Cit-.sup.C═O; or (d) a tetrapeptide residue selected from: .sup.NH-Gly-Gly-Phe-Gly .sup.C═O; and .sup.NH-Gly-Phe-Gly-Gly .sup.C═O.

3. The compound according to either claim 1 or claim 2, wherein a is: (a) 0 to 3; or (b) 0 or 1; or (c) 0.

4. The compound according to any one of claims 1 to 3, wherein b1 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g) 8.

5. The compound according to any one of claims 1 to 3, wherein b2 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g) 8.

6. The compound according to any one of claims 1 to 5, wherein Y is H.

7. The compound according to any one of claims 1 to 6, wherein X.sup.1 and X.sup.2 are the same.

8. The compound according to any one of claims 1 to 7, wherein c1 is: (a) 0 to 3, (b) 1 or 2; or (c) 2.

9. The compound according to any one of claims 1 to 8, wherein c2 is: (a) 0 to 3; (b) 1 or 2; or (c) 2.

10. The compound according to any one of claims 1 to 9, wherein c1 and c2 are the same.

11. The compound according to any one of claims 1 to 10, wherein X.sup.3 is —C(═O)—.

12. The compound according to any one of claims 1 to 11, wherein d1 is: (a) 0 to 3; (b) 1 or 2; or (c) 2.

13. The compound according to any one of claims 1 to 12, wherein d2 is: (a) 0 to 3; (b) 1 or 2; or (c) 2; or (d) 0.

14. The compound according to any one of claims 1 to 13, wherein e is: (a) 0 to 8; (b) 0; (c) 2; (d) 4; or (e) 8.

15. The compound according to any one of claims 1 to 11, wherein d1+d2 is 2 and e is 0.

16. The compound according to claim 14, wherein each a is 0, each b1 is 0, each b2 is 2, c1 is 2, c2=2, X3=—C(═O)—, d1 is 2, d2 is 0 and e is 0.

17. The compound according to any one of claims 1 to 16, wherein G.sup.L is selected from ##STR00046## ##STR00047## ##STR00048## where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

18. A compound according to claim 17, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

19. 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 a Drug Linker unit that is of formula III: ##STR00049## where X.sup.1, X.sup.2, X.sup.3, X.sup.4, c1 and c2 are as defined in any one of claims 1 to 16; G.sup.LL is a linker connected to a Ligand Unit; and p is an integer of from 1 to 20.

20. The conjugate according to claim 19, wherein G.sup.LL is selected from: ##STR00050## ##STR00051## ##STR00052## where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

21. The conjugate according to claim 20, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

22. The conjugate according to either claim 19 or 20, wherein the Ligand Unit is an antibody or an active fragment thereof.

23. The conjugate according to claim 22, wherein the p is an integer from 1 to about 10.

24. A mixture of conjugates according to either claim 22 or 23, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 2 to about 20.

25. A pharmaceutical composition comprising the conjugate or mixture of any one of claims 20 to 24 and a pharmaceutically acceptable diluent, carrier or excipient.

26. The conjugate or mixture according to any one of claims 19 to 24, or the pharmaceutical composition according to claim 25, for use in the treatment of a proliferative disease in a subject.

27. The conjugate, mixture or pharmaceutical composition according to claim 26, wherein the disease is cancer.

28. Use of a conjugate or mixture according to any one of claims 19 to 24, or the pharmaceutical composition according to claim 25 in a method of medical treatment.

29. A method of medical treatment comprising administering to a patient the pharmaceutical composition of claim 25.

30. The method of claim 29 wherein the method of medical treatment is for treating cancer.

Description

EXAMPLES

[0317] Flash chromatography was performed using a Biotage® Isolera™ and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed 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.

[0318] Extraction and chromatography solvents were bought and used without further purification from VWR U.K or Fisher Scientific, U.K..

[0319] All fine chemicals were purchased from Sigma-Aldrich, Lancaster or BDH unless otherwise stated.

[0320] LC/MS conditions

[0321] Method A

[0322] Positive mode electrospray mass spectrometry was performed using a Waters Aquity H-class SQD2.

[0323] Mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). 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. Flow rate was 0.8 mL/minute. Detection was at 254 nm. Columns: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

[0324] Method B

[0325] The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

[0326] 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. Flow rate was 0.8 mL/minute. Wavelength detection range: 190 to 800 nm. Columns: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

[0327] Method C

[0328] The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

[0329] Initial composition 5% B held over 1 min, then increase from 5% B to 100% B over a 9 min period. The composition was held for 2 min at 100% B, then returned to 5% B in 0.10 minutes and hold there for 3 min. Total gradient run time equals 15 min. Flow rate 0.6 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: 50° C. Column: ACE Excel 2 C18-AR, 2 p, 3.0×100 mm.

[0330] HPLC Conditions

[0331] Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was carried out on a Shimadzu Prominence™ machine using a Phenomenex™ Gemini NX 5μ C18 column (at 50° C.) dimensions: 150×21.2 mm. Eluents used were solvent A (H.sub.2O with 0.1% formic acid) and solvent B (CH.sub.3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: Initial composition 13% B increased to 30% B over a 3 minutes period, then increased to 45% B over 8 minutes and again to 100% over 6 minutes before retunning to 13% over 2 min and hold for 1 min. The total duration of the gradient run was 20.0 minutes. Flow rate was 20.0 mL/minute and detection was at 254 and 223 nm.

[0332] NMR Method

[0333] Proton NMR chemical shift values were measured on the delta scale at 400 MHz using a Bruker AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz.

Synthesis of Key Intermediates

[0334] ##STR00027##

a) N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I2)

[0335] 5,6,7,8-tetrahydronaphthalen-1-amine I1 (8.54 g, 58.0 mmol) was dissolved in dichloromethane (80 mL). Triethylamine (18 mL, 129 mmol) was added and the mixture cooled to 0° C. Dropwise, acetic anhydride (11.5 mL, 122 mmol) was added, upon completion of the addition, the reaction mixture was warmed to rt and stirred for 45 min, whereupon LCMS indicated the reaction was complete. The mixture was diluted with CH.sub.2Cl.sub.2, washed with H.sub.2O, sat. NaHCO.sub.3, 10% citric acid, the organic phase dried over MgSO.sub.4 and concentrated in vacuo. The off-white solid was triturated with 1:3 Et.sub.2O/isohexane to afford 12 (10.8 g, 57.1 mmol, 98% Yield) as a white solid which was used without further purification. LC/MS (method A): retention time 1.44 mins (ES+) m/z 190 [M+H].sup.+

b) N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (13)

[0336] N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I2 (1.00 g, 5.2840 mmol) was added portion-wise to sulfuric acid (15 mL, 281 mmol) at −5° C. Sodium nitrate (450 mg, 5.2945 mmol) was added portion-wise to the reaction mixture and stirred for 30 min at −5° C. whereupon LCMS indicated no further reaction progress. The reaction mixture was poured onto ice with external cooling, the aqueous mixture extracted with CH.sub.2Cl.sub.2, the organic phase dried over MgSO.sub.4 and purified by Isolera (10-80% EtOAc in isohexane) to afford a mixture of N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 and N-(2-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (956 mg, 4.0811 mmol, 77% Yield) as a white/yellow solid. LC/MS (method A): retention time 1.53 mins (ES+) m/z 235 [M+H].sup.+.

c) N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I4)

[0337] N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 (1.01 g, 4.31 mmol) was dissolved in acetone (30 mL). Magnesium sulfate in water (3.9 mL, 5.9 mmol, 1.5 mol/L) was added and the mixture was cooled to 0° C. Potassium permanganate (2.07 g, 13.0 mmol) was added portionwise to the reaction mixture and the mixture warmed to rt and stirred for 50 min, whereupon TLC indicated the reaction was complete. The reaction mixture was filtered through Celite, the solids washed with CHCl.sub.3 and the resulting organic mixture washed with H.sub.2O, brine, dried over MgSO.sub.4 and purified by isolera (20-50% EtOAc in isohexane) to afford a mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg, 2.86 mmol, 66%) as a white/yellow solid. LC/MS (method A): retention time 1.44 mins (ES+) m/z 190 [M+H].sup.+

d) 8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one (I5)

[0338] A mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg, 2.8561 mmol) and 6N hydrochloric acid (7 mL) were stirred at 80° C. for 2.5 h, whereupon LCMS indicated the reaction was complete. The reaction mixture was cooled in an ice bath and 6N NaOH solution was added until the pH was basic. The aqueous mixture was extracted with CH.sub.2Cl.sub.2, the organic phase dried over MgSO.sub.4 and concentrated in vacuo. Isolera (0-50% EtOAc in isohexane) afforded 8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one 15 (320 mg, 1.552 mmol, 54% Yield) as a yellow/orange solid. LC/MS (method A): retention time 1.54 mins (ES+) m/z 207 [M+H].sup.+

e) 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I6)

[0339] 8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one I5 (430 mg, 2.0854 mmol) was dissolved in dichloromethane (20 mL). Pyridine (340 μL, 4.20 mmol) was added and the mixture cooled to 0° C. Trifluoroacetic anhydride (590 μL, 4.197 mmol) was added and stirred for 30 min, whereupon LCMS indicated the reaction was complete. The mixture was diluted with CH.sub.2Cl.sub.2, washed with H.sub.2O, the organic phase dried over MgSO.sub.4 and concentrated in vacuo to afford 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I6 (630 mg, 2.0846 mmol, >99% Yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.86 min (ES+) m/z 301X [M−H].sup.−

f) N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide (I7)

[0340] Zinc (2.73 g, 41.7 mmol) was suspended in methanol (80 mL), formic acid (4 mL) and water (4 mL) and the mixture cooled to 0° C. 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I6 (568 mg, 2.0865 mmol) was added portion-wise and the mixture stirred at 0° C. for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was filtered, the filtrate diluted with EtOAc and washed with sat NaHCO.sub.3. The organic phase was dried over MgSO.sub.4 and concentrated in vacuo to afford N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide 17 (568 mg, 2.0865 mmol, >99% Yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.65 min (ES+) m/z 273 [M+H].sup.+

g) N-(4-acetamido-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide (I8)

[0341] N-(8-amino-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I7 (568 mg, 2.0865 mmol) was dissolved in dichloromethane (20 mL). Triethylamine (580 μL, 4.16 mmol) then acetyl chloride (297 μL, 4.173 mmol) were added and the mixture stirred for 30 min, whereupon LCMS indicated the reaction was complete. The reaction mixture was diluted with CH.sub.2Cl.sub.2, washed with H.sub.2O, the organic phase dried over MgSO.sub.4 and concentrated in vacuo to afford N-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I8 (655 mg, 2.084 mmol, >99% yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.55 min (ES+) m/z 315 [M+H].sup.+

h) N-(4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I9)

[0342] N-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I8 (2.77 g, 8.81 mmol) was dissolved in methanol (240 mL) and water (17 mL). Potassium carbonate (4.88 g, 35.3 mmol) was added and the mixture stirred for 1.5 h at 50° C., whereupon LCMS indicated the reaction was complete. The reaction mixture was cooled, concentrated in vacuo, dissolved in 10% MeOH in CH.sub.2Cl.sub.2 and washed with H.sub.2O. The organic phase was dried over MgSO.sub.4 and purified by isolera chromatography (2-15% MeOH in CH.sub.2Cl.sub.2) to afford N-(8-amino-1-oxo-tetralin-5-yl)acetamide 19 (1.20 g, 5.50 mmol, 62.3% Yield) as a yellow solid. LC/MS (method A): retention time 0.98 min (ES+) m/z 219 [M+H].sup.+

##STR00028##

i) (S)—N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo[de]pyrano[3,4′:6,7]indolizino[1,2-b]quinolin-4-yl)acetamide (I10)

[0343] N-(8-amino-1-oxo-tetralin-5-yl)acetamide I9 (641 mg, 2.94 mmol, 1.0 eq.), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione A3 (840 mg, 3.19 mmol, 1.1 eq.) and PPTS (740 mg, 2.95 mmol, 1.0 eq.) were dissolved in toluene (60 mL) and stirred at reflux for 3 h, whereupon LCMS indicated 19 had been consumed. The reaction mixture was cooled and concentrated in vacuo. The resulting solids were triturated with acetonitrile, then acetone to afford 110 as a brown solid with minor TsOH contamination (1.26 g, 96%). LC/MS (method A): retention time 1.32 mins (ES+) m/z 447 [M+H].sup.+

j) (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H, 13H-benzo[de]pyrano[3,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione (I11)

[0344] (S)—N-(9-ethyl-9-hydroxy-10, 13-dioxo-2,3,9, 10,13, 15-hexahydro-1H, 12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)acetamide (I10) (1.26 g, 2.83 mmol, 1.0 eq.) was dissolved in hydrochloric acid (6 mol/L) in H.sub.2O (12 mL) and the mixture stirred for 5 h at 80° C., whereupon LCMS indicated 110 had been consumed. The reaction mixture was diluted with H.sub.2O and concentrated in vacuo to afford (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H, 13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione I11 (1.51 g, 2.85 mmol, 90 mass %, 101% Yield) as a red crystaline solid. LC/MS (method A): retention time 1.36 mins (ES+) m/z 405 [M+H].sup.+.

##STR00029## ##STR00030##

a) 6,8-Difluoro-5-nitro-1-tetralone (I13)

[0345] To a dust of 6,8-difluoro-1-tetralone I12 (15 g, 82.3 mmol) was added dropwise concentrated H.sub.2SO.sub.4 (90 mL) at 0° C. To the resulting mixture was added KNOB (8.2 g, 90.1 mmol) in portion-wise at 0° C. The reaction mixture was stirred at 0° C. for 2 h. The reaction was quenched with ice-water (200 mL) and then extracted with EtOAc (400 mL×3). The combined organic layers were washed with aqueous NaHCO.sub.3 (400 mL) and brine (400 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc=100:1) to afford compound I13 (8.1 g, 43% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 6.98 (t, J=10.0 Hz, 1 H), 3.01-2.98 (m, 2H), 2.72-2.68 (m, 2H), 2.21-2.05 (m, 2H).

b) 5-Amino-6,8-difluoro-1-tetralone (I14)

[0346] To a mixture of compound I13 (9.1 g, 39.6 mmol) in EtOH/H.sub.2O (8:1, 270 mL) were added NH.sub.4Cl (6.4 g, 0.12 mol) and dust Fe (17.6 g, 0.32 mol). The reaction mixture was stirred at 80° C. for 2 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. The residue was diluted with water (50 mL) and then extracted with EtOAc (200 mL×3). The combined organic layers were washed with brine (200 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether/EtOAc=8:1) to afford compound I14 (7.3 g, 94% yield). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ ppm 7.04 (t, J=11.6 Hz, 1H), 5.05 (br s, 2H), 2.71-2.2.68 (m, 2H), 2.5 (m, 2H), 2.03-1.98 (m, 2H).

c) 5-Acetylamino-6,8-difluoro-1-tetralone (I15)

[0347] To a solution of compound I14 (7.3 g, 37 mmol) and Et.sub.3N (4.5 g, 44.4 mmol) in DCM (100 mL) was added dropwise Ac.sub.2O (4.5 g, 44.4 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=300:1) to afford compound I15 (5.3 g, 60% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 6.84 (t, J=10 Hz, 1H), 6.75 (br s, 1H), 2.89-2.86 (m, 2H), 2.66-2.63 (m, 2H), 2.25 (s, 3H), 2.10-2.06 (m, 2H).

d) 5-Acetylamino-6-fluoro-8-amino-1-tetralone (I16)

[0348] To a solution of compound I15 (5.2 g, 21.7 mmol) in DMSO (50 mL) was added 25% aqueous NH.sub.4OH (80 mL) at room temperature. The reaction mixture was stirred at 130° C. for 16 h. The mixture was cooled to room temperature and then extracted with EtOAc (200 mL×5). The combined organic layers were washed with brine (200 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=100:1) to afford compound I16 (1.5 g, 30% yield) as a brownish solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ ppm 9.16 (s, 1H), 6.42 (d, J=12.4 Hz, 1H), 2.66 (m, 2H), 2.55-2.48 (m, 2H), 2.00 (s, 3H), 1.88-1.85 (m, 2H).

e) (S)—N-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)acetamide I17

[0349] Compound I16 (150 mg, 0.635 mmol), 168 mg (0.638 mmol) of (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione A3, and 168 mg (0.668 mmol) of pyridinium p-toluenesulfonate were mixed in 30 mL of anhydrous toluene. Equipped with a Dean-Stark trap, the reaction was heated with at 130° C. for 4 h. There was a water layer in the condenser. The solvent was evaporated, and the residue was precipitated into 14 mL of acetone and centrifuged to get 180 mg of the desired product as a brown solid. The residue on the flask wall was washed off with acetone and collected to give 60 mg of the desired product as a brown solid. The combined yield of the crude product 117 was 82%. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=464; .sup.1H NMR (400 MHZ, DMSO-d.sub.6): signals for the desired product, δ ppm 9.77 (s, 1H), 7.72 (d, J=11.1 Hz, 1H), 7.25 (s, 1H), 5.36 (s, 2H), 5.17 (s, 2H), 3.09 (t, J=5.5 Hz, 2H), 2.91 (t, J=5.5 Hz, 2H), 2.22 (s, 1H), 2.08 (s, 3H), 1.96 (m, 2H), 1.80 (m, 2H), 0.81 (t, J=7.3 Hz, 3H).

f) (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione I18

[0350] 60 mg of crude compound I17 was dissolved in 0.5 mL of HCl (37%), and the reaction was carried out in a sealed tube in a microwave reactor at 100° C. for 1 h. The solvent was evaporated, and the residue was dissolved in 1 mL of NMP and purified on Prep-HPLC with 0.1% TFA in water as A solvent and 0.1% TFA in acetonitrile as B solvent. The fractions containing the desired product were collected and frozen. After lyophilization, the reaction afforded 28 mg (42%) of the desired product 118 as an orange solid. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=422; .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ ppm 7.56 (d, J=12.4 Hz, 1H), 7.14 (s, 1H), 5.34 (s, 2H), 5.10 (s, 2H), 2.99 (t, J=6.1 Hz, 2H), 2.78 (t, J=6.1 Hz, 2H), 1.95 (t, J=5.8 Hz, 2H), 1.79 (m, 2H), 1.40-1.00 (m, 3H), 0.81 (t, J=7.4 Hz, 3H).

Example 1

[0351] ##STR00031## ##STR00032##

a) Allyl ((S)-3-methyl-1-oxo-1-(((S)-1-oxo-((5-oxo-4-(2,2,2-trifluoroacetamido)-5,6,7,8-tetrahydronaphthalen-1-yl)amino)propan-2-yl)amino)butan-2-yl)carbamate (A1)

[0352] DCC (6.54 g, 31.7 mMol) and HOPO (3.36 g, 30.2 mMol) were added to a solution of alloc-Val-Ala-OH (9.09 g, 31.7 mmol) and 17 (7.85 g, 28.8 mMol) in CH.sub.2Cl.sub.2 (300 mL) at 25° C.. The resulting mixture was left to stir overnight. The white solid that formed during the reaction was filtered out and washed with cold CH.sub.2Cl.sub.2. The filtrate was washed with water (150 mL) and brine (150 mL). The organic layer was dried over MgSO.sub.4, filtered and evaporated. The crude product was purified by silica gel chromatography (Hex/EtOAc, 60:40). Product A1 isolated was contaminated with co-eluting DCU (21.1 g, 140% yield). LC/MS (Method B): ES.sup.+=1.81 min, m/z 527.6 [M+H].sup.+.

b) Allyl ((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (A2)

[0353] Protected aniline A1 (18 g, 34.19 mMol) was solubilised in a mixture of MeOH and H.sub.2O 10:1 (165 mL) and K.sub.2CO.sub.3 was added (10 g, 72.36 mMol). The mixture was stirred at 50° C. until complete. The mixture was vacced down to almost dryness and the residue was taken up with CH.sub.2Cl.sub.2 and washed with H.sub.2O and brine, before being dried over MgSO.sub.4, filtered and evaporated. The crude product was purified by silica gel chromatography (CHCl.sub.3/MeOH, 100% to 7:3). The isolated product A2 was contaminated with a co-eluting impurity (10.71 g, 73% yield). LC/MS (Method B): ES.sup.+=1.46 min, m/z 431.7 [M+H].sup.+.

c) Allyl ((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methylbutan-2-yl)carbamate (A4)

[0354] Aniline A2 (450 mg, 1.045 mMol), lactone A3 (280 mg, 1.064 mMol) and pyridinium p-toluenesulfonate (273 mg, 1.086 mMol) were solubilised in toluene (20 mL) and the mixture was heated to 130° C. (high reflux). Every now and then a few drops of MeOH is added to help solubilise the mixture. After 7h the crude reaction was vacced down to dryness. The crude product was purified by silica gel chromatography (CHCl.sub.3/MeOH, 100% to 95:5) to give product A4 (360 mg, 52.3% yield). LC/MS (Method B): ES.sup.+=1.51 min, m/z 658.8 [M+H].sup.+.

d) Allyl (S)-2-amino-N—((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (A5)

[0355] Excess piperidine was added (642 μL) to a solution of A4 (543 mg, 0.82 mMol) and PdP(Ph.sub.3).sub.4 (89 mg, 0.08 mMol) in CH.sub.2Cl.sub.2 (15 mL). The mixture was allowed to stir at room temperature for 20 min, at which point the reaction had gone to completion (as monitored by LC/MS). The reaction mixture was diluted with CH.sub.2Cl.sub.2 (25 mL) and the organic phase was washed with H.sub.2O (25 mL) and brine (25 mL). The organic phase was dried over MgSO.sub.4, filtered and excess solvent removed by rotary evaporation under reduced pressure to afford crude product A5 which was used as such in the next step. LC/MS (Method B): ES.sup.+=1.15 min, m/z 574.6 [M+H].sup.+.

e) (S)-2-((2S,5S)-16-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-5-isopropyl-2-methyl-1,4,7-trioxo-10,13,19,22-tetraoxa-3,6,16-triazapentacosan-25-amido)-N—((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide (1)

[0356] EDCI.HCl (0.26 mmol, 2.1 eq) was added to a solution of A6 (purchased from Broadpharm) (0.122 mmol, 1.0 eq) in DCM (25 mL) and the resulting mixture stirred at room temperature for 60 min. A5 (0.26 mmol, 2.1 eq) was added and stirring continued for a further 2 hrs. The reaction mixture was evaporated to dryness and the residue purified by prep HPLC (30-40% MeCN/water+0.05% formic acid over 10 mins) to leave the product 1 as a white solid. Yield=23 mg (12%). LC/MS (Method B): rt 1.54 min m/z 1601.2 [M+H].sup.+.

Example 2—Conjugation

[0357] Herceptin-C239i antibody

[0358] Herceptin antibodies were engineered to have cysteine inserted between the 239 and 240 positions were produced following the methods described in Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516 (DOI: 5 10.1021/acs.molpharmaceut.6b00995).

[0359] A 50 mM solution of (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 2.67 micromoles, 53.3 μL) to a 3.3 mL solution of Herception-C239i antibody (10 mg, 67 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 3.0 mg/mL. 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 30 mM Histidine, 30 mM Arginine pH 6.8 and 1 mM EDTA to remove all the excess reducing agent. A 50 mM solution of dehydroascorbic acid (DHAA, 30 molar equivalent/antibody, 2.0 micromoles, 40 μL) in DMSO was added and the reoxidation mixture was allowed to react for 3 hours at room temperature with gentle (60 rpm) shaking at an antibody concentration of 3.0 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 1 was added as a DMSO solution (10 molar equivalent/antibody, 0.5 micromole, in 0.375 mL DMSO) to 3.375 mL of this reoxidised antibody solution (7.5 mg, 50 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 (2.5 micromoles, 25 μL at 100 mM), then purified on an AKTA™ Start FPLC using a GE Healthcare HiLoad™ 26/600 column packed with Superdex 200 PG, eluting with 2.6 mL/min PBS. Fractions corresponding to ConjA monomer peak were pooled, concentrated using a 15 mL Amicon Ultracell 30 kDa MWCO spin filter, sterile-filtered and analysed.

[0360] UHPLC analysis on a Shimadzu Prominence system using a Thermo Scientific MAbPac 50 mm×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjA at 214 nm and 330 nm (Compound 1 specific) shows unconjugated light chains and a mixture of unconjugated heavy chains and heavy chains attached to a single molecule of Compound 1, consistent with a drug-per-antibody ratio (DAR) of 3.88 molecules of Compound 1 per antibody (since each molecule of Compound 1 contains two drugs).

[0361] 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 98%. UHPLC SEC analysis gives a concentration of final ConjA at 2.13 mg/mL in 2.5 mL, obtained mass of ConjA is 5.32 mg (71% yield).

Example 3—ADC In Vitro Assay

[0362] The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2×10.sup.5/ml, dispensed (50 pl per well) into 96-well flat-bottom plates.

[0363] 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 8x 10-fold dilutions of stock ADC were made in a 24-well plate by serial transfer of 100 pl into 900 pl of cell culture medium. ADC dilution was dispensed (50 pl per well) into 4 replicate wells of the 96-well plate, containing 50 pl cell suspension seeded the day previously. Control wells received 50 pl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37° C. in a CO2-gassed incubator for the exposure time.

[0364] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 pl per well) into each well and incubated for 4 hours at 37° C. in the CO.sub.2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC.sub.50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.

[0365] ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87. MDA-MB-468 and NCI-N87 were cultured in RPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum.

TABLE-US-00001 EC.sub.50 (μg/mL) NCI-N87 MDA-MB-468 ConjA 0.0492 >10

STATEMENTS OF INVENTION

[0366] 1. A compound with the formula I:

##STR00033##

[0367] where X.sup.1 and X.sup.2 are independently selected from a group of formula Ia:

##STR00034##

[0368] Q is:

##STR00035##

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

[0369] a=0 to 5, b1=0 to 16 and b2=0 to 16, wherein at least b1 or b2=0 (i.e. only one of b1 and b2 may not be 0);

[0370] Y is H or F;

[0371] c1 is 0 to 5;

[0372] c2 is 0 to 5;

[0373] X.sup.3 is —CH.sub.2— or —C(═O)—;

[0374] X.sup.4 is .sup.X3—(CH.sub.2).sub.d1—(C.sub.2H.sub.4O).sub.e—(CH.sub.2).sub.d2—.sup.GL, where d1 is 0 to 5, d2 is 0 to 5 and e is 0 to 16;

[0375] and

[0376] G.sup.L is a linker for connecting to a Ligand Unit.

[0377] 2. The compound according to statement 1, wherein Q is an amino acid residue.

[0378] 3. The compound according to statement 2, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0379] 4. The compound according to statement 1, wherein Q is a dipeptide residue.

[0380] 5. The compound according to statement 4, wherein Q is selected from: [0381] .sub.NH-Phe-Lys-.sup.C═O, [0382] .sup.NH-Val-Ala-.sup.C═O, [0383] .sup.NH-Val-Lys-.sup.C═O, [0384] .sup.NH-Ala-Lys-.sup.C═O, [0385] .sup.NH-Val-Cit-.sup.C═O, [0386] .sup.NH-Phe-Cit-.sup.C═O, [0387] .sup.NH-Leu-Cit-.sup.C═O, [0388] .sup.NH-Ile-Cit-.sup.C═O, [0389] .sup.NH-Phe-Arg-.sup.C═O, [0390] .sup.NH-Trp-Cit-.sup.C═O, and [0391] .sup.NH-Gly-Val-.sup.C═O.

[0392] 6. The compound according to statement 5, wherein Q is selected from .sup.NH-Phe-Lys-.sup.C═O, .sup.NH-Val-Cit-.sup.C═O and .sup.NH-Val-Ala-.sup.C═O.

[0393] 7 The compound according to statement 1, wherein Q is a tripeptide residue.

[0394] 8. The compound according to statement 7, wherein Q is selected from .sup.NH-Glu-Val-Ala-.sup.C═O,.sup.NH-Glu-Val-Cit-.sup.C═O, .sup.NH-αGlu-Val-Ala-.sup.C═O and .sup.NH-αGlu-Val-Cit-.sup.C═O.

[0395] 9. The compound according to statement 1, wherein Q is a tetrapeptide residue.

[0396] 10. The compound according to statement 9, wherein Q is selected from: [0397] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O; and [0398] .sup.NH-Gly-Phe-Gly-Gly .sup.C═O.

[0399] 11. The compound according to statement 10, wherein Q is: [0400] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O.

[0401] 12. The compound according to any one of statements 1 to 11, wherein a is 0 to 3.

[0402] 13. The compound according to statement 12, wherein a is 0 or 1.

[0403] 14. The compound according to statement 12, wherein a is 0.

[0404] 15. The compound according to any one of statements 1 to 14, wherein b1 is 0 to 8.

[0405] 16. The compound according to statement 15, wherein b1 is 0.

[0406] 17. The compound according to statement 15, wherein b1 is 2.

[0407] 18. The compound according to statement 15, wherein b1 is 3

[0408] 19. The compound according to statement 15, wherein b1 is 4.

[0409] 20. The compound according to statement 15, wherein b1 is 5.

[0410] 21. The compound according to statement 15, wherein b1 is 8.

[0411] 22. The compound according to any one of statements 1 to 14 and 16, wherein b2 is 0 to 8.

[0412] 23. The compound according to statement 22, wherein b2 is 0.

[0413] 24. The compound according to statement 22, wherein b2 is 2.

[0414] 25. The compound according to statement 22, wherein b2 is 3.

[0415] 26. The compound according to statement 22, wherein b2 is 4.

[0416] 27. The compound according to statement 22, wherein b2 is 5.

[0417] 28. The compound according to statement 22, wherein b2 is 8.

[0418] 29. The compound according to any one of statements 1 to 28, wherein Y is H.

[0419] 30. The compound according to any one of statements 1 to 28, wherein Y is F.

[0420] 31. The compound according to any one of statements 1 to 30, wherein X.sup.1 and X.sup.2 are the same.

[0421] 32. The compound according to any one of statements 1 to 31, wherein c1 is 0 to 3.

[0422] 33. The compound according to statement 32, wherein c1 is 1 or 2.

[0423] 34. The compound according to statement 33, wherein c1 is 2.

[0424] 35. The compound according to any one of statements 1 to 34, wherein c2 is 0 to 3.

[0425] 36. The compound according to statement 35, wherein c2 is 1 or 2.

[0426] 37. The compound according to statement 36, wherein c2 is 2.

[0427] 38. The compound according to any one of statements 1 to 34, wherein c1 and c2 are the same.

[0428] 39. The compound according to any one of statements 1 to 38, wherein X.sup.3 is —CH.sub.2—.

[0429] 40. The compound according to any one of statements 1 to 38, wherein X.sup.3 is —C(═O)—.

[0430] 41. The compound according to any one of statements 1 to 40, wherein d1 is 0 to 3.

[0431] 42. The compound according to statement 41, wherein d1 is 1 or 2.

[0432] 43. The compound according to statement 42, wherein d1 is 2.

[0433] 44. The compound according to any one of statements 1 to 43, wherein d2 is 0 to 3.

[0434] 45. The compound according to statement 44, wherein d2 is 1 or 2.

[0435] 46. The compound according to statement 44, wherein d2 is 0.

[0436] 47. The compound according to any one of statements 1 to 40, wherein d1+d2 is 0 to 3.

[0437] 48. The compound according to statement 47, wherein d1+d2 is 2.

[0438] 49. The compound according to any one of statements 1 to 48, wherein e is 0 to 8.

[0439] 50. The compound according to statement 49, wherein e is 0.

[0440] 51. The compound according to statement 49, wherein e is 2.

[0441] 52. The compound according to statement 49, wherein e is 4.

[0442] 53. The compound according to statement 49, wherein e is 8.

[0443] 54. The compound according to any one of statements 1 to 53, wherein e+the largest value of b1 or b2 is no more than 16.

[0444] 55. The compound according to statement 54, wherein e+the largest value of b1 or b2 is no more than 8.

[0445] 56. The compound according to any one of statements 1 to 55, wherein each a is 0, each b1 is 0, each b2 is 2, c1 is 2, c2 is 2, X.sup.3=—C(═O)—, d1 is 2, d2 is 0 and e is 0.

[0446] 57. The compound according to any one of statements 1 to 56, wherein G.sup.L is selected from

##STR00036## ##STR00037## ##STR00038##

[0447] where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

[0448] 58. A compound according to statement 57, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

[0449] 59. A compound according to statement 57, wherein G.sup.L is G.sup.L1-1.

[0450] 60. A conjugate of formula IV:

L−(D.sup.L).sub.P  (IV)

[0451] or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit (i.e., a targeting agent), D.sup.L is a Drug Linker unit that is of formula III:

##STR00039##

[0452] where X.sup.1, X.sup.2, X.sup.3, X.sup.4, c1 and c2 are as defined in any one of statements 1 to 56;

[0453] G.sup.LL is a linker connected to a Ligand Unit; and

[0454] p is an integer of from 1 to 20.

[0455] 61. The conjugate according to statement 60, wherein G.sup.LL is selected from:

##STR00040## ##STR00041## ##STR00042##

[0456] where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

[0457] 62. The conjugate according to statement 61, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

[0458] 63. The conjugate according to statement 62, wherein G.sup.LL is G.sup.LL1-1.

[0459] 64. The conjugate according to any one of statements 60 to 63, wherein the Ligand Unit is an antibody or an active fragment thereof.

[0460] 65. The conjugate according to statement 64, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.

[0461] 66. The conjugate according to statement 65, 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)-(89):

[0462] (1) BMPR1B;

[0463] (2) E16;

[0464] (3) STEAP1;

[0465] (4) 0772P;

[0466] (5) MPF;

[0467] (6) Napi3b;

[0468] (7) Sema 5b;

[0469] (8) PSCA hlg;

[0470] (9) ETBR;

[0471] (10) MSG783;

[0472] (11) STEAP2;

[0473] (12) TrpM4;

[0474] (13) CRIPTO;

[0475] (14) CD21;

[0476] (15) CD79b;

[0477] (16) FcRH2;

[0478] (17) HER2;

[0479] (18) NCA;

[0480] (19) MDP;

[0481] (20) IL20R-alpha;

[0482] (21) Brevican;

[0483] (22) EphB2R;

[0484] (23) ASLG659;

[0485] (24) PSCA;

[0486] (25) GEDA;

[0487] (26) BAFF-R;

[0488] (27) CD22;

[0489] (28) CD79a;

[0490] (29) CXCR5;

[0491] (30) HLA-DOB;

[0492] (31) P2X5;

[0493] (32) CD72;

[0494] (33) LY64;

[0495] (34) FcRH1;

[0496] (35) IRTA2;

[0497] (36) TENB2;

[0498] (37) PSMA—FOLH1;

[0499] (38) SST;

[0500] (38.1) SSTR2;

[0501] (38.2) SSTR5;

[0502] (38.3) SSTR1;

[0503] (38.4) SSTR3;

[0504] (38.5) SSTR4;

[0505] (39) ITGAV;

[0506] (40) ITGB6;

[0507] (41) CEACAM5;

[0508] (42) MET;

[0509] (43) MUC1;

[0510] (44) CA9;

[0511] (45) EGFRvIII;

[0512] (46) CD33;

[0513] (47) CD19;

[0514] (48) IL2RA;

[0515] (49) AXL;

[0516] (50) CD30—TNFRSF8;

[0517] (51) BCMA—TNFRSF17;

[0518] (52) CT Ags—CTA;

[0519] (53) CD174 (Lewis Y)—FUT3;

[0520] (54) CLEC14A;

[0521] (55) GRP78—HSPA5;

[0522] (56) CD70;

[0523] (57) Stem Cell specific antigens;

[0524] (58) ASG-5;

[0525] (59) ENPP3;

[0526] (60) PRR4;

[0527] (61) GCC—GUCY2C;

[0528] (62) Liv-1—SLC39A6;

[0529] (63) 5T4;

[0530] (64) CD56—NCMA1;

[0531] (65) CanAg;

[0532] (66) FOLR1;

[0533] (67) GPNMB;

[0534] (68) TIM-1—HAVCR1;

[0535] (69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

[0536] (70) B7-H4—VTCN1;

[0537] (71) PTK7;

[0538] (72) CD37;

[0539] (73) CD138-SDC1;

[0540] (74) CD74;

[0541] (75) Claudins—CLs;

[0542] (76) EGFR;

[0543] (77) Her3;

[0544] (78) RON-MST1R;

[0545] (79) EPHA2;

[0546] (80) CD20-MS4A1;

[0547] (81) Tenascin C—TNC;

[0548] (82) FAP;

[0549] (83) DKK-1;

[0550] (84) CD52;

[0551] (85) CS1—SLAMF7;

[0552] (86) Endoglin—ENG;

[0553] (87) Annexin A1—ANXA1;

[0554] (88) V-CAM (CD106)-VCAM1;

[0555] (89) ASCT2 (SLC1A5).

[0556] 67. The conjugate according to any one of statements 64 to 66, wherein the antibody or antibody fragment is a cysteine-engineered antibody.

[0557] 68. The conjugate according to any one of statements 64 to 67, wherein p is an integer from 1 to about 10.

[0558] 69. The conjugate according to statement 68, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0559] 70. A mixture of conjugates according to any one of statements 64 to 69, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 2 to about 20.

[0560] 71. The conjugate or mixture according to any one of statements 60 to 70, for use in therapy.

[0561] 72. A pharmaceutical composition comprising the conjugate or mixture of any one of statements 60 to 70 and a pharmaceutically acceptable diluent, carrier or excipient.

[0562] 73. The conjugate or mixture according to any one of statements 60 to 70, or the pharmaceutical composition according to statement 72, for use in the treatment of a proliferative disease in a subject.

[0563] 74. The conjugate, mixture or pharmaceutical composition according to statement 73, wherein the disease is cancer.

[0564] 75. Use of a conjugate or mixture according to any one of statements 60 to 70, or the pharmaceutical composition according to statement 72 in a method of medical treatment.

[0565] 76. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 72.

[0566] 77. The method of statement 76 wherein the method of medical treatment is for treating cancer.

[0567] 78. The method of statement 77, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.

[0568] 79. Use of a conjugate or mixture according to any one of statements 60 to 670 in a method of manufacture of a medicament for the treatment of a proliferative disease.

[0569] 80. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of conjugate or mixture according to any one of statements 60 to 70, or the pharmaceutical composition according to statement 72.