COMPOUNDS AND CONJUGATES THEREOF

Abstract

A conjugate comprising the following topoisomerase inhibitor derivative (A*): with a linker for connecting to a Ligand Unit, wherein the linker is attached in a cleavable manner to the amino residue. The Ligand Unit is preferably an antibody. Also provided is A* with the linking unit attached, and intermediates for their synthesis, as well as the released warhead.

##STR00001##

Claims

1. A compound with the formula I: ##STR00085## and salts and solvates thereof, wherein R.sup.L is a linker for connection to a Ligand Unit, which is selected from: (ia): ##STR00086## wherein Q is: ##STR00087## where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue;  X is: ##STR00088## where a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, wherein at least b1 or b2=0 and at least c1 or c2=0; G.sup.L is a linker for connecting to a Ligand Unit; (ib): ##STR00089## 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.

2. The compound according to claim 1, wherein R.sup.L is of formula Ia.

3. The compound according to claim 2, 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 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.

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

5. The compound according to any one of claims 2 to 4, 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.

6. The compound according to any one of claims 2 to 4, 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.

7. The compound according any to one of claims 2 to 6, wherein: (i) c1 is: (a) 0; or (b) 1; and (ii) c2 is: (a) 0; or (b) 1; wherein at least one of c1 and c2 is 0.

8. The compound according to any one of claims 2 to 7, wherein d is: (a) 0 to 3; or (b) 1 or 2; or (c) 2; or (d) 5.

9. The compound according to any one of claims 2 to 8, wherein: (a) a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is 0, 2, 3, 4, 5 or 8; or (b) a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is 0, 2, 3, 4, 5 or 8; or (c) a is 0, b1 is 0, c1 iso, c2 is 0 and d is 1, and b2 is 0, 2, 3, 4, 5 or 8; or (d) b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1 or 5; or (e) a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0, 2, 3, 4, 5 or 8.

10. The compound according to any one of claims 2 to 9, wherein G.sup.L is selected from ##STR00090## ##STR00091## where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

11. A compound according to claim 10, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

12. The compound according to claim 1, wherein R.sup.L is of formula Ib, and: (a) both R.sup.L1 and R.sup.L2 are H; or (b) R.sup.L1 is H and R.sup.L2 is methyl; or (c) both R.sup.L1 and R.sup.L2 are methyl; or (d) wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group; or (e) wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

13. 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: ##STR00092## R.sup.LL is a linker connected to the Ligand unit selected from (ia′): ##STR00093## where Q and X are as defined in any one of claims 1 to 9 and G.sup.LL is a linker connected to a Ligand Unit; and (ib′): ##STR00094## where R.sup.L1 and R.sup.L2 are as defined in either claim 1 or claim 12; and p is an integer of from 1 to 20.

14. The conjugate according to claim 13, wherein G.sup.LL is selected from: ##STR00095## ##STR00096## where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

15. The conjugate according to claim 14, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

16. The conjugate according to any one of claims 13 to 15, wherein the Ligand Unit is an antibody or an active fragment thereof.

17. The conjugate according to claim 16, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 10.

18. A mixture of conjugates according to either claim 16 or claim 17, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

19. A pharmaceutical composition comprising the conjugate or mixture of any one of claims 13 to 18 and a pharmaceutically acceptable diluent, carrier or excipient.

20. The conjugate or mixture according to any one of claims 13 to 18, or the pharmaceutical composition according to claim 19, for use in the treatment of a proliferative disease in a subject.

21. The conjugate, mixture or pharmaceutical composition according to claim 20, wherein the disease is cancer.

22. Use of a conjugate or mixture according to any one of claims 13 to 18, or the pharmaceutical composition according to claim 19 in a method of medical treatment.

23. A method of medical treatment comprising administering to a patient the pharmaceutical composition of claim 19.

24. The method of claim 23 wherein the method of medical treatment is for treating cancer.

25. The compound A: ##STR00097##

26. A compound with the formula VI: ##STR00098## where Q is as in either claim 1 or 3.

Description

EXAMPLES

[0366] The column chromatography on silica gel was performed using Qingdao Hailang silica gel or using a Biotage® Isolera™ and fractions checked for purity using thin-layer chromatography (TLC). TLC was performed using Huanghai HSF254 silica gel or Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on glass plate. Visualisation of TLC was achieved with UV light. Extraction and chromatography solvents, and all fine chemicals were bought and used without further purification from SINOPHARM (China), VWR (US), or Sigma-Aldrich (US) unless otherwise stated. 6,8-Difluoro-3,4-dihydronaphthalen-1(2H)-one was obtained from Bide Pharmatech Ltd.

[0367] Reverse-phase purification was performed on the Waters Prep HPLC system composed of Waters 2767, Waters 2545, Waters 515 HPLC pumps, WATERS SFO, WATERS 2424, Acquity QDa with MassLynx program.

[0368] Analytical LC/MS conditions were as follows: Positive mode electrospray mass spectrometry was performed using a Waters Acquity H-class SQD2. Mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). Gradient for 5-minute run: Initial composition 5% B held over 1 minute, then increased from 5% B to 95% B over a 3 minutes period. The composition was held for 30 seconds at 95% B, then returned to 5% B in 30 seconds and held there for 84 seconds. The total duration of the gradient run was 5.0 minutes. Flow rate was 0.8 mL/minute. Columns: Agilent ZORBAX Extend 80A 1.8 μm 2.1×50 mm at 45° C.

[0369] Conditions for 3 minutes rum: Flow rate was 0.3 mL/minute. Detection was at 210 nm. Columns: Waters Acquity UPLC® BEH Shield C18 1.7 μm 2.1×50 mm at 35° C. fitted with Waters Acquity UPLC® BEH Shield C18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

Example 1

[0370] ##STR00036## ##STR00037##

[0371] a) 6,8-Difluoro-5-nitro-1-tetralone A2 To a dust of 6,8-difluoro-1-tetralone A1 (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 KNO.sub.3 (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 A2 (8.1 g, 43% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): δ ppm 6.98 (t, J=10.0 Hz, 1H), 3.01-2.98 (m, 2H), 2.72-2.68 (m, 2H), 2.21-2.05 (m, 2H).

[0372] b) 5-Amino-6,8-difluoro-1-tetralone A3

[0373] To a mixture of compound A2 (9.1 g, 39.6 mmol) in EtOH/H.sub.2O (8:1, 270 mL) were added NH.sub.4C.sub.1 (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 A3 (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).

[0374] c) 5-Acetylamino-6,8-difluoro-1-tetralone A4

[0375] To a solution of compound A3 (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 A4 (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).

[0376] d) 5-Acetylamino-6-fluoro-8-amino-1-tetralone A5

[0377] To a solution of compound A4 (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 A5 (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).

[0378] 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 A7

[0379] Compound A5 (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 A6, 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 A7 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).

[0380] 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 1

[0381] 60 mg of crude compound A7 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 1 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 2

[0382] ##STR00038##

[0383] a) 5,8-Diamino-6-fluoro-1-tetralone A8

[0384] A solution of 5-acetylamino-6-fluoro-8-amino-1-tetralone A5 (1.0 g, 4.2 mmol) in 6N HCl (50 mL) was refluxed for 4 h. The mixture was concentrated under reduced pressure. The residue was added to saturated aqueous NaHCO.sub.3 (60 mL) slowly. The resulting mixture was extracted with EtOAc (100 mL×3). The combined organic layers were washed with brine (100 mL), dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure to afford compound A8 (0.7 g, 90% yield) as a yellow solid.

[0385] (Microwave Method) 240 mg of 5-acetylamino-6-fluoro-8-amino-1-tetralone A5 (1.06 mmol) was dissolved in 3 mL HCl (37%) and reacted in microwave reactor at 100° C. for 1 h. The mixture was concentrated under reduced pressure. The residue was added to saturated aqueous NaHCO.sub.3 (10 mL) slowly. The resulting mixture was extracted with EtOAc (15 mL×3). The combined organic layers were washed with brine (20 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated under reduced pressure to afford compound A8 (180 mg, 87% yield).

[0386] b) 5-Allocglycine-8-amino-6-fluoro-1-tetralone A9

[0387] To a solution of compound A8 (0.7 g, 3.8 mmol) and Alloc-Gly-OH (0.7 g, 4.2 mmol) in THF (50 mL) were added Et.sub.3N (0.4 g, 4.2 mmol), HOBt (0.6 g, 4.2 mmol) and EDCI (0.9 g, 4.6 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (100 mL) and then washed with saturated aqueous NaHCO.sub.3 (50 mL) and brine (50 mL). The organic phase was dried over anhydrous MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=200:1) to afford the compound A9 (0.52 g, 41% yield) as an off-white solid.

[0388] .sup.1H NMR (400 MHz, DMSO-d6): δ ppm 9.15 (s, 1H), 7.53 (t, J=6.0 Hz, 1H), 6.41 (d, J=12.4 Hz, 1H), 5.92-5.88 (m, 1H), 5.33-5.28 (m, 1H), 5.20-5.17 (m, 1H), 4.51-4.49 (m, 2H), 3.78 (d, J=6.0 Hz, 1H), 2.65 (t, J=6.0 Hz, 1H), 2.55-2.49 (m, 2H), 1.87-1.84 (m, 2H).

[0389] c) Allyl (S)-(2-((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)amino)-2-oxoethyl)carbamate A10

[0390] 250 mg (0.746 mmol) of compound A9, 200 mg (0.760 mmol) of (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione A6, and 200 mg (0.796 mmol) of pyridinium p-toluenesulfonate were dissolved in 30 mL of anhydrous toluene. Equipped with a Dean-Stark trap, the reaction was heated at 130° C. for 4 h. The solvent was evaporated, and the residue was precipitated into acetone to afford 250 mg of the desired product as a brown solid after centrifugated and dried under vacuum. The residue on the flask wall was washed with acetone and concentrated to give 110 mg of the compound A10 as a brown solid. The yield of the crude product was 87%. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=563; .sup.1H NMR (400 MHz, DMSO-d6): δ ppm: signals for the desired product, 9.88 (s 1H), 7.83 (d, J=11 Hz, 1H), 7.63 (t, J=6.1 Hz, 1H), 7.33 (s, 1H), 5.99-5.88 (m, 1H), 5.44 (s, 2H), 5.32 (dd, J=6.4 Hz, 1H), 5.26 (s, 2H), 5.20 (dd, J=Hz, 1H), 4.53 (d, J=5.3 Hz, 2H), 3.93 (d, J=6 Hz, 2H), 3.18 (t, J=5.7 Hz, 2H), 2.97 (t, J=5.3 Hz, 2H), 2.23 (s, 1H), 2.03 (m, 2H), 1.88 (m, 2H), 0.88 (t, J=7.4 Hz, 3H).

[0391] d) (9H-Fluoren-9-yl)methyl (2-((2-((S)-1-((2-(((S)-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 yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate A12

[0392] A11 was synthesised as follows:

[0393] Fmoc-GGF (500 mg, 0.997 mmol, synthesized by standard solution peptide synthetic method) and 276 mg (1.50 mmol) of pentafluorophenol were dissolved in 20 mL of NMP. To this suspension, 0.33 mL of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (1.8 mmol) was added, and the reaction was stirred at room temperature overnight. The progress of the reaction was monitored with LC-MS.

[0394] 50 mg (0.089 mmol) of the compound A10, 103 mg (0.0887 mmol) of Pd(PPh.sub.3).sub.4 and 145 μL (0.899 mmol) of triethylsilane were dissolved in 2 mL of NMP. To the mixture, added 4 mL (0.2 mmol) of the activated acid solution A11. The progress of the reaction was monitored by LC-MS. The reaction mixture was precipitated into ether (2 vials of 15 mL) and centrifuged to give compound A12. The solid was air-dried and used without further purification.

[0395] e) (S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((S)-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)amino)-2-oxoethyl)-3-phenylpropanamide A13

[0396] Crude compound A12 was dissolved in 2 mL of NMP, added 2 mL of 20% 4-methylpiperidine (3.0 mmol). The reaction mixture was stirred at room temperature, and the progress was monitored by LC-MS. After the reaction was completed, the reaction mixture was 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/lyophilized to give 23 mg (35%) of compound A13 as a yellow solid.

[0397] LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=741; .sup.1H NMR (400 MHz, DMSO-d6): δ ppm 9.74 (s, 1H), 8.51 (t, J=5.5 Hz, 1H), 8.43 (t, J=5.5 Hz, 1H), 8.30 (d, J=8.2 Hz, 1H), 7.91 (br, s, 2H+H.sup.+), 7.76 (d, J=11 Hz, 1H), 7.26 (s, 1H), 7.21-7.15 (m, 4H), 7.14-7.07 (m, 1H), 5.37 (s, 2H), 5.21 (s, 2H), 4.55 (m, 1H), 3.98 (m, 2H), 3.82 (dd, J=16.8, 5.6 Hz, 1H), 3.64 (dd, J=16.8, 5.6 Hz, 1H), 3.48 (m, 2H), 3.11 (t, J=5.6 Hz, 2H), 3.05 (dd, J=13.9, 4.4 Hz, 1H), 2.91 (t, J=5.3 Hz, 2H), 2.73 (dd, J=13.8, 9.9 Hz, 1H), 1.96 (m, 2H), 1.80 (m, J=7.4 Hz, 2H), 0.81 (t, J=7.4 Hz, 3H).

[0398] f) 1-(3-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((2-(((S)-1-((2-(((S)-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)amino)-2-oxoethyl)amino)-1-oxo phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide 2

[0399] 15 mg (0.020 mmol) of compound A13 and 15 mg (0.022 mmol) of Mal-PEG8-NHS ester A14 were dissolved in 1 mL of NMP, and 14 μL (0.10 mmol) of TEA was added to the solution. The reaction was stirred at room temperature. The progress of the reaction was monitored with LC/MS. After the complete consumption of the amine, the reaction mixture was filtered 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/frozen/lyophilized to give 14 mg (53%) of the desired product as a yellow solid.

[0400] LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=1315; .sup.1H NMR (400 MHz, DMSO-d6): δ ppm 9.64 (s, 1H), 8.43 (t, J=5.6 Hz, 1H), 8.12-8.06 (m, 2H), 7.94 (t, J=4.6 Hz, 2H), 7.76 (d, J=11 Hz, 1H), 7.26 (s, 1H), 7.21-7.15 (m, 4H), 7.14-7.07 (m, 1H), 6.93 (s, 2H), 5.37 (s, 2H), 5.20 (s, 2H), 4.51-4.46 (m, 1H), 3.95 (m, 2H), 3.72 (d, J=6.0 Hz, 1H), 3.68 (d, J=6.0 Hz, 2H), 3.60 (d, J=5.6 Hz, 2H), 3.44-3.41 (m, PEG and H.sub.2O signals overlapped), 3.29 (t, J=6.0 Hz, 2H), 3.14-3.00 (m, 5H), 2.91 (t, J=6.1 Hz, 2H), 2.78 (m, 1H), 2.31 (t, J=6.5 Hz, 2H), 2.26 (t, J=7.2 Hz, 2H), 1.96 (m, 2H), 1.80 (m, 2H), 0.81 (t, J=7.2 Hz, 3H).

[0401] General Information for Example 3

[0402] 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. Extraction and chromatography solvents were bought and used without further purification from VWR U.K. All fine chemicals were purchased from Sigma-Aldrich unless otherwise stated. Pegylated reagents were obtained from Quanta biodesign US via Stratech UK.

[0403] Analytical LC/MS conditions were as follows: Positive mode electrospray mass spectrometry was performed using a Waters Aquity H-class SQD2.

[0404] Mobile phases used were solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). 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. 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.

Example 3

[0405] ##STR00039##

[0406] a) Alternative synthesis of (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 A7

[0407] Compound A5 (136 mg, 0.57569 mmol) and trione A6 (167 mg, 0.63 mmol) were dissolved in toluene (20 mL) before 4-methylbenzenesulfonate; pyridin-1-ium (149 mg, 0.59 mmol) was added and the mixture stirred at reflux for 3.5 h. LCMS indicated the reaction was complete. The reaction mixture was concentrated in vacuo and triturated with MeCN to afford compound A7 (220 mg, 0.4746 mmol, 82.45% Yield) as a beige solid, which was used without further purification. The MeCN washings were concentrated in vacuo and purified by isolera chromatography (0-5% MeOH in CH.sub.2Cl.sub.2) to afford a further 20 mg of compound A7 after isolera purification (0-5% MeOH in CH.sub.2Cl.sub.2) as a brown solid. LCMS: RT=1.41 min, 464.5 [M+H]+.

[0408] b) Alternative synthesis of (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 1

[0409] Compound A7 (220 mg, 0.474 mmol) was dissolved in 5M HCl.sub.aq (15 mL, 75 mmol, 5 mol/L) and the mixture stirred for 4 h at 80° C., whereupon LCMS indicated that all the starting material had been consumed. The reaction mixture was concentrated in vacuo to afford Compound 1.2HCl (235 mg, 0.475 mmol, 100.2% Yield) as a red solid. The product was used as crude in the next step. LCMS: RT=1.49 min, no mass.

##STR00040##

[0410] c) In-situ formation of [(2R)-2-[(2-nitrophenyl)disulfanyl]propyl] carbonochloridate A16

[0411] (2R)-2-[(3-nitro-2-pyridyl)disulfanyl]propan-1-ol A15 (14 mg, 0.057 mmol) was dissolved in CH.sub.2Cl.sub.2 (0.5 mL, 8 mmol). Pyridine (5.0 μL, 0.062 mmol), then triphosgene (6 mg, 0.020 mmol) were added and the mixture stirred under argon for 30 min, whereupon LCMS (Et.sub.2NH quench) indicated the reaction was complete. LCMS: RT=1.94 min, 346.4 [M+Et.sub.2NH].sup.+

[0412] d) (R)-2-((3-nitropyridin-2-yl)disulfaneyl)propyl((S)-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)carbamate 3

[0413] In a separate flask, Compound 1.2HCl (22 mg, 0.044 mmol) was dissolved in CH.sub.2Cl.sub.2 (1 mL, 15.60 mmol, 100 mass %), DIPEA (45 μL, 0.258 mmol) and pyridine (22 μL, 0.272 mmol). The chloroformate reaction mixture was added to the aniline solution and the mixture stirred for 30 min, whereupon LCMS indicated that the chloroformate had been consumed, but no compound 3 was observed. More triphosgene was added to the reaction and stirred for 20 min, whereupon LCMS indicated the presence of a small amount of product. More triphosgene was added and the mixture stirred for 1 h, whereupon LCMS indicated major component was compound 3. The reaction mixture was concentrated in vacuo and purified by isolera chromatography (0-4% MeOH in CH.sub.2Cl.sub.2), then reverse-phase isolera chromatography (0-60% eluent B in eluent A) to afford pure compound 3 (8 mg, 0.01153 mmol, 25.91% yield) as a yellow solid after freeze drying.

[0414] Eluent A=0.01% HCO.sub.2H in H.sub.2O

[0415] Eluent B=0.01% HCO.sub.2H in MeCN

[0416] LCMS: RT=1.95 min, 694.6 [M+H].sup.+.

Example 4

[0417] ##STR00041##

[0418] a) 5-Fmoc-alanine-6-fluoro-8-amino-1-tetralone A17

[0419] 164 mg (0.84 mmol) of 5,8-diamino-6-fluoro-1-tetralone A8 was dissolved in 6 mL of THF, and 315 mg (1.01 mmol, 1.2 eq.) of Fmoc-Ala-OH, and 138 mg of HOAt (1.01 mmol, 1.2 eq.) were added to the solution. 275 μL (1.24 mmol) of EDCI and 142 μL (1.02 mmol) of Et.sub.3N were then added to the solution. The reaction mixture was stirred at room temperature. The progress of the reaction was monitored by LC/MS. After 4 hours, the reaction mixture was stored in the freezer. The reaction mixture was worked-up with 50 mL EtOAc/50 mL H.sub.2O, followed by washing the organic layer with H.sub.2O, then brine, and was subsequently dried over Na.sub.2SO.sub.4. The crude product was purified on silica column with dichloromethane/methanol to give 260 mg of the desired product. LCMS ESI [M+H]=488.93; calculated 488.20

[0420] b) A18

[0421] 210 mg of 5-Fmoc-alanine-6-fluoro-8-amino-1-tetralone A17 (0.43 mmol), 114 mg of trione A6 (0.43 mmol), and 109 mg of pyridinium p-toluenesulfonate (0.43 mmol) were dissolved in 30 mL of anhydrous toluene. With a Dean-Stark trap, the reaction was heated with an oil bath at 130° C. for 4 hours, resulting in a water layer in the condenser. The solution was decanted and dried under reduced pressure to give 270 mg of the desired product. The solvent of the solution was evaporated and dissolved in 0.5 mL NMP and precipitated into 14 mL of diethyl ether. Centrifugation gave a brown solid which was washed with ether again. The resulting solid was dried to give a further 30 mg of the crude product. The total crude desired product (300 mg, 97% yield) was used without further purification. LCMS ESI [M+H]=716.01; calculated 715.26

[0422] c) A19

[0423] 220 mg (0.31 mmol) of A18 was dissolved in 2 mL NMP, and 150 μL (1.28 mmol) of 4-methylpiperidine was added to the solution. The reaction mixture was stirred at room temperature, and the progress of the reaction was monitored by LC-MS. After the reaction was completed, the reaction mixture was purified with 0.1% TFA water/0.1% TFA acetonitrile. The fractions containing the desired product were collected, combined, then frozen, and gave 42 mg (28% yield) of the desired product after lyophilization. LCMS ESI [M+H]=493.23; calculated 493.19

[0424] d) A20

[0425] 23 mg (0.046 mmol) of A19 was dissolved in 0.5 mL of NMP. 35 mg (0.11 mmol) of Boc-Val-NHS and 20 μL (0.12 mmol) of DIPEA were added to the above solution. The reaction mixture was stirred at room temperature and the progress of the reaction was checked by LC-MS. After the reaction was completed, the product was precipitated into ether, and washed with ether twice. The residue was air-dried to provide 32 mg (99% yield) of a brown solid. LCMS ESI [M+H]=693.67; calculated 692.31

[0426] e) A21

[0427] Crude A20 was treated with 0.1 mL TFA in 0.3 mL DCM and the progress of the reaction was monitored by LC-MS. After the reaction was completed, DCM and trifluoroacetic acid were removed under vacuum. The residue was dried under the vacuum overnight to give 27 mg (98% yield) of the crude product. LCMS ESI [M+H]=592.04; calculated 592.26.

[0428] .sup.1HNMR (DMSO-d.sub.6): δ ppm 10.07 (s, 1H), 8.78 (d, J=6.9 Hz, 1H), 8.10 (d, J=4.1 Hz, 3H), 7.82 (d, J=11.0 Hz, 1H), 7.32 (s, 1H), 6.53 (s, br, 1H), 5.43 (s, 2H), 5.27 (s, 2H), 4.67 (q, J=6.7 Hz, 1H), 4.67 (q, J=7.0 Hz, 1H), 3.63 (q, J=5.2 Hz, 1H), 3.17 (t, J=5.9 Hz, 2H), 2.96 (t, J=5.7 Hz, 2H), 2.14-2.07 (m, 1H), 2.05-1.94 (m, 2H), 1.87 (p, J=7.3 Hz, 2H), 1.46 (d, J=7.1 Hz, 3H), 0.96 (dd, J=6.8, 4.2 Hz, 6H), 0.88 (t, J=7.3 Hz, 3H).

[0429] f) 4

[0430] 12 mg (0.017 mmol) of Mal-PEG8-NHS A14 was dissolved in 1 mL NMP. 10.3 mg (0.017 mmol) of crude A21 and 12 μL (0.0094 mmol) of DIPEA was added to the above solution. The progress of the reaction was monitored by LC-MS. After the consumption of the starting material A21, the reaction mixture was acidified with 8 μL of TFA, then purified with 0.1% TFA water/0.1% TFA acetonitrile to give the desired product 11 mg (54% yield) after lyophilization. LCMS ESI [M+H]=1166.09; calculated 1165.52

[0431] .sup.1HNMR (DMSO-d.sub.6): δ ppm 9.86 (s, 1H), 8.26 (d, J=6.7 Hz, 1H), 8.00 (t, J=5.5 Hz, 1H), 7.90 (d, J=8.7 Hz, 1H), 7.80 (d, J=11 Hz, 1H), 7.32 (s, 1H), 7.00 (s, 2H), 5.43 (s, 2H), 5.26 (s, 2H), 4.54 (q, J=6.7 Hz, 1H), 4.26 (dd, J=8.2, 6.7 Hz, 1H), 3.81-3.48 (m, overlapped with H.sub.2O), 3.35 (t, J=6.0 Hz, 2H), 3.20-3.10 (m, 4H), 2.96 (t, 2H), 2.40 (t, J=6.3 Hz, 1H), 2.32 (m, 2H), 2.06-1.93 (m, 3H), 1.93-1.80 (m, 2H), 1.41 (d, J=7.1 Hz, 3H), 0.91-0.80 (m, 9H).

Example 5

[0432] ##STR00042##

[0433] 22 mg (0.037 mmol) of A21 and 14 mg (0.045 mmol) of Mal-Caproyl-NHS A22 were dissolved in 0.5 mL of NMP, and 12 μL (0.068 mmol) of DIPEA was added to this solution. The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by LC-MS. After the reaction was completed, the reaction was quenched with 12 μL of trifluoroacetic acid, and was purified on prep-HPLC with 0.1% TFA water/0.1% TFA acetonitrile to give 10 mg (34% yield) of the desired product after lyophilization. LCMS ESI [M+H]=785.88; calculated 785.33. .sup.1HNMR (DMSO-d.sub.6): δ ppm 9.86 (s, 1H), 8.23 (d, J=6.7 Hz, 1H), 7.84 (d, J=8.7 Hz, 1H), 7.80 (d, J=11 Hz, 1H), 7.32 (s, 1H), 6.98 (s, 2H), 6.55-6.50 (m, 1H), 5.43 (s, 2H), 5.26 (s, 2H), 4.53 (q, J=7.0 Hz, 1H), 4.22 (dd, J=8.7, 6.7 Hz, 1H), 3.16 (t, J=6.0 Hz, 2H), 2.96 (t, 2H), 2.22-2.07 (m, 3H), 2.04-1.94 (m, 3H), 1.93-1.81 (m, 2H), 1.49-1.43 (m, 4H), 1.40 (d, J=7.1 Hz, 3H), 1.15 (q, J=7.5 Hz, 2H), 0.92-0.82 (m, 9H).

Example 6

[0434] ##STR00043##

[0435] 27 mg of A13 (0.0365 mmol) and 13 mg of Mal-Caproyl-NHS A22 (0.04217 mmol) were dissolved in 0.5 mL of NMP, and 10 μL of DIPEA was added to the reaction mixture. The reaction was stirred at room temperature and monitored by LC-MS. After the reaction was completed, the reaction mixture was purified on prep-HPLC with 0.1% TFA/ACN to give 9 mg (26%) of the desired product after lyophilization. LCMS (0.1% formic acid/acetonitrile) ESI [M+H]=933.29; calculated 933.36. .sup.1HNMR (DMSO-d.sub.6): δ ppm 9.70 (s, 1H), 8.49 (d, J=5.8 Hz, 1H), 8.15 (d, J=8.0 Hz, 1H), 8.05 (t, J=5.7 Hz, 1H), 8.01 (t, J=5.7 Hz, 1H), 7.82 (d, J=11.0 Hz, 1H), 7.32 (s, 1H), 7.26 (s, 2H), 7.24 (s, 2H), 7.21-7.15 (m, 1H), 6.98 (s, 2H), 6.56 (br, 1H), 5.43 (s, 2H), 5.26 (s, 2H), 4.58-4.52 (m, 1H), 4.02 (dt, J=16.9, 6.0 Hz, 2H), 3.76 (dd, J=16.7, 5.9 Hz, 1H), 3.65 (d, J=5.7 Hz, 2H), 3.60 (dd, J=16.7, 5.4 Hz, 1H), 3.34 (t, J=7.1 Hz, 2H), 3.17 (t, J=5.7 Hz, 2H), 3.10 (dd, J=13.7, 4.3 Hz, 1H), 2.97 (t, J=5.4 Hz, 2H), 2.84 (dd, J=13.7, 9.7 Hz, 1H), 2.08 (t, J=7.5 Hz, 2H), 2.02 (t, J=5.7 Hz, 2H), 1.87 (dq, J=7.3 Hz, 2H), 1.44 (dt, J=7.3 Hz, 4H), 1.20-1.12 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).

Example 7—Conjugation

[0436] Classical Conjugation

[0437] An anti-HER2 antibody, derived from trastuzumab, and a negative control antibody, NIP228, were used as the full-length antibodies to prepare ADCs. The reduction of antibodies was carried out by mixing the antibodies with 50 mM tris-(2-carboxyethyl)-phosphine (TCEP) in 1×PBS, 1 mM EDTA, pH 7.2 at 37° C., and the reaction mixture was shaken for 1 h. The reduced antibodies were then used for conjugation using 5 molar excess of compound 2 in dimethyl sulfoxide (Sigma-Aldrich). The volume of the buffer was adjusted to reach 10% final DMSO concentration for the conjugation solution. The conjugation was carried out at room temperature with shaking for 1 h. This method was used to produce: [0438] Conjugate Her2-2 [0439] Conjugate Nip228-2 [0440] Conjugate Her2-4 [0441] Conjugate Nip228-4 [0442] Conjugate Her2-5 [0443] Conjugate Nip228-5 [0444] Conjugate Her2-6 [0445] Conjugate Nip228-6

[0446] Engineered Conjugation

[0447] Herceptin and Nip228 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:

[0448] 510.1021/acs.molpharmaceut.6b00995). These antibodies were prepared using 50 mM tris-(2-carboxyethyl)-phosphine (TCEP) and reduced with 50 mM in PBS 1×, 1 mM EDTA, pH 7.2 at 37° C. with shaking for 3 h. The uncapping antibodies were dialysed with conjugation buffer (PBS 1×, 1 mM EDTA, pH 7.2) at 4° C. overnight. The recovered antibodies were then used for oxidation using 20 molar excess of 50 mM dehydroascorbic acid (dhAA) at room temperature with shaking for 4 h. The reduced antibodies were then used for conjugation using 8 molar excess of payload over antibody prepared in 100% dimethyl sulfoxide (10% final DMSO concentration, Sigma-Aldrich). The conjugation was carried out with shaking at room temperature for 1 h. This method was used to produce: [0449] Conjugate Her2*-2 [0450] Conjugate Nip228*-2

[0451] Purification

[0452] After conjugation, ADCs were purified on ceramic hydroxyapatite HPLC (CHT) to remove free compound 2 and other contaminants. The purification was carried out using 5 mL Bio-Scale Mini CHT Type II, 40 μm Cartridge column (Bio-Rad) and an AKTA Pure system (GE Healthcare). ADCs were diluted at a 1:3 ratio in pure water before loading. After loading and washing with two column volumes of buffer A, ADCs were eluted using a linear gradient of 50% buffer B for 30 min. (Buffer A: 10 mM Sodium phosphate buffer, pH7.0; Buffer B: 10 mM sodium phosphate/2M sodium chloride, pH7.0). SEC was used to characterize fractions containing ADCs. The fractions were concentrated to about 1 mg/mL of ADCs. SEC was used to analyze the monomeric content, aggregates, and fragments of ADCs. Data collection and process were carried out using MassHunter software (Agilent). The ADCs were filtered using a 0.22 mm syringe filter (Pall Corporation) to remove potential endotoxin contamination. Aliquots of the ADCs were stored at −80° C. for future use.

[0453] Conjugate Her2-2 had a DAR of 8.0, whilst Conjugate Nip228-2 had a DAR of 7.79.

[0454] Conjugate Her2-4 had a DAR of 8.0, whilst Conjugate Nip228-4 had a DAR of 7.88.

[0455] Conjugate Her2-5 had a DAR of 8.0, whilst Conjugate Nip228-5 had a DAR of 8.0.

[0456] Conjugate Her2-6 had a DAR of 7.91, whilst Conjugate Nip228-6 had a DAR of 8.0.

[0457] Conjugate Her2*-2 had a DAR of 2.0, and Conjugate Nip228*-2 had a DAR of 2.0.

Example 8—Further Conjugation

[0458] A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline pH 7.4 (PBS) was added (40 molar equivalent/antibody, 11.2 micromoles, 1.12 mL) to a 20 mL solution of antibody (Herceptin engineered to have cysteine inserted between the 239 and 240 positions) (42 mg, 280 nanomoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibody concentration of 2.1 mg/mL. The reduction mixture was allowed to react at room temperature for 16 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, 30 molar equivalent/antibody, 7.0 micromoles, 141 μL) in DMSO was added to 22 mL of this reduced buffer exchanged antibody (35.2 mg, 235 nanomoles) and the reoxidation mixture was allowed to react for 2 hours and 30 minutes at room temperature with gentle (60 rpm) shaking at an antibody concentration of 1.6 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. Compound 3 was added as a DMSO solution (20 molar equivalent/antibody, 2.2 micromole, in 1.29 mL DMSO) to 10.5 mL of this reoxidised antibody solution (16.8 mg, 112 nanomoles) pH adjusted with 1.16 mL of 1 M Sodium Bicarbonate for a 10% (v/v) final DMSO concentration and 10% (v/v) final sodium bicarbonate concentration. The solution left to react at room temperature for 2 hours with gentle shaking. Then the conjugation was quenched by addition of N-acetyl cysteine (11 micromoles, 112 μL at 100 mM), then purified and buffer exchanged into 25 mM Histidine 205 mM Sucrose pH 6.0 buffer using a 50 mL Amicon Ultracell 50 kDa MWCO spin filter, sterile-filtered and analysed. UHPLC analysis on a Shimadzu Prominence system using a Sepax Proteomix HIC Butyl-NP5 4.6×35 mm 5 μm column eluting with a gradient of 25 mM sodium phosphate, 1.5 M ammonium sulphate pH 7.4 buffer and 20% acetonitrile (v/v) in 25 mM sodium phosphate pH 7.4 buffer on intact sample of Conjugate Her2*-3 at 214 nm and 330 nm (Compound 3 specific) showed unconjugated and conjugated antibody attached to one or two molecules of Compound 3, consistent with a drug-per-antibody ratio (DAR) of 1.48 molecules of Compound 3 per antibody.

[0459] 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 Conjugate Her2*-3 at 280 nm shows a monomer purity of 98%. UHPLC SEC analysis gives a concentration of final Conjugate Her2*-3 at 1.38 mg/mL in 8.6 mL, obtained mass of Conjugate Her2*-3 is 11.9 mg (71% yield).

Example 9—In-Vitro Cytotoxicity Test—Compounds

[0460] Killing of human tumor cell lines was evaluated in vitro using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, Wis.). Briefly, 3×10.sup.3 cells in 80 mL RPMI+10% FBS were added to the inner wells of white-walled 96-well plates (Corning® Costar®, Fisher Scientific, Waltham, Mass.). The following cell lines were tested: A549, HCT116 and SKBR3. The test compounds were diluted to a 5Ax stock (125 μM) in RPMI+10% FBS. Treatments were then serially diluted 1:10 in RPMI+10% FBS. 20 mL of this series was added to the cells in triplicate, resulting in a 9-point dose curve of test compound ranging from 25 mM at the highest concentration to 2.5×10.sup.−7 mM at the lowest. DMSO (vehicle) and media-only controls also were included. Plates were incubated at 37° C., 5% CO.sub.2 for 72 hours. At the end of the incubation period, 100 mL of the Substrate Solution (Promega, Madison Wis.) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, Mass.). Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, Calif.).

[0461] Exatecan:

##STR00044##

[0462] was included in the assay for comparison with Compound 1.

TABLE-US-00001 IC.sub.50 (nM) Exatecan Compound 1 A549 2.449 0.2484 SKBR3 0.181 0.09575 HCT116 0.9956 0.1644

Example 10—In-Vitro Cytotoxicity Test of ADCs

[0463] For the ADCs in-vitro cytotoxicity test, the same protocol as that of small molecules was used. HER2-expressing human cell lines breast cancer cell lines SKBR-3 (ATCC) and NCI-N87 (ATCC) were used in in-vitro cytotoxicity assay. An MDA-MB-468 (ATCC) breast cancer cell line that does not express HER2 was used as a negative control. Five-fold serial dilution of each ADCs (starting at 300 μg/mL) were added to each well in triplicate. The cells treated with ADCs were cultured for six days. At the end of the incubation period, 100 mL of the Substrate Solution (Promega, Madison Wis.) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, Mass.). Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, Calif.).

TABLE-US-00002 EC.sub.50 (μg/mL) Her2-2 NIP228-2 Her2*-2 NIP228*-2 SKBR3 0.0004781 84.91 0.002179 10.06 NCI-N87 0.001003 ~77610 0.01878 ~10637 MDA-MB-468 2.849 ~275569 ~137570 466.0

Example 11—Further In-Vitro Cytotoxicity Test of ADC

[0464] The concentration and viability of cells from a sub-confluent (80-90% confluency) T75 flask are measured by trypan blue staining, and counted using the LUNA-II™ Automated Cell Counter. Cells were diluted to 2×10.sup.5/ml, dispensed (50 μl per well) into 96-well flat-bottom plates.

[0465] 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 per well) into 4 replicate wells of the 96-well plate, containing 50 μl cell suspension seeded the day previously. 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.

[0466] At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20 μl per well) into each well and incubated for 4 hours at 37° C. in the CO.sub.2-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival was calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control untreated wells (100%). IC.sub.50 was determined from the dose-response data using GraphPad Prism using the non-linear curve fit algorithm: sigmoidal dose-response curve with variable slope.

[0467] 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-00003 EC.sub.50 (μg/mL) Her2*-3 NCI-N87 0.09328 MDA-MB-468 ~0.9772

Example 12—In Vivo Studies in Mouse Xenograft Models (JIMT-1)

[0468] Mice

[0469] Female SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcolcrCrl, Charles River) were ten weeks old with body weight (BW) range of 17.3 to 26.3 g on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o′cobs™ Laboratory Animal Bedding in static microisolators on a 12-hour light cycle at 20-22° C. (68-72° F.) and 40-60% humidity. Charles River Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals concerning restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. The animal care and use program at Charles River 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.

[0470] Tumor Cell Culture

[0471] JIMT-1 human breast carcinoma cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 100 units/mL penicillin G sodium, 100 μg/mL streptomycin sulfate, 25 μg/mL gentamicin, and 2 mM glutamine. Cell cultures were maintained in tissue culture flasks in a humidified incubator at 37° C., in an atmosphere of 5% CO.sub.2 and 95% air.

[0472] In Vivo Implantation and Tumor Growth

[0473] The JIMT-1 tumor cells used for implantation were harvested during log phase growth and resuspended in 50% Matrigel® Matrix (Corning®) in phosphate-buffered saline (PBS) at a concentration of 1×10.sup.8 cells/mL. Each test mouse was injected subcutaneously in the right flank with 1×10.sup.7 JIMT-1 cells (0.1 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 150 to 250 mm.sup.3. Tumors were measured twice weekly in two dimensions using calipers, and volume was calculated using the formula:

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

[0474] where w=width and l=length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[0475] Twenty-one days after tumor implantation, designated as Day 1 of the study, animals with individual tumor volumes ranging from 172 to 221 mm.sup.3 were sorted into nine groups (n=8) with a group mean tumor volumes of 199 to 202 mm.sup.3.

[0476] Treatment

[0477] Treatment began on Day 1 in nine groups of female SCID mice (n=8) with established subcutaneous JIMT-1 xenografts (172-221 mm.sup.3). Each test agent was evaluated at 3 mg/kg administered intravenously (i.v.) in a single injection on Day 1 (qd×1). A vehicle-treated group served as the control for tumor engraftment and growth.

[0478] Tumors were measured twice per week until the study was ended on Day 78. Each mouse was euthanized when its tumor reached the endpoint volume of 1000 mm.sup.3 or on the final day, whichever came first. The time to endpoint (TTE) was calculated for each mouse by the following equation:

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

[0479] where TTE is expressed in days, endpoint volume is expressed in mm3, b is the intercept, and m is the slope of the line obtained by linear regression of a log-transformed tumor growth data set. 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.

[0480] Treatment efficacy may be determined from the tumor volumes of animals remaining in the study on the last day. The MTV (n) was defined as the median tumor volume on the last day of the study in the number of animals remaining (n) whose tumors had not attained the endpoint volume.

[0481] Treatment efficacy may also be determined from the incidence and magnitude of regression responses observed during the study. Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume was 50% or less of its Day 1 volume for three consecutive measurements during the course of the study, and equal to or greater than 13.5 mm.sup.3 for one or more of these three measurements. In a CR response, the tumor volume was less than 13.5 mm.sup.3 for three consecutive measurements during the course of the study. An animal with a CR response at the termination of a study was additionally classified as a tumor-free survivor (TFS). Animals were monitored for regression responses.

[0482] Results

[0483] All regimens were well tolerated. The median TTE for controls was 39.4 days, establishing a maximum possible TGD of 38.6 days (98%) for the 78-day study.

TABLE-US-00004 Statistical Group n Agent Median TTE T-C % TGD Significance 1 8 vehicle 39.4 — — — 2 8 Her2-2 78.0 38.6 98 *** 3 8 Her2-4 78.0 38.6 98 *** 4 8 Her2-6 78.0 38.6 98 *** 5 8 Her2-5 78.0 38.6 98 *** 6 8 NIP228-2 56.9 17.5 44 *** 7 8 NIP228-4 49.9 10.5 27 *** 8 8 NIP228-6 60.2 20.8 53 *** 9 8 NIP228-5 45.9 6.5 16 *

TABLE-US-00005 MTV(n) Regressions Deaths Group Agent Day 78 PR CR TFS Mean BW Nadir TR NTR 1 vehicle — 0 0 0 −1.5% Day 33 0 0 2 Her2-2 255 (8) 3 5 1 −3.7% Day 50 0 0 3 Her2-4 226 (8) 4 4 0 −1.0% Day 4 0 0 4 Her2-6 550 (7) 6 2 0 −5.0% Day 40 0 0 5 Her2-5 365 (8) 3 5 0 −10.4% Day 75 0 0 6 NIP228-2 — 0 0 0 −0.6% Day 5 0 0 7 NIP228-4 — 1 0 0 −3.0% Day 43 0 0 8 NIP228-6 — 1 0 0 −1.0% Day 4 0 0 9 NIP228-5 — 0 0 0 −4.8% Day 43 0 0

[0484] The four Trastuzumab-ADCs produced the maximal TGD of 98%, with each showing both partial and complete tumour regressions.

Example 12— In Vivo Studies in Mouse Xenograft Models (NCI-N87)

[0485] Mice

[0486] Female SCID mice (Fox Chase SCID®, CB17/Icr-Prkdcscid/IcolcrCrl, Charles River) were twelve weeks old with a body weight (BW) range of 15.9 to 26.4 g on Day 1 of the study. The animals were fed ad libitum water (reverse osmosis, 1 ppm CI), and NIH 31 Modified and Irradiated Lab Diet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice were housed on irradiated Enrich-o′cobs™ Laboratory Animal Bedding in static microisolators 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.

[0487] 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.

[0488] Tumor Cell Culture

[0489] Human NCI-N87 gastric carcinoma 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% CO.sub.2 and 95% air.

[0490] In Vivo Implantation and Tumor Growth

[0491] The NCI-N87 tumor cells used for implantation were harvested during log phase growth and resuspended in 50% Matrigel® Matrix (Corning®) in phosphate buffered saline (PBS) at a concentration of 1×108 cells/mL. Each test mouse was injected subcutaneously in the right flank with 1×107 NCI-N87 cells (0.1 mL cell suspension), and tumor growth was monitored as the average size approached the target range of 150 to 250 mm.sup.3. Tumors were measured twice weekly in two dimensions using calipers, and volume was calculated using the formula:

[00003]$\mathrm{Tumor}\mathrm{Volume}\left({\mathrm{mm}}^3\right)=\frac{w^2\times l}{2}$

[0492] where w=width and l=length, in mm, of the tumor. Tumor weight may be estimated with the assumption that 1 mg is equivalent to 1 mm.sup.3 of tumor volume.

[0493] Forty days after tumor implantation, designated as Day 1 of the study, animals with individual tumor volumes ranging from 144 to 256 mm.sup.3 were sorted into nine groups (n=8) with group mean tumor volumes of 190 to 192 mm.sup.3.

[0494] Treatment

[0495] Treatment began on Day 1 in nine groups of female SCID mice (n=8) with established subcutaneous NCI-N87 xenografts (190 to 192 mm.sup.3). Each test agent was evaluated at 3 mg/kg administered intravenously (i.v.) in a single injection on Day 1 (qd×1). A vehicle-treated group served as the control for tumor engraftment and growth.

[0496] Tumors were measured twice per week until the study was ended on Day 59. Each mouse was euthanized when its tumor reached the endpoint volume of 800 mm.sup.3 or on the final day, whichever came first. Tumor progression was slow and all evaluable animals remained on study on the final day. Since the no animals reached the tumor volume endpoint, evaluation of efficacy utilized percent tumor growth inhibition (% TGI) on the last day of the study. The MTV (n), the median tumor volume for the number of animals, n, on the final day (Day 59), was determined for each group for the total tumor volume. % TGI was defined as the difference between the MTV of the designated control group (Group 1) and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:

%TGI=[1−(MTV.sub.drug treated/MTV.sub.control)]×100

[0497] Treatment efficacy may also be determined from the tumor volumes of animals remaining in the study on the last day and from the number and magnitude of regression responses. The MTV (n) is defined as the median tumor volume on the final day (Day 59) in the number of evaluable animals remaining, n.

[0498] Treatment may cause partial regression (PR) or complete regression (CR) of the tumor in an animal. In a PR response, the tumor volume is 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 is less than 13.5 mm.sup.3 for three consecutive measurements during the study. Animals were scored only once during the study for a PR or CR event and only as CR if both PR and CR criteria were satisfied.

[0499] Results

[0500] All regimens were acceptably tolerated. Control tumors exhibited slow, progressive growth, but did not attain the 800 mm.sup.3 analysis endpoint by study end. Tumor growth inhibition was evaluated on the final day of the study (Day 59).

TABLE-US-00006 MTV(n) Statistical Group n Agent Day 59 % TGI Significance 1 8 vehicle 550 (8) — — 2 8 Her2-2  3 (8) 99 *** 3 8 Her2-4  1 (8) 100 *** 4 8 Her2-6  5 (8) 99 *** 5 8 Her2-5  4 (8) 99 *** 6 8 NIP228-2 446 (8) 19 ns 7 8 NIP228-4 405 (8) 26 ns 8 8 NIP228-6 493 (8) 10 ns 9 8 NIP228-5 466 (8) 15 ns

TABLE-US-00007 Regressions Deaths Group Agent PR CR Mean BW Nadir TR NTR 1 vehicle 0 0 −7.0% Day 52 0 0 2 Her2-2 1 7 −10.1% Day 52 0 0 3 Her2-4 0 8 −10.3% Day 56 0 0 4 Her2-6 2 6 −10.6% Day 56 0 0 5 Her2-5 0 8 −7.6% Day 59 0 0 6 NIP228-2 0 0 −9.3% Day 56 0 0 7 NIP228-4 0 0 −10.0% Day 59 0 0 8 NIP228-6 0 0 −6.4% Day 59 0 0 9 NIP228-5 0 0 −5.8% Day 59 0 0

[0501] All Trastuzumab-ADC treatments produced statistically significant Day 59 TGI compared to vehicle-treated controls (P<0.001).

STATEMENTS OF INVENTION

[0502] 1. A compound with the formula I:

##STR00045##

[0503] and salts and solvates thereof, wherein R.sup.L is a linker for connection to a Ligand Unit, which is selected from: [0504] (ia):

##STR00046## [0505] wherein [0506] Q is:

##STR00047##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; [0507] X is:

##STR00048## [0508] where a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, wherein at least b1 or b2=0 and at least c1 or c2=0; [0509] G.sup.L is a linker for connecting to a Ligand Unit; [0510] (ib):

##STR00049## [0511] 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 [0512] e is 0 or 1.

[0513] 2. The compound according to statement 1, wherein R.sup.L is of formula Ia.

[0514] 3. The compound according to statement 2, wherein Q is an amino acid residue.

[0515] 4. The compound according to statement 3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0516] 5. The compound according to statement 2, wherein Q is a dipeptide residue.

[0517] 6. The compound according to statement 5, wherein Q is selected from: [0518] .sup.NH-Phe-Lys-.sup.C═O, [0519] .sup.NH-Val-Ala-.sup.C═O, [0520] .sup.NH-Val-Lys-.sup.C═O, [0521] .sup.NH Ala-Lys-.sup.C═O, [0522] .sup.NH-Val-Oft-.sup.C═O, [0523] .sup.NH-Phe-Cit-.sup.C═O, [0524] .sup.NH-Leu-Cit-.sup.C═O, [0525] .sup.NH-Ile-Cit-.sup.C═O, [0526] .sup.NH-Phe-Arg-.sup.C═O, [0527] .sup.NH-Trp-Cit-.sup.C═O, and [0528] .sup.NH-Gly-Val-.sup.C═O.

[0529] 7. The compound according to statement 6, wherein Q is selected from .sup.NH-Phe-Lys-.sup.C═O, .sup.NH-Val-Cit-.sup.C═O.sub.and .sup.NH-Val-Ala-.sup.C═O.

[0530] 8. The compound according to statement 2, wherein Q is a tripeptide residue.

[0531] 9. The compound according to statement 8, wherein Q is selected from: [0532] .sup.NH-Glu-Val-Ala-.sup.C═O, [0533] .sup.NH-Glu-Val-Cit-.sup.C═O, [0534] .sup.NH-αGlu-Val-Ala-.sup.C═O, and [0535] NH-αGlu-Val-Cit-.sup.C═O.

[0536] 10. The compound according to statement 2, wherein Q is a tetrapeptide residue.

[0537] 11. The compound according to statement 10, wherein Q is selected from: [0538] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O; and [0539] .sup.NH-Gly-Phe-Gly-Gly .sup.C═O.

[0540] 12. The compound according to statement 11, wherein Q is: [0541] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O.

[0542] 13. The compound according to any one of statements 2 to 12, wherein a is 0 to 3.

[0543] 14. The compound according to statement 13, wherein a is 0 or 1.

[0544] 15. The compound according to statement 13, wherein a is 0.

[0545] 16. The compound according to any one of statements 2 to 15, wherein b1 is 0 to 8.

[0546] 17. The compound according to statement 16, wherein b1 is 0.

[0547] 18. The compound according to statement 16, wherein b1 is 2.

[0548] 19. The compound according to statement 16, wherein b1 is 3.

[0549] 20. The compound according to statement 16, wherein b1 is 4.

[0550] 21. The compound according to statement 16, wherein b1 is 5.

[0551] 22. The compound according to statement 16, wherein b1 is 8.

[0552] 23. The compound according to any one of statements 2 to 15 and 17, wherein b2 is 0 to 8.

[0553] 24. The compound according to statement 23, wherein b2 is 0.

[0554] 25. The compound according to statement 23, wherein b2 is 2.

[0555] 26. The compound according to statement 23, wherein b2 is 3.

[0556] 27. The compound according to statement 23, wherein b2 is 4.

[0557] 28. The compound according to statement 23, wherein b2 is 5.

[0558] 29. The compound according to statement 23, wherein b2 is 8.

[0559] 30. The compound according to any one of statements 2 to 29, wherein c1 is 0.

[0560] 31. The compound according to any one of statements 2 to 29, wherein c1 is 1.

[0561] 32. The compound according to any one of statements 2 to 31, wherein c2 is 0.

[0562] 33. The compound according to any one of statements 2 to 30, wherein c2 is 1.

[0563] 34. The compound according to any one of statements 2 to 33, wherein d is 0 to 3.

[0564] 35. The compound according to statement 34, wherein d is 1 or 2.

[0565] 36. The compound according to statement 34, wherein d is 2.

[0566] 37. The compound according to any one of statements 2 to 33, wherein d is 5.

[0567] 38. The compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is from 0 to 8.

[0568] 39. The compound according to statement 38, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0569] 40. The compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is from 0 to 8.

[0570] 41. The compound according to statement 40, wherein b1 is 0, 2, 3, 4, 5 or 8.

[0571] 42. The compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 is from 0 to 8.

[0572] 43. The compound according to statement 42, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0573] 44. The compound according to any one of statements 2 to 12, wherein b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is from 1 to 5.

[0574] 45. The compound according to statement 41, wherein the other of a and d is 1 or 5.

[0575] 46. The compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is from 0 to 8.

[0576] 47. The compound according to statement 46, wherein b1 is 0, 2, 3, 4, 5 or 8.

[0577] 48. The compound according to any one of statements 2 to 47, wherein G.sup.L is selected from

##STR00050## ##STR00051##

[0578] where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

[0579] 49. A compound according to statement 48, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

[0580] 50. A compound according to statement 48, wherein G.sup.L is G.sup.L1-1.

[0581] 51. The compound according to statement 1, wherein R.sup.L is of formula Ib.

[0582] 52. The compound according to statement 51, wherein both R.sup.L1 and R.sup.L2 are H.

[0583] 53. The compound according to statement 51, wherein R.sup.L1 is H and R.sup.L2 is methyl.

[0584] 54. The compound according to statement 51, wherein both R.sup.L1 and R.sup.L2 are methyl.

[0585] 55. The compound according to statement 51, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.

[0586] 56. The compound according to statement 51, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

[0587] 57. The compound according to any one of statements 51 to 56, wherein e is 0.

[0588] 58. The compound according to any one of statements 51 to 56, wherein e is 1.

[0589] 59. A conjugate of formula IV:

L-(D.sup.L).sub.p  (IV′)

[0590] 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:

##STR00052##

[0591] R.sup.LL is a linker connected to the Ligand unit selected from

[0592] (ia′):

##STR00053##

[0593] where Q and X are as defined in any one of statements 1 to 47 and G.sup.LL is a linker connected to a Ligand Unit; and

[0594] (ib′):

##STR00054##

[0595] where R.sup.L1 and R.sup.L2 are as defined in any one of statements 1 and 52 to 56; and

[0596] p is an integer of from 1 to 20.

[0597] 60. The conjugate according to statement 59, wherein G.sup.LL is selected from:

##STR00055## ##STR00056##

[0598] where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

[0599] 61. The conjugate according to statement 60, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

[0600] 62. The conjugate according to statement 61, wherein G.sup.LL is G.sup.LL1-1.

[0601] 63. The conjugate according to any one of statements 59 to 62, wherein the Ligand Unit is a Cell Binding Agent.

[0602] 64. The conjugate according to any one of statements 59 to 62, wherein the Ligand Unit is an antibody or an active fragment thereof.

[0603] 65. The conjugate according to statement 64, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.

[0604] 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):

[0605] (1) BMPR1B;

[0606] (2) E16;

[0607] (3) STEAP1;

[0608] (4) 0772P;

[0609] (5) MPF;

[0610] (6) Napi3b;

[0611] (7) Sema 5b;

[0612] (8) PSCA hlg;

[0613] (9) ETBR;

[0614] (10) MSG783;

[0615] (11) STEAP2;

[0616] (12) TrpM4;

[0617] (13) CRIPTO;

[0618] (14) CD21;

[0619] (15) CD79b;

[0620] (16) FcRH2;

[0621] (17) HER2;

[0622] (18) NCA;

[0623] (19) MDP;

[0624] (20) IL20R-alpha;

[0625] (21) Brevican;

[0626] (22) EphB2R;

[0627] (23) ASLG659;

[0628] (24) PSCA;

[0629] (25) GEDA;

[0630] (26) BAFF-R;

[0631] (27) CD22;

[0632] (28) CD79a;

[0633] (29) CXCR5;

[0634] (30) HLA-DOB;

[0635] (31) P2X5;

[0636] (32) CD72;

[0637] (33) LY64;

[0638] (34) FcRH1;

[0639] (35) IRTA2;

[0640] (36) TENB2;

[0641] (37) PSMA—FOLH1;

[0642] (38) SST;

[0643] (38.1) SSTR2;

[0644] (38.2) SSTR5;

[0645] (38.3) SSTR1;

[0646] (38.4) SSTR3;

[0647] (38.5) SSTR4;

[0648] (39) ITGAV;

[0649] (40) ITGB6;

[0650] (41) CEACAM5;

[0651] (42) MET;

[0652] (43) MUC1;

[0653] (44) CA9;

[0654] (45) EGFRvIII;

[0655] (46) CD33;

[0656] (47) CD19;

[0657] (48) IL2RA;

[0658] (49) AXL;

[0659] (50) CD30-TNFRSF8;

[0660] (51) BCMA—TNFRSF17;

[0661] (52) CT Ags—CTA;

[0662] (53) CD174 (Lewis Y)—FUT3;

[0663] (54) CLEC14A;

[0664] (55) GRP78-HSPA5;

[0665] (56) CD70;

[0666] (57) Stem Cell specific antigens;

[0667] (58) ASG-5;

[0668] (59) ENPP3;

[0669] (60) PRR4;

[0670] (61) GCC—GUCY2C;

[0671] (62) Liv-1-SLC39A6;

[0672] (63) 5T4;

[0673] (64) CD56-NCMA1;

[0674] (65) CanAg;

[0675] (66) FOLR1;

[0676] (67) GPNMB;

[0677] (68) TIM-1-HAVCR1;

[0678] (69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

[0679] (70) B7-H4-VTCN1;

[0680] (71) PTK7;

[0681] (72) CD37;

[0682] (73) CD138-SDC1;

[0683] (74) CD74;

[0684] (75) Claudins—CLs;

[0685] (76) EGFR;

[0686] (77) Her3;

[0687] (78) RON—MST1R;

[0688] (79) EPHA2;

[0689] (80) CD20-MS4A1;

[0690] (81) Tenascin C—TNC;

[0691] (82) FAP;

[0692] (83) DKK-1;

[0693] (84) CD52;

[0694] (85) CS1-SLAMF7;

[0695] (86) Endoglin—ENG;

[0696] (87) Annexin A1—ANXA1;

[0697] (88) V-CAM (CD106)—VCAM1;

[0698] (89) ASCT2 (SLC1A5).

[0699] 67. The conjugate according to any one of statements 64 to 66, wherein the antibody or antibody fragment is a cysteine-engineered antibody.

[0700] 68. The conjugate according to any one of statements 64 to 61, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 10.

[0701] 69. The conjugate according to statement 62, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0702] 70. A mixture of conjugates according to any one of statements 64 to 63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

[0703] 71. The conjugate or mixture according to any one of statements 59 to 70, for use in therapy.

[0704] 72. A pharmaceutical composition comprising the conjugate or mixture of any one of statements 59 to 70 and a pharmaceutically acceptable diluent, carrier or excipient.

[0705] 73. The conjugate or mixture according to any one of statements 59 to 70, or the pharmaceutical composition according to statement 66, for use in the treatment of a proliferative disease in a subject.

[0706] 74. The conjugate, mixture or pharmaceutical composition according to statement 73, wherein the disease is cancer.

[0707] 75. Use of a conjugate or mixture according to any one of statements 59 to 70, or the pharmaceutical composition according to statement 72 in a method of medical treatment.

[0708] 76. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement 72.

[0709] 77. The method of statement 76 wherein the method of medical treatment is for treating cancer.

[0710] 78. The method of statement 77, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.

[0711] 79. Use of a conjugate or mixture according to any one of statements 59 to 70 in a method of manufacture of a medicament for the treatment of a proliferative disease.

[0712] 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 59 to 70, or the pharmaceutical composition according to statement 72.

[0713] 81. The compound A:

##STR00057##

[0714] 82. The compound of claim 81 as a single enantiomer or in an enantiomerically enriched form.

[0715] 83. A compound with the formula VI:

##STR00058##

[0716] where Q is as in any one of statements 1 and 3 and 12.

[0717] Statements of Invention from 1.sup.st Priority Application (P1)

[0718] P1-1. A compound with the formula I:

##STR00059##

[0719] and salts and solvates thereof, wherein R.sup.L is a linker for connection to a cell binding agent, which is selected from: [0720] (ia):

##STR00060## [0721] wherein [0722] Q is:

##STR00061##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; [0723] X is:

##STR00062## [0724] where a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, wherein at least b1 or b2=0; [0725] G.sup.L is a linker for connecting to a Ligand Unit; [0726] (ib):

##STR00063## [0727] 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 [0728] e is 0 or 1.

[0729] P1-2. The compound according to statement P1-1, wherein R.sup.L is of formula Ia.

[0730] P1-3. The compound according to statement P1-2, wherein Q is an amino acid residue.

[0731] P1-4. The compound according to statement P1-3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0732] P1-5. The compound according to statement P1-2, wherein Q is a dipeptide residue.

[0733] P1-6. The compound according to statement P1-5, wherein Q is selected from: [0734] .sup.NH-Phe-Lys-.sup.C═O, [0735] .sup.NH-Val-Ala-.sup.C═O, [0736] .sup.NH-Val-Lys-.sup.C═O, [0737] .sup.NH Ala-Lys-.sup.C═O, [0738] .sup.NH-Val-Cit-.sup.C═O, [0739] .sup.NH-Phe-Cit-.sup.C═O, [0740] .sup.NH-Leu-Cit-.sup.C═O, [0741] .sup.NH-Ile-Cit-.sup.C═O, [0742] .sup.NH-Phe-Arg-.sup.C═O, [0743] .sup.NH-Trp-Cit-.sup.C═O, and [0744] .sup.NH-Gly-Val-.sup.C═O.

[0745] P1-7. The compound according to statement P1-6, wherein Q is selected from .sup.NH-Phe-Lys-.sup.C═O, NH—Val-Cit-.sup.C═O and .sup.NH-Val-Ala-.sup.C═O.

[0746] P1-8. The compound according to statement P1-2, wherein Q is a tripeptide residue.

[0747] P1-9. The compound according to statement P1-8, wherein Q is selected from: [0748] .sup.NH-Glu-Val-Ala-.sup.C═O, [0749] .sup.NH-Glu-Val-Cit-.sup.C═O, [0750] .sup.NH-αGlu-Val-Ala-.sup.C═O, and [0751] .sup.NH-αGlu-Val-Cit-.sup.C═O.

[0752] P1-10. The compound according to statement P1-2, wherein Q is a tetrapeptide residue.

[0753] P1-11. The compound according to statement P1-10, wherein Q is selected from: [0754] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O; and [0755] .sup.NH-Gly-Phe-Gly-Gly .sup.C═O.

[0756] P1-12. The compound according to statement P1-11, wherein Q is: [0757] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O.

[0758] P1-13. The compound according to any one of statements P1-2 to P1-12, wherein a is 0 to 3.

[0759] P1-14. The compound according to statement P1-13, wherein a is 0 or 1.

[0760] P1-15. The compound according to statement P1-13, wherein a is 0.

[0761] P1-16. The compound according to any one of statements P1-2 to P1-15, wherein b1 is 0 to 8.

[0762] P1-17. The compound according to statement P1-16, wherein b1 is 0.

[0763] P1-18. The compound according to statement P1-16, wherein b1 is 2.

[0764] P1-19. The compound according to statement P1-16, wherein b1 is 3.

[0765] P1-20. The compound according to statement P1-16, wherein b1 is 4.

[0766] P1-21. The compound according to statement P1-16, wherein b1 is 5.

[0767] P1-22. The compound according to statement P1-16, wherein b1 is 8.

[0768] P1-23. The compound according to any one of statements P1-2 to P1-15 and P1-17, wherein b2 is 0 to 8.

[0769] P1-24. The compound according to statement P1-23, wherein b2 is 0.

[0770] P1-25. The compound according to statement P1-23, wherein b2 is 2.

[0771] P1-26. The compound according to statement P1-23, wherein b2 is 3.

[0772] P1-27. The compound according to statement P1-23, wherein b2 is 4.

[0773] P1-28. The compound according to statement P1-23, wherein b2 is 5.

[0774] P1-29. The compound according to statement P1-23, wherein b2 is 8.

[0775] P1-30. The compound according to any one of statements P1-2 to P1-29, wherein c is 0.

[0776] P1-31. The compound according to any one of statements P1-2 to P1-29, wherein c is 1.

[0777] P1-32. The compound according to any one of statements P1-2 to P1-31, wherein d is 0 to 3.

[0778] P1-33. The compound according to statement P1-32, wherein d is 1 or 2.

[0779] P1-34. The compound according to statement P1-32, wherein d is 2.

[0780] P1-35. The compound according to any one of statements P1-2 to P1-12, wherein a is 0, b1 is 0, c is 1 and d is 2, and b2 is from 0 to 8.

[0781] P1-36. The compound according to statement P1-35, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0782] P1-37. The compound according to any one of statements P1-2 to P1-12, wherein a is 1, b2 is 0, c is 0 and d is 0, and b1 is from 0 to 8.

[0783] P1-38. The compound according to statement P1-37, wherein b1 is 0, 2, 3, 4, 5 or 8.

[0784] P1-39. The compound according to any one of statements P1-2 to P1-12, wherein a is 0, b1 is 0, c is 0 and d is 1, and b2 is from 0 to 8.

[0785] P1-40. The compound according to statement P1-39, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0786] P1-41. The compound according to any one of statements P1-2 to P1-12, wherein b1 is 0, b2 is 0, c is 0, one of a and d is 0, and the other of a and d is from 1 to 5.

[0787] P1-42. The compound according to statement P1-41, wherein the other of a and d is 1 or 5.

[0788] P1-43. The compound according to any one of statements P1-2 to P1-42, wherein G.sup.L is selected from

##STR00064## ##STR00065##

[0789] where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

[0790] P1-44. A compound according to statement P1-43, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2

[0791] P1-45. A compound according to statement P1-43, wherein G.sup.L is G.sup.L1-1.

[0792] P1-46. The compound according to statement P1-1, wherein R.sup.L is of formula Ib.

[0793] P1-47. The compound according to statement 4 P1-6, wherein both R.sup.L1 and R.sup.L2 are H.

[0794] P1-48. The compound according to statement P1-46, wherein R.sup.L1 is H and R.sup.L2 is methyl.

[0795] P1-49. The compound according to statement P1-46, wherein both R.sup.L1 and R.sup.L2 are methyl.

[0796] P1-50. The compound according to statement P1-46, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.

[0797] P1-51. The compound according to statement P1-46, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

[0798] P1-52. The compound according to any one of statements v46 to P1-51, wherein e is 0.

[0799] P1-53. The compound according to any one of statements P1-46 to P1-51, wherein e is 1.

[0800] P1-54. 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 (i.e., a targeting agent), D.sup.L is a Drug Linker unit that is of formula III:

##STR00066##

[0801] R.sup.LL is a linker connected to the Ligand unit selected from

[0802] (ia′):

##STR00067##

[0803] where Q and X are as defined in any one of statements P1-1 to P1-42 and G.sup.LL is a linker connected to a Ligand Unit; and

[0804] (ib′):

##STR00068##

[0805] where R.sup.L1 and R.sup.L2 are as defined in any one of statements P1-1 and P1-47 to P1-51; and

[0806] p is an integer of from 1 to 20.

[0807] P1-55. The conjugate according to statement P1-54, wherein G.sup.LL is selected from:

##STR00069## ##STR00070##

[0808] where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

[0809] P1-56. The conjugate according to statement P1-55, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

[0810] P1-57. The conjugate according to statement P1-56, wherein G.sup.LL is G.sup.LL1-1.

[0811] P1-58. The conjugate according to any one of statements P1-54 to P1-57, wherein the Ligand Unit is an antibody or an active fragment thereof.

[0812] P1-59. The conjugate according to statement P1-58, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.

[0813] P1-60. The conjugate according to statement P1-59, 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):

[0814] (1) BMPR1B;

[0815] (2) E16;

[0816] (3) STEAP1;

[0817] (4) 0772P;

[0818] (5) MPF;

[0819] (6) Napi3b;

[0820] (7) Sema 5b;

[0821] (8) PSCA hlg;

[0822] (9) ETBR;

[0823] (10) MSG783;

[0824] (11) STEAP2;

[0825] (12) TrpM4;

[0826] (13) CRIPTO;

[0827] (14) CD21;

[0828] (15) CD79b;

[0829] (16) FcRH2;

[0830] (17) HER2;

[0831] (18) NCA;

[0832] (19) MDP;

[0833] (20) IL20R-alpha;

[0834] (21) Brevican;

[0835] (22) EphB2R;

[0836] (23) ASLG659;

[0837] (24) PSCA;

[0838] (25) GEDA;

[0839] (26) BAFF-R;

[0840] (27) CD22;

[0841] (28) CD79a;

[0842] (29) CXCR5;

[0843] (30) HLA-DOB;

[0844] (31) P2X5;

[0845] (32) CD72;

[0846] (33) LY64;

[0847] (34) FcRH1;

[0848] (35) IRTA2;

[0849] (36) TENB2;

[0850] (37) PSMA— FOLH1;

[0851] (38) SST;

[0852] (38.1) SSTR2;

[0853] (38.2) SSTR5;

[0854] (38.3) SSTR1;

[0855] (38.4) SSTR3;

[0856] (38.5) SSTR4;

[0857] (39) ITGAV;

[0858] (40) ITGB6;

[0859] (41) CEACAM5;

[0860] (42) MET;

[0861] (43) MUC1;

[0862] (44) CA9;

[0863] (45) EGFRvIII;

[0864] (46) CD33;

[0865] (47) CD19;

[0866] (48) IL2RA;

[0867] (49) AXL;

[0868] (50) CD30-TNFRSF8;

[0869] (51) BCMA—TNFRSF17;

[0870] (52) CT Ags—CTA;

[0871] (53) CD174 (Lewis Y)—FUT3;

[0872] (54) CLEC14A;

[0873] (55) GRP78-HSPA5;

[0874] (56) CD70;

[0875] (57) Stem Cell specific antigens;

[0876] (58) ASG-5;

[0877] (59) ENPP3;

[0878] (60) PRR4;

[0879] (61) GCC—GUCY2C;

[0880] (62) Liv-1-SLC39A6;

[0881] (63) 5T4;

[0882] (64) CD56-NCMA1;

[0883] (65) CanAg;

[0884] (66) FOLR1;

[0885] (67) GPNMB;

[0886] (68) TIM-1-HAVCR1;

[0887] (69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

[0888] (70) B7-H4-VTCN1;

[0889] (71) PTK7;

[0890] (72) CD37;

[0891] (73) CD138-SDC1;

[0892] (74) CD74;

[0893] (75) Claudins— CLs;

[0894] (76) EGFR;

[0895] (77) Her3;

[0896] (78) RON—MST1R;

[0897] (79) EPHA2;

[0898] (80) CD20-MS4A1;

[0899] (81) Tenascin C—TNC;

[0900] (82) FAP;

[0901] (83) DKK-1;

[0902] (84) CD52;

[0903] (85) CS1-SLAMF7;

[0904] (86) Endoglin—ENG;

[0905] (87) Annexin A1—ANXA1;

[0906] (88) V-CAM (CD106)—VCAM1;

[0907] (89) ASCT2 (SLC1A5).

[0908] P1-61. The conjugate according to any one of statements P1-58 to P1-60, wherein the antibody or antibody fragment is a cysteine-engineered antibody.

[0909] P1-62. The conjugate according to any one of statements P1-58 to P1-61, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 10.

[0910] P1-63. The conjugate according to statement P1-62, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0911] P1-64. A mixture of conjugates according to any one of statements P1-58 to P1-63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

[0912] P1-65. The conjugate or mixture according to any one of statements P1-54 to P1-64, for use in therapy.

[0913] P1-66. A pharmaceutical composition comprising the conjugate or mixture of any one of statements P1-54 to P1-64 and a pharmaceutically acceptable diluent, carrier or excipient.

[0914] P1-67. The conjugate or mixture according to any one of statements P1-54 to P1-64, or the pharmaceutical composition according to statement P1-66, for use in the treatment of a proliferative disease in a subject.

[0915] P1-68. The conjugate, mixture or pharmaceutical composition according to statement P1-67, wherein the disease is cancer.

[0916] P1-69. Use of a conjugate or mixture according to any one of statements P1-54 to P1-64, or the pharmaceutical composition according to statement P1-66 in a method of medical treatment.

[0917] P1-70. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement P1-66.

[0918] P1-71. The method of statement P1-70 wherein the method of medical treatment is for treating cancer.

[0919] P1-72. The method of statement P1-71, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.

[0920] P1-73. Use of a conjugate or mixture according to any one of statements P1-54 to P1-64 in a method of manufacture of a medicament for the treatment of a proliferative disease.

[0921] P1-74. 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 P1-54 to P1-64, or the pharmaceutical composition according to statement P1-66.

[0922] P1-75. The compound A:

##STR00071##

[0923] P1-76. The compound of claim P1-75 as a single enantiomer or in an enantiomerically enriched form.

[0924] Statements of Invention from 2.sup.nd Priority Application (P2)

[0925] P2-1. A compound with the formula I:

##STR00072##

[0926] and salts and solvates thereof, wherein R.sup.L is a linker for connection to a cell binding agent, which is selected from: [0927] (ia):

##STR00073## [0928] wherein [0929] Q is:

##STR00074##

where Q.sup.X is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; [0930] X is:

##STR00075## [0931] where a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, wherein at least b1 or b2=0; [0932] G.sup.L is a linker for connecting to a Ligand Unit; [0933] (ib):

##STR00076## [0934] 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 [0935] e is 0 or 1.

[0936] P2-2. The compound according to statement P2-1, wherein R.sup.L is of formula Ia.

[0937] P2-3. The compound according to statement P2-2, wherein Q is an amino acid residue.

[0938] P2-4. The compound according to statement P2-3, wherein Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0939] P2-5. The compound according to statement P2-2, wherein Q is a dipeptide residue.

[0940] P2-6. The compound according to statement P2-5, wherein Q is selected from: [0941] .sup.NH-Phe-Lys-.sup.C═O, [0942] .sup.NH-Val-Ala-.sup.C═O, [0943] .sup.NH-Val-Lys-.sup.C═O, [0944] .sup.NH Ala-Lys-.sup.C═O, [0945] .sup.NH-Val-Cit-.sup.C═O, [0946] .sup.NH-Phe-Cit-.sup.C═O, [0947] .sup.NH-Leu-Cit-.sup.C═O, [0948] .sup.NH-Ile-Cit-.sup.C═O, [0949] NH-Phe-Arg-.sup.C═O, [0950] .sup.NH-Trp-Cit-.sup.C═O, and [0951] .sup.NH-Gly-Val-.sup.C═O.

[0952] P2-7. The compound according to statement P2-6, 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.

[0953] P2-8. The compound according to statement P2-2, wherein Q is a tripeptide residue.

[0954] P2-9. The compound according to statement P2-8, wherein Q is selected from: [0955] .sup.NH-Glu-Val-Ala-.sup.C═O, [0956] .sup.NH-Glu-Val-Cit-.sup.C═O, [0957] .sup.NH-αGlu-Val-Ala-.sup.C═O, and [0958] .sup.NH-αGlu-Val-Cit-.sup.C═O.

[0959] P2-10. The compound according to statement P2-2, wherein Q is a tetrapeptide residue.

[0960] P2-11. The compound according to statement P2-10, wherein Q is selected from: [0961] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O; and [0962] .sup.NH-Gly-Phe-Gly-Gly .sup.C═O.

[0963] P2-12. The compound according to statement P2-11, wherein Q is: [0964] .sup.NH-Gly-Gly-Phe-Gly .sup.C═O.

[0965] P2-13. The compound according to any one of statements P2-2 to P2-12, wherein a is 0 to 3.

[0966] P2-14. The compound according to statement P2-13, wherein a is 0 or 1.

[0967] P2-15. The compound according to statement P2-13, wherein a is 0.

[0968] P2-16. The compound according to any one of statements P2-2 to P2-15, wherein b1 is 0 to 8.

[0969] P2-17. The compound according to statement P2-16, wherein b1 is 0.

[0970] P2-18. The compound according to statement P2-16, wherein b1 is 2.

[0971] P2-19. The compound according to statement P2-16, wherein b1 is 3.

[0972] P2-20. The compound according to statement P2-16, wherein b1 is 4.

[0973] P2-21. The compound according to statement P2-16, wherein b1 is 5.

[0974] P2-22. The compound according to statement P2-16, wherein b1 is 8.

[0975] P2-23. The compound according to any one of statements P2-2 to P2-15 and P2-17, wherein b2 is 0 to 8.

[0976] P2-24. The compound according to statement P2-23, wherein b2 is 0.

[0977] P2-25. The compound according to statement P2-23, wherein b2 is 2.

[0978] P2-26. The compound according to statement P2-23, wherein b2 is 3.

[0979] P2-27. The compound according to statement P2-23, wherein b2 is 4.

[0980] P2-28. The compound according to statement P2-23, wherein b2 is 5.

[0981] P2-29. The compound according to statement P2-23, wherein b2 is 8.

[0982] P2-30. The compound according to any one of statements P2-2 to P2-29, wherein c is 0.

[0983] P2-31. The compound according to any one of statements P2-2 to P2-29, wherein c is 1.

[0984] P2-32. The compound according to any one of statements P2-2 to P2-31, wherein d is 0 to 3.

[0985] P2-33. The compound according to statement P2-32, wherein d is 1 or 2.

[0986] P2-34. The compound according to statement P2-32, wherein d is 2.

[0987] P2-35. The compound according to any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 1 and d is 2, and b2 is from 0 to 8.

[0988] P2-36. The compound according to statement P2-35, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0989] P2-37. The compound according to any one of statements P2-2 to P2-12, wherein a is 1, b2 is 0, c is 0 and d is 0, and b1 is from 0 to 8.

[0990] P2-38. The compound according to statement P2-37, wherein b1 is 0, 2, 3, 4, 5 or 8.

[0991] P2-39. The compound according to any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 0 and d is 1, and b2 is from 0 to 8.

[0992] P2-40. The compound according to statement P2-39, wherein b2 is 0, 2, 3, 4, 5 or 8.

[0993] P2-41. The compound according to any one of statements P2-2 to P2-12, wherein b1 is 0, b2 is 0, c is 0, one of a and d is 0, and the other of a and d is from 1 to 5.

[0994] P2-42. The compound according to statement P2-41, wherein the other of a and d is 1 or 5.

[0995] P2-43. The compound according to any one of statements P2-2 to P2-42, wherein G.sup.L is selected from

##STR00077## ##STR00078##

[0996] where Ar represents a C.sub.5-6 arylene group, and X represents C.sub.1-4 alkyl.

[0997] P2-44. A compound according to statement P2-43, wherein G.sup.L is selected from G.sup.L1-1 and G.sup.L1-2.

[0998] P2-45. A compound according to statement P2-43, wherein G.sup.L is G.sup.L1-1.

[0999] P2-46. The compound according to statement P2-1, wherein R.sup.L is of formula Ib.

[1000] P2-47. The compound according to statement P2-46, wherein both R.sup.L1 and R.sup.L2 are H.

[1001] P2-48. The compound according to statement P2-46, wherein R.sup.L1 is H and R.sup.L2 is methyl.

[1002] P2-49. The compound according to statement P2-46, wherein both R.sup.L1 and R.sup.L2 are methyl.

[1003] P2-50. The compound according to statement P2-46, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclopropylene group.

[1004] P2-51. The compound according to statement P2-46, wherein R.sup.L1 and R.sup.L2 together with the carbon atom to which they are bound form a cyclobutylene group.

[1005] P2-52. The compound according to any one of statements P2-46 to P2-51, wherein e is 0.

[1006] P2-53. The compound according to any one of statements P2-46 to P2-51, wherein e is 1.

[1007] P2-54. 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 (i.e., a targeting agent), D.sup.L is a Drug Linker unit that is of formula III:

##STR00079##

[1008] R.sup.LL is a linker connected to the Ligand unit selected from

[1009] (ia′):

##STR00080##

[1010] where Q and X are as defined in any one of statements P2-1 to P2-42 and G.sup.LL is a linker connected to a Ligand Unit; and

[1011] (ib′):

##STR00081##

[1012] where R.sup.L1 and R.sup.L2 are as defined in any one of statements P2-1 and P2-47 to P2-51; and

[1013] p is an integer of from 1 to 20.

[1014] P2-55. The conjugate according to statement P2-54, wherein G.sup.LL is selected from:

##STR00082## ##STR00083##

[1015] where Ar represents a C.sub.5-6 arylene group and X represents C.sub.1-4 alkyl.

[1016] P2-56. The conjugate according to statement P2-55, wherein G.sup.LL is selected from G.sup.LL1-1 and G.sup.LL1-2.

[1017] P2-57. The conjugate according to statement P2-56, wherein G.sup.LL is G.sup.LL1-1.

[1018] P2-58. The conjugate according to any one of statements P2-54 to P2-57, wherein the Ligand Unit is an antibody or an active fragment thereof.

[1019] P2-59. The conjugate according to statement P2-58, wherein the antibody or antibody fragment is an antibody or antibody fragment for a tumour-associated antigen.

[1020] P2-60. The conjugate according to statement P2-59, 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):

[1021] (1) BMPR1B;

[1022] (2) E16;

[1023] (3) STEAP1;

[1024] (4) 0772P;

[1025] (5) MPF;

[1026] (6) Napi3b;

[1027] (7) Sema 5b;

[1028] (8) PSCA hlg;

[1029] (9) ETBR;

[1030] (10) MSG783;

[1031] (11) STEAP2;

[1032] (12) TrpM4;

[1033] (13) CRIPTO;

[1034] (14) CD21;

[1035] (15) CD79b;

[1036] (16) FcRH2;

[1037] (17) HER2;

[1038] (18) NCA;

[1039] (19) MDP;

[1040] (20) IL20R-alpha;

[1041] (21) Brevican;

[1042] (22) EphB2R;

[1043] (23) ASLG659;

[1044] (24) PSCA;

[1045] (25) GEDA;

[1046] (26) BAFF-R;

[1047] (27) CD22;

[1048] (28) CD79a;

[1049] (29) CXCR5;

[1050] (30) HLA-DOB;

[1051] (31) P2X5;

[1052] (32) CD72;

[1053] (33) LY64;

[1054] (34) FcRH1;

[1055] (35) IRTA2;

[1056] (36) TENB2;

[1057] (37) PSMA—FOLH1;

[1058] (38) SST;

[1059] (38.1) SSTR2;

[1060] (38.2) SSTR5;

[1061] (38.3) SSTR1;

[1062] (38.4) SSTR3;

[1063] (38.5) SSTR4;

[1064] (39) ITGAV;

[1065] (40) ITGB6;

[1066] (41) CEACAM5;

[1067] (42) MET;

[1068] (43) MUC1;

[1069] (44) CA9;

[1070] (45) EGFRvIII;

[1071] (46) CD33;

[1072] (47) CD19;

[1073] (48) IL2RA;

[1074] (49) AXL;

[1075] (50) CD30-TNFRSF8;

[1076] (51) BCMA—TNFRSF17;

[1077] (52) CT Ags—CTA;

[1078] (53) CD174 (Lewis Y)—FUT3;

[1079] (54) CLEC14A;

[1080] (55) GRP78-HSPA5;

[1081] (56) CD70;

[1082] (57) Stem Cell specific antigens;

[1083] (58) ASG-5;

[1084] (59) ENPP3;

[1085] (60) PRR4;

[1086] (61) GCC—GUCY2C;

[1087] (62) Liv-1-SLC39A6;

[1088] (63) 5T4;

[1089] (64) CD56-NCMA1;

[1090] (65) CanAg;

[1091] (66) FOLR1;

[1092] (67) GPNMB;

[1093] (68) TIM-1-HAVCR1;

[1094] (69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

[1095] (70) B7-H4-VTCN1;

[1096] (71) PTK7;

[1097] (72) CD37;

[1098] (73) CD138-SDC1;

[1099] (74) CD74;

[1100] (75) Claudins—CLs;

[1101] (76) EGFR;

[1102] (77) Her3;

[1103] (78) RON-MST1R;

[1104] (79) EPHA2;

[1105] (80) CD20-MS4A1;

[1106] (81) Tenascin C—TNC;

[1107] (82) FAP;

[1108] (83) DKK-1;

[1109] (84) CD52;

[1110] (85) CS1-SLAMF7;

[1111] (86) Endoglin—ENG;

[1112] (87) Annexin A1—ANXA1;

[1113] (88) V-CAM (CD106)—VCAM1;

[1114] (89) ASCT2 (SLC1A5).

[1115] P2-61. The conjugate according to any one of statements P2-58 to P2-60, wherein the antibody or antibody fragment is a cysteine-engineered antibody.

[1116] P2-62. The conjugate according to any one of statements P2-58 to P2-61, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about 10.

[1117] P2-63. The conjugate according to statement P2-62, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[1118] P2-64. A mixture of conjugates according to any one of statements P2-58 to P2-63, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about 10.

[1119] P2-65. The conjugate or mixture according to any one of statements P2-54 to P2-64, for use in therapy.

[1120] P2-66. A pharmaceutical composition comprising the conjugate or mixture of any one of statements P2-54 to P2-64 and a pharmaceutically acceptable diluent, carrier or excipient.

[1121] P2-67. The conjugate or mixture according to any one of statements P2-54 to P2-64, or the pharmaceutical composition according to statement P2-66, for use in the treatment of a proliferative disease in a subject.

[1122] P2-68. The conjugate, mixture or pharmaceutical composition according to statement P2-67, wherein the disease is cancer.

[1123] P2-69. Use of a conjugate or mixture according to any one of statements P2-54 to P2-64, or the pharmaceutical composition according to statement P2-66 in a method of medical treatment.

[1124] P2-70. A method of medical treatment comprising administering to a patient the pharmaceutical composition of statement P2-66.

[1125] P2-71. The method of statement P2-70 wherein the method of medical treatment is for treating cancer.

[1126] P2-72. The method of statement P2-71, wherein the patient is administered a chemotherapeutic agent, in combination with the conjugate.

[1127] P2-73. Use of a conjugate or mixture according to any one of statements P2-54 to P2-64 in a method of manufacture of a medicament for the treatment of a proliferative disease.

[1128] P2-74. 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 P2-54 to P2-64, or the pharmaceutical composition according to statement 66.

[1129] P2-75. The compound A:

##STR00084##

[1130] P2-76. The compound of claim P2-75 as a single enantiomer or in an enantiomerically enriched form.