Antibody drug conjugates

Abstract

Drug conjugates of formula [D-(X)b-(AA)w-(L)-]n-Ab wherein: D is a drug moiety having the following formula (I) or a pharmaceutically acceptable salt, ester, solvate, tautomer or stereoisomer thereof, wherein: A is selected from (II) and (III) R.sub.1, R.sub.2 and R.sub.3 is H, OR.sub.a, OCOR.sub.a, OCO—OR.sub.a, alkyl, alkenyl, alkynyl, etc.; R.sub.3′ is, COR.sub.a, COOR.sub.a, CONR.sub.aR.sub.b, etc; each of R.sub.4 to R.sub.10 and R.sub.12 is alkyl, alkenyl or alkynyl; R.sub.11 is H, COR.sub.a, COOR.sub.a, alkyl, alkenyl or alkynyl, or R.sub.11 and R.sub.12+N+C atoms to which they are attached may form a heterocyclic group; each of R.sub.13 and R.sub.14 is H, COR.sub.a, COOR.sub.a, alkyl, alkenyl or alkynyl; each R.sub.a and R.sup.b is H, alkyl, alkenyl, alkynyl, etc.; each dotted line represents an optional additional bond; X is an extending group; AA is an amino acid unit; L is a linker group; w is 0 to 12; b is 0 or 1; A bis a moiety comprising at least one antigen binding site, and n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(L)-] to the moiety comprising at least one antigen binding site and is in the range from 1 to 20, are useful in the treatment of cancer. ##STR00001##

Claims

1. A drug conjugate, selected from the formulas (IV) and (V): ##STR00136## wherein: n is the ratio of the group [D-(X)b-(AA)w-(L)-] to the Ab moiety and is in the range from 1 to 5; L is a linker as defined in between AAw and Ab in formulas (IV) or (V); R.sub.19 is C.sub.3-C.sub.6 alkylene-; M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7 carbocyclo)-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00137## wherein R.sub.22 is isopropyl, R.sub.23 is —(CH.sub.2).sub.3NHCONH.sub.2, wherein the wavy lines indicate the point of covalent attachments to (X)b if any, or the drug moiety (the wavy line to the left) and to the linker (the wavy line to the right); X is an extending group selected from the group consisting of CONH—(C.sub.2-C.sub.4 alkylene)NH, —CONH—(C.sub.2-C.sub.4 alkylene)NH—COO—CH.sub.2-(phenylene)-NH—, —CONH—(C.sub.2-C.sub.4 alkylene)S—, —CONH—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)NHCO (C.sub.1-C.sub.3 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)NH— and —(C.sub.2-C.sub.4 alkylene)NH—COO—CH.sub.2-(phenylene)-NH—; b is an integer of 0 or 1; D is a drug moiety, or a pharmaceutically acceptable salt or stereoisomer thereof selected from the following group: ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## wherein the wavy lines indicate the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or the linker; Ab is an antibody moiety selected from trastuzumab, rituximab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD13 antibody, or an antigen binding fragment thereof.

2. The drug conjugate according to claim 1, wherein the Ab moiety is selected from Rituximab and an anti-CD4 antibody.

3. The drug conjugate according to claim 1, wherein the Ab moiety is Trastuzumab.

4. A drug conjugate selected from the formulas (IV) and (V): ##STR00145## wherein: n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(L)-], wherein L is a linker as defined in formulas (IV) or (V), to the Ab moiety and is in the range from 1 to 5 R.sub.19 is —C.sub.3-C.sub.6 alkylene-; M is —C.sub.1-C.sub.3 alkylene-(C.sub.5-C.sub.7 carbocyclo)-; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00146## wherein R.sub.22 is isopropyl, R.sub.23 is —(CH.sub.2).sub.3NHCONH.sub.2, wherein the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or the drug moiety (the wavy line to the left) and to the linker (the wavy line to the right); X is an extending group selected from the group consisting of —CONH—(C.sub.2-C.sub.4 alkylene)NH—, —CONH—(C.sub.2-C.sub.4 alkylene)NH—COO—CH.sub.2-(phenylene)-NH—, —CONH—(C.sub.2-C.sub.4 alkylene)S—, —CONH—(C.sub.2-C.sub.4 alkylene)NHCO(C.sub.1-C.sub.3 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)NHCO (C.sub.1-C.sub.3 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)S—, —(C.sub.2-C.sub.4 alkylene)NH— and —(C.sub.2-C.sub.4 alkylene)NH—COO—CH.sub.2-(phenylene)-NH—; b is an integer of 0 or 1; wherein D is the drug moiety, or a pharmaceutically acceptable salt or stereoisomer thereof selected from: ##STR00147## wherein the wavy lines indicate the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or the linker; Ab is an antibody moiety selected from trastuzumab, rituximab, an anti-CD4 antibody, an anti-CD5 antibody, and an anti-CD13 antibody, or an antigen binding fragment thereof.

5. The drug conjugate according to claim 1, of formula (IV): ##STR00148## wherein: R.sub.19 is —C.sub.5 alkylene-; b is 1; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00149## wherein R.sub.22 is isopropyl, R.sub.23 is —(CH.sub.2).sub.3NHCONH.sub.2, and the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or the drug moiety (the wavy line to the left) and to the linker (the wavy line to the right); and X is an extending group selected from —CONH(CH.sub.2).sub.3NHCOOCH.sub.2-phenylene-NH— and —CONH(CH.sub.2).sub.3NH—; or of formula (V) ##STR00150## wherein M is -methyl-cyclohexylene-; b is 1; w is 0; and X is an extending group selected from —CONH(CH.sub.2).sub.3—S— and —CONH(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S—; D is a drug moiety, or a pharmaceutically acceptable salt-or stereoisomer thereof selected from: ##STR00151## wherein the wavy lines indicate the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or the linker; n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(L)-] wherein L is as defined in formulas (IV) or (V) to the Ab moiety and is in the range from 1 to 5.

6. The drug conjugate according to claim 5, wherein the Ab moiety is selected from Rituximab and an anti-CD4 antibody.

7. The drug conjugate according to claim 5, wherein the Ab moiety is Trastuzumab.

8. The drug conjugate according to claim 1, of formula (IV): ##STR00152## wherein R.sub.19 is —C.sub.5 alkylene-; n is the ratio of the group [D-(X).sub.b-(AA).sub.w-(L)-] wherein L is as defined in (IV) to the moiety and is in the range from 3 to 5; b is 1; w is 0 or 2, and where w is 2, then (AA).sub.w is of formula (III): ##STR00153## wherein R.sub.22 is isopropyl, R.sub.23 is —(CH.sub.2).sub.3NHCONH.sub.2, and the wavy lines indicate the point of covalent attachments to (X).sub.b if any, or the drug moiety (the wavy line to the left) and to the linker (the wavy line to the right); and X is an extending group selected from —(CH.sub.2).sub.3NHCOOCH.sub.2-phenylene-NH—, and —(CH.sub.2).sub.3NH—; or of formula (V) ##STR00154## wherein M is -methyl-cyclohexylene-; b is 1; w is 0; and X is an extending group selected from —(CH.sub.2).sub.3S— and —(CH.sub.2).sub.3NHCO(CH.sub.2).sub.2S—; D is a drug moiety, or a pharmaceutically acceptable salt or stereoisomer thereof selected from: ##STR00155## wherein the wavy lines indicate the point of covalent attachment to (X).sub.b if any, or (AA).sub.w if any, or the linker.

9. The drug conjugate according to claim 8, wherein the Ab moiety is selected from Rituximab and an anti-CD4 antibody.

10. The drug conjugate according to claim 8, wherein the Ab moiety is Trastuzumab.

11. The antibody drug conjugate according to claim 5, selected from the group consisting of: ##STR00156## wherein each of ##STR00157## is selected from Trastuzumab, Rituximab, an anti-CD4 antibody, an anti-CD5 antibody and an anti-CD13 antibody, or an antigen binding fragment thereof.

12. The drug conjugate according to claim 4, selected from the group consisting of: ##STR00158## wherein each of ##STR00159## is selected from Trastuzumab, Rituximab, an anti-CD4 antibody, an anti-CD5 antibody and an anti-CD13 antibody, or an antigen binding fragment thereof.

13. The drug conjugate according to claim 11, wherein the Ab moiety is selected from Rituximab and an anti-CD4 antibody.

14. The drug conjugate according to claim 12, wherein the Ab moiety is selected from Rituximab and an anti-CD4 antibody.

15. The drug conjugate according to claim 11, wherein the Ab moiety comprising at least one antigen binding site is Trastuzumab.

16. The drug conjugate according to claim 12, wherein the Ab moiety comprising at least one antigen binding site is Trastuzumab.

17. A method for the treatment of cancer comprising administering an effective amount of a drug conjugate according to claim 1 to a patient in need thereof.

18. The method for the treatment of cancer according to claim 17, wherein the cancer is selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukemia, multiple myeloma, lymphoma and ovarian cancer.

19. A pharmaceutical composition comprising a drug conjugate according to claim 1 and a pharmaceutically acceptable carrier.

20. The method according to claim 17, wherein the cancer is selected from breast cancer, leukemia, lymphoma and ovarian cancer.

21. The drug conjugate according to claim 4, wherein the Ab moiety an anti-CD4 antibody.

22. The drug conjugate according to claim 4, wherein the Ab moiety is Trastuzumab.

23. The drug conjugate according to claim 4, wherein the Ab moiety is Rituximab.

24. A method for the treatment of cancer comprising administering an effective amount of a drug conjugate according to claim 4 to a patient in need thereof, wherein the cancer is selected from lung cancer, colorectal cancer, breast cancer, pancreas carcinoma, kidney cancer, leukemia, multiple myeloma, lymphoma and ovarian cancer.

25. The method according to claim 24, wherein the Ab moiety is an anti-CD4 antibody.

26. The method according to claim 24, wherein the Ab moiety is Trastuzumab.

27. The method according to claim 24, wherein the Ab moiety is Rituximab.

28. A pharmaceutical composition comprising a drug conjugate according to claim 4 and a pharmaceutically acceptable carrier.

29. A drug conjugate according to claim 1 wherein n is 3 to 5.

30. A drug conjugate according to claim 4, wherein n is 3 to 5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention is diagrammatically illustrated, by way of example, in the accompanying drawings in which:

(2) FIG. 1 is a schematic illustration of one process according to the present invention wherein conjugation to the antibody is via free thiol groups;

(3) FIG. 2 is a schematic illustration of another process according to the present invention wherein conjugation to the antibody is via free lysine groups;

(4) FIG. 3 is a representative dose response curves for ADC1 against various cancer cell lines;

(5) FIG. 4 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC1 at 10 or 1 μg/mL;

(6) FIG. 5 is a representative dose response curves for ADC2 against various cancer cell lines;

(7) FIG. 6 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC2 at 10 or 1 μg/mL;

(8) FIG. 7 is a representative dose response curves for ADC3 against various cancer cell lines;

(9) FIG. 8 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC3 at 10 or 1 μg/mL;

(10) FIG. 9 is a representative dose response curves for ADC4 against various cancer cell lines;

(11) FIG. 10 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC4 at 10 or 1 μg/mL;

(12) FIG. 11 is a representative dose response curves of ADC5 against various cancer cell lines;

(13) FIG. 12 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC5 at 10 or 1 μg/mL;

(14) FIG. 13 is a representative dose response curves of ADC6 against various cancer cell lines;

(15) FIG. 14 shows histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC6 at 10 or 1 μg/mL;

(16) FIG. 15 is a representative dose response curves of ADC7 against various cancer cell lines;

(17) FIG. 16 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC7 at 50 or 1 μg/mL;

(18) FIG. 17 is a representative dose response curves of ADC8 against various cancer cell lines;

(19) FIG. 18 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC8 at 50 or 10 μg/mL;

(20) FIG. 19 is a representative dose response curves of ADC9 against various cancer cell lines;

(21) FIG. 20 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC9 at 50 or 0.1 μg/mL;

(22) FIG. 21 is a representative dose response curves of ADC10 against various cancer cell lines;

(23) FIG. 22 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC10 at 50 or 1 μg/mL;

(24) FIG. 23 is a representative dose response curves of ADC11 against various cancer cell lines;

(25) FIG. 24 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC11 at 50 or 1 μg/mL;

(26) FIG. 25 is a representative dose response curves of ADC12 against various cancer cell lines;

(27) FIG. 26 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC12 at 1 or 0.1 μg/mL;

(28) FIG. 27 is a representative dose response curves of ADC13 against various cancer cell lines;

(29) FIG. 28 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC13 at 1 or 0.1 μg/mL;

(30) FIG. 29 is a representative dose response curves of ADC14 against various cancer cell lines;

(31) FIG. 30 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC14 at 1 μg/mL;

(32) FIG. 31 is a representative dose response curves of ADC16 against various cancer cell lines;

(33) FIG. 32 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC16 at 1 μg/mL and 0.1 μg/mL;

(34) FIG. 33 is a representative dose response curves of ADC17 against various cancer cell lines;

(35) FIG. 34 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC17 at 1 μg/mL and 0.1 μg/mL;

(36) FIG. 35 is a representative dose response curves of ADC14 against two Raji cell clones;

(37) FIG. 36 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC14 at 10 μg/mL;

(38) FIG. 37 is a representative dose response curves of ADC15 against two Raji cell clones; and

(39) FIG. 38 shows histograms showing the percentage of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC15 at 10 μg/mL.

EXAMPLES

(40) The present invention is further illustrated by way of the following, non-limiting examples. In the examples, the following abbreviations are used: CDI, 1,1′-carbonyldiimidazole DIPEA, diisopropylethylamine Hex, hexane EtOAc, ethyl acetate DCM, dichloromethane NMP, N-methyl-2-pyrrolidone DMF, dimethylformamide EDC, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride EDTA, ethylenediaminetetraacetic acid MeOH, methanol DTT, dithiothreitol Py, pyridine THF, tetrahydrofuran TCEP, Tris[2-carboxyethyl]phosphine hydrochloride MC, 6-maleimidocaproyl Fmoc, 9-fluorenylmethoxycarbonyl Cit, citrulline Val, valine DMSO, dimethylsulfoxide Trt, triphenylmethyl HOBt, 1-hydroxybenzotriazole DIPCDI, N,N′-diisopropylcarbodiimide TFA, trifluoroacetic acid PABOH, 4-aminobenzyl alcohol bis-PNP, bis(4-nitrophenyl) carbonate NAC, N-Acetylcysteine SEC, size-exclusion chromatography HPLC, high performance liquid chromatography ADC, antibody drug conjugate ATCC, American Type Culture Collection DMEM, Dulbecco's Modified Eagle's Medium RPMI, Rosmell Park Memorial Institute medium ITS, Insulin-transferrin-sodium selenite media supplement FCS, Fetal Calf Serum SRB, sulforhodamine B PBS, phosphate buffered saline DR, dose-response UV, ultraviolet SMCC, Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate LAR, Linker to Antibody Ratio

Preparative Example

Preparation of Compound 9: MC-Val-Cit-PABC-PNP

(41) ##STR00117##

(a) Preparation of Compound 10: MC-Val-Cit-OH

(42) ##STR00118##

(43) Cl-TrtCl-resin (20 g, 1.49 mmol/g) (Iris Biotech, Ref.: BR-1065, 2-Chlorotrityl chloride resin (200-400 mesh, 1% DVB, 1.0-1.6 mmol/g), CAS 42074-68-0) was placed in a filter plate. 100 mL of DCM was added to the resin and the mixture was stirred for 1 h. The solvent was eliminated by filtration under vacuum. A solution of Fmoc-Cit-OH (11.83 g, 29.78 mmol) and DIPEA (17.15 mL, 98.45 mmol) in DCM (80 mL) was added and the mixture was stirred for 10 min. After that DIPEA (34.82 mmol, 199.98 mmol) was added and the mixture was stirred for 1 h. The reaction was terminated by addition of MeOH (30 mL) after stirring for 15 minutes. The Fmoc-Cit-O-TrtCl-resin produced as a result was subjected to the following washing/treatments: DCM (5×mL×0.5 min), DMF (5×50 mL×0.5 min), piperidine:DMF (1:4, 1×1 min, 2×10 min), DMF (5×50 mL×0.5 min), DCM (5×50 mL×0.5 min). The final piperidine wash gave NH.sub.2-Cit-O-TrtCl-resin. The loading was calculated: 1.15 mmol/g.

(44) The NH.sub.2-Cit-O-TrtCl-resin produced above was washed with DMF (5×50 mL×0.5 min) and a solution of Fmoc-Val-OH (31.22 g, 91.98 mmol), HOBt (11.23 g, 91.98 mmol) in DMF (100 mL) was added to the NH.sub.2-Cit-O-TrtCl-resin, stirred and DIPCDI (14.24 mL, 91.98 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5×50 mL×0.5 min). The Fmoc-Val-Cit-O-TrtCl-resin thus produced was treated with piperidine:DMF (1:4, 1×1 min, 2×10 min) and washed with DMF (5×50 mL×0.5 min). The final piperidine wash gave NH.sub.2—Val-Cit-O-TrtCl-resin.

(45) A solution of 6-maleimidocaproic acid (MC-OH) (9.7 g, 45.92 mmol), HOBt (6.21 g, 45.92 mmol) in DMF (100 mL) was added to the NH.sub.2—Val-Cit-O-TrtCl-resin produced above, stirred and DIPCDI (7.12 mL, 45.92 mmol) was added and the mixture was stirred for 1.5 h. The reaction was terminated by washing with DMF (5×50 mL×0.5 min) and DCM (5×50 mL×0.5 min).

(46) The peptide was cleaved from the resin by treatments with TFA:DCM (1:99, 5×100 mL). The resin was washed with DCM (7×50 mL×0.5 min). The combined filtrates were evaporated to dryness under reduced pressure and the solid obtained was triturated with Et.sub.2O and filtrated to obtain Compound 10 (7.60 g, 71%) as a white solid.

(47) .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 12.47 (s, 1H), 8.13 (d, J=7.3 Hz, 1H), 7.74 (d, J=9.0 Hz, 1H), 6.99 (s, 2H), 5.93 (s, 1H), 5.35 (s, 2H), 4.20 (dd, J=9.0, 6.8 Hz, 1H), 4.15-4.07 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.00-2.88 (m, 2H), 2.21-2.12 (m, 1H), 2.11-2.03 (m, 1H), 1.98-1.86 (m, 1H), 1.74-1.62 (m, 1H), 1.61-1.50 (m, 1H), 1.50-1.31 (m, 6H), 1.21-1.11 (m, 2H), 0.84 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H).

(48) ESI-MS m/z: Calcd. for C.sub.21H.sub.33N.sub.5O.sub.7: 467.2. Found: 468.3 (M+H).sup.+.

(b) Preparation of Compound 11: MC-Val-Cit-PABOH

(49) ##STR00119##

(50) To a solution of Compound 10 (1.6 g, 3.42 mmol) and 4-aminobenzyl alcohol (PABOH) (0.84 g, 6.84 mmol) in DCM (60 mL) was added a solution of HOBt (0.92 g, 6.84 mmol) in DMF (5 mL). DIPCDI (1.05 mL, 6.84 mmol) was added, the reaction mixture was stirred for 2 h at 23° C., Et.sub.2O (150 mL) was added, and the solid obtained was filtrated in a filter plate under vacuum to obtain Compound 11 (1.31 g, 67%).

(51) .sup.1H NMR (500 MHz, DMSO-d.sub.6): δ 9.88 (s, 1H), 8.03 (d, J=7.6 Hz, 1H), 7.77 (dd, J=12.2, 8.5 Hz, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.21 (d, J=8.2 Hz, 2H), 6.99 (s, 3H), 6.01-5.92 (m, 1H), 5.39 (s, 2H), 5.07 (s, 1H), 4.41 (s, 2H), 4.39-4.31 (m, 1H), 4.23-4.12 (m, 1H), 3.36 (t, J=7.0 Hz, 2H), 3.06-2.97 (m, 1H), 2.96-2.90 (m, 1H), 2.22-2.03 (m, 2H), 2.01-1.88 (m, 1H), 1.76-1.62 (m, 1H), 1.63-1.28 (m, 6H), 1.25-1.11 (m, 2H), 0.84 (d, J=6.9 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H).

(52) ESI-MS m/z: Calcd. for C.sub.28H.sub.40N.sub.6O.sub.7: 572.3. Found: 573.3 (M+H).sup.+.

(53) ##STR00120##

(54) To a solution of Compound 11 (500 mg, 0.87 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23° C. and poured onto a silica gel column (DCM:CH.sub.3OH, from 50:1 to 10:1) to afford pure target Compound 9 (364 mg, 57%).

(55) R.sub.f=0.40 (CH.sub.2Cl.sub.2:CH.sub.3OH, 9:1).

(56) .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): δ 9.45 (s, 1H), 8.23 (d, J=8.3 Hz, 2H), 7.59 (d, J=8.5 Hz, 2H), 7.35 (d, J=8.3 Hz, 2H), 7.34 (d, J=8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J=10.5, 5.4 Hz, 1H), 4.15 (d, J=7.2 Hz, 1H), 3.46 (dd, J=8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J=8.3, 6.6 Hz, 2H), 2.06-1.97 (m, 1H), 1.90-1.83 (m, 1H), 1.73-1.46 (m, 7H), 1.34-1.20 (m, 2H), 0.91 (d, J=6.7 Hz, 3H), 0.90 (d, J=6.7 Hz, 3H).

(57) .sup.13C NMR (125 MHz, CDCl.sub.3/CD.sub.3OD) δ 174.4, 172.4, 171.1, 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1, 129.9, 129.5, 125.2, 121.8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1, 26.2, 26.2, 25.1, 19.1, 18.1.

(58) ESI-MS m/z: Calcd. for C.sub.35H.sub.43N.sub.7O.sub.1: 737.3. Found: 738.3 (M+H).sup.+.

Example 1

Preparation of Compound 1

(59) ##STR00121##

(a) Preparation of Compound 3

(60) To a solution of Compound 2 (Compound 30a, prepared as described in WO 2007144423, the contents of which are incorporated herein by reference) (1.014 g, 1.8 mmol) in DCM (45 mL) was added 1,1′-carbonyldiimidazole (876 mg, 5.4 mmol). After being stirred at 23° C. overnight, the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures, from 99:1 to 85:15) to yield pure Compound 3 (1.176 g, 86%).

(61) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.39 (d, J=9.0 Hz, 1H), 8.12 (bs, 1H), 7.40 (bs, 1H), 7.30 (t, J=11.5 Hz, 1H), 7.08 (bs, 1H), 6.91 (t, J=12.0 Hz, 1H), 6.86 (t, J=10.0 Hz, 1H), 6.22 (d, J=9.0 Hz, 1H), 6.18 (d, J=11.0 Hz, 1H), 5.67 (d, J=12.0 Hz, 1H), 5.63-5.61 (m, 2H), 5.28 (d, J=11.5 Hz, 1H), 4.94-4.91 (m, 1H), 4.81-4.76 (m, 1H), 4.42 (d, J=9.0 Hz, 1H), 4.23-4.19 (m, 1H), 3.66 (s, 3H), 2.87-2.82 (m, 1H), 2.58-2.46 (m, 3H), 2.42-2.35 (m, 3H), 2.08 (s, 3H), 1.83 (s, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.06 (s, 9H).

(62) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.5, 166.4, 161.5, 148.7, 145.2, 140.4, 137.6, 137.0, 134.4, 133.9, 133.0, 130.9, 124.2, 123.9, 121.0, 120.5, 117.03, 110.0, 108.1, 104.1, 81.7, 77.8, 60.4, 55.4, 37.2, 34.5, 26.6, 26.3, 21.0, 17.1, 16.6.

(63) ESI-MS m/z: Calcd. for C.sub.34H.sub.45ClN.sub.4O.sub.7: 656.30. Found: 657.3 (M+H).sup.+.

(b) Preparation of Compound 4

(64) To a solution of Compound 3 (1.160 g, 1.78 mmol) in DCM (45 mL), prepared as described in step (a) above, was added propane 1,3-diamine (0.19 mL, 2.22 mmol). The reaction mixture was stirred at 23° C. overnight, and then concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiNH.sub.2, DCM:CH.sub.3OH, from 100:0 to 97:3) to obtain pure Compound 4 (800 mg, 68%).

(65) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.90 (d, J=11.7 Hz, 1H), 7.34-7.26 (m, 1H), 6.99-6.74 (m, 2H), 6.50 (d, J=9.0 Hz, 1H), 6.15 (d, J=12.9 Hz, 1H), 5.83 (t, J=11.5 Hz, 1H), 5.70 (d, J=11.5 Hz, 1H), 5.68-5.57 (m, 2H), 5.27 (d, J=9.4 Hz, 1H), 4.80 (q, J=8.3 Hz, 1H), 4.52-4.44 (m, 2H), 4.24-4.17 (m, 1H), 3.66 (s, 3H), 3.39-3.17 (m, 2H), 2.93-2.82 (m, 1H), 2.78 (t, J=6.5 Hz, 2H), 2.50-2.34 (m, 2H), 2.34-2.24 (m, 2H), 2.19-1.99 (m, 2H), 2.06 (s, 3H), 1.84 (s, 3H), 1.72-1.50 (m, 2H), 1.16 (d, J=6.6 Hz, 3H), 1.04 (s, 9H).

(66) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.4, 166.1, 161.5, 156.7, 145.2, 139.9, 137.1, 134.0, 133.9, 131.8, 124.3, 124.2, 122.5, 120.9, 108.1, 105.5, 81.8, 74.3, 60.6, 55.4, 39.81, 39.30 37.2, 34.7, 33.1, 31.5, 29.6, 26.7, 26.2, 21.0, 17.1, 16.6.

(67) ESI-MS m/z: Calcd. for C.sub.34H.sub.51ClN.sub.4O.sub.7: 662.30. Found: 663.3 (M+H).sup.+.

(68) To a solution of Compound 4 (52 mg, 0.078 mmol), prepared as described in step (b) above, and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (27.1 mg, 0.088 mmol) in DCM (2 mL) was added DIPEA (15 μL, 0.086 mmol). The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure target Compound 1 (29.7 mg, 44%).

(69) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.86 (d, J=10.8 Hz, 1H), 7.30 (t, J=11.6 Hz, 1H), 6.90 (td, J=11.5, 1.2 Hz, 1H), 6.78 (t, J=9.7 Hz, 1H), 6.68 (bs, 2H), 6.63 (d, J=9.4 Hz, 1H), 6.51 (t, J=6.4 Hz, 1H), 6.16 (d, J=11.8 Hz, 1H), 5.76 (t, J=6.4 Hz, 1H), 5.72 (d, J=11.6 Hz, 1H), 5.65 (dd, J=6.4, 2.9 Hz, 1H), 5.62-5.57 (m, 1H), 5.29 (d, J=11.1 Hz, 1H), 4.81 (q, J=8.3 Hz, 1H), 4.52-4.48 (m, 2H), 4.24 (ddd, J=11.4, 7.3, 4.3 Hz, 1H), 3.66 (s, 3H), 3.50 (t, J=7.3 Hz, 2H), 3.33-3.10 (m, 4H), 2.93-2.81 (m, 1H), 2.45-2.31 (m, 5H), 2.17 (t, J=7.6 Hz, 2H), 2.10-1.98 (m, 1H), 2.07 (s, 3H), 1.84 (s, 3H), 1.72-1.54 (m, 8H), 1.16 (d, J=6.6 Hz, 3H), 1.05 (s, 9H).

(70) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 173.8, 170.8, 168.3, 166.5, 161.6, 157.1, 145.1, 140.4, 137.5, 134.2, 134.1, 134.0, 131.9, 124.2, 124.0, 122.5, 120.6, 108.3, 106.0, 81.8, 74.5, 60.6, 55.4, 37.7, 37.6, 37.2, 36.3, 36.1, 34.7, 33.4, 31.0, 29.8, 28.3, 26.7, 26.3, 26.2, 25.6, 21.0, 17.2, 16.6.

(71) ESI-MS m/z: Calcd. for C.sub.44H.sub.62ClN.sub.5O.sub.10: 855.42. Found: 856.5 (M+H).sup.+.

Example 2

Preparation of Compound 5

(72) ##STR00122##

(a) Preparation of Compound 7

(73) To a solution of Compound 6 (Compound 30b, prepared as described in WO 2007144423, the contents of which are incorporated herein by reference) (750 mg, 1.42 mmol) in DCM (35.5 mL) was added 1,1′-carbonyldiimidazole (691 mg, 4.26 mmol). After being stirred at 23° C. overnight, the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 7 (717 mg, 81%).

(74) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.24 (d, J=11.0 Hz, 1H), 8.09 (s, 1H), 7.40 (s, 1H), 7.36-7.23 (m, 1H), 7.05 (s, 1H), 6.95-6.83 (m, 2H), 6.34 (d, J=9.2 Hz, 1H), 6.14 (d, J=11.8 Hz, 1H), 5.74-5.57 (m, 3H), 5.43-5.34 (m, 1H), 5.28 (d, J=10.2 Hz, 1H), 4.98-4.88 (m, 1H), 4.78 (q, J=7.8 Hz, 1H), 4.47 (d, J=9.2 Hz, 1H), 4.25-4.16 (m, 1H), 3.64 (s, 3H), 2.92-2.76 (m, 1H), 2.59-2.37 (m, 6H), 1.83 (s, 3H), 1.64 (d, J=6.7 Hz, 3H), 1.14 (d, J=6.7 Hz, 3H), 1.03 (s, 9H).

(b) Preparation of Compound 8

(75) To a solution of Compound 7 (1.68 g, 2.7 mmol), prepared as described in step (a) above, in DCM (80 mL) was added propane 1,3-diamine (0.27 mL, 3.24 mmol). The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, DCM:CH.sub.3OH, from 100:0 to 97:3) to obtain Compound 8 (854 mg, 50%).

(76) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.90 (d, J=11.7 Hz, 1H), 7.39-7.18 (m, 1H), 6.92-6.84 (m, 2H), 6.50 (d, J=9.0 Hz, 1H), 6.15 (d, J=12.9 Hz, 1H), 5.75-5.67 (m, 2H), 5.66-5.54 (m, 2H), 5.46-5.33 (m, 1H), 5.26 (d, J=10.3 Hz, 1H), 4.83 (q, J=8.3 Hz, 1H), 4.50-4.48 (m, 2H), 4.30-4.04 (m, 1H), 3.66 (s, 3H), 3.39-3.17 (m, 2H), 2.93-2.82 (m, 1H), 2.78 (t, J=6.5 Hz, 2H), 2.50-2.34 (m, 3H), 2.34-2.24 (m, 2H), 2.19-1.99 (m, 1H), 1.83 (s, 3H), 1.72-1.50 (m, 2H), 1.62 (d, J=6.7 Hz, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.04 (s, 9H).

(77) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.3, 166.2, 161.6, 157.1, 145.1, 139.9, 137.1, 134.0, 133.9, 126.9, 124.9, 124.2, 123.9, 120.9, 108.2, 106.3, 81.8, 75.0, 60.6, 55.4, 39.6, 37.2, 34.7, 32.8, 31.5, 31.1, 29.6, 26.7, 26.2, 17.1, 16.6, 12.9.

(78) ESI-MS m/z: Calcd. for C.sub.34H.sub.52N.sub.4O.sub.7: 628.4. Found: 629.5 (M+H).sup.+.

(79) To a solution of Compound 8 (150 mg, 0.24 mmol), prepared as described in step (b) above, in DCM (8 mL) at 23° C. was added 6-maleimidohexanoic acid N-hydroxysuccinimide ester (88.3 mg, 0.28 mmol). The reaction mixture was stirred at 23° C. for 18 h, and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure target Compound 5 (75 mg, 38%).

(80) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.87 (d, J=10.7 Hz, 1H), 7.32-7.22 (m, 1H), 6.89 (t, J=11.6 Hz, 1H), 6.78 (t, J=9.7 Hz, 1H), 6.68 (s, 2H), 6.61 (d, J=9.4 Hz, 1H), 6.54 (t, J=6.0 Hz, 1H), 6.15 (d, J=11.6 Hz, 1H), 5.77-5.51 (m, 2H), 5.64 (dd, J=6.4, 3.0 Hz, 1H), 5.60-5.55 (m, 1H), 5.38 (ddd, J=13.0, 8.8, 6.6 Hz, 1H), 5.28 (d, J=10.0 Hz, 1H), 4.83 (q, J=8.3 Hz, 1H), 4.59-4.44 (m, 2H), 4.23 (ddd, J=11.5, 7.2, 4.4 Hz, 1H), 3.65 (s, 3H), 3.49 (t, J=7.2 Hz, 2H), 3.29-3.12 (m, 4H), 2.87-2.81 (m, 1H), 2.48-2.32 (m, 5H), 2.18-2.09 (m, 3H), 1.88-1.82 (m, 1H), 1.83 (s, 3H), 1.67-1.55 (m, 7H), 1.62 (d, J=6.8 Hz, 3H), 1.15 (d, J=6.7 Hz, 3H), 1.04 (s, 9H).

(81) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 173.6, 170.8, 168.2, 166.4, 161.6, 157.4, 145.2, 140.2, 137.4, 134.2, 134.1, 134.0, 127.0, 124.9, 123.9, 120.7, 108.3, 106.5, 81.8, 75.3, 60.6, 55.4, 37.7, 37.6, 37.2, 36.3, 36.0, 34.7, 31.8, 31.6, 31.1, 29.9, 28.3, 26.7, 26.4, 26.2, 25.2, 22.6, 17.2, 16.6.

(82) ESI-MS m/z: Calcd. for C.sub.44H.sub.63N.sub.5O.sub.10: 821.5. Found: 822.4 (M+H).sup.+.

Example 3

Preparation of Compound 12

(83) ##STR00123##

(a) Preparation of Compound 12

(84) DIPEA (25 μL, 0.14 mmol) was added to a solution of Compound 9 (94.5 mg, 0.13 mmol), prepared as shown in the Preparative Example above, and Compound 4 (85 mg, 0.13 mmol), prepared as described in Example 1(b) above, in NMP (6.5 mL) at 23° C. After 9 h the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, DCM:CH.sub.3OH, from 100:0 to 90:10). Finally, purification of target Compound 12 (35.7 mg, 22%) was achieved by semipreparative HPLC (Symmetry 018, 7 μm, 19×150 mm, gradient H.sub.2O+CH.sub.3CN, flow 15 mL/min, UV detection).

(85) .sup.1H NMR (500 MHz, CDCl.sub.3/CD.sub.3OD): δ 7.49 (d, J=8.1 Hz, 2H), 7.22 (d, J=8.1 Hz, 2H), 7.19 (t, J=11.8 Hz, 1H), 6.96 (dd, J=23.5, 8.9 Hz, 1H), 6.84 (t, J=11.5 Hz, 1H), 6.70-6.64 (m, 1H), 6.64 (s, 2H), 6.10 (d, J=11.6 Hz, 1H), 5.93 (t, J=6.2 Hz, 1H), 5.82 (t, J=6.2 Hz, 1H), 5.69 (d, J=11.4 Hz, 1H), 5.61 (dd, J=6.3, 3.1 Hz, 1H), 5.54 (t, J=7.8 Hz, 1H), 5.22 (d, J=9.7 Hz, 1H), 4.96 (s, 2H), 4.75 (q, J=8.1 Hz, 1H), 4.55-4.49 (m, 2H), 4.43-4.36 (m, 1H), 4.23-4.10 (m, 2H), 3.59 (s, 3H), 3.44 (t, J=7.2 Hz, 2H), 3.18-3.04 (m, 8H), 2.82-2.72 (m, 1H), 2.49-2.34 (m, 3H), 2.27 (t, J=7.1 Hz, 2H), 2.18 (t, J=7.2 Hz, 2H), 2.16-2.06 (m, 1H), 2.01-1.95 (m, 1H), 2.00 (s, 3H), 1.87-1.79 (m, 1H), 1.78 (s, 3H), 1.73-1.40 (m, 11H), 1.35-1.20 (m, 2H), 1.09 (d, J=10.0 Hz, 3H), 0.96 (s, 9H), 0.87 (dd, J=6.8, 4.3 Hz, 6H).

(86) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 174.1, 172.2, 171.0, 170.3, 168.8, 166.8, 162.1, 160.2, 157.0, 156.9, 144.9, 140.2, 137.7, 137.5, 134.0, 132.4, 131.7, 128.7, 124.0, 123.4, 122.4, 120.4, 119.8, 111.5, 108.6, 107.0, 81.9, 73.8, 68.6, 66.2, 60.3, 58.8, 55.4, 53.0, 37.6, 37.5, 37.1, 35.9, 34.6, 33.2, 30.6, 29.9, 29.2, 28.0, 26.5, 26.2, 26.0, 25.0, 22.6, 20.8, 19.1, 18.2, 17.04, 16.4.

(87) ESI-MS m/z: Calcd. for C.sub.63H.sub.89ClN.sub.10O.sub.15: 1260.6. Found: 1261.6 (M+H).sup.+.

Example 4

Preparation of Compound 13

(88) ##STR00124##

(a) Preparation of Compound 14

(89) To a solution of Compound 4 (110 mg, 0.17 mmol), prepared as described in Example 1(b) above, and 3-(methyldisulfanyl)propanoic acid (34 mg, 0.22 mmol) in DCM (5 mL) were added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (47.8 mg, 0.22 mmol) and N,N′-diisopropylethylamine (3.8 μL, 0.22 mmol). The reaction mixture was stirred at 23° C. for 6 h, diluted with H.sub.2O and extracted with DCM. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 14 (123 mg, 93%).

(90) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.88 (d, J=10.8 Hz, 1H), 7.29-7.24 (m, 1H), 6.90 (t, J=11.5 Hz, 1H), 6.82 (t, J=9.1 Hz, 1H), 6.63 (t, J=6.1 Hz, 1H), 6.49 (d, J=9.4 Hz, 1H), 6.16 (dd, J=11.5, 1.5 Hz, 1H), 5.70 (d, J=11.5 Hz, 1H), 5.68-5.51 (m, 3H), 5.29 (d, J=9.7 Hz, 1H), 4.81 (q, J=8.2 Hz, 1H), 4.52 (d, J=9.5 Hz, 1H), 4.52-4.43 (m, 1H), 4.24 (ddd, J=11.5, 7.3, 4.3 Hz, 1H), 3.66 (s, 3H), 3.37-3.21 (m, 3H), 3.21-3.12 (m, 1H), 2.97 (t, J=7.2 Hz, 2H), 2.90-2.81 (m, 1H), 2.60 (t, J=7.2 Hz, 2H), 2.49-2.35 (m, 3H), 2.39 (s, 3H), 2.33 (t, J=7.0 Hz, 2H), 2.14-2.07 (m, 1H), 2.07 (s, 3H), 1.84 (s, 3H), 1.73-1.64 (m, 2H), 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(91) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 171.6, 168.2, 166.4, 161.6, 157.2, 145.2, 140.3, 137.4, 134.2, 134.0, 131.9, 124.4, 124.1, 122.4, 120.7, 108.3, 105.6, 81.8, 74.8, 60.6, 60.4, 55.5, 37.8, 37.2, 36.2, 35.6, 34.7, 33.1, 31.0, 29.8, 26.7, 26.2, 23.0, 21.0, 17.2, 16.6.

(b) Preparation of Compound 13

(92) A solution of Compound 14 (100 mg, 0.125 mmol), prepared as described in step (a) above, in a mixture of EtOAc (4.3 mL) and CH.sub.3OH (4.3 mL) was treated with a solution of dithiothreitol (154.8 mg, 1.0 mmol) in 0.05 M potassium phosphate buffer (4.3 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer (13 mL) at pH 6.0 containing 2 mM EDTA and then extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The crude obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure target Compound 13 (35 mg, 37%).

(93) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.91 (d, J=10.8 Hz, 1H), 7.27-7.24 (m, 1H), 6.91 (t, J=11.5 Hz, 1H), 6.82 (t, J=9.7 Hz, 1H), 6.67 (t, J=6.1 Hz, 1H), 6.49 (d, J=9.4 Hz, 1H), 6.16 (d, J=11.6 Hz, 1H), 5.71 (d, J=11.6 Hz, 1H), 5.66-5.57 (m, 3H), 5.29 (d, J=9.9 Hz, 1H), 4.84 (q, J=8.3 Hz, 1H), 4.51 (d, J=9.5 Hz, 1H), 4.50-4.45 (m, 1H), 4.24-4.20 (m, 1H), 3.66 (s, 3H), 3.36-3.12 (m, 4H), 2.90-2.71 (m, 3H), 2.64-2.24 (m, 7H), 2.14-2.04 (m, 1H), 2.06 (s, 3H), 1.83 (s, 3H), 1.73-1.68 (m, 2H), 1.15 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(94) ESI-MS m/z: Calcd. for C.sub.37H.sub.55ClN.sub.4O.sub.8S: 750.3. Found: 773.2 (M+Na).sup.+.

Example 5

Preparation of Compound 15

(95) ##STR00125##

(a) Preparation of Compound 16

(96) To a solution of Compound 2 (Compound 30a, prepared as described in WO 2007144423, the contents of which are incorporated herein by reference) (300 mg, 0.53 mmol) in DCM (5 mL) were added pyridine (85 μL, 1.06 mmol) and 4-nitrophenyl chloroformate (214.7 mg, 1.06 mmol) at 0° C. The reaction mixture was stirred at 23° C. for 1.5 h, diluted with citric acid 10% and extracted with DCM. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure Compound 16 (307 mg, 80%).

(97) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.29 (d, J=9.2 Hz, 2H), 8.08 (d, J=10.9 Hz, 1H), 7.44 (d, J=9.1 Hz, 2H), 7.27-7.22 (m, 1H), 6.92-6.83 (m, 2H), 6.20 (d, J=9.2 Hz, 1H), 6.17 (dd, J=11.6, 1.4 Hz, 1H), 5.67-5.58 (m, 3H), 5.29 (d, J=10.0 Hz, 1H), 4.84 (q, J=8.2 Hz, 1H), 4.77-4.72 (m, 1H), 4.41 (d, J=9.3 Hz, 1H), 4.22 (ddd, J=11.5, 7.5, 4.4 Hz, 1H), 3.67 (s, 3H), 2.89-2.82 (m, 1H), 2.54-2.33 (m, 6H), 2.10 (d, J=1.2 Hz, 3H), 1.85 (d, J=1.3 Hz, 3H), 1.17 (d, J=6.7 Hz, 3H), 1.02 (s, 9H).

(98) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.4, 166.1, 161.5, 155.3, 152.5, 145.5, 145.2, 140.4, 137.6, 134.3, 134.0, 133.2, 125.3, 124.4, 124.1, 121.8, 121.2, 120.4, 108.1, 104.6, 81.8, 79.1, 60.4, 55.5, 37.3, 34.7, 32.7, 30.1, 26.6, 26.3, 21.1, 17.2, 16.6.

(b) Preparation of Compound 17

(99) To a solution of Compound 16 (156.3 mg, 0.21 mmol) in DCM (2.5 mL) were added a suspension of 3-aminopropane-1-thiol hydrochloride (44.8 mg, 0.26 mmol) in DCM (2.5 mL), triethylamine (58 μL, 0.34 mmol) and DMF (0.1 mL) at 23° C. The reaction mixture was stirred at 23° C. for 3 h, diluted with H.sub.2O and extracted with DCM. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 17 (80 mg, 95%).

(100) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.68 (d, J=10.6 Hz, 1H), 7.28 (t, J=11.6 Hz, 1H), 6.89 (t, J=11.5 Hz, 1H), 6.76 (t, J=9.6 Hz, 1H), 6.65 (d, J=9.1 Hz, 1H), 6.13 (d, J=11.7 Hz, 1H), 5.87-5.51 (m, 4H), 5.28 (d, J=5.0 Hz, 1H), 4.77 (q, J=8.2 Hz, 1H), 4.61-4.39 (m, 2H), 4.29-4.00 (m, 1H), 3.65 (s, 3H), 3.31-3.18 (m, 2H), 2.98-2.77 (m, 1H), 2.68 (t, J=7.4 Hz, 2H), 2.55-2.22 (m, 6H), 2.04 (s, 3H), 2.00-1.80 (m, 2H), 1.83 (s, 3H), 1.15 (d, J=6.6 Hz, 3H), 1.05 (s, 9H).

(101) ESI-MS m/z: Calcd. for C.sub.68H.sub.98C.sub.12N.sub.60O.sub.14S.sub.2: 1356.6. Found: 1357.3 (M+H).sup.+.

(102) A solution of Compound 17 (59.4 mg, 0.044 mmol) in a mixture of EtOAc (1.5 mL) and CH.sub.3OH (1.5 mL) was treated with a solution dithiothreitol (0.35 mL, 0.35 mmol) in 0.05 M potassium phosphate buffer (1.5 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer at pH 6.0 containing 2 mM EDTA and the extracted with EtOAc (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure target Compound 15 (31 mg, 59%).

(103) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.66 (d, J=10.7 Hz, 1H), 7.29 (t, J=11.2 Hz, 1H), 6.91 (t, J=11.5 Hz, 1H), 6.83 (t, J=9.7 Hz, 1H), 6.38 (d, J=9.4 Hz, 1H), 6.17 (d, J=11.8 Hz, 1H), 5.70 (d, J=11.4 Hz, 1H), 5.65-5.51 (m, 2H), 5.34 (t, J=6.3 Hz, 1H), 5.29 (d, J=10.0 Hz, 1H), 4.82 (q, J=8.3 Hz, 1H), 4.56-4.48 (m, 1H), 4.45 (d, J=9.3 Hz, 1H), 4.22 (ddd, J=11.4, 7.5, 4.3 Hz, 1H), 3.67 (s, 3H), 3.31 (q, J=6.4 Hz, 2H), 2.88-2.83 (m, 1H), 2.55 (q, J=7.7 Hz, 2H), 2.47-2.30 (m, 5H), 2.12-2.07 (m, 1H), 2.08 (s, 3H), 1.88-1.76 (m, 5H), 1.17 (d, J=6.6 Hz, 3H), 1.06 (s, 9H).

(104) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.2, 166.2, 161.5, 156.7, 145.2, 140.2, 137.3, 134.2, 134.0, 132.0, 124.4, 124.2, 122.3, 120.8, 108.1, 105.5, 81.8, 74.5, 60.6, 55.4, 39.6, 37.3, 34.6, 33.9, 33.3, 30.8, 26.7, 26.3, 21.8, 21.1, 17.2, 16.7.

(105) ESI-MS m/z: Calcd. for C.sub.34H.sub.50ClN.sub.3O.sub.7S: 679.3. Found: 702.4 (M+Na).sup.+.

Example 6

Preparation of Compound 18

(106) ##STR00126##

(a) Preparation of Compound 19

(107) To a solution of Compound 8 (280 mg, 0.45 mmol), prepared as described in Example 2(b) above, and 3-(methyldisulfanyl)propanoic acid (88 mg, 0.58 mmol) in DCM (7.5 mL) were added N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (126 mg, 0.58 mmol) and N,N′-diisopropylethylamine (0.1 mL, 0.58 mmol). The reaction mixture was stirred at 23° C. for 3 h, diluted with H.sub.2O and extracted with DCM. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 19 (240 mg, 71%).

(108) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.91 (d, J=10.8 Hz, 1H), 7.34-7.20 (m, 1H), 6.89 (t, J=11.4 Hz, 1H), 6.83-6.72 (m, 2H), 6.51 (d, J=9.5 Hz, 1H), 6.16 (d, J=11.3 Hz, 1H), 5.70 (d, J=11.5 Hz, 1H), 5.64 (dd, J=6.1, 3.3 Hz, 1H), 5.61-5.55 (m, 2H), 5.47-5.33 (m, 1H), 5.28 (d, J=9.3 Hz, 1H), 4.84 (q, J=8.3 Hz, 1H), 4.51 (d, J=9.6 Hz, 1H), 4.52-4.47 (m, 1H), 4.27-4.19 (m, 1H), 3.66 (s, 3H), 3.37-3.21 (m, 3H), 3.21-3.12 (m, 1H), 2.96 (t, J=7.2 Hz, 2H), 2.90-2.81 (m, 1H), 2.60 (t, J=7.2 Hz, 2H), 2.43-2.35 (m, 5H), 2.39 (s, 3H), 2.16-2.04 (m, 1H), 1.84 (s, 3H), 1.70-1.61 (m, 5H), 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(109) .sup.13C NMR (125 MHz, CDCl.sub.3) δ 171.5, 168.1, 166.3, 157.4, 145.2, 140.3, 137.4, 134.2, 134.0, 127.0, 124.9, 124.0, 120.8, 108.3, 106.3, 81.8, 75.5, 60.6, 55.4, 53.4, 37.8, 37.2, 36.2, 35.9, 34.7, 33.1, 31.8, 31.2, 29.8, 26.7, 26.2, 23.0, 17.2, 16.6, 13.0.

(110) ESI-MS m/z: Calcd. for C.sub.38H.sub.58N.sub.4O.sub.8S.sub.2: 763.4. Found: 762.4 (M+H).sup.+.

(b) Preparation of Compound 18

(111) A solution of Compound 19 (240 mg, 0.31 mmol), prepared as described in step (a) above, in a mixture of EtOAc (15 mL) and CH.sub.3OH (22 mL) was treated with a solution dithiothreitol (0.79 mL of 1.0 M solution, 0.79 mmol) in 0.05 M potassium phosphate buffer (17.4 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer (21 mL) at pH 6.0 containing 2 mM EDTA and the extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The crude obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure target Compound 18 (105 mg, 47%)

(112) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.93 (d, J=10.6 Hz, 1H), 7.30-7.22 (m, 1H), 6.90 (t, J=11.6 Hz, 1H) 6.86-6.72 (m, 2H), 6.48 (d, J=9.4 Hz, 1H), 6.16 (d, J=11.6 Hz, 1H), 5.70 (d, J=11.4 Hz, 1H), 5.65-5.55 (m, 3H), 5.48-5.34 (m, 1H), 5.29 (d, J=9.9 Hz, 1H), 4.84 (q, J=9.4, 8.7 Hz, 1H), 4.52 (d, J=9.5 Hz, 1H), 4.57-4.44 (m, 1H), 4.32-4.17 (m, 1H), 3.66 (s, 3H), 3.39-3.20 (m, 3H), 3.22-3.09 (m, 1H), 2.90-2.75 (m, 3H), 2.50 (t, J=6.9, 2H), 2.45-2.28 (m, 5H), 2.16-2.08 (m, 1H), 1.83 (s, 3H), 1.72-1.65 (m 2H), 1.64 (d, J=6.6 Hz, 3H) 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(113) ESI-MS m/z: Calcd. for C.sub.37H.sub.56N.sub.4O.sub.8S: 716.4. Found: 717.3 (M+H).sup.+.

Example 7

Preparation of Compound 25

(114) ##STR00127##

(a) Preparation of Compound 23

(115) To a solution of Compound 22 (333 mg, 0.63 mmol) (Compound 71, prepared as described in WO 2009080761, the contents of which are incorporated herein by reference) in CH.sub.2Cl.sub.2 (12.5 mL) was added 1,1′-carbonyldiimidazole (308 mg, 1.90 mmol). After being stirred at 23° C. overnight, the reaction mixture was concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure Compound 23 (344 mg, 88%).

(116) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.14 (s, 1H), 8.11 (d, J=10.8 Hz, 1H), 7.43 (s, 1H), 7.28 (t, J=11.6 Hz, 1H), 7.08 (dd, J=1.7, 0.9 Hz, 1H), 6.96-6.81 (m, 2H), 6.26 (d, J=9.3 Hz, 1H), 6.17 (d, J=11.6 Hz, 1H), 5.66 (dt, J=11.4, 1.4 Hz, 1H), 5.62 (dd, J=5.9, 3.5 Hz, 1H), 5.28 (d, J=10.0 Hz, 1H), 5.04-4.89 (m, 1H), 4.80 (q, J=8.3 Hz, 1H), 4.40 (d, J=9.3 Hz, 1H), 4.21 (ddd, J=10.3, 7.5, 5.3 Hz, 1H), 3.65 (s, 3H), 2.91-2.78 (m, 1H), 2.68-2.49 (m, 3H), 2.45-2.31 (m, 2H), 1.89 (t, J=2.5 Hz, 3H), 1.84 (s, 3H), 1.15 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(117) ESI-MS m/z: Calcd. for C.sub.34H.sub.44N.sub.4O.sub.7: 620.32. Found: 621.3 (M+H).sup.+.

(b) Preparation of Compound 24

(118) To a solution of Compound 23 (0.130 g, 0.21 mmol)), prepared as described in step (a) above, in CH.sub.2Cl.sub.2 (3.5 mL) was added propane 1,3-diamine (0.022 mL, 0.26 mmol). The reaction mixture was stirred at 23° C. for 6 hours and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, DCM:CH.sub.3OH, from 100:0 to 97:3) to obtain Compound 24 (120 mg, 91%).

(119) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.71 (d, J=10.7 Hz, 1H), 7.28 (t, J=11.6 Hz, 1H), 6.91-6.77 (m, 2H), 6.44 (d, J=9.4 Hz, 1H), 6.14 (d, J=11.6 Hz, 1H), 5.77-5.57 (m, 3H), 5.27 (d, J=10.0 Hz, 1H), 4.83 (q, J=8.3 Hz, 1H), 4.57-4.53 (m, 1H), 4.46 (d, J=12.4 Hz, 1H), 4.31-4.11 (m, 1H), 3.65 (s, 3H), 3.31-3.24 (m, 2H), 2.93-2.67 (m, 3H), 2.56-2.24 (m, 6H), 2.16 (s, 3H), 1.83 (s, 3H), 1.63 (dd, J=9.6, 3.5 Hz, 2H), 1.15 (d, J=6.6 Hz, 3H), 1.04 (s, 9H).

(120) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.3, 166.1, 161.6, 156.5, 145.2, 140.0, 137.2, 134.1, 134.0, 124.3, 124.2, 120.9, 108.1, 106.0, 81.8, 78.4, 74.5, 73.2, 60.6, 55.4, 39.8, 39.2, 37.3, 34.8, 33.0, 30.9, 30.2, 26.7, 26.3, 17.2, 16.7, 3.6.

(121) ESI-MS m/z: Calcd. for C.sub.34H.sub.50N.sub.4O.sub.7: 626.37. Found: 627.3 (M+H).sup.+.

(122) To a solution of Compound 24 (40 mg, 0.064 mmol)), prepared as described in step (b) above, in CH.sub.2Cl.sub.2 (2 mL) was added 6-maleimidohexanoic acid N-hydroxysuccinimide ester (21.6 mg, 0.07 mmol). The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 25 (33.5 mg, 64%).

(123) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.67 (d, J=10.7 Hz, 1H), 7.29-7.23 (m, 1H), 6.90 (t, J=11.5 Hz, 1H), 6.80 (t, J=9.6 Hz, 1H), 6.68 (s, 2H), 6.46 (d, J=9.4 Hz, 1H), 6.42 (bs, 1H), 6.16 (d, J=11.6 Hz, 1H), 5.73-5.67 (m, 2H), 5.64 (dd, J=6.2, 3.1 Hz, 1H), 5.30 (d, J=9.6 Hz, 1H), 4.86 (q, J=8.4 Hz, 1H), 4.63-4.54 (m, 1H), 4.44 (d, J=9.4 Hz, 1H), 4.30-4.18 (m, 1H), 3.66 (s, 3H), 3.50 (t, J=7.2 Hz, 2H), 3.34-3.10 (m, 3H), 2.85 (dt, J=9.9, 6.9 Hz, 1H), 2.54-2.37 (m, 4H), 2.36-2.28 (m, 1H), 2.16 (t, J=7.6 Hz, 2H), 1.84 (d, J=1.3 Hz, 3H), 1.83-1.81 (m, 3H), 1.70-1.51 (m, 8H), 1.34-1.23 (m, 2H), 1.16 (d, J=6.6 Hz, 3H), 1.05 (s, 9H).

(124) .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.4, 170.8, 168.1, 166.4, 161.6, 157.0, 145.2, 140.3, 137.5, 134.3, 134.1, 133.9, 124.2, 120.7, 108.2, 106.2, 81.8, 78.4, 74.3, 73.5, 60.7, 55.4, 37.7, 37.6, 37.3, 36.4, 35.9, 34.6, 32.8, 30.3, 29.9, 28.3, 26.7, 26.4, 26.2, 25.4, 25.2, 24.4, 17.2, 16.6, 3.6.

(125) ESI-MS m/z: Calcd. for C.sub.44H.sub.61ClN.sub.5O.sub.10: 819.44. Found: 820.4 (M+H).sup.+.

Example 8

Preparation of Compound 27

(126) ##STR00128##

(a) Preparation of Compound 26

(127) To a solution of Compound 24 (70 mg, 0.11 mmol), prepared as described in Example 7(b) above, in CH.sub.2Cl.sub.2 (2 mL) was added 3-(methyldisulfanyl)propanoic acid N-hydroxysuccinimide ester (36.2 mg, 0.12 mmol). The reaction mixture was stirred at 23° C. for 16 h and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 26 (46.3 mg, 61%) as a solid white.

(128) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.72 (d, J=10.8 Hz, 1H), 7.29-7.19 (m, 1H), 6.90 (t, J=11.3 Hz, 1H), 6.80 (t, J=9.7 Hz, 1H), 6.69 (t, J=6.1 Hz, 1H), 6.47 (d, J=9.4 Hz, 1H), 6.16 (d, J=11.0 Hz, 1H), 5.69 (d, J=11.5 Hz, 1H), 5.64 (dd, J=6.3, 3.1 Hz, 1H), 5.30 (d, J=0.5 Hz, 1H), 4.86 (q, J=8.4 Hz, 1H), 4.60-4.54 (m, 1H), 4.46 (d, J=9.4 Hz, 1H), 4.23 (ddd, J=11.5, 7.4, 4.8 Hz, 1H), 3.66 (s, 3H), 3.33-3.22 (m, 3H), 3.19-3.14 (m, 1H), 2.96 (t, J=7.2 Hz, 2H), 2.66-2.54 (m, 1H), 2.59 (t, J=7.2 Hz, 2H), 2.48-2.42 (m, 5H), 2.40 (s, 3H), 2.38-2.28 (m, 1H) 1.83 (s, 3H), 1.82 (s, 3H), 1.73-1.64 (m, 2H), 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(129) .sup.13C NMR (100 MHz, CDCl.sub.3): δ 171.5, 168.1, 166.4, 165.2, 157.0, 145.1, 140.4, 137.5, 134.3, 134.0, 124.20, 124.0, 120.7, 108.2, 106.2, 81.8, 78.4, 74.2, 73.6, 60.7, 55.4, 37.8, 37.3, 35.9, 34.6, 33.1, 30.3, 29.8, 26.7, 26.2, 24.4, 23.0, 17.2, 16.6, 3.6.

(b) Preparation of Compound 27

(130) A solution of Compound 26 (44.3 mg, 0.064 mmol), prepared as described in step (a) above, in a mixture of EtOAc (3.6 mL) and CH.sub.3OH (3.6 mL) was treated with a solution of dithiothreitol (0.19 mL of 1.0 M solution, 0.19 mmol) in 0.05 M potassium phosphate buffer (3.6 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer (21 mL) at pH 6.0 containing 2 mM EDTA and then extracted with ethyl acetate. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The crude obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure target Compound 27 (33.6 mg, 74%) as a solid white.

(131) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.73 (d, J=10.7 Hz, 1H), 7.27 (t, J=11.5 Hz, 1H), 6.91 (td, J=11.5, 1.1 Hz, 1H), 6.94-6.86 (m, 1H), 6.85-6.77 (m, 1H), 6.43 (d, J=9.4 Hz, 1H), 6.17 (dd, J=12.0, 1.6 Hz, 1H), 5.71 (d, J=11.4 Hz, 1H), 5.65 (dd, J=6.5, 2.9 Hz, 1H), 5.54 (t, J=6.3 Hz, 1H), 5.30 (d, J=10.5 Hz, 1H), 4.84 (q, J=8.3 Hz, 1H), 4.53 (d, J=9.5 Hz, 1H), 4.48-4.44 (m, 1H), 4.24 (ddd, J=11.5, 7.3, 4.2 Hz, 1H), 3.67 (s, 3H), 3.336-3.22 (m, 3H), 3.21-3.10 (m, 1H), 2.89-2.84 (m, 1H), 2.80 (dt, J=8.2, 6.8 Hz, 2H), 2.49 (t, J=7.2 Hz, 2H), 2.45-2.36 (m, 5H), 2.31-2.28 (m, 1H), 1.84 (s, 3H), 1.83 (s, 3H), 1.72-1.69 (m, 2H), 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(132) ESI-MS m/z: Calcd. for C.sub.37H.sub.54N.sub.4O.sub.8S: 714.37. Found: 737.3 (M+Na)+.

Example 9

Preparation of Antibody-Drug Conjugate ADC1 with Trastuzumab and Compound 1

(a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab (Compound 20)

(133) A Trastuzumab (Trastuzumab purchased from Roche as a white lyophilised powder for the preparation of a concentrated solution for infusion) solution (9.52 mL, 200 mg, 1.38 μmol) was diluted to a concentration of 5 mg/mL with 20 mM histidine/acetate buffer (pH 5.5, 30.5 mL) followed by a pH adjustment with phosphate/EDTA buffer (pH 8.4, 13 mL). Partial reduction of the disulfide bonds in the antibody was performed by the addition of a 5.17 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (689 μL, 3.562 μmol, 2.6 eq.) The reduction reaction was left to stir for 90 min at 20° C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.1, very close to the value of 4.0, as planned.

(b) Preparation of ADC1

(134) To the solution of partially reduced Trastuzumab Compound 20 (24.98 mL, 93.0 mg, 0.64 μmol), prepared as described in Example 7(a) above, in DMSO was added (1.25 mL) followed by addition of a freshly prepared solution of Compound 1, prepared as described in Example 1, (10 mM in DMSO, 366 μL, 3.66 μmol, 5.7 eq.). Upon addition of Compound 1, the solution turned very turbid, hence DMSO (1 mL) was additionally added. The conjugation reaction was stirred for 30 min at 20° C. and the turbidity vanished during the conjugation reaction. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 366 μL, 3.66 μmol) followed by stirring the solution for 20 min. The quenched conjugation reaction was purified by Vivaspin centrifugation and the buffer was exchanged with the final PBS formulation buffer. The final target product ADC1 was concentrated to a final concentration of 8.56 mg/mL as determined by UV and 7.4 mL (63.3 mg, 0.43 μmol, 68.0%) ADC solution was obtained. SEC HPLC runs were performed to determine the purity of the product (61.4%).

(135) ADC1 was further purified by preparative gel filtration chromatography on an Äkta purifier system using a HiLoad 16/600 superdex 200 column due to the presence of high amounts of aggregates. After pooling, the final concentration (1.6 mg/mL) was determined by UV and the purity (90.9%) of the final drug products was determined by SEC HPLC to yield 8.65 mL (13.7 mg, 0.09 μmol, 14.7%) of the ADC solution (ADC1).

Example 10

Preparation of Antibody-Drug Conjugate ADC2 with Trastuzumab and Compound 5

(a) Partial Reduction of Trastuzumab to Give Partially Reduced Trastuzumab (Compound 20)

(136) A Trastuzumab solution (14.29 mL, 300 mg, 2.06 μmol) was diluted to a concentration of 5 mg/mL with 20 mM histidine/acetate buffer (pH, 5.5, 45.74 mL) followed by a pH adjustment with phosphate/EDTA buffer (pH 8.4, 14.4 mL). Partial reduction of the disulfide bonds in the antibody was performed by addition of a 5 mM tris[2-carboxyethyl]phosphine hydrochloride (TCEP) solution (1.07 mL, 5.36 μmol, 2.6 eq.). The reduction reaction was left to stir for 90 min at 20° C. Immediately after the reduction, an Ellman assay was performed to give a Free Thiol to Antibody ratio (FTAR) of 4.1, very close to the value of 4.0, as planned.

(b) Preparation of ADC2

(137) To the solution of partially reduced Trastuzumab Compound 20 (23.6 mL, 93.8 mg, 0.645 μmol), prepared as described in Example 8(a) above, DMSO was added (1.18 mL) followed by the addition of freshly prepared solution of Compound 5, prepared as described in Example 2, (10 mM in DMSO, 368 μL, 3.68 μmol, 5.7 eq.). The drug-linker was carefully added in 10 portions. After the sixth portion, the solution turned slightly turbid and the turbidity did not vanish during the conjugation reaction and quench. The conjugation reaction was stirred for 30 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 368 μL, 3.68 μmol) by stirring the solution for 46 min. The solution was filtered over a 0.2 μm syringe filter. The quenched conjugation reaction was concentrated to 16.5 mg/mL by Vivaspin centrifugation and was purified over NAP-25 columns. SEC HPLC runs were performed to determine the purity of the product (36.1%).

(138) ADC2 was further purified by preparative gel filtration chromatography on an Äkta purifier system using a HiLoad 16/600 superdex 200 column due to the presence of high amounts of aggregates. After pooling, the final concentration (3.7 mg/mL) was determined by UV and the purity (78.3%) of the final target ADC was determined by SEC HPLC to yield 5.3 mL (19.4 mg, 19.4%) of the ADC solution (ADC2).

Example 11

Preparation of Antibody-Drug Conjugate ADC3 with Trastuzumab and Compound 12

(a) Preparation of ADC3

(139) To the solution of partially reduced Trastuzumab Compound 20 (23.6 mL, 93.8 mg, 0.645 μmol), prepared as described in Example 10(a) above, DMSO was added (1.18 mL), followed by the addition of freshly prepared solution of Compound 12, prepared as described in Example 3, (10 mM in DMSO, 369 μL, 3.69 μmol, 5.7 eq.). The drug-linker was carefully added in 10 portions, nevertheless the solution started to turn turbid after the third portion. High turbidity was observed during addition of the last two portions. The solution did not clear until the filtration step. The conjugation reaction was stirred for 31 min at 20° C. The excess of drug was quenched by addition of N-acetylcysteine (NAC) (10 mM, 369 μL, 3.69 μmol) by stirring the reaction mixture for 50 min. The quenched conjugation reaction solution was filtered over a 0.2 μm syringe filter and concentrated to 14.2 mg/mL by Vivaspin centrifugation. Then it was purified over NAP-25 columns. SEC HPLC runs were performed to determine the purity of the product (34.2%).

(140) ADC3 was further purified by preparative gel filtration chromatography on an Äkta purifier system using a HiLoad 16/600 superdex 200 column due to the presence of high amounts of aggregates. After pooling, the final concentration (2.3 mg/mL) was determined by UV and the purity (78.6%) of the final drug products was determined by SEC HPLC to yield 7.3 mL (16.6 mg, 16.6%) of the ADC solution (ADC3).

Example 12

Preparation of Antibody-Drug Conjugates ADCs 4, 5 and 6 with Trastuzumab and Compounds 13, 15 and 18 Respectively

(a) SMCC Conjugation to Trastuzumab (Compound 21)

(141) The buffer of the Trastuzumab solution (262 mg, 1.8 μmol) was exchanged by phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 6.5) using NAP-25 columns. To the pooled Trastuzumab solution in glass reactors (16-17 g/L) DMSO was added (5%). The linker conjugation was started by adding SMCC (20.0 mM, 8.0 eq.) to the Trastuzumab solution. The reaction was stirred at 18° C. for 3 hours. The reaction mixture was then purified over NAP-25 columns to give Compound 21. A reversed Ellman assay was performed to determine a LAR of 3.7.

(b) Conjugation of Compounds 13, 15 and 18 to Trastuzumab-MCC: Preparation of ADC4, ADC5 and ADC6

(142) For the conjugation reaction, in a first step the antibody conjugate solution Compound 21, was diluted with phosphate buffer (50 mM phosphate, 2 mM EDTA, pH 6.5) to a concentration of 10 g/L. Then DMSO (5%) was added to the Compound 21 solution. The conjugation reactions with compounds 13, 15 and 18 were carried out by slowly adding the drug (10 mM, 6.3-6.6 eq.) to the Compound 21 solution and stirring for four hours at 18° C. After the conjugation reaction was complete, the reaction mixtures were 0.2 μm filtered and again purified over NAP-25 columns with a buffer exchange into 1×PBS buffer. SEC HPLC runs were performed to determine the purity of the product and the concentration of the final product was measured by UV.

(143) ADC from sample preparation with compound 15 was isolated with good purity (74.5%) and a yield of 56% (49 mg) was obtained and did not require further purification. The final concentration (5.7 mg/mL) of the ADC5 solution (87 mL) was determined by UV.

(144) However in the two sample preparations with compound 13 and 18, low molecular species were present. These species had a very similar retention time to the product peak and were not well separated on the SEC column. In order to remove possible remainders of drugs still present in the solution, the solutions were passed again over NAP-25 columns. The chromatograms after the first and second NAP-25 purification were identical, therefore species did not arise from free drugs still present in the solution and must be of larger origin. The samples were then further purified by gel filtration chromatography on a size exclusion column.

(145) After pooling, the final concentrations (2.8 and 3.9 mg/mL) were determined by UV and the purity (62.3% and 50.9%) of the final drug products was determined by SEC HPLC to yield 6.7 mL (19.3 mg, 22.0%) of the ADC4 solution and 6.8 mL (26.7 mg, 30.5%) of the ADC6 solution, respectively.

Example 13

Preparation of Antibody-Drug Conjugates ADCs 7 and 8 with Trastuzumab and Compounds 25 and 27

(a) General Procedures

(146) The antibody concentration was checked spectrophotometrically by monitoring its absorbance at 280 nm using a molar extinction coefficient of 2.18E5 M.sup.−1 cm.sup.−1 and a molecular weight of 150 kDa. Buffers used in these processes were either buffer A (50 mM sodium phosphate pH 6.5 with 2 mM EDTA) or buffer B (50 mM sodium phosphate pH 8.0) or phosphate saline buffer (“PBS”). Drug to antibody ratio (“DAR”) was deduced from the linker to antibody ratio (“LAR”) in the case of conjugation via Lys, or from the free Cys per mol of antibody ratio in the case of Cys-targeted conjugation, assuming that the conjugation reaction of the drug-linker to either the maleimide connector or to free Cys was quantitative. Both determinations were based on the colorimetric reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (“DTNB”) with free thiol groups to form a colored thionitrobenzoate adduct. For LAR determination, the adduct was preformed by mixing equal volumes of a 200 μM solution of DTNB in buffer B with a 200 μM solution of N-acetyl-cysteine in the same buffer. 75 μL of this mixture were then mixed with 75 μL of the test sample and after a 1 h incubation the absorbance at 412 nm was determined spectrophotometrically and the resulting value was compared to those obtained from a standard curve using known concentrations of 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (“SMCC”) to obtain the concentration of maleimides in the sample. This concentration is then referred to the antibody concentration to calculate the LAR. Likewise, free Cys were determined by mixing 50 μL of the test sample with 150 μL of 133 μM DTNB in buffer B, monitoring absorbance at 412 nm and comparing the resulting value with those obtained from a standard curve using known concentrations of Cys: the deduced concentration of free Cys in the test sample is then referred to the antibody concentration to calculate the ratio.

(b) Preparation of the Antibody-Drug Conjugates

(147) When the cytotoxic payload was conjugated to Cys residues (as with Compound 25 for the preparation of ADC7) the antibody was previously reduced with Tris(2-carboxyethyl)phosphine hydrochloride (“TCEP”). Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer B was mixed with the appropriate amount of a 5 mM solution of TCEP in water to keep the reducing agent in a 2.5-fold excess over the antibody. The mixture was incubated and stirred for 60 min at 20° C. and afterwards a small aliquot of the resulting reduced antibody was removed to calculate the free Cys to antibody ratio, while the remaining sample was mixed with the appropriate volume of a 10 mM solution of Compound 25 in DMSO to reach a 6-fold excess of the compound over the antibody: considering that the reduced antibody usually presents less than 6 free Cys per protein molecule, the molar ratio of the compound to the accessible free Cys is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v) and the mixture was incubated for 30 min at 20° C. Afterwards N-Acetyl-cysteine was added to quench the reaction, using the appropriate volume of a 10 mM solution in water to match the concentration of the drug-linker. The resulting conjugate was finally purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 2 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

(148) When the cytotoxic payload was conjugated to Lys residues (as with Compound 27 for the preparation of ADC8), the antibody was previously activated with SMCC. Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer A was mixed with the appropriate amount of a 20 mM solution of SMCC in DMSO to keep the activating reagent in a 8-fold excess over the antibody. DMSO was added if necessary to reach a final DMSO concentration of 5% (v/v). The mixture was incubated and stirred for 3 h at 18° C. and the excess of SMCC was then removed by gel filtration chromatography on Sephadex G-25 using PD-10 columns from GE Healthcare. A small aliquot of the resulting activated antibody was removed to calculate the LAR and the remaining sample was mixed with the appropriate volume of a 10 mM solution of Compound 27 in DMSO to reach a 8-fold excess of the compound over the antibody: considering that LAR value never exceeds 8, this ensures that the molar ratio of the compound to the accessible reacting sites is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v). The mixture was incubated for 4 h at 18° C. and the resulting conjugate was purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 2 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

Example 14

Preparation of Anti-CD4, Anti-CD5 and Anti-CD13 Monoclonal Antibodies

(149) Anti-CD4, anti-CD5 and anti-CD13 monoclonal antibodies were obtained following well known procedures commonly used in the art. Briefly BALB/c mice were immunized with HPB-ALL cells (for the ultimate production of anti-CD4 antibody) or with human T-cells activated with a mixture of phorbol 12-myristate 13-acetate and commercially available anti-CD3 monoclonal antibody as described by Cebrian et al. (1988, J. Exp. Med. 168:1621-1637) (for the ultimate production of anti-CD5 antibody) or with human endothelial cells isolated from umbilical cord (for the ultimate production of anti-CD13 antibody). To that end, 1.5E7 of the corresponding cells were injected to the mice intraperitoneally on days −45 and −30 and intravenously on day −3. On day 0 spleen from these animals were removed and spleen cells were fused with SP2 mouse myeloma cells at a ratio of 4:1 according to standard techniques to produce the corresponding hybridomas and distributed on 96-well tissue culture plates (Costar Corp., Cambridge, Mass.). After 2 weeks hybridoma culture supernatants were harvested and their reactivity against the cell line used in the immunization step was tested by flow cytometry. Positive supernatants were assayed by immunofluorescence staining the corresponding cells used as antigens. Hybridomas showing a specific staining, immunoprecipitation pattern and cell distribution were selected and cloned and subcloned by limiting dilution.

(150) Once the clones were selected, cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37° C. during 3-4 days until the medium turned pale yellow. At that point, two thirds of the medium volume were removed, centrifuged at 1,000×g for 10 min to pellet the cells and the supernatant was either centrifuged again for further cleaning at 3,000×g for 10 min or filtered through 22 μm pore size membranes. The clarified supernatant was subjected to precipitation with 55% saturation ammonium sulphate and the resulting pellet was resuspended in 100 mM Tris-HCl pH 7.8 (1 mL per 100 mL of the original clarified supernatant) and dialyzed at 4° C. for 16-24 h against 5 L of 100 mM Tris-HCl pH 7.8 with 150 mM NaCl, changing the dialyzing solution at least three times. The dialyzed material was finally loaded onto a Protein A-Sepharose column and the corresponding monoclonal antibody was eluted with 100 mM sodium citrate pH 3.0 or alternatively with 1M glycine pH 3.0. Those fractions containing the antibody were neutralized with 2M Tris-HCl pH 9.0 and finally dialyzed against PBS and stored at −80° C. until its use.

Example 15

Preparation of Antibody-Drug Conjugates ADCs 9, 10 and 11 with Anti-CD13 and Compounds 1, 12 and 13

(a) General Procedures

(151) In all the methods reported herein the antibody concentration was checked spectrophotometrically by monitoring its absorbance at 280 nm using a molar extinction coefficient of 2.25E5 M.sup.−1 cm.sup.−1 and a molecular weight of 150 kDa. Buffers used in these processes were either buffer A (50 mM sodium phosphate pH 6.5 with 2 mM EDTA) or buffer B (50 mM sodium phosphate pH 8.0) or phosphate saline buffer (“PBS”). Drug to antibody ratio (“DAR”) was deduced from the linker to antibody ratio (“LAR”) in the case of conjugation via Lys, or from the free Cys per mol of antibody ratio in the case of Cys-targeted conjugation, assuming that the conjugation reaction of the drug-linker to either the maleimide connector or to free Cys was quantitative. Both determinations were based on the colorimetric reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (“DTNB”) with free thiol groups to form a colored thionitrobenzoate adduct. For LAR determination, the adduct was preformed by mixing equal volumes of a 200 μM solution of DTNB in buffer B with a 200 μM solution of N-acetyl-cysteine in the same buffer. 75 μL of this mixture were then mixed with 75 μL of the test sample and after a 1 h incubation the absorbance at 412 nm was determined spectrophotometrically and the resulting value was compared to those obtained from a standard curve using known concentrations of 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (“SMCC”) to obtain the concentration of maleimides in the sample. This concentration is then referred to the antibody concentration to calculate the LAR. Likewise, free Cys were determined by mixing 50 μL of the test sample with 150 μL of 133 μM DTNB in buffer B, monitoring absorbance at 412 nm and comparing the resulting value with those obtained from a standard curve using known concentrations of Cys: the deduced concentration of free Cys in the test sample is then referred to the antibody concentration to calculate the ratio.

(b) Preparation of the Antibody-Drug Conjugates

(152) When the cytotoxic payload was conjugated to Cys residues (as with Compound 1 for the preparation of ADC9 or Compound 12 for the preparation of ADC10) the antibody was previously reduced with Tris(2-carboxyethyl)phosphine hydrochloride (“TCEP”). Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer B was mixed with the appropriate amount of a 5 mM solution of TCEP in water to keep the reducing agent in a 2.5-fold excess over the antibody. The mixture was incubated and stirred for 60 min at 20° C. and afterwards a small aliquot of the resulting reduced antibody was removed to calculate the free Cys to antibody ratio, while the remaining sample was mixed with the appropriate volume of a 10 mM solution of the drug linker (Compound 1 for ADC9 or Compound 12 for ADC10) in DMSO to reach a 6-fold excess of the compound over the antibody: considering that the reduced antibody usually presents less than 6 free Cys per protein molecule, the molar ratio of the compound to the accessible free Cys is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v) and the mixture was incubated for 30 min at 20° C. Afterwards N-Acetyl-cysteine was added to quench the reaction, using the appropriate volume of a 10 mM solution in water to match the concentration of the drug-linker. The resulting conjugate was finally purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 2 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

(153) When the cytotoxic payload was conjugated to Lys residues (as with Compound 13 for the preparation of ADC11), the antibody was previously activated with SMCC. Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer A was mixed with the appropriate amount of a 20 mM solution of SMCC in DMSO to keep the activating reagent in a 8-fold excess over the antibody. DMSO was added if necessary to reach a final DMSO concentration of 5% (v/v). The mixture was incubated and stirred for 3 h at 18° C. and the excess of SMCC was then removed by gel filtration chromatography on Sephadex G-25 using PD-10 columns from GE Healthcare. A small aliquot of the resulting activated antibody was removed to calculate the LAR and the remaining sample was mixed with the appropriate volume of a 10 mM solution of Compound 13 in DMSO to reach a 8-fold excess of the compound over the antibody: considering that LAR value never exceeds 8, this ensures that the molar ratio of the compound to the accessible reacting sites is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v). The mixture was incubated for 4 h at 18° C. and the resulting conjugate was purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 2 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

Example 16

Preparation of Antibody-Drug Conjugates ADCs 12 and 13 with Rituximab and Compounds 1 and 12

(a) General Procedures

(154) The antibody concentration was checked spectrophotometrically by monitoring its absorbance at 280 nm using a molar extinction coefficient of 2.45E5 M.sup.−1 cm.sup.−1 and a molecular weight of 150 kDa. Buffers used in these processes were either buffer B (50 mM sodium phosphate pH 8.0) or phosphate saline buffer (“PBS”). Drug to antibody ratio (“DAR”) was deduced from the free Cys per mol of antibody ratio, assuming that the conjugation reaction of the drug-linker to free Cys was quantitative. The determination was based on the colorimetric reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (“DTNB”) with free thiol groups to form a colored thionitrobenzoate adduct. 50 μL of the test sample were mixed with 150 μL of 133 μM DTNB in buffer B, absorbance at 412 nm was then measured and the resulting value compared with those obtained from a standard curve using known concentrations of Cys. The deduced concentration of free Cys in the test sample is then referred to the antibody concentration to calculate the ratio.

(b) Preparation of the Antibody-Drug Conjugates

(155) Prior to conjugation to the drug-linkers via Cys, the antibody was reduced with Tris(2-carboxyethyl)phosphine hydrochloride (“TCEP”). Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer B was mixed with the appropriate amount of a 5 mM solution of TCEP in water to keep the reducing agent in a 2.5-fold excess over the antibody. The mixture was incubated and stirred for 60 min at 20° C. and afterwards a small aliquot of the resulting reduced antibody was removed to calculate the free Cys to antibody ratio, while the remaining sample was mixed with the appropriate volume of a 10 mM solution of the drug linker (Compound 1 for ADC12 or Compound 12 for ADC13) in DMSO to reach a 6-fold excess of the compound over the antibody: considering that the reduced antibody usually presents less than 6 free Cys per protein molecule, the molar ratio of the compound to the accessible free Cys is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v) and the mixture was incubated for 30 min at 20° C. Afterwards N-Acetyl-cysteine was added to quench the reaction, using the appropriate volume of a 10 mM solution in water to match the concentration of the drug-linker. The resulting conjugate was finally purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 1 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

Example 17

Preparation of Antibody-Drug Conjugates ADCs 14 and 15 with Anti-CD5 and Compounds 1 and 12

(a) General Procedures

(156) The antibody concentration was checked spectrophotometrically by monitoring its absorbance at 280 nm using a molar extinction coefficient of 2.25E5 M.sup.−1 cm.sup.−1 and a molecular weight of 150 kDa. Buffers used in these processes were either buffer B (50 mM sodium phosphate pH 8.0) or phosphate saline buffer (“PBS”). Drug to antibody ratio (“DAR”) was deduced from the free Cys per mol of antibody ratio, assuming that the conjugation reaction of the drug-linker to free Cys was quantitative. The determination was based on the colorimetric reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (“DTNB”) with free thiol groups to form a colored thionitrobenzoate adduct. 50 μL of the test sample were mixed with 150 μL of 133 μM DTNB in buffer B, absorbance at 412 nm was then measured and the resulting value compared with those obtained from a standard curve using known concentrations of Cys. The deduced concentration of free Cys in the test sample is then referred to the antibody concentration to calculate the ratio.

(b) Preparation of the Antibody-Drug Conjugates

(157) Prior to conjugation to the drug-linkers via Cys, the antibody was reduced with Tris(2-carboxyethyl)phosphine hydrochloride (“TCEP”). Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer B was mixed with the appropriate amount of a 5 mM solution of TCEP in water to keep the reducing agent in a 2.5-fold excess over the antibody. The mixture was incubated and stirred for 60 min at 20° C. and afterwards a small aliquot of the resulting reduced antibody was removed to calculate the free Cys to antibody ratio, while the remaining sample was mixed with the appropriate volume of a 10 mM solution of the drug linker (Compound 1 for ADC14 or Compound 12 for ADC15) in DMSO to reach a 6-fold excess of the compound over the antibody: considering that the reduced antibody usually presents less than 6 free Cys per protein molecule, the molar ratio of the compound to the accessible free Cys is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v) and the mixture was incubated for 30 min at 20° C. Afterwards N-Acetyl-cysteine was added to quench the reaction, using the appropriate volume of a 10 mM solution in water to match the concentration of the drug-linker. The resulting conjugate was finally purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 1 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

Example 18

Preparation of Antibody-Drug Conjugates ADCs 16 and 17 with Anti-CD4 and Compounds 1 and 12

(a) General Procedures

(158) The antibody concentration was checked spectrophotometrically by monitoring its absorbance at 280 nm using a molar extinction coefficient of 2.25E5 M.sup.−1 cm.sup.−1 and a molecular weight of 150 kDa. Buffers used in these processes were either buffer B (50 mM sodium phosphate pH 8.0) or phosphate saline buffer (“PBS”). Drug to antibody ratio (“DAR”) was deduced from the free Cys per mol of antibody ratio, assuming that the conjugation reaction of the drug-linker to free Cys was quantitative. The determination was based on the colorimetric reaction of 5,5′-dithiobis(2-nitrobenzoic acid) (“DTNB”) with free thiol groups to form a colored thionitrobenzoate adduct. 50 μL of the test sample were mixed with 150 μL of 133 μM DTNB in buffer B, absorbance at 412 nm was then measured and the resulting value compared with those obtained from a standard curve using known concentrations of Cys. The deduced concentration of free Cys in the test sample is then referred to the antibody concentration to calculate the ratio.

(b) Preparation of the Antibody-Drug Conjugates

(159) Prior to conjugation to the drug-linkers via Cys, the antibody was reduced with Tris(2-carboxyethyl)phosphine hydrochloride (“TCEP”). Briefly, a 70 μM (10.5 mg/mL) solution of the antibody in buffer B was mixed with the appropriate amount of a 5 mM solution of TCEP in water to keep the reducing agent in a 2.5-fold excess over the antibody. The mixture was incubated and stirred for 60 min at 20° C. and afterwards a small aliquot of the resulting reduced antibody was removed to calculate the free Cys to antibody ratio, while the remaining sample was mixed with the appropriate volume of a 10 mM solution of the drug linker (Compound 1 for ADC16 or Compound 12 for ADC17) in DMSO to reach a 6-fold excess of the compound over the antibody: considering that the reduced antibody usually presents less than 6 free Cys per protein molecule, the molar ratio of the compound to the accessible free Cys is never below 1. DMSO was added if needed to keep its concentration at 5% (v/v) and the mixture was incubated for 30 min at 20° C. Afterwards N-Acetyl-cysteine was added to quench the reaction, using the appropriate volume of a 10 mM solution in water to match the concentration of the drug-linker. The resulting conjugate was finally purified from the rest of the reagents by gel filtration in Sephadex G-25 using PD-10 columns from GE Healthcare. The presence of aggregates was checked by analytical size exclusion chromatography using an Äkta FPLC system equipped with a Superdex-100 10/300 column running an isocratic method with PBS at 1 ml/min: if the area of the peak corresponding to aggregates exceeded 10% of the total peak area, monomers were purified using the same chromatography system with a Superdex 200 16/600 preparative column running the same method described above. Final ADC concentration was determined spectrophotometrically by monitoring its absorbance at 280 nm using the same molar extinction coefficient than that of the parental antibody: if the ADC concentration was below 1 mg/mL it was concentrated using Vivaspin devices from GE Healthcare and the new concentration was again determined as above.

Example 19

Synthesis of a Compound of Formula D-X-(AA).SUB.w.-L.SUB.1

Preparation of Compound 28

(160) ##STR00129##

(a) Preparation of Compound 28

(161) DIPEA (10 μL, 0.06 mmol) was added to a solution of Compound 9 (13 mg, 0.02 mmol), prepared as shown in the Preparative Example above, and Compound 8 (20 mg, 0.02 mmol), prepared as described in Example 2 above, in NMP (6.5 mL) at 23° C. After 9 h the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, DCM:CH.sub.3OH, from 100:0 to 90:10) to afford pure target Compound 28 (9 mg, 38%).

(162) .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.3OD): δ 9.40 (s, 1H), 8.92 (d, J=10.6 Hz, 1H), 7.67 (d, J=7.7 Hz, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.21 (d, J=7.9 Hz, 2H), 7.16 (t, J=11.9 Hz, 1H), 6.97 (t, J=9.6 Hz, 2H), 6.82 (t, J=11.4 Hz, 1H), 6.67-6.64 (m, 1H), 6.64 (s, 2H), 6.08 (d, J=11.7 Hz, 1H), 5.68 (d, J=11.4 Hz, 1H), 5.62-5.58 (m, 1H), 5.54-5.47 (m, 1H), 5.35-5.29 (m, 1H), 5.20 (d, J=9.9 Hz, 1H), 4.94 (s, 2H), 4.77 (q, J=8.1 Hz, 1H), 4.54-4.45 (m, 2H), 4.37 (d, J=9.2 Hz, 1H), 4.20-4.12 (m, 1H), 4.08 (t, J=7.8 Hz, 1H), 3.58 (s, 3H), 3.42 (t, J=7.2 Hz, 2H), 3.18-3.00 (m, 7H), 2.81-2.75 (m, 1H), 2.35-2.30 (m, 3H), 2.29-2.25 (m, 3H), 2.17 (t, J=7.2 Hz, 2H), 2.14-2.06 (m, 1H), 2.04-1.92 (m, 1H), 1.86-1.74 (m, 1H), 1.76 (s, 3H), 1.61-1.42 (m, 10H), 1.54 (d, J=6.3 Hz, 3H), 1.30-1.14 (m, 4H), 1.08 (d, J=6.4 Hz, 3H), 0.94 (s, 9H), 0.86 (dd, J=6.8, 4.3 Hz, 6H).

(163) .sup.13C NMR (100 MHz, CDCl.sub.3): δ 173.0, 172.1, 171.0, 170.2, 168.5, 167.1, 162.0, 161.3, 157.2, 157.0, 145.0, 140.2, 137.7, 137.5, 137.1, 134.0, 132.4, 128.8, 126.9, 124.9, 124.4, 124.3, 124.0, 120.5, 119.8, 108.6, 107.3. 81.9, 74.8, 66.1, 60.4, 58.8, 55.4, 37.6, 37.2, 36.0, 34.8, 31.9, 31.6, 30.9, 30.7, 30.0, 29.7, 29.3, 28.1, 26.5, 26.2, 26.1, 25.1, 19.2, 18.3, 17.1, 16.5, 12.9.

(164) ESI-MS m/z: Calcd. for C.sub.63H.sub.90N.sub.10O.sub.15: 1226.7. Found: 1267.4 (M+H).sup.+.

Example 20

Synthesis of a Compound of Formula D-X-(AA).SUB.w.-H

Preparation of Compound 36

(165) ##STR00130##

(a) Preparation of Compound 29

(166) To a solution of Compound 6 (1.01 g, 1.91 mmol) (Compound 30b, prepared as described in WO 2007144423, the contents of which are incorporated herein by reference) in DCM (40 mL) were added pyridine (0.31 mL, 3.82 mmol) and 4-nitrophenyl chloroformate (769.7 mg, 3.82 mmol) at 0° C. The reaction mixture was stirred at 23° C. for 1.5 h, diluted with citric acid 10% and extracted with DCM. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure Compound 29 (783 mg, 59%).

(167) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.26 (d, J=9.2 Hz, 2H), 8.02 (d, J=10.9 Hz, 1H), 7.43 (d, J=9.2 Hz, 2H), 7.22 (t, J=9.2 Hz, 1H), 6.92-6.76 (m, 2H), 6.21 (d, J=9.3 Hz, 1H), 6.17-6.12 (m, 1H), 5.73-5.64 (m, 1H), 5.65-5.56 (m, 2H), 5.46-5.38 (m, 1H), 5.27 (d, J=9.9 Hz, 1H), 4.86 (q, J=8.2 Hz, 1H), 4.76 (p, J=6.2 Hz, 1H), 4.40 (d, J=9.3 Hz, 1H), 4.21 (ddd, J=10.7, 7.6, 4.9 Hz, 1H), 3.66 (s, 3H), 2.89-2.79 (m, 1H), 2.59-2.29 (m, 6H), 1.83 (d, J=1.3 Hz, 3H), 1.61 (s, 3H), 1.16 (d, J=6.7 Hz, 3H), 1.00 (s, 9H).

(b) Preparation of Compound 33

(168) ##STR00131##

Preparation of Compound 30

(169) To a solution of 3-bromopropylamine hydrobromide (1.22 g, 2.98 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added 4-methoxytriphenylmethyl chloride (5.89 g, 19.1 mmol) and DIPEA (6.3, mL, 36.38 mmol). The reaction mixture was stirred at 23° C. overnight, diluted with H.sub.2O and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 30 (8.16 g, 100%) as a solid white.

(170) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.50-7.42 (m, 4H), 7.39-7.33 (m, 2H), 7.29-7.22 (m, 4H), 7.22-7.15 (m, 2H), 6.81 (d, J=8.9 Hz, 2H), 3.78 (s, 3H), 3.56 (t, J=6.8 Hz, 2H), 2.26 (t, J=6.7 Hz, 2H), 2.07-1.96 (m, 2H).

Preparation of Compound 31

(171) To a solution of Compound 30 (1.49 g, 3.63 mmol) in ethanol (36 mL) was added potassium ethylxanthogenate (1.46 g, 9.08 mmol). The reaction mixture was stirred at 23° C. overnight and the precipitated potassium bromide was then filtered from the solution. After the filtrate was evaporated under reduced pressure, and the solid residue was triturated with Hexane. The resulting solid was eliminated by filtration and the filtrate was evaporated and purified by flash chromatography (SiO.sub.2, Hex/EtOAc mixtures) to give Compound 31 (1.31 g, 80%).

(172) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.50-7.42 (m, 4H), 7.39-7.33 (m, 2H), 7.29-7.22 (m, 4H), 7.22-7.15 (m, 2H), 6.81 (d, J=8.9 Hz, 2H), 4.62 (q, J=7.1 Hz, 2H), 3.78 (s, 3H), 3.32-3.20 (dd, J=7.7, 6.9 Hz, 2H), 2.23 (t, J=6.7 Hz, 2H), 1.91-1.80 (m, 2H), 1.41 (t, J=7.1 Hz, 3H).

Preparation of Compound 32

(173) To a solution of Compound 31 (3 g, 6.64 mmol) in DCM (10 mL) was added 1-propylamine (4.4 mL, 66.4 mmol). The reaction mixture was stirred at 23° C. for 10 min and concentrated under vacuum. The residue obtained was used in the next step without further purification. It was dissolved in dry methanol (50 mL) and cooled to 0° C. Methyl methanethiosulfonate (9.7 mL, 7.97 mmol) was added and the solution was stirred for 16 h at 23° C. The solvent was removed in vacuum, the residual oil was dissolved in dichloromethane, washed with pH 7 buffer and brine, dried and evaporated. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield Compound 32 (1, 6 g, 59%).

(174) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.52-7.44 (m, 4H), 7.38 (d, J=8.9 Hz, 2H), 7.32-7.22 (m, 4H), 7.19 (d, J=7.3 Hz, 2H), 6.82 (d, J=8.9 Hz, 2H), 3.79 (s, 3H), 2.90-2.72 (m, 2H), 2.39 (s, 3H), 2.24 (t, J=6.7 Hz, 2H), 1.88 (p, J=6.9 Hz, 2H).

Preparation of Compound 33

(175) To a solution of Compound 32 (1.22 g, 2.98 mmol) in CH.sub.2Cl.sub.2 (30 mL) was added dichloroacetic acid (0.9 mL, 10.9 mmol). The reaction mixture was stirred at 23° C. for 20 min and diluted with water. The organic layer was extracted and the aqueous phase was basificated with KOH 10%. Then it was extracted thoroughly with dichloromethane (3×), and the combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated to give Compound 33 (409 mg, 100%).

(176) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 2.83-2.75 (m, 2H), 2.41 (s, 3H), 1.88-1.81 (m, 2H).

(177) To a solution of Compound 29 (230.2 mg, 0.33 mmol), prepared as described in step (a) above, in CH.sub.2Cl.sub.2 (10 mL) was added Compound 33 (50 mg, 0.36 mmol) and DIPEA (0.06 mL, 0.36 mmol). The reaction mixture was stirred at 23° C. for 3 h, diluted with H.sub.2O and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 34 (150 mg, 66%).

(178) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.68 (d, J=10.7 Hz, 1H), 7.31 (t, J=6.0 Hz, 1H), 6.90 (dd, J=12.7, 10.2 Hz, 1H), 6.81 (dd, J=10.9, 8.5 Hz, 1H), 6.38 (d, J=9.4 Hz, 1H), 6.16 (d, J=11.7 Hz, 1H), 5.69 (d, J=11.5 Hz, 1H), 5.65-5.52 (m, 3H), 5.41-5.37 (m, 1H), 5.29-5.56 (d, J=8.9 Hz, 1H), 4.83 (q, J=8.3 Hz, 1H), 4.54-4.50 (m, 1H), 4.46 (d, J=9.4 Hz, 1H), 4.24-4.19 (m, 1H), 3.65 (s, 3H), 3.36-3.16 (m, 2H), 2.86-2.82 (m, 1H), 2.70 (t, J=7.2 Hz, 2H), 2.44-2.35 (m, 5H), 2.40 (s, 3H), 2.19-2.04 (m, 1H), 1.98-1.83 (m, 2H), 1.84 (s, 3H), 1.63 (d, J=6.6 Hz, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.05 (s, 9H).

(179) ESI-MS m/z: Calcd. for C.sub.35H.sub.53N.sub.3O.sub.7S.sub.2: 691.33. Found: 692.4 (M+H).sup.+.

(d) Preparation of Compound 35

(180) To a solution of Compound 29 (121.3 mg, 0.17 mmol), prepared as described in step (a) above, in DCM (2.5 mL) were added a suspension of 3-aminopropane-1-thiol hydrochloride (37.2 mg, 0.29 mmol) in DCM (2.5 mL), DIPEA (59 μL, 0.34 mmol) and DMF (0.1 mL) at 23° C. The reaction mixture that was stirred at 23° C. for 7 h, diluted with H.sub.2O and extracted with EtOAc. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 35 (40.5 mg, 37%).

(181) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.63 (d, J=10.6 Hz, 1H), 7.34-7.21 (m, 1H), 6.88 (t, J=11.4 Hz, 1H), 6.76 (t, J=9.6 Hz, 1H), 6.68 (d, J=9.3 Hz, 1H), 6.13 (d, J=11.6 Hz, 1H), 5.7-5.67 (m, 3H), 5.66-5.48 (m, 3H), 4.81 (q, J=8.1 Hz, 1H), 4.66-4.50 (m, 1H), 4.46 (d, J=9.2 Hz, 1H), 4.25-4.18 (m, 1H), 3.64 (s, 3H), 3.27-3.34 (m, 1H), 2.88-2.77 (m, 1H), 2.66 (t, J=7.2 Hz, 2H), 2.42-2.30 (m, 5H), 2.22-2.06 (m, 2H), 1.89-1.74 (m, 5H), 1.62 (d, J=6.6 Hz, 3H), 1.15 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(182) ESI-MS m/z: Calcd. for C.sub.68H.sub.100N.sub.6O.sub.14S.sub.2: 1288.67. Found: 1289.4 (M+H).sup.+.

(e) Preparation of Compound 36

(183) A solution of Compound 35 (40.5 mg, 0.03 mmol) in a mixture of EtOAc (1.5 mL) and CH.sub.3OH (1.5 mL) was treated with a solution dithiothreitol (0.36 mL, 0.36 mmol) in 0.05 M potassium phosphate buffer (1.2 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer at pH 6.0 containing 2 mM EDTA and the extracted with EtOAc (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for HPLC (SiO.sub.2, Hex:EtOAc mixtures) to yield Compound 36 (15 mg, 38%).

(184) .sup.1H NMR (300 MHz, CDCl.sub.3): δ 8.70 (d, J=10.7 Hz, 1H), 7.35-7.21 (m, 1H), 6.89 (t, J=11.4 Hz, 1H), 6.80 (t, J=9.8 Hz, 1H), 6.42 (d, J=9.4 Hz, 1H), 6.16 (d, J=11.3 Hz, 1H), 5.75-5.48 (m, 3H), 5.52-5.13 (m, 3H), 4.83 (q, J=8.3 Hz, 1H), 4.65-4.38 (m, 2H), 4.32-4.16 (m, 1H), 3.65 (s, 3H), 3.29 (q, J=6.6 Hz, 2H), 2.85 (dt, J=9.8, 6.9 Hz, 1H), 2.54 (q, J=7.3 Hz, 2H), 2.48-2.26 (m, 5H), 2.19-2.01 (m, 1H), 1.89-1.74 (m, 5H), 1.63 (dd, J=6.8, 1.7 Hz, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.05 (s, 9H).

(185) ESI-MS m/z: Calcd. for C.sub.34H.sub.51N.sub.3O.sub.7S: 645.85. Found: 668.4 (M+Na).sup.+.

Example 21

Alternative Synthesis of Compound 14

(186) ##STR00132##

(a) Preparation of Compound 37

(187) To a solution of N-Fmoc-1,3-propanediamine hydrobromide (377 mg, 1 mmol) in CH.sub.2Cl.sub.2 (15 mL) was added DIPEA (0.52 mL, 3 mmol) and 6-Maleimidohexanoic acid N-hydroxysuccinimide ester (323.7 mg, 1.1 mmol). The reaction mixture was stirred at 23° C. overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 37 (430 mg, 100%) as a white solid.

(188) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.76 (dd, J=7.6, 1.0 Hz, 2H), 7.64-7.55 (m, 2H), 7.45-7.37 (m, 2H), 7.31 (td, J=7.5, 1.2 Hz, 2H), 6.16 (bs, 1H), 5.24 (bs, 1H), 4.42 (d, J=6.9 Hz, 2H), 4.21 (t, J=6.8 Hz, 1H), 3.27 (dq, J=18.3, 6.3 Hz, 4H), 2.99 (t, J=7.0 Hz, 2H), 2.62 (t, J=7.0 Hz, 2H), 2.41 (s, 3H), 1.71-1.59 (m, 2H).

(b) Preparation of Compound 38

(189) To a solution of 37 (430 mg, 1 mmol), prepared as described in step (a) above, in CH.sub.2Cl.sub.2 (8 mL) was added diethylamine (1.4 mL, 13.5 mmol). The reaction mixture was stirred at 23° C. for 6 h and concentrated under vacuum. The residue obtained was triturated with Et.sub.2O and filtrated to obtain Compound 38 (148 mg, 71%) as a white solid.

(190) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 3.29 (td, J=6.5, 2.3 Hz, 2H), 3.01-2.86 (m, 2H), 2.78 (t, J=6.6 Hz, 2H), 2.57 (t, J=7.1 Hz, 2H), 2.37 (s, 3H), 1.69 (p, J=6.6 Hz, 2H).

(191) To a solution of Compound 16 (60 mg, 0.08 mmol), prepared as described in Example 5(a) above, in CH.sub.2Cl.sub.2 (2 mL) was added a solution of Compound 38 (58 mg, 0.28 mmol), prepared as described in step (b) above, and DIPEA (0.1 mL, 0.56 mmol) in CH.sub.2Cl.sub.2 (2 mL). The reaction mixture was stirred at 23° C. for 3 h, diluted with H.sub.2O and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 14 (60.5 mg, 95%).

(192) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.88 (d, J=10.8 Hz, 1H), 7.29-7.24 (m, 1H), 6.90 (t, J=11.5 Hz, 1H), 6.82 (t J=9.1 Hz, 1H), 6.63 (t, J=6.1 Hz, 1H), 6.49 (d, J=9.4 Hz, 1H), 6.16 (dd, J=11.5, 1.5 Hz, 1H), 5.70 (d, J=11.5 Hz, 1H), 5.68-5.51 (m, 3H), 5.29 (d, J=9.7 Hz, 1H), 4.81 (q, J=8.2 Hz, 1H), 4.52 (d, J=9.5 Hz, 1H), 4.52-4.43 (m, 1H), 4.24 (ddd, J=11.5, 7.3, 4.3 Hz, 1H), 3.66 (s, 3H), 3.37-3.21 (m, 3H), 3.21-3.12 (m, 1H), 2.97 (t, J=7.2 Hz, 2H), 2.90-2.81 (m, 1H), 2.60 (t, J=7.2 Hz, 2H), 2.49-2.35 (m, 3H), 2.39 (s, 3H), 2.33 (t, J=7.0 Hz, 2H), 2.14-2.07 (m, 1H), 2.07 (s, 3H), 1.84 (s, 3H), 1.73-1.64 (m, 2H), 1.16 (d, J=6.7 Hz, 3H), 1.05 (s, 9H).

(193) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 171.6, 168.2, 166.4, 161.6, 157.2, 145.2, 140.3, 137.4, 134.2, 134.0, 131.9, 124.4, 124.1, 122.4, 120.7, 108.3, 105.6, 81.8, 74.8, 60.6, 60.4, 55.5, 37.8, 37.2, 36.2, 35.6, 34.7, 33.1, 31.0, 29.8, 26.7, 26.2, 23.0, 21.0, 17.2, 16.6.

Example 22

Alternative Synthesis of Compound 15

(194) ##STR00133##

(a) Preparation of Compound 39

(195) To a solution of Compound 16 (178.7 mg, 0.25 mmol), prepared as described in Example 5(a) above, in CH.sub.2Cl.sub.2 (2.5 mL) was added Compound 33 (120 mg, 0.88 mmol), prepared as described in Example 20(b) above, and DIPEA (0.05 mL, 0.25 mmol). The reaction mixture was stirred at 23° C. for 2 h, diluted with H.sub.2O and extracted with CH.sub.2Cl.sub.2. The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to afford pure Compound 39 (100 mg, 56%).

(196) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 8.65 (d, J=10.7 Hz, 1H), 7.28 (t, J=11.6 Hz, 1H), 6.90 (t, J=11.5 Hz, 1H), 6.82 (t, J=9.6 Hz, 1H), 6.37 (d, J=9.4 Hz, 1H), 6.17 (d, J=11.7 Hz, 1H), 5.69 (d, J=11.4 Hz, 1H), 5.64-5.57 (m, 2H), 5.34 (t, J=6.2 Hz, 1H), 5.29 (d, J=10.0 Hz, 1H), 4.81 (q, J=8.3 Hz, 1H), 4.54-4.49 (m, 1H), 4.45 (d, J=9.4 Hz, 1H), 4.28-4.17 (m, 1H), 3.66 (s, 3H), 3.37-3.21 (m, 2H), 2.85 (dt, J=9.7, 6.9 Hz, 1H), 2.71 (t, J=7.2 Hz, 2H), 2.44-2.30 (m, 5H), 2.38 (s, 3H) 2.13-2.06 (m, 1H), 2.07 (s, 3H), 1.93 (p, J=6.9 Hz, 2H), 1.84 (s, 3H), 1.16 (d, J=6.6 Hz, 3H), 1.06 (s, 9H).

(197) ESI-MS m/z: Calcd. for C.sub.35H.sub.52ClN.sub.3O.sub.7S.sub.2: 725.9. Found: 748.3 (M+Na).sup.+.

(b) Preparation of Compound 15

(198) A solution of Compound 39 (90 mg, 0.12 mmol), prepared as described in Example 5(a) above, in a mixture of EtOAc (6.7 mL) and CH.sub.3OH (6.7 mL) was treated with a dithiothreitol solution (0.36 mL, 0.36 mmol) in 0.05 M potassium phosphate buffer (6.7 mL) at pH 7.5 containing 2 mM ethylenediaminetetraacetic acid (EDTA). The mixture was stirred at 23° C. for 4 h. The reaction was treated with a solution of 0.2 M potassium phosphate buffer at pH 6.0 containing 2 mM EDTA and the extracted with EtOAc (×3). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (SiO.sub.2, Hex:EtOAc mixtures) to yield pure target Compound 15 (40.2 mg, 46%).

(199) .sup.1H NMR (500 MHz, CDCl.sub.3): δ 8.66 (d, J=10.7 Hz, 1H), 7.29 (t, J=11.2 Hz, 1H), 6.91 (t, J=11.5 Hz, 1H), 6.83 (t, J=9.7 Hz, 1H), 6.38 (d, J=9.4 Hz, 1H), 6.17 (d, J=11.8 Hz, 1H), 5.70 (d, J=11.4 Hz, 1H), 5.65-5.51 (m, 2H), 5.34 (t, J=6.3 Hz, 1H), 5.29 (d, J=10.0 Hz, 1H), 4.82 (q, J=8.3 Hz, 1H), 4.56-4.48 (m, 1H), 4.45 (d, J=9.3 Hz, 1H), 4.22 (ddd, J=11.4, 7.5, 4.3 Hz, 1H), 3.67 (s, 3H), 3.31 (q, J=6.4 Hz, 2H), 2.88-2.83 (m, 1H), 2.55 (q, J=7.7 Hz, 2H), 2.47-2.30 (m, 5H), 2.12-2.07 (m, 1H), 2.08 (s, 3H), 1.88-1.76 (m, 5H), 1.17 (d, J=6.6 Hz, 3H), 1.06 (s, 9H).

(200) .sup.13C NMR (125 MHz, CDCl.sub.3): δ 168.2, 166.2, 161.5, 156.7, 145.2, 140.2, 137.3, 134.2, 134.0, 132.0, 124.4, 124.2, 122.3, 120.8, 108.1, 105.5, 81.8, 74.5, 60.6, 55.4, 39.6, 37.3, 34.6, 33.9, 33.3, 30.8, 26.7, 26.3, 21.8, 21.1, 17.2, 16.7.

(201) ESI-MS m/z: Calcd. for C.sub.34H.sub.50ClN.sub.3O.sub.7S: 679.3. Found: 702.4 (M+Na).sup.+.

Examples Demonstrating the Cytotoxicity of the Antibody-Drug Conjugates of the Present Invention

(202) Bioassays for the Detection of Antitumor Activity

(203) The aim of the assay was to evaluate the in vitro cytostatic (ability to delay or arrest tumor cell growth) or cytotoxic (ability to kill tumor cells) activity of the samples being tested.

(204) Cell Lines and Cell Culture

(205) All tumor cell lines used in this study were obtained from the American Type Culture Collection (ATCC), unless otherwise indicated; BT-474 (ATCC HTB-20, Breast Ductal Carcinoma), SK-BR-3 (ATCC HTB-30, Breast Adenocarcinoma) and HCC-1954 (ATCC CRL-2338, Breast Ductal Carcinoma), all HER2+; MDA-MB-231 (ATCC HTB-26, Breast Adenocarcinoma) and MCF-7 (ATCC HTB-22 Breast Adenocarcinoma, pleural effusion), all HER2−; SK-OV-3 (ATCC HTB-77, Ovary Adenocarcinoma), HER2+; NB-4 (Acute Promyelocytic Leukemia, APL, CD13+, M. Lanotte et al. (1991) NB4, a maturation inducible cell line with t(15; 17) marker isolated from a human acute promyelocytic leukemia (M3). Blood 77, 1080-1086), (CD13+) and U937 (ATCC CRL-1593.2, Histiocytic Lymphoma, CD13+ and CD4+); Raji (ATCC CCL-86, Burkitt's Lymphoma) (CD13−, CD20+, CD5−, and CD4−); RPMI-8226 (ATCC CRM-CCL-155, Multiple Myeloma) (CD13−, CD20−, CD5− and CD4−); Karpas-299 (DSMZ ACC-31, Non-Hodgkin's Lymphoma) (CD20−, CD5+, and CD4+); MOLT-4 (ATCC CRL-1582, Acute Lymphoblastic Leukemia, CD5+). In addition, the two Raji cell (ATCC CCL-86, Burkitt's Lymphoma) clones used in this study Raji-clone #10 (high CD5 expression) and Raji-clone18 (null CD5 expression), were provided by Dr. Juan M. Zapata (Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC-UAM, Madrid, Spain). Cells were maintained at 37° C., 5% CO.sub.2 and 95% humidity in Dulbecco's Modified Eagle's Medium (DMEM) (for MCF and MDA-MB-231 cells), RPMI-1640 (for SK-BR-3, HCC-1954, NB-4, U937, Raji, RPMI-8226, Karpas-299, MOLT-4, Raji-clone #10 and Raji-clone #18 cells), RPMI-1640+1% ITS (for BT-474 cells) or McCOyS (for SK-OV-3 cells), all media supplemented with 10% Fetal Calf Serum (FCS) and 100 units/mL penicillin and streptomycin.

(206) Cytotoxicity Assay

(207) For adherent cells: A colorimetric assay using sulforhodamine B (SRB) was adapted for quantitative measurement of cell growth and cytotoxicity, as described in V. Vichai and K. Kirtikara (2006) Sulforhodamine B colorimetric assay for cytotoxicity screening. Nature Protocols, 1, 1112-1116. Briefly, cells were seeded in 96-well microtiter plates and allowed to stand for 24 hours in drug-free medium before treatment with vehicle alone or the indicated compounds for 72 hours. For quantification, cells were washed twice with phosphate buffered saline (PBS), fixed for 15 min in 1% glutaraldehyde solution, rinsed twice with PBS, stained in 0.4% SRB-1% acetic acid solution for 30 min, rinsed several times with 1% acetic acid solution and air-dried. SRB was then extracted in 10 mM trizma base solution and the optical density measured at 490 nm in a microplate spectrophotometer. Cell survival was expressed as percentage of control, untreated cell survival.

(208) For suspension cells: A standard metabolic assay using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was adapted for quantitative measurement of cell growth and cytotoxicity, as described in T. Mosmann (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Meth., 65, 55-63. Briefly, MTT solution was added to the cell cultures at a final concentration of 0.5 mg/mL, and incubated for 1 to 4 hours at 37° C. until formazan crystals are formed. Culture medium is carefully removed from the cell cultures and formazan crystals resuspended in 100 μL DMSO. After mixing to assure solubilization, the quantity of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer. Cell survival was expressed as percentage of control, untreated cell survival.

(209) The IC.sub.50 value refers to the concentration of compound inducing a 50% of cell death as compared to the control cell survival.

Bioactivity Example 1—Cytotoxicity of ADC1 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(210) The in vitro cytotoxicity of ADC1 along with the parent cytotoxic Compounds 1 and 4 and Trastuzumab was evaluated against different human breast cancer cell lines over-expressing or not the HER2 receptor, including BT-474, HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER negative cells). SK-OV-3, a HER2+ ovarian cancer cell line, was also included in the study as a non-breast tissue cell model. Standard dose-response (DR) curves for 72 hours were performed.

(211) Cytotoxicity of Trastuzumab

(212) First of all, the in vitro cytotoxicity of Trastuzumab was assayed against the different tumor cell lines. In triplicate DR curves ranging from 5.0E01 to 2.6E−03 μg/mL (3.4E−07-1.8E−11 M), in two independent experiments, Trastuzumab was completely inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their HER2 status (see Table 3).

(213) TABLE-US-00003 TABLE 3 Summary of the in vitro cytotoxicity of Trastuzumab Trastuzumab Breast cells Ovary cells HER2+ HER2− HER2+ BT-474 HCC1954 SK-BR3 MCF7 MDA-MB-231 SK-OV-3 IC50 (ug/mL) >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 IC50 (Molar) >3.44E−07 >3.44E−07 >3.44E−07 >3.44E−07 >3.44E−07 >3.44E−07
Cytotoxicity of Compound 4

(214) The cytotoxicity of the intermediate Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 1E−01 to 2.6E−05 μg/mL (1.5E−07 to 3.9E−11 M).

(215) The cytotoxicity of this compound, in two independent experiments, was relatively homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 8.9E−05 to 1.7E−03 μg/mL (1.34E−10 to 2.6E−09 M), with the mean IC.sub.50 value across the whole cell panel being 5.69E−04 μg/mL (8.57E−10 M). In addition, the cytotoxicity of Compound 4 was independent of the HER2 status of the tumor cell lines (see Table 4).

(216) TABLE-US-00004 TABLE 4 Summary of the in vitro cytotoxicity of Compound 4 Compound 4 Breast cells Ovary cells HER2+ HER2− HER2+ BT-474 HCC1954 SK-BR3 MCF7 MDA-MB-231 SK-OV-3 IC50 (ug/mL) 1.74E−03 1.30E−04 8.90E−05 4.15E−04 3.10E−04 7.30E−04 IC50 (Molar) 2.62E−09 1.96E−10 1.34E−10 6.26E−10 4.68E−10 1.10E−09
Cytotoxicity of Compound 1

(217) The activity of parent Compound 1 was assayed using the same conditions as above, from 1E−01 to 2.6E−05 μg/mL (1.1E−07 to 3.0E−11 M). The cytotoxicity of this compound, in two independent experiments, was also relatively homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 8.9E−04 to 6.4E−03 μg/mL (1.04E−09 to 7.47E−09 M), with the mean IC.sub.50 value across the whole cell panel being 3.41E−03 μg/mL (3.98E−09 M). The maleimide linker seemed to slightly decrease the cytotoxic effect of the compound. Again, the cytotoxicity of Compound 1 was independent of the HER2 status of the tumor cell lines (see Table 5).

(218) TABLE-US-00005 TABLE 5 Summary of the in vitro cytotoxicity of Compound 1 Compound 1 Breast cells Ovary cells HER2+ HER2− HER2+ BT-474 HCC1954 SK-BR3 MCF7 MDA-MB-231 SK-OV-3 IC50 (ug/mL) 6.40E−03 9.60E−04 8.90E−04 3.70E−03 2.80E−03 5.70E−03 IC50 (Molar) 7.47E−09 1.12E−09 1.04E−09 4.32E−09 3.27E−09 6.66E−09
Cytotoxicity of ADC1

(219) The cytotoxicity of ADC1 was assayed against the different cell lines. To ensure the appropriate range of concentrations, the conjugate was assayed in six different, triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1, 0.1, 0.01 and 0.001 μg/mL (equivalent to 3.3E−07, 6.6E−08, 6.6E−09, 6.6E−10, 6.6E−11 and 6.6E−12 molar concentration), in two independent experiments.

(220) A representative DR curve is shown in FIG. 3.

(221) After adjusting the different DR curves, the mean IC.sub.50 value calculated for the ADC1 against the different cell lines is shown in Table 6 below. ADC1 showed a cytotoxicity relatively similar to that of the parent compound Compound 1 alone and, importantly, a clear specificity against HER2+ expressing cells. We assume, therefore, that the conjugate was actually acting through the interaction of the mAb with the membrane associated HER2 receptor on the tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue. Among the HER2 positive cell lines, there were significant differences in sensitivity against ADC1. The most sensitive cell lines were HCC-1954 and SK-BR-3, showing IC.sub.50s of 3.88E−02 and 2.45E−02 μg/mL (equivalent to 2.581E−10 and 1.63E−10 M), followed by BT-474 cells, which showed a significantly higher IC.sub.50 value of 7.4E−01 μg/mL (equivalent to 4.93E−09 M). The ovarian cell line SK-OV-3 showed an even higher IC.sub.50 value of 7.0E+00 μg/mL (equivalent to 4.67E−08 M). The two HER negative cells showed a similar sensitivity in the order of 2.0E+01 μg/mL (equivalent to around 1.0E−07 M) (see Table 6).

(222) TABLE-US-00006 TABLE 6 Summary of the in vitro cytotoxicity of ADC1 Compound ADC1 Breast cells Ovary cells Cell line HER2+ HER2− HER2+ HER2 status BT-474 HCC1954 SK-BR3 MCF7 MDA-MB-231 SK-OV-3 IC50 (ug/mL) 7.40E−01 3.88E−02 2.45E−02 1.20E+01 2.00E+01 7.00E+00 Mean IC50 (ug/mL) HER2 (breast) positive cells 2.68E−01 Mean IC50 (ug/mL) HER2 (breast) negative cells 1.60E+01 IC50 (M) 4.93E−09 2.58E−10 1.63E−10 7.97E−08 1.33E−07 4.67E−08 Mean IC50 (M) HER2 (breast) positive cells 1.79E−09 Mean IC50 (M) HER2 (breast) negative cells 1.07E−07

(223) Thus, the most responsive HER2 positive cell lines were around 300-800 times more sensitive that the HER2 negative cell lines, indicating the specificity of the conjugate against the HER2 expressing cells.

(224) To graphically compare the cytotoxicity of the mAb Trastuzumab alone with that of the conjugate ADC1, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC at 10 or 1 μg/mL, are shown in FIG. 4. As can be seen from FIG. 4, at an equal concentration of 10 μg/mL, the mAb Trastuzumab alone showed little or no cytotoxicity (<20% max) against any of the cell lines tested, independently of their HER2 status. In contrast, ADC1 showed a potent cytotoxicity against the HER2 expressing cells, HCC-1954 and SK-BR-3 and, to a lesser extent, BT-474 and SK-OV-3, inducing a mean inhibition of the cell survival of 88%, 82%, 52% and 47% respectively, as compared to the control cells. At this concentration, ADC1 displayed some cytotoxicity against the HER negative cells MCF-7 and MDA-MB-231, with mean percentages of cell survival inhibition of 38% and 32%, respectively. At a concentration of 1 μg/mL, ADC1 conjugate showed a somewhat similar cytotoxicity against the HER2 positive cells to that observed at 10 μg/mL, but in this case without detectable effects on HER2 negative cells (FIG. 4).

(225) These results clearly demonstrated the remarkable cytotoxicity and specificity of ADC1 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 2—Cytotoxicity of ADC2 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(226) The in vitro cytotoxicity of ADC2, a Trastuzumab-Compound 5 ADC, along with the parent cytotoxic Compounds 5 and 8 and the mAb Trastuzumab was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(227) Cytotoxicity of Compound 8

(228) The cytotoxicity of the intermediate Compound 8 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−04 μg/mL (1.6E−06 to 4.0E−10 M).

(229) The cytotoxicity of this compound, in two independent experiments, was relatively homogenous along the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 3.40E−03 to 6.75E−03 μg/mL (5.4E−09 to 1.0E−08 M), with the mean IC.sub.50 value across the whole cell panel being 5.53E−03 μg/mL (equivalent to 8.79E−09 M). In addition, the cytotoxicity of Compound 8 was independent of the HER2 status of the tumor cell lines (Table 7).

(230) TABLE-US-00007 TABLE 7 Summary data of the in vitro cytotoxicity of Compound 8 Compound 8 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 5.40E−03 3.40E−03 6.75E−03 6.55E−03 IC50 (Molar) 8.59E−09 5.41E−09 1.07E−08 1.04E−08
Cytotoxicity of Compound 5

(231) The activity of Compound 5, the modified Compound 8 carrying the maleimide linker, was assayed in the same conditions as above, from 01E+00 to 2.6E−04 μg/mL (1.2E−06 to 3.1E−10 M).

(232) The cytotoxicity of this compound, in two independent experiments, was also relatively homogenous along the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 1.9E−02 to 7.7E−02 μg/mL (2.32E−08 to 9.41E−08 M), being the mean IC.sub.50 value across the whole cell panel 4.33E−02 μg/mL (5.26E−08 M). The presence of the maleimide linker in Compound 5 slightly decreased the cytotoxicity of the compound as compared to Compound 8. Also, the cytotoxicity of Compound 5 seemed to be independent of the HER2 status of the tumor cell lines (Table 8).

(233) TABLE-US-00008 TABLE 8 Summary data of the in vitro cytotoxicity of Compound 5 Compound 5 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 1.95E−02 1.90E−02 7.75E−02 5.70E−02 IC50 (Molar) 2.38E−08 2.32E−08 9.41E−08 6.94E−08
Cytotoxicity of ADC2

(234) The cytotoxic activity of the ADC2 was assayed against the different cell lines. Just to assure the appropriate range of concentrations, the conjugate was assayed in five different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1, 0.1, and 0.01 μg/mL, in two independent experiments. A representative DR curve is shown in FIG. 5. After adjusting all the different DR curves, the mean IC.sub.50 values calculated for the ADC2 against the different cell lines are shown in Table 9. The conjugate ADC2 showed specificity against the HER2+ expressing cells, HCC-1954 and SK-BR-3, in which the compound demonstrated a cytotoxicity similar to that of the parent Compound 5, with mean IC.sub.50 values of 5.8E+00 and 2.2E−01 μg/mL (equivalent to 4.0E−08 and 1.5E−09 M), respectively. The two HER-cell lines, MCF-7 and MDA-MB-231, were virtually unresponsive to ADC2 in the range of concentrations tested, not reaching an IC.sub.50 value (>5.0E+01 μg/mL) (see FIG. 5 and Table 9).

(235) We assume, therefore, that the conjugate was actually acting through the interaction of the mAb with the membrane associated HER2 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(236) TABLE-US-00009 TABLE 9 Summary data of the in vitro cytotoxicity of ADC2 (Trastuzumab-Compound 5 ADC) ADC2 (Trastuzumab-Compound 5 ADC) Breast cells Cell line HER2+ HER2− HER2 status HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 5.80E+00 2.20E−01 >5.0E+01 >5.0E+01 Mean IC50 (ug/mL) HER2 positive cells 3.01E+00 Mean IC50 (ug/mL) HER2 negative cells >5.0E+01 IC50 (M) 4.00E−08 1.52E−09 >3.4E−07 >3.4E−07 Mean IC50 (M) HER2 positive cells 2.08E−08 Mean IC50 (M) HER2 negative cells >3.4E−07

(237) To graphically compare the cytotoxicity of the Trastuzumab alone with that of the conjugate ADC2, histograms showing the percentages of cell survival after treatment of the different cell lines with Trastuzumab alone (10 μg/mL) or the ADC at 10 or 1 μg/mL, are shown in FIG. 6. At a concentration of 10 μg/mL, the mAb Trastuzumab alone showed no cytotoxicity against any of the cell lines tested, independently of their HER2 status. In contrast, ADC2 conjugate presented a significant and specific cytotoxicity against HER2 expressing cells HCC-1954 and SK-BR-3, inducing a mean inhibition of the cell survival of 57% and 78%, respectively, as compared to the control cells.

(238) At a concentration of 1 μg/mL, ADC2 conjugate showed a somewhat similar cytotoxicity against the HER2 positive cells to that observed at 10 μg/mL, again, without detectable effects on HER2 negative cells (FIG. 6).

(239) These results clearly demonstrated the remarkable cytotoxicity and specificity of ADC2 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 3—Cytotoxicity of ADC3 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(240) The in vitro cytotoxicity of ADC3, along with the parent cytotoxic Compounds 12 and 4 and the mAb Trastuzumab was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(241) Cytotoxicity of Compound 4

(242) The cytotoxicity of the parent Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−07 to 3.9E−12 M).

(243) The cytotoxicity of Compound 4, in two independent experiments, was homogenous along the different cell lines tested, with IC.sub.50 values in the picomolar range, from 1.16E−04 to 2.80E−04 μg/mL (1.75E−10 to 4.23E−10 M), with the mean IC.sub.50 value across the whole cell panel being 1.97E−04 μg/mL (equivalent to 2.96E−10 M). In addition, the cytotoxicity of Compound 4 was independent of the HER2 status of the tumor cell lines (Table 10).

(244) TABLE-US-00010 TABLE 10 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 1.20E−04 1.16E−04 2.80E−04 2.70E−04 IC50 (Molar) 1.81E−10 1.75E−10 4.23E−10 4.07E−10
Cytotoxicity of Compound 12

(245) The activity of Compound 12, the modified Compound 4 carrying the cleavable peptidic linker, was assayed in the same experimental conditions than above, in the range of concentrations from 01E+01 to 2.6E−03 μg/mL (7.9E−06 to 2.0E−09 M).

(246) The cytotoxicity of Compound 12, in two independent experiments, was also relatively homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 7.60E−03 to 3.05E−02 μg/mL (6.02E−09 to 2.42E−08 M), with the mean IC.sub.50 value across the whole cell panel being 1.63E−02 μg/mL (1.29E−08 M) (Table 11). The presence of the peptidic linker in Compound 12 had a negative effect on the cytotoxicity of the compound, as compared to Compound 4. The cytotoxicity of Compound 12 was rather independent of the HER2 status of the tumor cell lines.

(247) TABLE-US-00011 TABLE 11 Summary data of the in vitro cytotoxicity of Compound 12 Compound 12 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 7.60E−03 9.05E−03 3.05E−02 1.80E−02 IC50 (Molar) 6.02E−09 7.18E−09 2.42E−08 1.43E−08
Cytotoxicity of ADC 3

(248) Finally, the cytotoxicity of the ADC3 was assayed against the different cell lines. To ensure the appropriate range of concentrations, the conjugate was assayed in six different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1, 0.1, 0.01 and 0.001 μg/mL (equivalent to 3.33E−07, 6.64E−08, 6.64E−09, 6.64E−10, 6.64E−11 and 6.64E−12 molar concentration), in two independent experiments. A representative DR curve is shown in FIG. 7. The mean IC.sub.50 values calculated for ADC3 against the different cell lines tested are shown in Table 12.

(249) The conjugate ADC3 clearly showed a significant specificity against HER2+ expressing cells, in which the compound demonstrated a potent cytotoxicity, similar to that of the parent Compound 4 (about 1 log more active than the intermediate Compound 12 carrying the peptidic linker). Both HER2+ cell lines, HCC-1954 and SK-BR-3, showed a comparable sensitivity against ADC3, with mean IC.sub.50 values of 8.83E−02 and 6.77E−02 μg/mL (equivalent to 5.86E−10 and 4.49E−10 M), respectively. The two HER negative cell lines, MCF-7 and MDA-MB-231, showed a significantly lower sensitivity against ADC3, with mean IC.sub.50 values of 9.40E+00 and >5.0E+01 μg/mL (equivalent to around 6.24E−08 M and >3.32E−07 M), respectively.

(250) FIG. 8 is a plot of the cytotoxicity of ADC3 against HER2 positive and negative breast cancer cells. It was found that HER2+ cell lines (mean IC.sub.50 7.80E−02 μg/mL) were at least >120 times more sensitive to ADC3 than the HER2 negative MCF-7 cells (mean IC.sub.50 9.40E+00 μg/mL), and far more sensitive than the MDA-MB-231 cells, clearly showing the specificity of ADC3 against the HER2 expressing cells (FIG. 7 and Table 12). We assume, therefore, that the conjugate was actually acting through the interaction of the mAb with the membrane associated HER2 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(251) TABLE-US-00012 TABLE 12 Summary data of the in vitro cytotoxicity of ADC3 (Trastuzumab-Compound 12). ADC3 (Trastuzumab-Compound 12 ADC) Breast cells Cell line HER2+ HER2− HER2 status HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (ug/mL) 8.83E−02 6.77E−02 9.40E+00 >5.0E+01 Mean IC50 (ug/mL) HER2 positive cells 7.80E−02 Mean IC50 (ug/mL) HER2 negative cells 9.40E+00 IC50 (M) 5.86E−10 4.49E−10 6.24E−08 >3.32E−07 Mean IC50 (M) HER2 positive cells 5.18E−10 Mean IC50 (M) HER2 negative cells 6.24E−08

(252) To graphically compare the cytotoxicity of the mAb Trastuzumab, alone with that of the conjugate ADC3, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC3 at 10 or 1 μg/mL, are shown in FIG. 8, which shows the cytotoxic activity of Trastuzumab vs ADC3 against different breast human cancer cell lines.

(253) At an equal concentration of 10 μg/mL, trastuzumab, alone, showed no cytotoxicity against none of the cell lines tested, independently of their HER2 status. In contrast, ADC3 conjugate showed a potent cytotoxicity against the HER2 expressing cells, HCC-1954 and SK-BR-3. In these cell lines, ADC3 exerted an inhibition of the cell survival of 83% and 84%, respectively, as compared to the control cells. At this concentration, ADC3 also had some effect on HER2 negative cells, MCF-7 and MDA-MB-231, producing a slight inhibition of cell survival of 33% and 20%, respectively. At a concentration of 1 μg/mL, ADC3 conjugate showed a similar cytotoxicity against the HER2 positive cells than that observed at 10 μg/mL, but without detectable effects on HER2 negative cells (FIG. 8). These results clearly demonstrated the remarkable cytotoxicity and specificity of ADC3 against HER2 expressing human tumor cells in vitro.

Bioactivity Example 4—Cytotoxicity of ADC4 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(254) The in vitro cytotoxicity of ADC4, along with the parent cytotoxic Compounds 13 and 4 and the mAb Trastuzumab was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER2 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(255) Cytotoxicity of Compound 4

(256) The cytotoxicity of the parent compound Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 1E−01 to 2.6E−05 μg/mL (1.5E−07 to 3.0E−11 M)

(257) The cytotoxicity of Compound 4, in two independent experiments, was homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 2.43E−04 to 4.45E−04 μg/mL (3.6E−10 to 6.7E−10 M), with the mean IC.sub.50 value across the whole cell panel 3.3E−04 μg/mL (equivalent to 4.98E−10 M). Thus, the cytotoxicity of Compound 4 was independent of the HER2 status of the tumor cell lines (Table 13).

(258) TABLE-US-00013 TABLE 13 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 2.98E−04 2.43E−04 4.45E−04 3.35E−04 IC50 (Molar) 4.49E−10 3.66E−10 6.71E−10 5.05E−10
Cytotoxicity of Compound 13

(259) The activity of Compound 13, the modified Compound 4 carrying the thiol containing group, was assayed in the same conditions than above, from 1E−01 to 2.6E−05 μg/mL (1.3E−07 to 2.0E−11 M)

(260) The cytotoxic activity of Compound 13, in two independent experiments, was also relatively homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 7.95E−04 to 2.63E−03 μg/mL (1.0E−09 to 3.5E−09 M), being the mean IC.sub.50 value across the whole cell panel 1.83E−03 μg/mL (2.44E−09 M) (Table 14). The presence of the thiol containing tail in Compound 13 slightly decreased (about 5 fold) the cytotoxic activity of the compound as compared to Compound 4. Also, the cytotoxicity of Compound 13 seemed to be independent of the HER2 status of the tumor cell lines (Table 14).

(261) TABLE-US-00014 TABLE 14 Summary data of the in vitro cytotoxicity of Compound 13 Compound 13 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.49E−03 7.95E−04 2.43E−03 2.63E−03 IC50 (Molar) 1.98E−09 1.06E−09 3.23E−09 3.50E−09
Cytotoxicity of ADC4

(262) The cytotoxicity of the ADC4 was assayed against the different cell lines. Just to assure the appropriate range of concentrations, the conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, in two independent experiments. A representative DR curve is shown in FIG. 9. After adjusting all the different DR curves, the mean IC.sub.50 values calculated for ADC4 against the different cell lines tested are shown in Table 15.

(263) The conjugate ADC4 showed specificity against the HER2+ expressing cells, HCC-1954 and SK-BR-3, in which the compound demonstrated a potent cytotoxicity similar to that of the parent Compounds 4 and 13, with mean IC.sub.50 values of 1.17E−01 and 4.80E−02 μg/mL, respectively. The two HER negative cell lines, MCF-7 and MDA-MB-231, showed a significant lower sensitivity against ADC4, with mean IC.sub.50 values of 5.35E+00 and 6.50E+00 μg/mL, respectively. It seemed that the conjugate was preferentially acting through the interaction of the mAb with the membrane associated HER2 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(264) TABLE-US-00015 TABLE 15 Summary data of the in vitro cytotoxicity of ADC4. ADC 4 HER2 status HER2+ HER2− Cell line HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.17E−01 4.80E−02 5.35E+00 6.50E+00 Mean IC50 (μg/mL) HER2 positive cells 8.24E−02 Mean IC50 (μg/mL) HER2 negative cells 5.93E+00

(265) To graphically compare the cytotoxicity of the mAb Trastuzumab, alone with that of the conjugate ADC4, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC4 at 10 or 1 μg/mL, are shown in FIG. 10. At a concentration of 10 μg/mL, the mAb trastuzumab alone, showed no cytotoxicity against any of the cell lines tested, independently of their HER2 status. In contrast, ADC4 conjugate presented a significant and specific cytotoxicity against the HER2 expressing cells, HCC-1954 and SK-BR-3, inducing a mean inhibition of the cell survival of 80% and 75%, respectively, as compared to the control cells. At this concentration, ADC4 also had effect on HER2 negative cells, MCF-7 and MDA-MB-231, producing an inhibition of cell survival of 53% and 40%, respectively. At a concentration of 1 μg/mL, the ADC4 conjugate showed a somewhat similar cytotoxicity against the HER2 positive cells than that observed at 10 μg/mL, but without detectable effects on HER2 negative cells (FIG. 10). These results clearly demonstrated the remarkable cytotoxicity and specificity of ADC4 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 5—Cytotoxicity of ADC5 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(266) The in vitro cytotoxicity of ADC5 along with the parent cytotoxic Compounds 15 and 40, was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER negative cells).

(267) Cytotoxicity of Compound 40

(268) ##STR00134##

(269) Compound 40 was prepared as described in WO2007144423 (Compound 1 in such patent application), the contents of which are incorporated herein by reference.

(270) The cytotoxicity of the parent Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 1E−02 to 2.6E−06 μg/mL (1.65E−08 to 4.29E−12 M).

(271) The cytotoxicity of Compound 40, in two independent experiments, was very homogenous along the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 4.90E−05 to 1.73E−04 μg/mL (8.10E−11 to 2.84E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.06E−04 μg/mL (equivalent to 1.75E−10 M). Thus the cytotoxicity of Compound 40 was independent of the HER2 status of the tumor cell lines (Table 16).

(272) TABLE-US-00016 TABLE 16 Summary data of the in vitro cytotoxicity of Compound 40. Compound 40 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 7.05E−05 4.90E−05 1.32E−04 1.73E−04 IC50 (Molar) 1.16E−10 8.10E−11 2.18E−10 2.84E−10
Cytotoxicity of Compound 15

(273) The activity of Compound 15, the modified Compound 40 carrying the thiol containing group, was assayed in the same conditions than above, from 1E−01 to 2.6E−05 μg/mL (1.47E−07 to 3.82E−11 M).

(274) The cytotoxicity of Compound 15, in two independent experiments, was also quite homogenous along the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 4.80E−04 to 1.49E−03 μg/mL (7.06E−10 to 2.19E−09 M), being the mean IC.sub.50 value across the whole cell panel 1.03E−03 μg/mL (1.51E−09 M). The presence of the thiol containing tail in Compound 15 slightly decreased (about 8 fold) the cytotoxic activity of the compound as compared to Compound 40. Also, the cytotoxicity of Compound 15 was independent of the HER2 status of the tumor cell lines. (Table 17)

(275) TABLE-US-00017 TABLE 17 Summary data of the in vitro cytotoxicity of Compound 15 Compound 15 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 6.75E−04 4.80E−04 1.45E−03 1.49E−03 IC50 (Molar) 9.94E−10 7.06E−10 2.14E−09 2.19E−09
Cytotoxicity of ADC 5

(276) The cytotoxicity of the ADC5 was assayed against the different cell lines. Just to assure the appropriate range of concentrations, the conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, in two independent experiments. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 11. After adjusting all the different DR curves, the mean IC.sub.50 values calculated for the ADC5 against the different cell lines tested are shown in Table 18.

(277) The conjugate ADC5 showed specificity against the HER2+ expressing cells, HCC-1954 and SK-BR-3, in which the compound demonstrated a cytotoxic activity similar to that of the parent compounds Compound 40 and Compound 15, with mean IC.sub.50 values of 1.13E−01 and 4.61E−02 μg/mL, respectively. The two HER negative cell lines, MCF-7 and MDA-MB-231, showed a significantly lower sensitivity against ADC5, with mean IC.sub.50 values of 1.23E+00 and 1.45E+00 μg/mL, respectively.

(278) It seemed that the conjugate ADC5 was preferentially acting through the interaction of the mAb with the membrane associated HER2 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(279) TABLE-US-00018 TABLE 18 Summary data of the in vitro cytotoxicity of ADC5. ADC5 HER2 status HER2+ HER2− Cell line HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.13E−01 4.61E−02 1.23E+00 1.45E+00 Mean IC50 (μg/mL) HER2 positive cells 7.96E−02 Mean IC50 (μg/mL) HER2 negative cells 1.34E+00

(280) To graphically compare the cytotoxic activity of the mAb Trastuzumab alone with that of the conjugate ADC5, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC5 at 10 μg/mL or 1 μg/mL, are shown in FIG. 12. At a concentration of 10 μg/mL, the mAb trastuzumab alone showed no cytotoxicity activity against any of the cell lines tested, independently of their HER2 status. In contrast, ADC5 conjugate presented a significant and specific cytotoxicity against HER2 expressing cells, HCC-1954 and SK-BR-3, inducing a mean inhibition of the cell survival of 82% and 72%, respectively, as compared to the control cells. At this concentration, ADC5 also had effect on HER2 negative cells, MCF-7 and MDA-MB-231, producing a inhibition of cell survival of 58% and 54%, respectively. At a concentration of 1 μg/mL, the ADC5 conjugate showed a relatively similar cytotoxic activity against the HER2 positive cells than that observed at 10 μg/mL (77% and 75%, respectively), but much less activity against HER2 negative cells, with an inhibition of cell survival of 24% and 15%, respectively (FIG. 12). These results demonstrated the remarkable cytotoxic activity and relative specificity of ADC5 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 6—Cytotoxicity of ADC6 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(281) The in vitro cytotoxicity of ADC6 along with the parent cytotoxic Compounds 18 and 8 was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER2 negative cells). Standard dose-response (DR) curves for hours were performed.

(282) Cytotoxicity of Compound 8

(283) The cytotoxicity of the parent Compound 8 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 1E+00 to 2.6E−04 μg/mL (1.59E−06 to 4.13E−10 M). The cytotoxic activity of this compound, in two independent experiments, was relatively homogenous across the different cell lines tested (slightly more active against SK-BR-3 cells), with IC.sub.50 values in the low nanomolar range, from 1.85E−03 to 9.50E−03 μg/mL (2.94E−09 to 1.51E−08 M), being the mean IC.sub.50 value across the whole cell panel 5.45E−03 μg/mL (equivalent to 8.67E−09 M). Thus, the cytotoxicity of Compound 8 seemed to be independent of the HER2 status of the tumor cell lines (Table 19).

(284) TABLE-US-00019 TABLE 19 Summary data of the in vitro cytotoxicity of Compound 8 Compound 8 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 5.20E−03 1.85E−03 9.50E−03 5.25E−03 IC50 (Molar) 8.27E−09 2.94E−09 1.51E−08 8.35E−09
Cytotoxicity of Compound 18

(285) The activity of Compound 18, the modified Compound 8 carrying the thiol containing group, was assayed in the same conditions than above, from 1E+00 to 2.6E−04 μg/mL (1.39E−06 to 3.63E−10 M). The cytotoxicity of this compound, in two independent experiments, was also quite homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 4.40E−03 to 1.85E−02 μg/mL (6.14E−09 to 2.58E−08 M), being the mean IC.sub.50 value across the whole cell panel 1.06E−02 μg/mL (1.48E−08 M). The presence of the thiol containing tail in Compound 18 had little effect on the activity of the compound, as compared to Compound 8. Also, the cytotoxicity of Compound 18 seemed to be rather independent of the HER2 status of the tumor cell line (Table 20).

(286) TABLE-US-00020 TABLE 20 Summary data of the in vitro cytotoxicity of Compound 18 Compound 18 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 8.05E−03 4.40E−03 1.85E−02 1.15E−02 IC50 (Molar) 1.12E−08 6.14E−09 2.58E−08 1.60E−08
Cytotoxicity of ADC6

(287) The cytotoxicity of the ADC6 was assayed against the different cell lines. Just to assure the appropriate range of concentrations, the conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, in two independent experiments. A representative DR is shown in FIG. 13. After adjusting all the different DR curves, the mean IC.sub.50 values calculated for ADC6 against the different cell lines tested are shown in Table 21.

(288) The conjugate ADC6, although limited, showed some specificity towards HER2+ expressing cells, HCC-1954 and SK-BR-3. In these cell lines, the conjugate was slightly less cytotoxic than the parent Compounds 8 and 18 alone (5.6 and 3.2 times respectively), with mean IC.sub.50 values of 1.04E+01 and 3.80E+00 μg/mL, respectively. The two HER negative cell lines, MCF-7 and MDA-MB-231, showed slightly lower sensitivity against ADC6 (5 fold less), with mean IC.sub.50 values of 3.50E+01 and 4.40E+01 μg/mL, respectively. It seemed that the conjugate ADC6 had some preference for HER2 expressing cells, acting through the interaction of the mAb with the membrane associated HER2 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(289) TABLE-US-00021 TABLE 21 Summary data of the in vitro cytotoxicity of ADC6. ADC6 HER2 status HER2+ HER2− Cell line HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.04E+01 3.80E+00 3.50E+01 4.40E+01 Mean IC50 (μg/mL) HER2 positive cells 7.12E+00 Mean IC50 (μg/mL) HER2 negative cells 3.95E+01

(290) To graphically compare the cytotoxic activity of the mAb Trastuzumab alone with that of the conjugate ADC6, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (10 μg/mL) or ADC6 at 10 or 1 μg/mL, are shown in FIG. 14. At a concentration of 10 μg/mL, the mAb trastuzumab alone showed no cytotoxic activity against any of the cell lines tested, independently of their HER2 status. In contrast, ADC6 conjugate presented specific cytotoxicity against HER2 expressing cells, HCC-1954 and SK-BR-3, inducing a mean inhibition of the cell survival of 57% and 70%, respectively, as compared to the control cells. At this concentration, ADC6 also had a residual effect on HER2 negative cells, MCF-7 and MDA-MB-231, producing an inhibition of cell survival of 9% and 7%, respectively. At a concentration of 1 μg/mL, the ADC6 conjugate still had cytotoxic activity against the HER2 positive cells, although less than that observed at 10 μg/mL (19% and 38%, respectively). At this concentration, ADC6 was completely inactive against HER2 negative cells (FIG. 14). These results demonstrated the preferential cytotoxic activity of ADC6 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 7—Cytotoxicity of ADC7 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(291) The in vitro cytotoxicity of ADC7 along with the parent cytotoxic Compounds 24, 25 and 41 was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER2 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(292) Cytotoxicity of Compound 41

(293) ##STR00135##

(294) Compound 41 was prepared as described in WO 2009/080761 (Compound 72 in such patent application), the contents of which are incorporated herein by reference.

(295) The cytotoxicity of the parent Compound 41 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 1E−02 to 2.6E−06 μg/mL (1.8E−08 to 4.6E−12 M). The cytotoxic activity of this compound, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 1.0E−04 to 2.6E−04 μg/mL (1.8E−10 to 4.6E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.6E−04 μg/mL (equivalent to 2.9E−10 M). Thus, the cytotoxicity of Compound 41 was independent of the HER2 status of the tumor cell lines (Table 22)

(296) TABLE-US-00022 TABLE 22 Summary data of the in vitro cytotoxicity of Compound 41. Compound 41 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.04E−04 1.10E−04 2.65E−04 1.80E−04 IC50 (Molar) 1.83E−10 1.93E−10 4.65E−10 3.16E−10
Cytotoxicity of Compound 24

(297) The activity of Compound 24 was assayed in the same conditions than above, from 1E+00 to 2.6E−04 μg/mL (1.6E−06 to 4.1E−10 M). The cytotoxic activity of this compound, in two independent experiments, was homogeneous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 9.0E−03 to 1.8E−02 μg/mL (1.4E−08 to 2.8E−08 M), being the mean IC.sub.50 value across the whole cell panel 1.5E−02 μg/mL (2.4E−08 M). The presence of the 1,3-propylenediamine group in Compound 24 significantly decreased (about 2 logs) the cytotoxic activity of the compound as compared to Compound 41. Also, the cytotoxicity of Compound 24 seemed to be independent of the HER2 status of the tumor cell lines (Table 23).

(298) TABLE-US-00023 TABLE 23 Summary data of the in vitro cytotoxicity of Compound 24. Compound 24 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 1.80E−02 9.00E−03 1.65E−02 1.75E−02 IC50 (Molar) 2.87E−08 1.44E−08 2.63E−08 2.79E−08
Cytotoxicity of Compound 25

(299) The activity of Compound 25, the modified Compound 24 carrying the MC linker, was assayed in the same conditions than above, from 1E−01 to 2.6E−05 μg/mL (1.2E−07 to 3.2E−11 M). The cytotoxic activity of this compound, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 2.5E−02 to 5.3E−02 μg/mL (3.1E−08 to 6.5E−08 M), being the mean IC.sub.50 value across the whole cell panel 4.1E−02 μg/mL (4.9E−08 M). The presence of the MC linker in Compound 25 very slightly decreased the cytotoxic activity of the compound as compared to Compound 24, particularly in MDA-MB-231 cells. The cytotoxicity of Compound 25 seemed to be independent of the HER2 status of the tumor cell lines (Table 24).

(300) TABLE-US-00024 TABLE 24 Summary data of the in vitro cytotoxicity of Compound 25 Compound 25 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 4.40E−02 2.55E−02 5.30E−02 >1.0E−01 IC50 (Molar) 5.37E−08 3.11E−08 6.46E−08 >1.22E−07
Cytotoxicity of ADC7

(301) The cytotoxicity of the ADC7 was assayed again the different cell lines. Just to assure the appropriate range of concentrations, the conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL in two independent experiments. A representative DR curve (starting concentration 10 μg/mL) is shown in FIG. 15. After adjusting all the different DR curves, the mean IC.sub.50 values calculated for the ADC7 against the difference cell lines tested are shown in Table 25.

(302) The conjugate ADC7 showed specificity against the HER2+ expressing cells, HCC-1954 and SK-BR-3, in which the compound demonstrated a cytotoxic activity nearly similar to that of the parent Compound 41, with mean IC.sub.50 values of 3.7E−01 and 8.9E−02 μg/mL, respectively. The two HER negative cell lines, MCF-7 and MDA-MB-231, were virtually unresponsive to ADC7. The conjugate seemed to be acting through the interaction of the mAb with the membrane associated HER2 receptor on positive tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(303) TABLE-US-00025 TABLE 25 Summary data of the in vitro cytotoxicity of ADC7 ADC7 HER2 status HER2+ HER2− Cell line HCC1954 SK-BR3 MCF7 MDA-MB-231 IC50 (μg/mL) 3.75E−01 8.97E−02 >5.0E+01 >5.0E+01 Mean IC50 (μg/mL) HER2 positive cells 2.32E−01 Mean IC50 (μg/mL) HER2 negative cells >5.0E+01

(304) To graphically compare the cytotoxic activity of the mAb Trastuzumab alone with that of the conjugate ADC7, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC7 at 50 or 1 μg/mL, are shown in FIG. 16. At a concentration of 50 μg/mL, the mAb trastuzumab alone, showed no significant cytotoxic activity against any of the cell lines tested, independently of their HER2 status. In contrast, ADC7 conjugate presented a significant and specific cytotoxicity against HER2 expressing cells, HCC-1954 and SK-BR-3, inducing a mean inhibition of the cell survival of 77% and 76%, respectively, as compared to the control cells. At this concentration, ADC7 only had a residual effect on HER2 negative cells, MCF-7 and MDA-MB-231, producing an inhibition of cell survival of 13% and 15%, respectively. A similar activity and specificity was detected at lower concentrations of ADC7 (as low as 1 μg/mL) in HCC-1954 and SK-BR-3 cells, producing an inhibition of the cell survival of 68% and 79%, respectively (FIG. 16). Together, these results clearly demonstrated the remarkable cytotoxic activity and specificity of ADC7 conjugate against HER2 expressing human tumor cells in vitro.

Bioactivity Example 8—Cytotoxicity of ADC8 and Related Reagents Against HER2 Positive and Negative Breast Cancer Cells

(305) The in vitro cytotoxic activity of ADC8 along with the parent cytotoxic Compounds 24, 27 and 41, was evaluated against different human breast cancer cell lines expressing or not the HER2 receptor, including HCC-1954 and SK-BR-3 (HER2 positive cells) and MDA-MB-231 and MCF-7 (HER negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(306) Cytotoxicity of Compound 41

(307) The cytotoxic activity of the parent Compound 41 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.8E−08 to 4.6E−12 M). The cytotoxic activity of this compound, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 7.5E−05 to 1.4E−04 μg/mL (1.3E−10 to 2.4E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.1E−04 μg/mL (equivalent to 1.9E−10 M). Thus, the cytotoxicity of Compound 41 was independent of the HER2 status of the tumor cell lines (Table 26).

(308) TABLE-US-00026 TABLE 26 Summary data of the in vitro cytotoxicity of Compound 41 Compound 41 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC.sub.50 (μg/mL) 7.55E−05 8.05E−05 1.39E−04 1.35E−04 IC.sub.50 (Molar) 1.33E−10 1.41E−10 2.44E−10 2.36E−10
Cytotoxicity of Compound 24

(309) The activity of Compound 24 (was assayed in the same conditions than above, from 01E+00 to 2.6E−04 μg/mL (1.6E−06 to 4.1E−10 M). The cytotoxic activity of this compound, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 9.0E−03 to 1.8E−02 μg/mL (1.4E−08 to 2.9E−08 M), being the mean IC.sub.50 value across the whole cell panel 1.5E−02 μg/mL (2.4E−08 M). The presence of the 1,3-propylenediamine group in Compound 24 significantly decreased (about 2 logs) the cytotoxic activity of the compound as compared to Compound 41. The cytotoxicity of Compound 24 seemed to be independent of the HER2 status of the tumor cell lines (Table 27).

(310) TABLE-US-00027 TABLE 27 Summary data of the in vitro cytotoxicity of Compound 24 Compound 24 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC.sub.50 (μg/mL) 1.80E−02 9.00E−03 1.65E−02 1.75E−02 IC.sub.50 (Molar) 2.87E−08 1.44E−08 2.63E−08 2.79E−08
Cytotoxicity of Compound 27

(311) The activity of Compound 27 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−04 μg/mL (1.4E−06 to 3.6E−10 M). The cytotoxic activity of Compound 27, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the micromolar range, from 1.05E−02 to 3.9E−02 μg/mL (1.5E−08 to 5.5E−08 M), being the mean IC.sub.50 value across the whole cell panel 2.5E−02 μg/mL (3.5E−08 M). The presence of the MPA linker in Compound 27 had no significant effect on the cytotoxic activity of the compound as compared to Compound 24. The activity of Compound 27 was independent of the HER2 status of the tumor cell lines (Table 28).

(312) TABLE-US-00028 TABLE 28 Summary data of the in vitro cytotoxicity of Compound 27 Compound 27 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC.sub.50 (μg/mL) 1.65E−02 1.05E−02 3.90E−02 3.45E−02 IC.sub.50 (Molar) 2.31E−08 1.47E−08 5.46E−08 4.83E−08
Cytotoxicity of ADC8

(313) The cytotoxic activity of the ADC8 was assayed against the different cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 mg/mL, in two independent experiments. A representative DR curve (maximum concentration of 10 μg/mL) is shown in FIG. 17. ADC8 showed some specificity against the HER2+ expressing cells, particularly in SK-BR-3, the most sensitive cell line. Except for these cells, which are around 4 times more sensitive than HER2 negative cells (IC.sub.50 2.3E−09M), ADC8 showed, similar cytotoxic activity, HER2 independent, than the parent Compound 27, with IC.sub.50 values in the nanomolar range (Table 29).

(314) TABLE-US-00029 TABLE 29 Summary data of the in vitro cytotoxicity of ADC8 ADC8 Breast cells HER2+ HER2− HCC1954 SK-BR3 MCF7 MDA-MB-231 IC.sub.50 (μg/mL) 3.83E+00 6.37E−01 7.75E+00 1.02E+01 Mean IC.sub.50 (μg/mL) HER2 positive cells 2.23E+00 Mean IC.sub.50 (μg/mL) HER2 negative cells 8.98E+00

(315) To graphically compare the cytotoxic activity of the mAb Trastuzumab alone with that of the conjugate ADC8, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC8 (50 or 10 μg/mL), are shown in FIG. 18. At 50 μg/mL, the mAb alone had no activity in any of the cell lines tested, except for SK-BR-3, in which it produced an inhibition of cell survival of less than 20%. ADC8, in turn, showed no significant specificity for the HER2+ cell lines, producing a strong inhibition of cell survival of more than 60% in all the cells analyzed. At a concentration of 10 μg/mL, ADC8 showed some, but little, specificity against HER2+ cells, producing a 78% inhibition of cell survival in HCC-1954 and SK-BR-3 cells, both HER2 positive, while having a smaller effect on HER2 negative cells, 59% and 41%, in MCF7 and MDA-MB-231, respectively. HER2 positive cells were, roughly, between 1.5 and 2 times more sensitive to ADC8 than HER2 negative cells.

Bioactivity Example 9—Cytotoxicity of ADC9 and Related Reagents Against CD13 Positive and Negative Human Tumor Cells

(316) The in vitro cytotoxic activity of ADC9 along with the parent cytotoxic Compounds 1 and 4, was evaluated against different human tumor cell lines expressing or not the CD13 receptor, including NB4 and U937 (CD13 positive cells) and Raji and RPMI-8226 (CD13 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(317) Cytotoxicity of the Anti-CD13 Mouse Monoclonal Antibody

(318) First of all, the in vitro cytotoxic activity of the anti-CD13 mouse mAb alone was assayed against the different tumor cell lines. In triplicate DR curves ranging from 5.0E+01 to 1.3E−02 μg/mL (3.3E−07-8.7E−11 M), in two independent experiments, the antibody was virtually inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their CD13 status (Table 30).

(319) TABLE-US-00030 TABLE 30 Summary data of the in vitro cytotoxic activity of the antiCD13 mouse mAb antiCD13 mouse mAb Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 IC.sub.50 (Molar) >3.33E−07 >3.33E−07 >3.33E−07 >3.33E−07
Cytotoxicity of Compound 4

(320) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4, in two independent experiments, was very homogenous across the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 7.9E−05 to 2.65E−03 μg/mL (1.2E−10 to 4.0E−09 M), being the mean IC.sub.50 value across the whole cell panel 8.4E−04 μg/mL (equivalent to 1.2E−09 M). Thus, the cytotoxicity of Compound 4 was rather independent of the CD13 status of the tumor cell lines (Table 31).

(321) TABLE-US-00031 TABLE 31 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 7.93E−05 2.78E−04 2.65E−03 3.58E−04 IC.sub.50 (Molar) 1.20E−10 4.19E−10 4.00E−09 5.39E−10
Cytotoxicity of Compound 1

(322) The activity of the Compound 1 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.1E−07 to 3.0E−11 M). The cytotoxic activity of Compound 1, in two independent experiments, was somewhat homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 8.0E−04 to 6.3E−03 μg/mL (9.4E−10 to 7.3E−09 M), being the mean IC.sub.50 value across the whole cell panel 2.8E−03 μg/mL (3.3E−09 M). The presence of the maleimide linker in Compound 1 does not alter very significantly the cytotoxic activity of the compound, as compared to Compound 4. In addition, the cytotoxicity of the compound was not related to the CD13 status of the tumor cell lines (Table 32).

(323) TABLE-US-00032 TABLE 32 Summary data of the in vitro cytotoxicity of Compound 1 Compound 1 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 8.05E−04 1.65E−03 6.25E−03 2.50E−03 IC.sub.50 (Molar) 9.39E−10 1.93E−09 7.31E−09 2.92E−09
Cytotoxicity of ADC9

(324) The cytotoxic activity of the ADC9 was assayed against the different cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 mg/mL, in two independent experiments. A representative DR curve (maximum concentration 0.1 μg/mL) is shown in FIG. 19.

(325) The conjugate ADC9 showed a significant specificity against CD13+ expressing cells, in which the compound demonstrated a cytotoxic activity similar, or slightly higher, to that of the parent Compounds 4 and 1. Both CD13+ cell lines, NB4 and U937, showed a comparable sensitivity against ADC9, with mean IC.sub.50 values of 8.7E−03 and 2.4E−02 μg/mL, respectively. The two CD13 negative cell lines, Raji and RPMI-8226, showed a significantly lower sensitivity against ADC9, with mean IC.sub.50 values of 1.6E+00 and 5.9E−01 μg/mL, respectively. Average, CD13+ cell lines (mean IC.sub.50 1.66E−02 μg/mL) were around 65 times more sensitive to ADC9 than the CD13− cells (mean IC.sub.50 1.08E+00 μg/mL). Comparing the activity of ADC9 in NB4 cells (the most sensitive) vs Raji cells (the least sensitive); it was found a difference of around 180 times. These results clearly showed the specificity of the conjugate against the CD13 expressing cells (Table 33). We assume, therefore, that the ADC9 was, at least in part, acting through the interaction of the mAb with the membrane associated CD13 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(326) TABLE-US-00033 TABLE 33 Summary data of the in vitro cytotoxicity of ADC9 ADC9 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 8.75E−03 2.44E−02 1.57E+00 5.92E−01 Mean IC.sub.50 (μg/mL) CD13 positive cells 1.66E−02 Mean IC.sub.50 (μg/mL) CD13 negative cells 1.08E+00

(327) To graphically compare the cytotoxic activity of the mAb alone with that of the conjugate ADC9, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or the ADC at 50 or 0.1 μg/mL, are shown in FIG. 20. At an equal concentration of 50 μg/mL, the anti-CD13 antibody alone, showed no cytotoxic activity against any of the cell lines tested, independently of their CD13 status. In contrast, ADC9 conjugate showed a potent cytotoxic activity against all the cell lines, inducing an inhibition of the cell survival of more than 80%. At a concentration of 0.1 μg/mL, the conjugate ADC9 showed a similar cytotoxic activity against the CD13 positive cells than that observed at 50 μg/mL, but without any detectable effect on CD13 negative cells. These results further demonstrated the remarkable cytotoxic activity and specificity of ADC9 against CD13 expressing human tumor cells in vitro.

Bioactivity Example 10—Cytotoxicity of ADC10 and Related Reagents Against CD13 Positive and Negative Human Tumor Cells

(328) The in vitro cytotoxic activity of ADC10 along with the parent cytotoxic Compounds 12 and 4, was evaluated against different human tumor cell lines expressing or not the CD13 receptor, including NB4 and U937 (CD13 positive cells) and Raji and RPMI-8226 (CD13 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(329) Cytotoxicity of Compound 4

(330) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4, in two independent experiments, was very homogenous across the different cell lines tested, with IC.sub.50 values in the low nanomolar range, from 7.9E−05 to 2.65E−03 μg/mL (1.2E−10 to 4.0E−09 M), being the mean IC.sub.50 value across the whole cell panel 8.4E−04 μg/mL (equivalent to 1.2E−09 M). Thus, the cytotoxicity of Compound 4 was rather independent of the CD13 status of the tumor cell lines (Table 34).

(331) TABLE-US-00034 TABLE 34 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 7.93E−05 2.78E−04 2.65E−03 3.58E−04 IC.sub.50 (Molar) 1.20E−10 4.19E−10 4.00E−09 5.39E−10
Cytotoxicity of Compound 12

(332) The activity of the Compound 12 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−04 μg/mL (7.9E−07 to 2.0E−10 M). The cytotoxic activity of Compound 12, in two independent experiments, was somewhat homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 4.4E−03 to 4.8E−02 μg/mL (3.5E−09 to 3.8E−08 M), being the mean IC.sub.50 value across the whole cell panel 2.0E−02 μg/mL (1.6E−08 M). The presence of the long linker in Compound 12 decreased (approx 1 log) the cytotoxic activity of the compound, as compared to Compound 4. In addition, the cytotoxicity of the compound was not related to the CD13 status of the tumor cell lines (Table 35).

(333) TABLE-US-00035 TABLE 35 Summary data of the in vitro cytotoxicity of Compound 12 Compound 12 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 4.45E−03 1.07E−02 4.80E−02 1.90E−02 IC.sub.50 (Molar) 3.53E−09 8.44E−09 3.80E−08 1.51E−08
Cytotoxicity of ADC10

(334) The cytotoxic activity of the ADC10 was assayed against the different cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 mg/mL, in two independent experiments. A representative DR curve (maximum concentration 1 μg/mL) is shown in FIG. 21.

(335) The conjugate ADC10 showed a significant specificity against CD13+ expressing cells, in which the compound demonstrated a cytotoxic activity similar, or slightly higher, to that of the parent Compounds 4 and 12. Both CD13+ cell lines, NB4 and U937, showed a comparable sensitivity against ADC10, with mean IC.sub.50 values of 7.2E−03 and 9.8E−03 μg/mL, respectively. The two CD13 negative cell lines, Raji and RPMI-8226, showed a significantly lower sensitivity against ADC10, with mean IC.sub.50 values of 1.0E+01 and 5.3E+00 μg/mL, respectively. Average, CD13+ cell lines (mean IC.sub.50 8.50E−03 μg/mL) were around 900 times more sensitive to ADC10 than the CD13− cells (mean IC.sub.50 7.83E+00 μg/mL). Comparing the activity of ADC10 in NB4 cells (the most sensitive) vs Raji cells (the least sensitive); it was found a difference of around 1440 times. These results clearly showed the specificity of ADC10 against CD13 expressing cells (Table 36). We assume, therefore, that the ADC10 was, at least in part, acting through the interaction of the mAb with the membrane associated CD13 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(336) TABLE-US-00036 TABLE 36 Summary data of the in vitro cytotoxicity of ADC10 ADC10 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 7.18E−03 9.81E−03 1.04E+01 5.30E+00 Mean IC.sub.50 (μg/mL) CD13 positive cells 8.50E−03 Mean IC.sub.50 (μg/mL) CD13 negative cells 7.83E+00

(337) To graphically compare the cytotoxic activity of the mAb alone with that of the conjugate ADC10, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or the ADC at 50 or 1 μg/mL, are shown in FIG. 22. At an equal concentration of 50 μg/mL, the anti-CD13 antibody alone, showed no cytotoxic activity against any of the cell lines tested, independently of their CD13 status. In contrast, ADC10 conjugate showed a potent cytotoxic activity against all the cell lines, inducing an inhibition of the cell survival of more than 80%, except for Raji cells, in which it produced a lower, but still important, inhibition of around 70%. At a concentration of 1 μg/mL, ADC10 showed a similar cytotoxic activity against the CD13 positive cells than that observed at 50 μg/mL, but without any detectable effect on CD13 negative cells. These results further demonstrated the remarkable cytotoxic activity and specificity of ADC10 against CD13 expressing human tumor cells in vitro.

Bioactivity Example 11—Cytotoxicity of ADC11 and Related Reagents Against CD13 Positive and Negative Human Tumor Cells

(338) The in vitro cytotoxic activity of ADC11 along with the parent cytotoxic Compounds 13 and 40, was evaluated against different human tumor cell lines expressing or not the CD13 receptor, including NB4 and U937 (CD13 positive cells) and Raji and RPMI-8226 (CD13 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(339) Cytotoxicity of Compound 40

(340) The cytotoxic activity of Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40, in two independent experiments, was homogenous across the different cell lines tested, with IC.sub.50 values in the low picomolar range, from 3.1E−05 to 1.7E−04 μg/mL (5.2E−11 to 2.8E−10 M), being the mean IC.sub.50 value across the whole cell panel 8.6E−05 μg/mL (equivalent to 1.4E−10 M). The cytotoxicity of Compound 40 was independent of the CD13 expression levels on the tumor cell lines (Table 37).

(341) TABLE-US-00037 TABLE 37 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 3.15E−05 7.10E−05 1.70E−04 7.05E−05 IC.sub.50 (Molar) 5.20E−11 1.17E−10 2.80E−10 1.16E−10
Cytotoxicity of Compound 13

(342) The activity of the Compound 13 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.3E−07 to 3.4E−11 M). The cytotoxic activity of Compound 13, in two independent experiments, was somewhat homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 1.7E−03 to 1.0E−02 μg/mL (2.7E−09 to 1.4E−08 M), being the mean IC.sub.50 value across the whole cell panel 4.3E−03 μg/mL (5.7E−09 M). The presence of the thiol containing tail in Compound 13 reduced (less than 1 log) the cytotoxic activity of the compound, as compared to Compound 40. The cytotoxicity of the compound was rather independent of the CD13 status of the tumor cell lines (Table 38).

(343) TABLE-US-00038 TABLE 38 Summary data of the in vitro cytotoxicity of Compound 13 Compound 13 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 1.70E−03 2.85E−03 1.03E−02 2.30E−03 IC.sub.50 (Molar) 2.26E−09 3.80E−09 1.37E−08 3.06E−09
Cytotoxicity of ADC11

(344) The cytotoxic activity of the ADC11 was assayed against the different cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, in two independent experiments. A representative DR curve (maximum concentration of 1 μg/mL) is shown in FIG. 23. ADC11 showed some, but little, specificity against the CD13 expressing cells. The conjugate had rather similar cytotoxic activity, except for Raji cells, which are slightly less sensitive, in all the cell lines tested. The activity of ADC11 was comparable to that of the parent Compound 13, with IC.sub.50 values in the low nanomolar range (Table 39).

(345) TABLE-US-00039 TABLE 39 Summary data of the in vitro cytotoxicity of ADC11 ADC11 Cell lines CD13+ CD13− NB-4 U937 Raji RPMI18226 IC.sub.50 (μg/mL) 2.27E−01 6.77E−01 3.48E+00 6.95E−01 Mean IC.sub.50 (μg/mL) CD13 positive cells 4.52E−01 Mean IC.sub.50 (μg/mL) CD13 negative cells 2.09E+00

(346) To graphically compare the cytotoxic activity of the Anti-CD13 mAb alone with that of the conjugate ADC11, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or the ADC11 (50 or 1 μg/mL), are shown in FIG. 24. At a concentration of 50 μg/mL, the anti-CD13 antibody alone, showed some cytotoxic activity against CD13+ cell lines, producing an inhibition of cell survival of around 30%. In CD13− cells, the antibody was virtually inactive. At the same concentration, ADC11 conjugate showed a potent cytotoxic activity against all the cell lines, with some, but very little, specificity against CD13 expressing cells, in which it induced a reduction of cell survival of nearly 100%. In CD13− cells, ADC11 induced more than 80% reduction in cell survival. At a concentration of 1 μg/mL, ADC11 showed more specificity against CD13+ cells, NB-4 and U937, in which it induced a reduction of cell survival of 99 and 85%, respectively. In CD13− cells, Raji and RPMI8226, the conjugate induced a reduction of cell survival of 38 and 60%, respectively.

Bioactivity Example 12—Cytotoxicity of ADC12 and Related Reagents Against CD20 Positive and Negative Human Tumor Cells

(347) The in vitro cytotoxic activity of ADC12 along with the parent cytotoxic Compounds 1, 4, and 40, was evaluated was assayed against different human cancer cell lines expressing or not the CD20 antigen, including Raji (CD20 positive cells); RPMI-8226 and Karpas-299 (CD20 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(348) Cytotoxicity of Rituximab

(349) First of all, the in vitro cytotoxic activity of the mAb alone Rituximab was assayed against different human cancer cell lines expressing or not the CD20 antigen, including Raji (CD20 positive cells); RPMI-8226 and Karpas-299 (CD20 negative cells). In triplicate DR curves spanning from 5.0E+01 to 2.62E−05 μg/mL (3.4E−07 to 1.7E−13 M), the antibody was rather inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their CD20 status (Table 40).

(350) TABLE-US-00040 TABLE 40 Summary data of the in vitro cytotoxicity of Rituximab Rituximab CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/mL) >5.0E+01 >5.0E+01 >5.0E+01 IC.sub.50 (Molar) >3.48E−07 >3.48E−07 >3.48E−07
Cytotoxicity of Compound 40

(351) The cytotoxic activity of the parent compound Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was very homogenous across the different cell lines tested, with IC.sub.50 values in the low subnanomolar range, from 8.6E−05 to 1.1E−04 μg/mL (1.4E−10 to 1.9E−10 M), being the mean IC.sub.50 value across the whole cell panel 9.6E−05 μg/mL (equivalent to 1.6E−10 M). The cytotoxicity of Compound 40 was independent of the CD20 status of the tumor cell lines (Table 41).

(352) TABLE-US-00041 TABLE 41 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/mL) 1.15E−04 8.65E−05 8.60E−05 IC.sub.50 (Molar) 1.90E−10 1.43E−10 1.42E−10
Cytotoxicity of Compound 4

(353) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 5.7E−04 to 1.4E−03 μg/mL (8.6E−10 to 2.1E−09 M), being the mean IC.sub.50 value across the whole cell panel 9.7E−04 μg/mL (1.5E−09 M). The presence of the amine containing group in Compound 4 slightly reduced the cytotoxic activity of the compound, as compared to Compound 40. The cytotoxic activity of Compound 4 was also independent of the CD20 status of the tumor cell lines (Table 42).

(354) TABLE-US-00042 TABLE 42 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 1.41E−03 5.70E−04 9.30E−04 mL) IC.sub.50 (Molar) 2.12E−09 8.59E−10 1.40E−09
Cytotoxicity of Compound 1

(355) The activity of the Compound 1 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.2E−07 to 3.0E−11 M). The cytotoxic activity of Compound 1, in two independent experiments, was very homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 1.6E−03 to 2.8E−03 μg/mL (1.9E−09 to 3.3E−09 M), being the mean IC.sub.50 value across the whole cell panel 2.1E−03 μg/mL (2.5E−09 M). The presence of the maleimide linker in Compound 1 does not significantly alter the cytotoxic activity of the compound, as compared to Compound 4. In addition, the cytotoxicity of the compound was also independent of the CD20 status of the tumor cell lines (Table 43).

(356) TABLE-US-00043 TABLE 43 Summary data of the in vitro cytotoxicity of Compound 1 Compound 1 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 2.85E−03 1.60E−03 1.90E−03 mL) IC.sub.50 (Molar) 3.33E−09 1.87E−09 2.22E−09
Cytotoxicity of ADC12

(357) The cytotoxic activity of the ADC12 was assayed against the different tumor cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, respectively. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 25. Although higher in CD20 positive Raji cells, ADC12 presented a relatively similar cytotoxic activity, in the nanomolar range, in all the cell lines tested. Raji cells (CD20+), showed a mean IC.sub.50 value of 9.5E−02 μg/mL, while the respective values for RPMI-8226 and Karpas-299 cells (both CD20−), were 4.0E−01 and 4.1E−01 μg/mL, respectively (Table 44). Thus, CD20 positive cells were slightly more sensitive (4 fold) to ADC12 than CD20 negative cells.

(358) TABLE-US-00044 TABLE 44 Summary data of the in vitro cytotoxicity of ADC12 ADC12 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 9.54E−02 3.97E−01 4.13E−01 mL) Mean IC.sub.50 (μg/mL) CD20 positive cells 9.54E−02 Mean IC.sub.50 (μg/mL) CD20 negative cells 4.05E−01

(359) To graphically compare the cytotoxic activity of the mAb Rituximab alone with that of the conjugate ADC12, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC12 (1 and 0.1 μg/mL), are shown in FIG. 26. Rituximab alone, at a concentration of 50 μg/mL, was virtually inactive in all the cell lines tested, independently of their CD20 status. In contrast, the ADC12, at a concentration of 1 μg/mL, showed potent cytotoxic activity in all the cell lines tested, causing more than 70% reduction in the cell survival after 72 hours of treatment. At a lower concentration, 0.1 μg/mL, ADC12 conjugate showed some specificity, inducing a reduction of cell survival of around 60% in CD20 positive cells (Raji), while being virtually inactive in CD20 negative cells (RPMI-8226 and Karpas-299).

Bioactivity Example 13—Cytotoxicity of ADC13 and Related Reagents Against CD20 Positive and Negative Human Tumor Cells

(360) The in vitro cytotoxic activity of ADC13 along with the parent cytotoxic Compounds 1, 4, and 40, was evaluated was assayed against different human cancer cell lines expressing or not the CD20 antigen, including Raji (CD20 positive cells); RPMI-8226 and Karpas-299 (CD20 negative cells). Standard dose-response (DR) curves for 72 hours were performed.

(361) Cytotoxicity of Compound 40

(362) The cytotoxic activity of the parent compound Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was very homogenous across the different cell lines tested, with IC.sub.50 values in the low subnanomolar range, from 8.6E−05 to 1.1E−04 μg/mL (1.4E−10 to 1.9E−10 M), being the mean IC.sub.50 value across the whole cell panel 9.6E−05 μg/mL (equivalent to 1.6E−10 M). The cytotoxicity of Compound 40 was independent of the CD20 status of the tumor cell lines (Table 45).

(363) TABLE-US-00045 TABLE 45 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 1.15E−04 8.65E−05 8.60E−05 mL) IC.sub.50 (Molar) 1.90E−10 1.43E−10 1.42E−10
Cytotoxicity of Compound 4

(364) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 5.7E−04 to 1.4E−03 μg/mL (8.6E−10 to 2.1E−09 M), being the mean IC.sub.50 value across the whole cell panel 9.7E−04 μg/mL (1.5E−09 M). The presence of the amine containing group in Compound 4 slightly reduced the cytotoxic activity of the compound, as compared to Compound 40. The cytotoxic activity of Compound 4 was also independent of the CD20 status of the tumor cell lines (Table 46).

(365) TABLE-US-00046 TABLE 46 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 1.41E−03 5.70E−04 9.30E−04 mL) IC.sub.50 (Molar) 2.12E−09 8.59E−10 1.40E−09
Cytotoxicity of Compound 12

(366) The activity of the Compound 12 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−04 μg/mL (7.9E−07 to 2.1E−10 M). The cytotoxic activity of Compound 12, in two independent experiments, was very homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 2.2E−02 to 6.7E−02 μg/mL (1.8E−08 to 5.5E−08 M), being the mean IC.sub.50 value across the whole cell panel 3.9E−02 μg/mL (3.1E−08 M). The presence of the maleimide linker in Compound 12 reduced the cytotoxic activity of the compound, as compared to Compound 4 and Compound 40. In addition, the cytotoxicity of the compound was also independent of the CD20 status of the tumor cell lines (Table 471.

(367) TABLE-US-00047 TABLE 47 Summary data of the in vitro cytotoxicity of Compound 12 Compound 12 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 6.95E−02 2.25E−02 2.50E−02 mL) IC.sub.50 (Molar) 5.51E−08 1.78E−08 1.98E−08
Cytotoxicity of ADC13

(368) The cytotoxic activity of the ADC13 was assayed against the different tumor cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, respectively. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 27. Although higher in CD20 positive Raji cells, ADC13 presented a rather similar cytotoxic activity, in the nanomolar range, in all the cell lines tested. Raji cells (CD20+), showed a mean IC.sub.50 value of 2.5E−01 μg/mL, while the respective values for RPMI-8226 and Karpas-299 cells (both CD20−), were 1.1E+00 μg/mL (Table 48). Thus, CD20 positive cells were slightly more sensitive (about 5 fold) to ADC13 than CD20 negative cells.

(369) TABLE-US-00048 TABLE 48 Summary data of the in vitro cytotoxicity of ADC13 ADC13 CD20+ CD20− Raji RPMI-8226 Karpas-299 Burkitt's Lymphoma Multiple Myeloma NHL IC.sub.50 (μg/ 2.53E−01 1.08E+00 1.07E+00 mL) Mean IC.sub.50 (μg/mL) CD20 positive cells 2.53E−01 Mean IC.sub.50 (μg/mL) CD20 negative cells 1.07E+00

(370) To graphically compare the cytotoxic activity of the mAb Rituximab alone with that of the conjugate ADC13, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC13 (1 and 0.1 μg/mL), are shown in FIG. 28. Rituximab alone, at a concentration of 50 μg/mL, was virtually inactive in all the cell lines tested, independently of their CD20 status. In contrast, ADC13 showed, at both 1 and 0.1 μg/mL, cytotoxic activity, with some specificity for CD20 expressing Raji cells. At 1 μg/mL, ADC13 caused, after 72 hours of treatment, more than 65% reduction in the cell survival of Raji cells (CD20+) while inducing a 35-45% in RPMI-8226 and Karpas-299 cells (CD20−), respectively. At 0.1 μg/mL, ADC13 conjugate showed more clear specificity, inducing a reduction of cell survival of around 50% in CD20+ cells, while being inactive in CD20− cells.

Bioactivity Example 14—Cytotoxicity of ADC14 and Related Reagents Against CD5 Positive and Negative Human Tumor Cells

(371) The in vitro cytotoxic activity of ADC14 along with the parent cytotoxic Compounds 1, 4, and 40, was evaluated against different human cancer cell lines expressing or not the CD5 antigen, including Karpas-299 and MOLT-4 (both CD5+); Raji and RPMI-8226 (both CD5−). Standard dose-response (DR) curves for 72 hours were performed.

(372) Cytotoxicity of Anti-CD5 mAb

(373) First of all, the in vitro cytotoxic activity of the anti-CD5 mouse mAb alone was assayed against different human cancer cell lines expressing or not the CD5 antigen, including Karpas-299 and MOLT-4 (both CD5+); Raji and RPMI-8226 (both CD5−). In triplicate DR curves spanning from 5.0E+01 to 1.3E−02 μg/mL (3.3E−07 to 8.7E−11 M), in two independent experiments, the antibody was virtually inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their CD5 status (Table 49).

(374) TABLE-US-00049 TABLE 49 Summary data of the in vitro cytotoxicity of Anti-CD5 mAb Anti-CD5 mAb Cell lines CD5+ CD5− Karpas-299 MOLT-4 Raji RPMI18226 IC.sub.50 (μg/mL) >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 IC.sub.50 (Molar) >3.3E−07 >3.3E−07 >3.3E−07 >3.3E−07
Cytotoxicity of Compound 40

(375) The cytotoxic activity of the parent Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was very homogenous across the different cell lines tested, with IC.sub.50 values in the low subnanomolar range, from 7.5E−05 to 3.6E−04 μg/mL (1.2E−10 to 5.9E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.6E−04 μg/mL (equivalent to 2.6E−10 M). The cytotoxicity of Compound 40 was independent of the CD5 expression levels in the tumor cell lines (Table 50).

(376) TABLE-US-00050 TABLE 50 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Cell lines CD5+ CD5− Karpas-299 MOLT-4 Raji RPMI18226 IC.sub.50 (μg/mL) 1.12E−04 9.35E−05 3.60E−04 7.55E−05 IC.sub.50 (Molar) 1.85E−10 1.54E−10 5.94E−10 1.25E−10
Cytotoxicity of Compound 4

(377) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 6.3E−04 to 2.7E−03 μg/mL (9.5E−10 to 4.1E−09 M), being the mean IC.sub.50 value across the whole cell panel 1.3E−03 μg/mL (1.9E−09 M). The presence of the amine containing group in Compound 4 reduced the cytotoxic activity of the compound (around 1 log), as compared to Compound 40 The cytotoxic activity of Compound 4 was also independent of the CD5 expression levels in the tumor cell lines (Table 51).

(378) TABLE-US-00051 TABLE 51 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Cell lines CD5+ CD5− Karpas-299 MOLT-4 Raji RPMI18226 IC.sub.50 (μg/mL) 9.15E−04 9.10E−04 2.70E−03 6.30E−04 IC.sub.50 (Molar) 1.38E−09 1.37E−09 4.07E−09 9.50E−10
Cytotoxicity of Compound 1

(379) The activity of Compound 1 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.2E−07 to 3.0E−11 M). The cytotoxic activity of Compound 1 was nearly homogenous across the different cell lines tested, except for Raji cells in which the compound was less active, with IC.sub.50 values in the nanomolar range, from 1.8E−03 to 1.1E−02 μg/mL (2.2E−09 to 1.3E−09 M), being the mean IC.sub.50 value across the whole cell panel 4.6E−03 μg/mL (5.3E−09 M). The presence of the maleimide containing linker in Compound 1 did not alter the cytotoxic activity of the compound, as compared to Compound 4. The cytotoxicity of the compound was also independent of the CD5 expression levels in the tumor cell lines (Table 52).

(380) TABLE-US-00052 TABLE 52 Summary data of the in vitro cytotoxicity of Compound 1 Compound 1 Cell lines CD5+ CD5− Karpas-299 MOLT-4 Raji RPMI18226 IC.sub.50 (μg/mL) 2.15E−03 3.35E−03 1.09E−02 1.85E−03 IC.sub.50 (Molar) 2.51E−09 3.91E−09 1.28E−08 2.16E−09
Cytotoxicity of ADC14

(381) The cytotoxic activity of the ADC14 was assayed against the different tumor cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, respectively. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 29. ADC14 showed some trend of selectivity against CD5 positive cells, although the mean IC.sub.50 values, in the medium nanomolar range, were relatively similar for all the cell lines tested, independently of the CD5 status (Table 53).

(382) TABLE-US-00053 TABLE 53 Summary data of the in vitro cytotoxicity of ADC14 ADC14 Cell lines CD5+ CD5− Karpas-299 MOLT-4 Raji RPMI18226 IC.sub.50 (μg/mL) 5.56E−01 6.18E−01 4.23E+00 5.47E+00 Mean IC.sub.50 (μg/mL) CD5 positive cells 5.87E−01 Mean IC.sub.50 (μg/mL) CD5 negative cells 2.39E+00

(383) To graphically compare the cytotoxic activity of the anti-CD5 mAb alone with that of the conjugate ADC14, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC14 (1 μg/mL), are shown in FIG. 30. The anti-CD5 mAb alone, at a concentration of 50 μg/mL, was virtually inactive in all the cell lines tested. ADC14, at a concentration of 1 μg/mL, showed specific cytotoxic activity against the CD5 positive cells, Karpas-299 and MOLT-4, inducing an inhibition in cell survival of around 84% and 70%, respectively, while being virtually inactive against CD5 negative cells (FIG. 30).

Bioactivity Example 15—Cytotoxicity of ADC16 and Related Reagents Against CD4 Positive and Negative Human Tumor Cells

(384) The in vitro cytotoxic activity of ADC16 along with the parent cytotoxic Compounds 1, 4, and 40, was evaluated against different human cancer cell lines expressing or not the CD4 antigen, including Karpas-299 and U937 (both CD4+); Raji and RPMI-8226 (both CD4−). Standard dose-response (DR) curves for 72 hours were performed.

(385) Cytotoxicity of Anti-CD4 mAb

(386) First of all, the in vitro cytotoxic activity of the anti-CD4 mouse mAb alone was assayed against different human cancer cell lines expressing or not the CD4 antigen, including Karpas-299 and U937 (both CD4+); Raji and RPMI-8226 (both CD4−). In triplicate DR curves spanning from 5.0E+01 to 1.3E−02 μg/mL (3.3E−07 to 8.7E−11 M), in two independent experiments, the antibody was virtually inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their CD4 status (Table 54).

(387) TABLE-US-00054 TABLE 54 Summary data of the in vitro cytotoxicity of Anti-CD4 mAb Anti-CD4 mAb Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) >5.0E+01 >5.0E+01 >5.0E+01 >5.0E+01 IC.sub.50 (Molar) >3.3E−07 >3.3E−07 >3.3E−07 >3.3E−07
Cytotoxicity of Compound 40

(388) The cytotoxic activity of the parent compound Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was homogenous across the different cell lines tested, with IC.sub.50 values in the low subnanomolar range, from 7.9E−05 to 2.8E−04 μg/mL (1.3E−10 to 4.7E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.5E−04 μg/mL (equivalent to 2.5E−10 M). The cytotoxicity of Compound 40 was independent of the CD4 expression levels in the tumor cell lines (Table 55).

(389) TABLE-US-00055 TABLE 55 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 1.30E−04 7.90E−05 1.20E−04 2.85E−04 IC.sub.50 (Molar) 2.15E−10 1.31E−10 1.98E−10 4.70E−10
Cytotoxicity of Compound 4

(390) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was also homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 6.1E−04 to 2.7E−03 μg/mL (9.2E−10 to 4.1E−09 M), being the mean IC.sub.50 value across the whole cell panel 1.2E−03 μg/mL (1.8E−09 M). The presence of the amine containing group in Compound 4 reduced the cytotoxic activity of the compound, as compared to Compound 40. The cytotoxic activity of Compound 4 was also independent of the CD4 expression levels in the tumor cell lines (Table 56).

(391) TABLE-US-00056 TABLE 56 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 9.10E−04 6.10E−04 6.35E−04 2.75E−03 IC.sub.50 (Molar) 1.38E−09 9.20E−10 9.60E−10 4.15E−09
Cytotoxicity of Compound 1

(392) The activity of the Compound 1 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.2E−07 to 3.0E−11 M). The cytotoxic activity of Compound 1 was also homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 1.5E−03 to 7.3E−03 μg/mL (1.7E−09 to 8.6E−09 M), being the mean IC.sub.50 value across the whole cell panel 3.4E−03 μg/mL (3.9E−09 M). The presence of the maleimide containing linker in Compound 1 did not alter the cytotoxic activity of the compound, as compared to Compound 4. The cytotoxicity of the compound was also independent of the CD4 expression levels in the tumor cell lines (Table 57).

(393) TABLE-US-00057 TABLE 57 Summary data of the in vitro cytotoxicity of Compound 1 Compound 1 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 2.35E−03 1.50E−03 2.30E−03 7.35E−03 IC.sub.50 (Molar) 2.75E−09 1.75E−09 2.69E−09 8.57E−09
Cytotoxicity of ADC16

(394) The cytotoxic activity of the ADC16 was assayed against the different tumor cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, respectively. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 31. ADC16 presented some specificity against CD4 positive cells, although the mean difference in sensitivity with respect to the CD4 negative cells was relatively low, approximately 7 fold (being the maximum difference between the less and the most sensitive cell line, Raji and Karpas-299, respectively, was around 14 times) (Table 58). Although showing a small therapeutic window, it was likely that, at least part of the cytotoxicity of ADC16 observed was mediated by the interaction of the mAb and the CD4 glycoprotein in the cell membrane of tumor cells.

(395) TABLE-US-00058 TABLE 58 Summary data of the in vitro cytotoxicity of ADC16 ADC16 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 4.70E−02 8.18E−02 2.60E−01 6.74E−01 Mean IC.sub.50 (μg/mL) CD4 positive cells 6.44E−02 Mean IC.sub.50 (μg/mL) CD4 negative cells 4.67E−01

(396) To graphically compare the cytotoxic activity of the anti-CD4 mAb alone with that of the conjugate ADC16, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC16 (1 and 0.1 μg/mL), are shown in FIG. 32. The anti-CD4 mAb alone, at a concentration of 50 mg/mL, was virtually inactive in all the cell lines tested. In contrast, ADC16, at a concentration of 1 μg/mL, showed potent cytotoxic activity in all the cell lines tested, independently of their CD4 status, causing more than 60% reduction (range 60-90%) in the cell survival after 72 hours of treatment. Even at a concentration of 0.1 μg/mL, ADC16 showed specific cytotoxic activity against the CD4 positive cells, Karpas-299 and U937, inducing an inhibition in cell survival of around 80% and 70%, respectively, while being inactive against CD4 negative cells (FIG. 32).

Bioactivity Example 16—Cytotoxicity of ADC17 and Related Reagents Against CD4 Positive and Negative Human Tumor Cells

(397) The in vitro cytotoxic activity of ADC17 along with the parent cytotoxic Compounds 12, 4, and 40, was evaluated against different human cancer cell lines expressing or not the CD4 antigen, including Karpas-299 and U937 (both CD4+); Raji and RPMI-8226 (both CD4−). Standard dose-response (DR) curves for 72 hours were performed.

(398) Cytotoxicity of Compound 40

(399) The cytotoxic activity of the parent Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was homogenous across the different cell lines tested, with IC.sub.50 values in the low subnanomolar range, from 7.9E−05 to 2.8E−04 μg/mL (1.3E−10 to 4.7E−10 M), being the mean IC.sub.50 value across the whole cell panel 1.5E−04 μg/mL (equivalent to 2.5E−10 M). The cytotoxicity of Compound 40 was independent of the CD4 expression levels in the tumor cell lines (Table 59).

(400) TABLE-US-00059 TABLE 59 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 1.30E−04 7.90E−05 1.20E−04 2.85E−04 IC.sub.50 (Molar) 2.15E−10 1.31E−10 1.98E−10 4.70E−10
Cytotoxicity of Compound 4

(401) The cytotoxic activity of Compound 4 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was also homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 6.1E−04 to 2.7E−03 μg/mL (9.2E−10 to 4.1E−09 M), being the mean IC.sub.50 value across the whole cell panel 1.2E−03 μg/mL (1.8E−09 M). The presence of the amine containing group in Compound 4 reduced the cytotoxic activity of the compound, as compared to Compound 40. The cytotoxic activity of Compound 4 was also independent of the CD4 expression levels in the tumor cell lines (Table 60).

(402) TABLE-US-00060 TABLE 60 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 9.10E−04 6.10E−04 6.35E−04 2.75E−03 IC.sub.50 (Molar) 1.38E−09 9.20E−10 9.60E−10 4.15E−09
Cytotoxicity of Compound 12

(403) The activity of the Compound 12 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−04 μg/mL (7.9E−07 to 2.1E−10 M). The cytotoxic activity of Compound 12, in two independent experiments, was relatively homogenous across the different cell lines tested, with IC.sub.50 values in the nanomolar range, from 7.3E−02 to 4.1E−01 μg/mL (5.8E−08 to 3.2E−07 M), being the mean IC.sub.50 value across the whole cell panel 1.7E−01 μg/mL (1.3E−07 M). The presence of the long maleimide containing linker in Compound 12 strongly reduced the cytotoxic activity of the compound, as compared to Compound 40 (nearly 3 log) and Compound 4 (nearly 2 log). In addition, the cytotoxicity of the compound was also independent of the CD4 expression levels of the tumor cell lines (Table 61).

(404) TABLE-US-00061 TABLE 61 Summary data of the in vitro cytotoxicity of Compound 12 Compound 12 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 7.65E−02 7.30E−02 1.15E−01 4.15E−01 IC.sub.50 (Molar) 6.07E−08 5.79E−08 9.11E−08 3.29E−07
Cytotoxicity of ADC17

(405) The cytotoxic activity of the ADC17 was assayed against the different tumor cell lines. The conjugate was assayed in four different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 50, 10, 1 and 0.1 μg/mL, respectively. A representative DR curve (starting concentration 1 μg/mL) is shown in FIG. 33. ADC17 presented specificity against CD4 positive cells, with a mean difference in sensitivity with respect to the CD4 negative cells around 40 fold (range between 11-64 fold) (Table 62). It was likely that a major part of the cytotoxic activity of ADC17 observed was mediated by the interaction of the mAb and the CD4 glycoprotein in the cell membrane of tumor cells.

(406) TABLE-US-00062 TABLE 62 Summary data of the in vitro cytotoxicity of ADC17 ADC17 Cell lines CD4+ CD4− Karpas-299 U937 RPMI18226 Raji IC.sub.50 (μg/mL) 4.50E−02 3.86E−02 5.24E−01 2.90E+00 Mean IC.sub.50 (μg/mL) CD4 positive cells 4.18E−02 Mean IC.sub.50 (μg/mL) CD4 negative cells 1.71E+00

(407) To graphically compare the cytotoxic activity of the anti-CD4 mAb alone with that of the conjugate ADC17, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC17 (1 and 0.1 μg/mL), are shown in FIG. 34. Anti-CD4 mAb alone, at a concentration of 50 μg/mL, was virtually inactive in all the cell lines tested. In contrast, ADC17, at a concentration of 1 ug/mL, showed potent cytotoxic activity in three out of the four cell lines tested (except Raji cells), causing more than 70% reduction (range 72-95%) in the cell survival after 72 hours of treatment. Even at a concentration of 0.1 μg/mL, ADC17 showed specific cytotoxic activity against the CD4 positive cells, Karpas-299 and U937, inducing an inhibition in cell survival of around 70% and 75%, respectively, while being quite inactive against CD4 negative cells (FIG. 34).

Bioactivity Example 17—Cytotoxicity of ADC14 and Related Reagents Against Raji Cell Clones with High or Null CD5 Expression

(408) The in vitro cytotoxic activity of ADC14 along with the parent cytotoxic Compounds 1, 4, and 40, was evaluated against Raji cell clones expressing or not the CD5 antigen. Standard dose-response (DR) curves for 72 hours were performed.

(409) Cytotoxicity of Anti-CD5 mAb

(410) The in vitro cytotoxic activity of the anti-CD5 mouse mAb alone was assayed against Raji cell clones expressing (C #10) or not (C #18) the CD5 antigen. In triplicate DR curves ranging from 5.0E+01 to 1.3E−02 μg/mL (3.3E−07-8.7E−11 M), in two independent experiments, the antibody was virtually inactive, not reaching the IC.sub.50 in any of the cell lines tested, independently of their CD5 status (Table 63).

(411) TABLE-US-00063 TABLE 63 Summary data of the in vitro cytotoxicity of Anti-CD5 mAb Anti-CD5 mAb Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) >5.0E+01 >5.0E+01 IC.sub.50 (Molar) >3.3E−07 >3.3E−07
Cytotoxicity of Compound 40

(412) The cytotoxic activity of the parent Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was relatively similar between the CD5 expressing (clone #10) and non expressing (clone #18) Raji cells, with mean IC.sub.50 values in the subnanomolar range, 4.95E−04 and 8.90E−04 μg/mL (equivalent to 8.17E−10 and 1.47E−09 M), respectively. Although slightly higher in CD5 positive cells, the cytotoxicity of Compound 40 seemed to be rather independent of the CD5 expression levels in the tumor cell lines (Table 64)

(413) TABLE-US-00064 TABLE 64 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) 4.95E−04 8.90E−04 IC.sub.50 (Molar) 8.17E−10 1.47E−09
Cytotoxicity of Compound 4

(414) The cytotoxic activity of Compound 4 was assayed in DR response curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was relatively similar between the CD5 expressing (clone #10) and non expressing (clone #18) Raji cells, although in the null cells the compound did not reach the IC.sub.50 value. In the CD5 positive cells, the compound showed a mean IC.sub.50 value of 9.9E−03 μg/mL (equivalent to 1.57E−08 M). Although slightly higher in CD5 positive cells, the cytotoxicity of Compound 4 seemed to be rather independent of the CD5 expression levels in the tumor cells lines (see IC.sub.20 values in Table 65 as a reference).

(415) TABLE-US-00065 TABLE 65 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.20 (μg/mL) 4.65E−03 6.77E−03 IC.sub.20 (Molar) 7.01E−09 1.02E−08 IC.sub.50 (μg/mL) 9.90E−03 >1.00E−02 IC.sub.50 (Molar) 1.49E−08 >1.51E−08
Cytotoxicity of Compound 1

(416) The activity of Compound 1 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−01 to 2.6E−05 μg/mL (1.2E−07 to 3.0E−11 M). The cytotoxic activity of Compound 1 differs between the two Raji cell clones, being more active (around 1 log) in CD5 overexpressing cells (clone #10) than in CD5 null cells (clone #18), with mean IC.sub.50 values of 2.9E−03 and 3.8E−02 μg/mL (equivalent to 3.4E−09 and 4.4E−08 M), respectively (Table 66). In this case the cytotoxicity of Compound 1 seemed not to be independent of the CD5 status of the tumor cell lines.

(417) TABLE-US-00066 TABLE 66 Summary data of the in vitro cytotoxicity of Compound 1 Compound 1 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) 2.90E−03 3.80E−02 IC.sub.50 (Molar) 3.39E−09 4.44E−08
Cytotoxicity of ADC14

(418) The cytotoxic activity of the ADC14 was assayed against the two Raji clones. The conjugated was assayed in three different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 10, 1 and 0.1 μg/mL. A representative DR curve (starting concentration 10 μg/mL) is shown in FIG. 35. ADC 14 showed specificity against CD5 overexpressing cells (clone #10), in which the compound demonstrated a cytotoxic activity similar to, or even higher than, that of the parent Compounds 1, 4 and 40. In CD5 expressing Raji cells, the conjugate showed a mean IC.sub.50 value of 1.6E−01 μg/mL. In CD5 null cells the conjugate was more than 50 fold less active than in CD5 positive cells, showing a mean IC.sub.50 value of 9.0E+00 μg/mL. Although with some reservations, due to some differential sensitivity observed between the two Raji cell clones against some parent compounds, these results indicated that ADC14 has specificity against CD5 expressing cells (Table 67). We assume, therefore, that ADC14 was, at least partially, acting through the interaction of the mAb with the membrane associated CD5 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(419) TABLE-US-00067 TABLE 67 Summary data of the in vitro cytotoxicity of ADC14 ADC14 Raji Cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) 1.65E−01 9.00E+00

(420) To graphically compare the cytotoxic activity of the anti-CD5 mAb alone with that of the conjugate ADC14, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC14 (10 μg/mL), are shown in FIG. 36. Anti-CD5 mAb alone, at a concentration of 50 μg/mL, was inactive against the two Raji cell clones, independently of their CD5 status. On the contrary ADC14, at a concentration of 10 μg/mL, showed potent and somehow selective cytotoxic activity against CD5 positive Raji cells (clone #10), causing a nearly 90% reduction in their cell survival after 72 hours of treatment. Under the same conditions, ADC14 caused a 30% reduction in the cell survival of CD5 null cells (clone #18) (FIG. 36).

Bioactivity Example 18—Cytotoxicity of ADC15 and Related Reagents Against Raji Cell Clones with High or Null CD5 Expression

(421) The in vitro cytotoxic activity of ADC15 along with the parent cytotoxic Compounds 12, 4, and 40, was evaluated against Raji cell clones expressing or not the CD5 antigen. Standard dose-response (DR) curves for 72 hours were performed.

(422) Cytotoxicity of Compound 40

(423) The cytotoxic activity of the parent Compound 40 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E−03 to 2.6E−07 μg/mL (1.7E−09 to 4.3E−13 M). The cytotoxic activity of Compound 40 was relatively similar between the CD5 expressing (clone #10) and non expressing (clone #18) Raji cells, with mean IC.sub.50 values in the subnanomolar range, 4.95E−04 and 8.90E−04 μg/mL (equivalent to 8.17E−10 and 1.47E−09 M), respectively. Although slightly higher in CD5 positive cells, the cytotoxicity of Compound 40 seemed to be rather independent of the CD5 expression levels in the tumor cell lines (Table 68).

(424) TABLE-US-00068 TABLE 68 Summary data of the in vitro cytotoxicity of Compound 40 Compound 40 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) 4.95E−04 8.90E−04 IC.sub.50 (Molar) 8.17E−10 1.47E−09
Cytotoxicity of Compound 4

(425) The cytotoxic activity of Compound 4 was assayed in DR response curves using ten serial dilutions (1/2.5 ratio) from 01E−02 to 2.6E−06 μg/mL (1.5E−08 to 4.0E−12 M). The cytotoxic activity of Compound 4 was relatively similar between the CD5 expressing (clone #10) and non expressing (clone #18) Raji cells, although in the null cells the compound did not reach the IC.sub.50 value. In the CD5 positive cells, the compound showed a mean IC.sub.50 value of 9.9E−03 μg/mL (equivalent to 1.57E−08 M). Although slightly higher in CD5 positive cells, the cytotoxicity of Compound 4 seemed to be rather independent of the CD5 expression levels in the tumor cell lines (see IC.sub.20 values in Table 69 as a reference).

(426) TABLE-US-00069 TABLE 69 Summary data of the in vitro cytotoxicity of Compound 4 Compound 4 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.20 (μg/mL) 4.65E−03 6.77E−03 IC.sub.20 (Molar) 7.01E−09 1.02E−08 IC.sub.50 (μg/mL) 9.90E−03 >1.00E−02 IC.sub.50 (Molar) 1.49E−08 >1.51E−08
Cytotoxicity of Compound 12

(427) The activity of Compound 12 was assayed in DR curves using ten serial dilutions (1/2.5 ratio) from 01E+00 to 2.6E−4 μg/mL (7.9E−07 to 2.1E−10 M). The cytotoxic activity of Compound 12 was relatively similar between the CD5 expressing (clone #10) and non expressing (clone #18) Raji cells, although in the null cells the compound did not reach the IC.sub.50 value. In the CD5 positive cells, the compound showed a mean IC.sub.50 value of 2.7E−01 μg/mL (equivalent to 2.15E−07 M). Although slightly higher in CD5 positive cells, the cytotoxicity of Compound 12 seemed to be rather independent of the CD5 expression levels in the tumor cell lines (see IC.sub.20 values in Table 70 as reference).

(428) TABLE-US-00070 TABLE 70 Summary data of the in vitro cytotoxicity of Compound 12 Compound 12 Raji cells C#10 (high CD5) C#18 (null CD5) IC.sub.20 (μg/mL) 1.50E−01 2.00E−01 IC.sub.20 (Molar) 1.19E−07 1.59E−07 IC.sub.50 (μg/mL) 2.70E−01 >1.00E+00 IC.sub.50 (Molar) 2.14E−07 >7.92E−07
Cytotoxicity of ADC15

(429) The cytotoxic activity of the ADC15 was assayed against the two Raji clones. The conjugate was assayed in three different concentration ranges, each in triplicate DR curves (ten serial dilutions, 1/2.5 ratio) starting from 10, 1 and 0.1 μg/mL. A representative DR curve (starting concentration 10 μg/mL) is shown in FIG. 37. ADC15 showed significant specificity against CD5 overexpressing cells (clone #10), in which the compound demonstrated a cytotoxic activity similar to that of the parent compound 40 and even higher than that of Compounds 4 and 12. In CD5 expressing Raji cells, the conjugate showed a mean IC.sub.50 value of 9.3E−01 μg/mL. In CD5 null cells the conjugate was much more than 10 fold less active than in CD5 positive cells, not reaching the IC.sub.50 value. Although with reservations, due to the potential sensitivity observed between the two Raji cell clones against the parent Compounds 4 and 12, these results indicate that ADC15 has specificity against CD5 expressing cells (Table 71). We can assume that ADC15 was, at least partially, acting through the interaction of the mAb with the membrane associated CD5 receptor on tumor cells, and subsequent intracellular delivery of the cytotoxic drug into the target tissue.

(430) TABLE-US-00071 TABLE 71 Summary data of the in vitro cytotoxicity of ADC15 ADC15 Raji Cells C#10 (high CD5) C#18 (null CD5) IC.sub.50 (μg/mL) 9.30E−01 >1.0E+01

(431) To graphically compare the cytotoxicity activity of the anti-CD5 mAb alone with that of the conjugate ADC15, histograms showing the percentages of cell survival after treatment of the different cell lines with the mAb alone (50 μg/mL) or ADC15 (10 μg/mL), are shown in FIG. 38. Anti-CD5 mAb alone, at a concentration of 50 μg/mL, was inactive against the two Raji cell clones, independently of their CD5 status. On the contrary, ADC15 at a concentration of 10 μg/mL showed potent and selective cytotoxic activity against CD5 positive Raji cells (clone #10), causing a 80% reduction in their cell survival after 72 hours of treatment. Under the same conditions, ADC15 was inactive on CD5 null cells (clone #18) (FIG. 38).

Antibody drug conjugates

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C07D309/30

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A61P35/02

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Abstract

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