Tetramaleimide linkers and use thereof

Abstract

The present invention is directed to tetramaleimide linkers and use thereof, more specifically to the compounds represented by formula I and their use in the preparation of antibody-drug conjugates (ADCs). The ADCs obtained from the tetramaleimide linkers have high homogeneity and stability, and could be used effectively for the treatment of various diseases including tumors. The definition of the groups in formula I is the same as that in the description. ##STR00001##

Claims

1. An antibody-drug conjugate of formula IV: ##STR00060## wherein L is an antibody or antibody fragment; A is optionally other linker than tetramaleimide linker, including cleavable and noncleavable linker; D is a drug molecule; four maleimide groups are simultaneously linked to the same antibody or antibody fragment; P and Q are each independently selected from CR.sup.10, N and aryl; S and T are each independently selected from C═O and O; X and Y are each independently selected from —C(O)N(R.sup.11)—, —N(R.sup.12)C(O)— and —O—; Z is selected from CR.sup.13, N and aryl; U is selected from C═O and O; J′ is selected from C═O, O and NR.sup.14; h, i, j, k, l, m, p, q, s, t, x, y, u and w are each independently selected from 0 and 1; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are each independently selected from C.sub.1-C.sub.6 alkylene, and C.sub.1-C.sub.6 alkylene containing O in the backbone; R.sup.10, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are each independently selected from H and C.sub.1-C.sub.6 alkyl; and n is an integer of 1 to 4.

2. An antibody-drug conjugate of formula IV according to claim 1, wherein the antibody targets cell surface receptors or tumor-related antigens.

3. An antibody-drug conjugate of formula IV according to claim 2, wherein the antibody is IgG1.

4. An antibody-drug conjugate of formula IV according to claim 3, wherein the drug is cytotoxic drug, anti-autoimmune disease drug, or anti-inflammation drug.

5. A pharmaceutical composition comprising an antibody-drug conjugate of formula IV according to claim 1 and pharmaceutically acceptable carriers.

6. A method of making an antibody-drug conjugates, comprising conjugating an antibody to a drug using a compound of formula I as a linker, ##STR00061## and pharmaceutically acceptable salts thereof, wherein P and Q are each independently selected from CR.sup.10, N and aryl; S and T are each independently selected from C═O and O; X and Y are each independently selected from —C(O)N(R.sup.11)—, —N(R.sup.12)C(O)— and —O—; Z is selected from CR.sup.13, N and aryl; U is selected from C═O and O; J is selected from —COOH, —OH and —NHR.sup.14; h, i, j, k, l, m, p, q, s, t, x, y, u and w are each independently selected from 0 and 1; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are each independently selected from C.sub.1-C.sub.6 alkylene and C.sub.1-C.sub.6 alkylene containing O in the backbone; R.sup.10, R.sup.11, R.sup.12, R.sup.13 and R.sup.14 are each independently selected from H and C.sub.1-C.sub.6 alkyl.

7. An antibody-drug conjugate of formula IV according to claim 1 wherein A has a formula of C-E.sub.e-F.sub.f or G.sub.g; wherein C is a cleavable linker; E and F are self-immolative linkers; e and f are each independently selected from an integer of 0 to 5; G is a noncleavable linker; g is an integer of 0 to 5.

8. The method according to claim 6, wherein: X and Y are each independently selected from —C(O)N(R.sup.11)—; x and y are each independently selected from 0 and 1; R.sup.11 is selected from H and C.sub.1-C.sub.6 alkyl.

9. The method according to claim 6, wherein: Z is selected from CR.sup.13, N and C.sub.6-C.sub.10 aryl; R.sup.13 is selected from H and C.sub.1-C.sub.6 alkyl.

10. The method according to claim 6, wherein: R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are each independently selected from C.sub.1-C.sub.6 alkylene; h, i, j and k are each independently selected from 0 and 1.

11. The method according to claim 6, wherein: S and T are each independently selected from C═O and O; R.sup.5 and R.sup.6 are each independently selected from C.sub.1-C.sub.6 alkylene; l and m are each independently selected from 0 and 1; s and t are each independently selected from 0 and 1.

12. The method according to claim 6, wherein: R.sup.7 and R.sup.8 are each independently selected from C.sub.1-C.sub.6 alkylene and C.sub.1-C.sub.6 alkylene containing O in the backbone; p and q are each independently selected from 0 and 1.

13. The method according to claim 6, wherein: U is selected from C═O and O; R.sup.9 is selected from C.sub.1-C.sub.6 alkylene; u and w are each independently selected from 0 and 1.

14. The method according to claim 6, wherein: J is selected from —COOH, OH and NH.sub.2.

15. The method according to claim 6, wherein the compounds are selected from: ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the native MS spectrum of H-5-vcMMAE of the invention.

(2) FIGS. 2a-2b illustrate the SDS-PAGE results of the antibody-drug conjugates based on tetramaleimide linkers, wherein 2a represents the SDS-PAGE result of H-1-vcMMAE to H-6-vcMMAE (corresponding to 1-6 respectively); 2b represents the SDS-PAGE result of H-7-vcMMAE to H-12-vcMMAE (corresponding to 7-12 respectively).

(3) FIGS. 3a-3l illustrate the HIC results of the antibody-drug conjugates, wherein 3a-3l correspond to H-1-vcMMAE to H-12-vcMMAE respectively; 3m corresponds to P-7-vcMMAE.

EXAMPLES

(4) The present invention will be further described in details with the following examples. However, it should be understood that these examples are used to illustrate the present invention, but should not be considered as limiting the scope of the invention. The unstated experiment conditions are generally according to routine conditions or conditions suggested by manufacturers. All reactions were conducted under nitrogen atmosphere, except for hydrogenation reaction.

(5) Unless otherwise defined, all of the professional and scientific terms used in the present invention have the same meaning as those familiar by the expertise in the art. Furthermore, any method or material similar or equal to those used in the present invention can be applied herein. The optimized methods and materials used in the present invention are only used for illustration while not for limitation.

(6) Abbreviation

(7) Ab antibody Ac acetyl ACN acetonitrile ADC antibody-drug conjugate BOC (Boc) tert-butoxycarbonyl Cbz benzyloxy carbonyl t-Bu tert-butyl DCM dichloromethane DIPEA diisopropylethylamine DMF N,N-dimethylformamide ELISA enzyme linked immunosorbent assay EtOAc ethyl acetate Eq (eq) equivalent g gram h hour HATU 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOSu N-hydroxy succinimide HIC hydrophobic interaction chromatography HPLC high performance liquid chromatography LC-MS liquid chromatography-mass spectrum mAb monoclonal antibody min minute mL milliliter MS mass spectrometry nm nanometer μg microgram μL microliter PE petroleum ether RP-HPLC reverse phase-high performance liquid chromatography prep-RP-HPLC preparative-reverse phase-high performance liquid chromatography rt room temperature R.sub.t retention time SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electropheresis SEC size exclusion chromatography TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TsCl p-tolyl chloride

(8) Unless otherwise stated, all of the anhydrous solvents are purchased from the suppliers and kept under nitrogen atmosphere. All other reagents and solvents purchased are of high purity and need not to be purified before use.

(9) The structure of the compound is identified by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR chemical shifts (δ) are given in 10.sup.−6 (ppm). NMR is determined by Bruker AVANCE III 500. The solvents are deuterated-dimethyl sulfoxide (DMSO-d.sub.6), deuterated-chloroform (CDCl.sub.3) and deuterated-methanol (CD.sub.3OD) with tetramethylsilane (TMS) as an internal standard.

(10) Liquid chromatography-mass spectrometry (LC-MS) is determined on Agilent 6110 (acid method) or 6120B (base method) mass spectrometers coupled with Hewlette-Packard Agilent 1200 HPLC.

(11) Method 1: Waters Sunfire C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.4 min. The flow rate is 2.0 mL/min, and the column temperature is 50° C.

(12) Method 2: Waters Sunfire C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.4 min. The flow rate is 2.3 mL/min, and the column temperature is 50° C.

(13) Method 3: Waters Sunfire C18 reverse phase column (3.0×30 mm, 2.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.5 min. The flow rate is 1.5 mL/min, and the column temperature is 50° C.

(14) Method 4: Waters Sunfire C18 reverse phase column (4.6×50 mm, 3.5 μm) is used in the acid HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile, containing 0.01% TFA) in A (water, containing 0.01% TFA) over 1.2 min. The flow rate is 2.0 mL/min, and the column temperature is 50° C.;

(15) Method 5: Waters Xbridge C18 reverse phase column (4.60×50 mm, 3.5 μm) is used in the base HPLC method for separation, and the eluting gradient is 5%-95% B (acetonitrile) in A (water, containing 10 mM ammonium bicarbonate) over 1.5 min. The flow rate is 2.0 mL/min, and the column temperature is 40° C.

(16) Purification by preparative HPLC is conducted on a Gilson instrument. Waters Sunfire C18 reverse phase column (250×19 mm, 10 μm) is used for separation.

(17) Method 6: The acid HPLC preparation method. Mobile phase: A is aqueous solution containing 0.1% TFA; B is ACN. The flow rate is 20 mL/min.

(18) SK-BR-3 human breast cancer cell is purchased from ATCC. Her2 antigen is purchased from Sino Biological Inc (Beijing). Antibody H (Herceptin Biosimilar, IgG1) is purchased from Genor Biopharma Co. Ltd. (Shanghai). Antibody P (Perjeta Biosimilar, IgG1) is purchased from Biochem partner Co. Ltd. (Shanghai). The enzyme labeled anti-antibody is purchased from Sigma (Shanghai). Substrate solution is purchased from Decent Biotech (Shanghai). Cell Counting Kit (CCK-8) cell proliferation-cytotoxicity assay kit is purchased from Dojindo (Shanghai).

Example 1

(19) Synthesis of Compound 1 (Linker 1)

(20) ##STR00045##

(21) (S)-4,5-Diaminopentanoic acid dihydrochloride (15) (10 mg, 49 μmol, prepared according to Tetrahedron Asymmetry, 1993, 4, 91-100) and compound 16 (38 mg, 98 μmol, prepared according to WO2014114207) were dissolved in DMF (0.5 mL), to which DIPEA (25 mg, 196 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 32%-40% B in 8 min.fwdarw.95% B in 4 min) to give compound 1 (12 mg) as a white solid.

(22) LC-MS (method 1): Rt=1.39 min; m/z (ES+) 681.1 (M+H).sup.+.

(23) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 6.80 (s, 4H), 6.78 (s, 4H), 4.20-4.12 (m, 2H), 4.01-3.96 (m, 2H), 3.92-3.87 (m, 1H), 3.71-3.66 (m, 2H), 3.37-3.32 (m, 1H), 3.11-3.07 (m, 1H), 2.42-2.30 (m, 4H), 2.28-2.08 (m, 6H), 1.83-1.76 (m, 1H), 1.66-1.59 (m, 1H).

Example 2

(24) Synthesis of Compound 2 (Linker 2)

(25) ##STR00046##

(26) 2-(1,3-Diamino-2-propoxy)acetic acid dihydrochloride (17) (10 mg, 45 μmol, preparation according to WO2014114207) and compound 16 (35 mg, 90 μmol) were dissolved in DMF (0.5 mL), to which DIPEA (23 mg, 180 μmol) was then added. The reaction mixture was stirred at room temperature for 4 h, and then purified by RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4) to give compound 2 (9 mg) as a white solid.

(27) LC-MS (method 2): Rt=1.63 min; m/z (ES+) 697.0 (M+H).sup.+.

(28) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 6.79 (s, 4H), 6.78-6.76 (m, 4H), 4.22 (s, 2H), 4.18-4.14 (m, 2H), 4.00-3.95 (m, 2H), 3.70-3.66 (m, 2H), 3.50-3.45 (m, 1H), 3.31-3.27 (m, 1H), 3.24-3.23 (d, 2H), 3.18-3.14 (m, 1H), 2.46-2.38 (m, 2H), 2.29-2.17 (m, 4H), 2.14-2.08 (m, 2H).

Example 3

(29) Synthesis of Compound 3 (Linker 3)

(30) ##STR00047##

(31) 4,5-Bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanoic acid (18) (10 mg, 65 μmol, prepared according to WO2014114207) and 3,5-diaminobenzoic acid (38 mg, 130 μmol) were dissolved in DMF (0.6 mL), to which HATU (62 mg, 160 μmol) and DIPA (18 mg, 140 μmol) were then added. The reaction mixture was stirred at room temperature for 18 h, and then purified by RP-HPLC (method 6: 40%-70% Bin 8 min.fwdarw.95% B in 4) to give compound 3 (6 mg) as a white solid.

(32) LC-MS (method 2): Rt=1.74 min; mz (ES+) 700.8 (M+H).sup.+.

(33) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 10.03 (s, 2H), 8.02 (s, 1H), 7.84 (s, 2H), 7.01 (s, 4H), 6.98 (s, 4H), 4.09-4.02 (m, 2H), 3.84-3.75 (m, 2H), 3.65-3.61 (m, 2H), 2.33-2.25 (m, 6H), 2.06-1.95 (m, 2H).

Example 4

(34) Synthesis of Compound 4 (Linker 4)

(35) ##STR00048##

(36) 2-(1,4-Diaminobutan-2-yloxy)acetic acid (19) (20 mg, 85 μmol, prepared according to WO2014114207) and compound 16 (66 mg, 170 μmol) were dissolved in DMF (0.4 mL), to which DIPEA (44 mg, 340 μmol) was then added. The reaction mixture was stirred at room temperature for 4 h, and then purified by RP-HPLC (method 6: 35%-60% B in 8 min.fwdarw.95% B in 4) to give compound 4 (9 mg) as a white solid.

(37) LC-MS (method 1): Rt=1.41 min; m/z (ES+) 711.1 (M+H).sup.+.

(38) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 6.80 (s, 4H), 6.77 (s, 4H), 4.21 (s, 2H), 4.18-4.12 (m, 2H), 4.00-3.94 (m, 2H), 3.70-3.65 (m, 2H), 3.55-3.51 (m, 1H), 3.49-3.35 (m, 1H), 3.30-3.14 (m, 3H), 2.46-2.38 (m, 2H), 2.29-2.17 (m, 4H), 2.15-2.08 (m, 2H), 1.68-1.63 (m, 2H).

Example 5

(39) Synthesis of Compound 5 (Linker 5)

(40) ##STR00049##

Step 1: Synthesis of 5-(bis(2-(benzyloxycarbonylamino)ethyl)amino)-5-oxopentanoic Acid (21)

(41) Bis(2-(benzyloxycarbonylamino)ethyl)amine hydrochloride (20) (815 mg, 2 mmol, prepared according to European Journal of Medicinal Chemistry, 2009, 44, 678-688) and TEA (0.70 mL, 5 mmol) were dissolved in DMF (5 mL), to which glutaric anhydride (228 mg in 1 mL DMF, 2 mmol) was then added dropwise. The reaction mixture was stirred at room temperature overnight, and then water (20 mL) was added. The mixture was extracted with DCM (15 mL×3), and the combined organic phase was sequentially washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH 30:1) to give compound 21 (872 mg) as a pale yellow solid.

(42) LC-MS (method 3): Rt=1.21 min; m/z (ES+) 486.3 (M+H).sup.+.

Step 2: Synthesis of 5-(bis(2-aminoethyl)amino)-5-oxopentanoic Acid Dihydrobromide (22)

(43) A solution of HBr in acetic acid (33%, 3 mL) was added dropwise to compound 21 (522 mg, 1.1 mmol), and then the reaction mixture was stirred at room temperature for 15 min. Diethyl ether (20 mL) was added to the mixture, and the yellow precipitate was separated by centrifugation. The solid was suspended in diethyl ether (10 mL), and then was collected by centrifugation. The two-step process was repeated three times, after which the solid obtained was dried in vacuo (60° C.) to give hydrobromide of compound 22 (350 mg) as a yellow solid.

(44) LC-MS (method 4): Rt=0.28 min; m/z (ES+) 218.0 (M+H).sup.+.

Step 3: Synthesis of Compound 5

(45) Compound 22 hydrobromide (45 mg, 119 μmol) and compound 16 (50 mg, 128 μmol) were dissolved in DMF (5 mL), to which DIPEA (37 mg, 287 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4) to give compound 5 (30 mg) as a white solid.

(46) LC-MS (method 2): Rt=1.62 min; m/z (ES+) 765.9 (M+H).sup.+.

(47) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.95 (t, 1H), 7.84 (t, 1H), 7.00 (s, 4H), 6.97 (d, 4H), 4.01-3.95 (m, 2H), 3.80-3.75 (m, 2H), 3.61-3.56 (m, 2H), 3.25-3.16 (m, 4H), 3.15-3.06 (m, 4H), 2.27 (t, 2H), 2.23-2.15 (m, 4H), 2.04-1.98 (m, 4H), 1.96-1.88 (m, 2H), 1.72-1.66 (m, 2H).

Example 6

(48) Synthesis of Compound 6 (Linker 6)

(49) ##STR00050##

Step 1: Synthesis of (2-(tert-butoxycarbonylamino)ethyl)(3-(tert-butoxycarbonyl-amino)propyl)amine (23)

(50) CDI (2.9 g, 18.0 mmoL), tert-butyl alcohol (1.33 g, 18.0 mmoL) and potassium hydroxide (24 mg, 0.43 mmoL) were sequentially added to toluene (30 mL), and the reaction mixture was stirred at 60° C. for 3 h. N-(2-aminoethyl)propane-1,3-diamine (1.0 g, 8.55 mmol) was added to the mixture, and then the reaction mixture was stirred at 60° C. for 3 h. The reaction mixture was cooled to room temperature, and concentrated under reduced pressure to remove the solvent. DCM (50 mL) was added to the residue, and then the mixture was washed with water (30 mL×3). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 23 (1.0 g) as colorless oil. The crude product was used directly in the next step without purification.

Step 2: Synthesis of 4-((2-(tert-butoxycarbonylamino)ethyl)(3-(tert-butoxy carbonyl-amino)propyl)amino)-4-oxobutanoic Acid (24)

(51) Compound 23 (1.0 g) was dissolved in DCM (15 mL), to which succinic anhydride (0.47 g, 4.73 mmol) and DIPEA (1.22 g, 9.46 mmol) were then sequentially added. The reaction mixture was stirred at room temperature for 18 h, and then washed with water (30 mL×2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 24 (1.2 g) as colorless oil. The crude product was used directly for next step without purification.

(52) LC-MS (method 5): Rt=1.68 min; m/z (ES+) 418.3 (M+H).sup.+.

Step 3: Synthesis of 4-((2-aminoethyl)(3-aminopropyl)amino)-4-oxobutanoic Acid (25)

(53) 1,4-dioxane (6 mL) and concentrated hydrochloric acid (3 mL) were sequentially added to compound 24 (1.2 g), and the reaction mixture was stirred at room temperature for 2 h. The mixture was concentrated under reduced pressure to remove the solvent. The residue was dissolved in toluene, and then concentrated under reduced pressure to remove the solvent (repeated for 3 times). The residue was dried in vacuo to give compound 25 (520 mg) as a pale yellow solid.

(54) LC-MS (method 5): Rt=0.32 min; m/z (ES+) 218.2 (M+H).sup.+.

Step 4: Synthesis of Compound 6

(55) Compound 25 (24 mg, 83 μmol) and compound 16 (65 mg, 166 μmol) were dissolved in DMF (0.4 mL), to which DIPEA (43 mg, 332 μmol) was then added. The reaction mixture was stirred at room temperature for 4 h, and then purified by RP-HPLC (method 6: 32%-60% B in 8 min.fwdarw.95% B in 4) to give compound 6 (8 mg) as a white solid.

(56) LC-MS (method 2): Rt=1.58 min; m/z (ES+) 766.2 (M+H).sup.+.

(57) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 6.80-6.78 (m, 8H), 4.19-4.13 (m, 2H), 4.01-3.95 (m, 2H), 3.70-3.67 (m, 2H), 3.48-3.36 (m, 5H), 3.30-3.06 (m, 3H), 2.68-2.62 (m, 4H), 2.44-2.38 (m, 2H), 2.25-2.06 (m, 6H), 1.82-1.70 (m, 2H).

Example 7

(58) Synthesis of Compound 7 (Linker 7)

(59) ##STR00051##

Step 1: Synthesis of bis(3-(tert-butoxycarbonylamino)propyl)amine (26)

(60) CDI (3.4 g, 21 mmoL), tert-butylalcohol (1.55 g, 21 mmoL) and potassium hydroxide (28 mg, 0.50 mmoL) were added to toluene (30 mL) sequentially, and the reaction mixture was stirred at 60° C. for 3 h. N-(3-aminopropyl)-1,3-propyl diamine (1.31 g, 10 mmol) was added to the mixture, and the reaction mixture was stirred at 60° C. for 3 h. The reaction mixture was cooled to room temperature, and concentrated to remove the solvent. To the residue was added DCM (50 mL), and then the mixture was washed with water (30 mL×3). The organic phase was dried oven anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 26 (1.0 g) as a white solid. The crude product was used directly in next step without purification.

Step 2: Synthesis of 4-(bis(3-(tert-butoxycarbonylamino)propyl)amino)-4-oxobutanoic Acid (27)

(61) Compound 26 (1.0 g) was dissolved in DCM (15 mL), to which succinic anhydride (0.36 g, 3.6 mmol) and DIPEA (0.78 g, 6.0 mmol) were then sequentially added. The reaction mixture was stirred at room temperature for 18 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH 15:1) to give compound 27 (420 mg) as colorless oil.

Step 3: Synthesis of 4-(bis(3-aminopropyl)amino)-4-oxobutanoic Acid (28)

(62) Compound 27 (420 mg) was dissolved in DCM (900 μL), and the solution was cooled to 0° C., to which TFA (300 uL) was added. The reaction mixture was stirred at room temperature for 3 h, and then concentrated under reduced pressure to remove the solvent. The residue was dissolved in toluene, and concentrated to remove the solvent (repeated 3 times). The residue was dried in vacuo to give compound 28 (480 mg) as pale yellow oil.

(63) LC-MS (method 4): Rt=0.28, 0.34 min; m/z (ES+) 232.2 (M+H).sup.+.

Step 4: Synthesis of Compound 7

(64) Compound 28 (60 mg, 130 μmol) and compound 16 (101 mg, 260 μmol) were dissolved in DMF (0.6 mL), to which DIPEA (67 mg, 520 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 35%-58% B in 8 min.fwdarw.95% B in 4) to give compound 7 (35 mg) as a white solid.

(65) LC-MS (method 2): Rt=1.47 min; m/z (ES+) 780.2 (M+H).sup.+.

(66) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 6.80-6.78 (m, 8H), 4.18-4.13 (m, 2H), 4.00-3.95 (m, 2H), 3.71-3.67 (m, 2H), 3.42-3.35 (m, 4H), 3.25-3.08 (m, 4H), 2.68-2.67 (m, 4H), 2.45-2.38 (m, 2H), 2.25-2.08 (m, 6H), 1.86-1.80 (m, 2H), 1.73-1.68 (m, 2H).

Example 8

(67) Synthesis of Compound 8 (Linker 8)

(68) ##STR00052##

Step 1: Synthesis of methyl 3,5-bis(2-(tert-butoxycarbonylamino)ethoxy)benzoate (29)

(69) Methyl 3,5-dihydroxybenzoate (200 mg, 1.19 mmol) and tert-butyl 2-bromoethylcarbamate (666 mg, 2.98 mmol) were dissolved in DMF (10 mL), to which potassium carbonate (411 mg, 2.98 mmol) was then added. The reaction mixture was stirred at 50° C. for 18 h, and then concentrated to remove the solvent. The residue was purified by silica gel column chromatography (eluent: PE/EA 10:1) to give compound 29 (450 mg) as colorless oil.

(70) LC-MS (method 1): Rt=1.97 min; m/z (ES+) 477.0 (M+Na).sup.+.

Step 2: Synthesis of 3,5-bis(2-aminoethoxy)benzoic Acid (30)

(71) Compound 29 (200 mg) was dissolved in 1,4-dioxane (1 mL), to which concentrated hydrochloric acid (1 mL) was then added. The reaction mixture was stirred at 80° C. for 2 h, and then concentrated under reduced pressure to remove the solvent. To the residue was added toluene (3 mL), and then the mixture was concentrated under reduced pressure to remove the solvent. The same process was repeated several times until a solid was obtained. The solid was suspended in ethyl acetate, and then collected by filtration. The solid was dried in vacuo to give compound 30 (100 mg) as a brown solid. The crude product was used directly in next step without purification.

(72) LC-MS (method 1): Rt=0.32 min; m/z (ES+) 241.0 (M+H).sup.+.

Step 3: Synthesis of Compound 8

(73) Compound 30 (10 mg, 32 μmol) and compound 16 (25 mg, 64 μmol) were dissolved in DMF (0.5 mL), to which DIPEA (17 mg, 128 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 35%-65% B in 8 min.fwdarw.95% B in 4) to give compound 8 (6 mg) as a white solid.

(74) LC-MS (method 4): Rt=1.34 min; m/z (ES+) 789.2 (M+H).sup.+.

(75) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.05 (t, 2H), 7.04 (d, 2H), 7.00 (s, 4H), 6.96 (s, 4H), 6.72 (t, 1H), 4.02-3.94 (m, 6H), 3.80-3.74 (m, 2H), 3.61-3.56 (m, 2H), 3.37-3.33 (m, 4H), 2.23-2.14 (m, 2H), 2.06 (t, 4H), 1.98-1.90 (m, 2H).

Example 9

(76) Synthesis of Compound 9 (Linker 9)

(77) ##STR00053##

Step 1: Synthesis of tert-butyl bis(2-(2-hydroxyethoxy)ethyl)carbamate (32)

(78) Bis(2-(2-hydroxyethoxy)ethyl)amine (31) (4.2 g, 21.8 mmol, prepared according to Journal of Organic Chemistry, 1995, 60, 6097-6102) and TEA were dissolved in DCM (30 mL), to which di-tert-butyl dicarbonate (5.69 g, 26.1 mmol) was then added. The reaction mixture was stirred at room temperature for 18 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH 30:1.fwdarw.15:1) to give compound 32 (2.3 g) as pale yellow oil.

(79) LC-MS (method 1): Rt=1.41 min; m/z (ES+) 316.1 (M+Na).sup.+.

Step 2: Synthesis of tert-butyl bis(2-(2-(tosyloxy)ethoxy)ethyl)carbamate (33)

(80) Compound 32 (2.3 g, 7.85 mmol) and TEA (3.17 g, 31.4 mmol) were dissolved in DCM (40 mL), to which TsCl (4.49 g, 23.6 mmol) was then added slowly. The reaction mixture was stirred at room temperature for 18 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: PE/EA 3:1) to give compound 33 (3.3 g) as colorless oil.

(81) LC-MS (method 4): Rt=1.88 min; m/z (ES+) 624.2 (M+Na).sup.+.

Step 3: Synthesis of bis(2-(2-(tosyloxy)ethoxy)ethyl)amine (34)

(82) Compound 33 (3.3 g, 5.49 mmol) was dissolved in DCM (9 mL), and the reaction mixture was cooled down to 0° C., to which TFA (3 mL) was then slowly added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure to remove the solvent. The residue was dissolved in DCM, and washed with saturated sodium bicarbonate. The aqueous phase was extracted by DCM (10 mL), and the combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 34 (2.5 g) as colorless oil. The crude product was used directly in next step without purification.

Step 4: Synthesis of 4-(bis(2-(2-(tosyloxy)ethoxy)ethyl)amino)-4-oxobutanoic Acid (35)

(83) Compound 34 (2.5 g, 4.99 mmol) was dissolved in DCM (15 mL), to which succinic anhydride (0.75 g, 7.48 mmol) and DIPEA (1.93 g, 15.0 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 h, and then washed with water (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give colorless oil. Further purification by silica gel column chromatography (eluent: DCM/MeOH 20:1) gave compound 35 (1.6 g) as colorless oil.

(84) LC-MS (method 4): Rt=1.64 min; m/z (ES+) 602.1 (M+H).sup.+.

Step 5: Synthesis of 4-(bis(2-(2-azidoethoxy)ethyl)amino)-4-oxobutanoic Acid (36)

(85) Compound 35 (1.6 g, 2.66 mmol) was dissolved in DMF (10 mL), to which sodium azide (0.52 g, 7.99 mmol) was then added. The reaction mixture was stirred at 50° C. for 5 h, and then concentrated under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (eluent: DCM/MeOH 20:1) to give compound 36 (600 mg) as colorless oil.

(86) LC-MS (method 1): Rt=1.55 min; m/z (ES+) 344.1 (M+H).sup.+.

Step 6: Synthesis of 4-(bis(2-(2-aminoethoxy)ethyl)amino)-4-oxobutanoic Acid (37)

(87) Compound 36 (600 mg, 1.75 mmol) was dissolved in THF (10 mL) and water (126 μL), to which triphenylphosphine (1.37 g, 5.25 mmol) was then added. The reaction mixture was stirred at room temperature for 18 h, while insoluble oil was found on the flask wall and bottom. THF was carefully removed, and the colorless oil was washed with THF (3 mL×3) and then added with water (10 mL). The solution was lyophilized to give compound 37 (500 mg) as a white solid.

(88) LC-MS (method 5): Rt=0.35, 0.46 min; m/z (ES+) 292.1 (M+H).sup.+.

Step 7: Synthesis of Compound 9

(89) Compound 37 (20 mg, 68 μmol) and compound 16 (53 mg, 137 μmol) were dissolved in DMF (0.6 mL), to which DIPEA (71 mg, 548 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4) to give compound 9 (9 mg) as a white solid.

(90) LC-MS (method 2): Rt=1.61 min; m/z (ES+) 840.0 (M+H).sup.+.

(91) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.83-7.79 (m, 2H), 7.00 (s, 4H), 6.97 (s, 4H), 4.00-3.94 (m, 2H), 3.79-3.74 (m, 2H), 3.60-3.56 (m, 2H), 3.50-3.47 (m, 4H), 3.39 (s, 4H), 3.36-3.29 (m, 4H), 3.15-3.09 (m, 4H), 2.55 (t, 2H), 2.39 (t, 2H), 2.21-2.14 (m, 2H), 2.03-2.01 (m, 4H), 1.95-1.88 (m, 2H).

Example 10

(92) Synthesis of Compound 10 (Linker 10)

(93) ##STR00054##

(94) Compound 38 (15 mg, 54 μmol, prepared according to WO2014114207) and compound 39 (44 mg, 108 μmol, prepared according to WO2014114207) were dissolved in DMF (0.6 mL), to which DIPEA (28 mg, 216 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 37%-58% B in 8 min.fwdarw.95% B in 4) to give compound 10 (15 mg) as a white solid.

(95) LC-MS (method 2): Rt=1.63 min; m/z (ES+) 784.0 (M+H).sup.+.

(96) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ11.98 (br s, 1H), 7.59 (t, 1H), 7.44 (t, 1H), 7.05 (s, 8H), 3.88 (s, 2H), 3.85 (s, 2H), 3.82-3.77 (m, 2H), 3.52-3.50 (m, 8H), 3.29-3.19 (m, 6H), 3.15-3.12 (m, 2H), 2.49 (t, 2H), 2.41 (t, 2H).

Example 11

(97) Synthesis of Compound 11 (Linker 11)

(98) ##STR00055##

(99) Compound 38 (15 mg, 54 μmol) and compound 40 (45 mg, 108 μmol, prepared according to WO2014114207) were dissolved in DMF (0.5 mL), to which DIPEA (28 mg, 216 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 38%-58% B in 8 min.fwdarw.95% B in 4) to give compound 11 (14 mg) as a white solid.

(100) LC-MS (method 2): Rt=1.65 min; m/z (ES+) 812.0 (M+H).sup.+.

(101) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ12.01 (br s, 1H), 7.75 (t, 1H), 7.62 (t, 1H), 7.04 (s, 4H), 7.00 (s, 4H), 4.02-3.95 (m, 2H), 3.84-3.79 (m, 2H), 3.58-3.45 (m, 10H), 3.33-3.19 (m, 8H), 2.53 (t, 2H), 2.40 (t, 2H), 1.66-1.56 (m, 4H).

Example 12

(102) Synthesis of Compound 12 (Linker 12)

(103) ##STR00056##

(104) Compound 38 (15 mg, 54 μmol) and compound 41 (50 mg, 108 μmol, prepared according to WO2014114207) were dissolved in DMF (0.5 mL), to which DIPEA (28 mg, 216 μmol) was then added. The reaction mixture was stirred at room temperature for 2 h, and then purified by RP-HPLC (method 6: 35%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 12 (14 mg) as a white solid.

(105) LC-MS (method 2): Rt=1.58 min; m/z (ES+) 894.0 (M+H).sup.+.

(106) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.97 (t, 1H), 7.82 (t, 1H), 7.04 (s, 4H), 6.96 (s, 4H), 3.58 (t, 4H), 3.52 (t, 4H), 3.39-3.38 (m, 8H), 3.29 (t, 2H), 3.23 (t, 2H), 3.19-3.16 (m, 2H), 3.12-3.08 (m, 2H), 2.51-2.50 (m, 2H), 2.41 (t, 2H), 2.35-2.32 (m, 4H), 2.23-2.18 (m, 4H).

Example 13

(107) Synthesis of Compound 13 (Linker 13)

(108) ##STR00057##

Step 1: Synthesis of tert-butyl 4-(bis(3-(2,2,2-trifluoroacetamido)propyl)amino)-4-oxobutylcarbamate (44)

(109) 4-(Tert-butoxycarbonylamino)butanoic acid (42) (203 mg, 1.0 mmol, prepared according to US2015/111864) and bis(3-(2,2,2-trifluoroacetamido)propyl)amine (43) (388 mg, 1.2 mmol, prepared according to WO2006/20779) were dissolved in DMF (3 mL), to which HATU (456 mg, 1.2 mmol) and DIPEA (258 mg, 2 mmol) were then added. The reaction mixture was stirred at room temperature for 3 h, and then concentrated. The residue was purified by RP-HPLC (method 6: 45%-75% B in 8 min.fwdarw.95% B in 4 min) to give compound 44 (250 mg) as a colorless colloidal solid.

(110) LC-MS (method 1): Rt=1.81 min; m/z (ES+) 409.0 (M+H).sup.+.

(111) .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.34 (br s, 1H), 7.949 (s, 1H), 5.03 (br s, 1H), 3.42-3.30 (m, 4H), 3.30-3.17 (m, 4H), 3.09 (s, 2H), 2.39-2.27 (m, 2H), 1.91-1.82 (m, 2H), 1.82-1.73 (m, 2H), 1.73-1.63 (m, 2H), 1.37 (s, 9H).

Step 2 and 3: Synthesis of tert-butyl 4-(bis(3-(4,5-bis(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)pentanamido)propyl)amino)-4-oxobutylcarbamate (46)

(112) Compound 44 (20 mg, 39 μmol) was dissolved in methanol (1 mL), to which aqueous ammonia (28%, 1 mL) was then added. The reaction mixture was refluxed for 4 h, and then concentrated to remove the solvent. The residue was dissolved in methanol again and concentrated (repeated three times).

(113) The thus-obtained intermediate 45 was dissolved in DMF (1 mL) and DCM (1 mL), to which compound 18 (35 mg, 0.12 mmol), HATU (60 mg, 0.158 mmol) and DIPEA (30 mg, 0.23 mmol) were then sequentially added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated to remove the solvent. The residue was purified by RP-HPLC (method 6: 50%-80% B in 8 min.fwdarw.95% B in 4 min) to give compound 46 (20 mg) as a white solid.

(114) LC-MS (method 3): Rt=1.33 min; m/z (ES+) 865.5 (M+H).sup.+.

(115) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.81 (t, 1H), 7.72 (t, 1H), 7.00 (s, 4H), 6.98 (s, 2H), 6.97 (s, 2H), 6.80 (t, 1H), 4.02-3.94 (m, 2H), 3.82-3.73 (m, 2H), 3.64-3.54 (m, 2H), 3.24-3.10 (m, 4H), 3.05-2.85 (m, 6H), 2.26-2.12 (m, 4H), 2.08-1.86 (m, 6H), 1.63-1.43 (m, 6H), 1.37 (s, 9H).

Step 4: Synthesis of Compound 13

(116) Compound 46 (20 mg, 23 μmol) was dissolved in DCM (3 mL), to which TFA (1 mL) was then added. The reaction mixture was stirred at room temperature for 1 h, and then concentrated to remove the solvent. The residue was purified by RP-HPLC (method 6: 30%-60% B in 8 min.fwdarw.95% B in 4 min) to give compound 13 in TFA salt form (10 mg) as a white solid.

(117) LC-MS (method 2): Rt=1.46 min; m/z (ES+) 765.0 (M+H).sup.+.

(118) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.83 (t, 1H), 7.78-7.68 (m, 4H), 7.01 (s, 4H), 6.99 (s, 2H), 6.98 (s, 2H), 4.02-3.94 (m, 2H), 3.82-3.74 (m, 2H), 3.63-3.55 (m, 2H), 3.19 (t, 4H), 3.06-2.88 (m, 4H), 2.86-2.77 (m, 2H), 2.37 (t, 2H), 2.26-2.12 (m, 2H), 2.09-1.87 (m, 6H), 1.80-1.71 (m, 2H), 1.64-1.46 (m, 4H).

Example 14

(119) Synthesis of Compound 14 (Linker 14)

(120) ##STR00058##

Step 1: Synthesis of 4-hydroxy-N,N-bis(3-(2,2,2-trifluoroacetamido)propyl)butanamide (48)

(121) Sodium 4-hydroxybutanoate (47) (126 mg, 1.0 mmol, prepared according to WO2014/152127) and bis(3-(2,2,2-trifluoroacetamido)propyl)amine 43 (388 mg, 1.2 mmol) were dissolved in DMF (3 mL), to which HATU (456 mg, 1.2 mmol) and DIPEA (258 mg, 2 mmol) were then added. The reaction mixture was stirred at room temperature for 3 h, and then concentrated. The residue was purified by RP-HPLC (method 6: 40%-70% B in 8 min.fwdarw.95% B in 4 min) to give compound 48 (68 mg) as a colorless colloidal solid.

(122) LC-MS (method 5): Rt=1.55 min; m/z (ES+) 410.0 (M+H).sup.+.

(123) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ9.48 (t, 1H), 9.37 (t, 1H), 3.39 (t, 2H), 3.31-3.05 (m, 8H), 2.29 (t, 2H), 1.81-1.70 (m, 2H), 1.70-1.57 (m, 4H).

Step 2, 3: Synthesis of Compound 14

(124) Compound 48 (20 mg, 49 μmol) was dissolved in methanol (1 mL), to which aqueous ammonia (28%, 1 mL) was then added. The reaction mixture was refluxed for 2 h, and then concentrated to remove the solvent. The residue was dissolved in methanol again and concentrated, while such process was repeated three times.

(125) The thus-obtained intermediate 49 was dissolved in DMF (1 mL) and DCM (1 mL), to which compound 18 (43 mg, 0.15 mmol), HATU (74 mg, 0.20 mmol) and DIPEA (38 mg, 0.29 mmol) were then sequentially added. The reaction mixture was stirred at room temperature for 2 h, and then concentrated to remove the solvent. The residue was purified by RP-HPLC (method 6: 35%-65% B in 8 min.fwdarw.95% B in 4 min) to give compound 14 (5 mg) as a white solid.

(126) LC-MS (method 2): Rt=1.62 min; m/z (ES+) 765.9 (M+H).sup.+.

(127) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ7.82 (t, 1H), 7.72 (t, 1H), 7.00 (s, 4H), 6.98 (s, 2H), 6.97 (s, 2H), 4.02-3.94 (m, 2H), 3.82-3.73 (m, 2H), 3.62-3.56 (m, 2H), 3.37 (t, 2H), 3.24-3.13 (m, 4H), 3.03-2.87 (m, 4H), 2.26 (t, 2H), 2.23-2.13 (m, 2H), 2.08-1.87 (m, 6H), 1.68-1.43 (m, 6H).

Example 15

(128) Synthesis of Tetramaleimide Linker-Drug (1-vcMMAE)

(129) ##STR00059##

(130) Compound 1 (4.1 mg, 6 μmol) and NH.sub.2-vcMMA (TFA salt, 6.7 mg, 6 mol, prepared according to WO2013/173337) were dissolved in DMF (300 μL), to which DIPEA (2.3 mg, 9 μmol) and HATU (3.4 mg, 9 μmol) were then sequentially added. The reaction mixture was stirred at room temperature for 18 h, and then purified by prep-RP-HPLC (method 6: 45%-75% Bin 8 min.fwdarw.95% B in 4 min) to give compound 1-vcMMAE (4.8 mg) as white powder.

(131) LC-MS (method 2): R.sub.t=1.84 min; m/z (ES.sup.+) 892.8 [½ (M+2H)].sup.+.

Example 16

(132) Synthesis of Other Tetramaleimide Linker-Drug

(133) Other tetramaleimide linker-drugs were synthesized via the similar method as that for 1-vcMMAE in example 15, except that compound 1 was replaced with linker compounds 2-12. The linker-drugs and their characterization data were listed in Table 1, wherein the linker-drug 2-vcMMAE to 12-vcMMAE were named according to the tetramaleimide linker compounds 2 to 12.

(134) TABLE-US-00002 TABLE 1 Linker-drugs of the invention and their characterizations LC-MS Compound Method; R.sub.t (min); m/z 1/2[M + 2H].sup.+  2-vcMMAE 2; 1.90; 900.9  3-vcMMAE 2; 2.02; 903.5  4-vcMMAE 4; 1.56; 908.9  5-vcMMAE 2; 1.82; 935.3  6-vcMMAE 2; 1.95; 936.0  7-vcMMAE 2; 1.95; 943.1  8-vcMMAE 2; 1.87; 947.3  9-vcMMAE 4; 1.55; 973.0 10-vcMMAE 4; 1.56; 945.5 11-vcMMAE 4; 1.57; 959.0 12-vcMMAE  4; 1.53; 1000.5

Example 17

(135) Preparation and Characterization of Antibody-Drug Conjugates

(136) Tris(2-carboxyethyl)phosphine (TCEP, 10 eq, stock solution 10 mM) was added to a solution of antibody H (IgG1) (2-10 mg/mL, containing 25 mM boric acid-sodium borate buffer, 25 mM NaCl and 1 mM diethylene triamine pentacetic acid (DTPA), pH 7.0-8.0). The reaction mixture was incubated at 37° C. in a shaker for 2 h, and then cooled to ˜10° C., followed by buffer-exchange with a PBS buffer (100 mM KH.sub.2PO.sub.4—K.sub.2HPO.sub.4, 100 mM NaCl, 1 mM DTPA, pH 7.0-8.0) via ultrafiltration (Merck Millipore Amicon® Ultra, 50000 MWCO) or gel-filtration. The solution was cooled at 10° C., to which DMSO and compound 1-vcMMAE prepared in example 15 (stock solution in DMSO, 3-6 equivalent) were sequentially added, in which the volume percent of DMSO was controlled at ˜15%. The conjugation reaction was conducted at 10° C. for 0.5 h.

(137) Excess cysteine solution was added to the reaction mixture to quench the unreacted compound 1-vcMMAE, and the quenching reaction was kept at 10° C. for 30 min. The reaction mixture was ultrafiltered (Merck Millipore Amicon® Ultra, 50000 MWCO) or gel-filtered to remove excess 1-vcMMAE-cysteine adducts and excess cysteine. The filtrate was sterile filtered through 0.22 μm filter (Merck Millex-GV Filter), and the solution of conjugate H-1-vcMMAE thus obtained was kept at 4° C.

(138) Conjugates H-2-vcMMAE to H-12-vcMMAE were prepared from antibody H according to the same method as above, except for replacing compound 1-vcMMAE with compounds 2-vcMMAE to 12-vcMMAE. Conjugate P-7-vcMMAE was prepared from antibody P according to the same method as above, except for replacing compound 1-vcMMAE with compound 7-vcMMAE.

(139) 1) Determination of Average DAR

(140) The average DAR was measured by UV absorption method (Clin. Cancer Res. 2004, 10, 7063-7070; WO 2011/039721). Agilent 1100 HPLC with the size-exclusion chromatography (SEC) column (TSKgel G3000SWXL, 7.8*300 mm, Tosoh Bioscience Shanghai) was used.
DAR=(ε.sub.Ab248−R*ε.sub.Ab280)/(R*ε.sub.D280−ε.sub.D248)
wherein, ε.sub.Ab248 and ε.sub.Ab280 are molar extinction coefficients for the antibody at 248 nm and 280 nm, respectively. ε.sub.D280 and ε.sub.D248 are molar extinction coefficients for vcMMAE at 248 nm and 280 nm, respectively. R=A.sub.248/A.sub.280, wherein A.sub.248 and A.sub.280 are the absorbances of the ADC at 248 nm and 280 nm, respectively (peak area of the monomer on SEC spectrum was used to represent the absorbance in the invention).

(141) The average DARs of the ADCs of the invention were listed in table 2.

(142) TABLE-US-00003 TABLE 2 The average DAR results of the ADCs in the invention (equivalent ratio of the linker-drug to antibody was 3) ADC Average DAR ADC Average DAR H-1-vcMMAE 1.99  H-7-vcMMAE 1.99 H-2-vcMMAE 1.99  H-8-vcMMAE N/A H-3-vcMMAE N/A  H-9-vcMMAE  1.96* H-4-vcMMAE 1.96 H-10-vcMMAE  2.46* H-5-vcMMAE 2.05 H-11-vcMMAE 2.75 H-6-vcMMAE 1.88 H-12-vcMMAE 2.51 P-7-vcMMAE 2.20*.sup.& N/A: not available. *equivalent ratio of the linker-drug to antibody was 4. .sup.&DAR was calculated from HIC method, see reference Anal. Chem. 2013, 85, 1699-1704.

(143) As shown in table 2, the average DARs of the ADCs of the invention could be well-controlled around 2, which is due to the accurate site and number control by the site-specific linkers of the invention.

(144) 2) Native MS

(145) 8 μL of PNGase F (New England Biolabs, USA) was added to 400 μg of conjugate H-5-vcMMAE, and the mixture was incubated at 37° C. overnight (15 h). The deglycosylated ADC sample was buffer-exchanged into ammonium acetate buffer (20 mM, pH 7.0), and the buffer exchange procedure was repeated for 5 times.

(146) The mass spectrometer used was high-resolution Orbitrap Exactive Plus EMR (Thermo Fisher Scientific, Germany), and the ion source is TriVersa NanoMate® (Advion, USA). The sample concentration was adjusted to 2 μg/μL, and direct injection was adopted. The mass data was collected under the positive ion mode, and the native mass data was analyzed by Protein Deconvolution 4.0 software (Thermo Fisher Scientific, Germany).

(147) The native MS spectrum of conjugate H-5-vcMMAE was shown in FIG. 1, which shows that DAR=2 was the main component of the product.

(148) 3) SDS-PAGE

(149) SDS-PAGE was measured using NuPAGE™, 4-12%, Bis-Tris Gel (Thermal Fisher) on XCell SureLock® Mini-Cell protein electrophoresis instrument (Thermal Fisher). A sample (≥10 μg by weight) was combined with loading buffer, and the mixture was heated at 70° C. in water bath for 10 min. The sample and standard protein (5 μL/hole) were added to the spacer gel comb holes sequentially, and the electrophoresis was conducted at 220 V for 50 min. The gel was removed, rinsed by deionized water, and then stained in SimplyBlue™ SafeStain (Thermal Fisher) on a shaker for 3 h. The stained gel was rinsed by deionized water for three times, and destained on a shaker for 4 h. The destained gel was transferred to an imager to record the gel image.

(150) The SDS-PAGE results are shown in FIGS. 2a-2b, which shows that the main components in sample H-1-vcMMAE to H-12-vcMMAE were full antibody HHLL (full ADC) and half antibody HL (full ADC lost heavy chain interaction). The result proves that the tetramaleimide linkers of the invention can crosslink the inter chains of the reduced antibody, and thus effectively control the number of drugs per antibody (DAR).

(151) 4) Hydrophobic Interaction Chromatography (HIC) Analysis

(152) HIC was measured on an Agilent 1100 chromatograph. TSKgel butyl-NPR column (4.6×35 mm, 2.5 μm, Tosoh Bioscience Shanghai) was applied as the immobile phase. The method was consisted of a linear gradient from 100% buffer A (50 mM potassium phosphate (pH 7.0)+1.5 M ammonium sulfate) to 100% buffer B (80% v/v 50 mM sodium phosphate (pH 7.0), 20% v/v isopropanol) over 25 minutes. The flow rate was 0.8 mL/min, the column temperature was 30° C., and the detection wavelengths were 230 and 280 nm.

(153) HIC analysis results are shown in FIGS. 3a-3m, which show that the main components of the ADC samples (H-1-vcMMAE to H-12-vcMMAE, and P-7-vcMMAE) are DAR=2 components. The result proves that the tetramaleimide linkers of the invention could be used to effectively control the DAR and distribution of the ADC product.

Test Example 1

(154) Determination of the Antigen Binding Ability of the ADCs of the Invention by Enzyme-Linked Immunosorbent Assay (ELISA)

(155) Indirect ELISA was used to analyze binding ability of the antibody or antibody-drug conjugate to the corresponding antigen. The Her2 antigen was immobilized on a solid-phase support (96 well microplate) by coating to form a solid-phase antigen, and then unbound antigen was removed by washing. Serial dilutions of test antibody or antibody-drug conjugate were added, wherein specific antibody or antibody-drug conjugate bound to the antigen and formed solid-phase antigen-antibody complexes. The antibody or antibody-drug conjugate unbound to the solid-phase antigen was removed by washing. The enzyme labeled anti-antibody was added to bind to the above-formed complexes. After washing, substrate solution was added, and the optical density was read by a microplate reader at 450 nm/630 nm, based on which the curve was drawn and the EC.sub.50 was calculated.

(156) The binding abilities of the ADCs of the invention to Her2 antigen were listed in Table 3.

(157) TABLE-US-00004 TABLE 3 The binding ability of the ADCs of the invention to Her2 antigen ADC EC.sub.50 (ng/mL) ADC EC.sub.50 (ng/mL) H 33.5 H-1-vcMMAE 14.5  H-7-vcMMAE 34.0 H-2-vcMMAE 15.7  H-8-vcMMAE 28.9 H-3-vcMMAE 33.2  H-9-vcMMAE 25.8 H-4-vcMMAE 15.7 H-10-vcMMAE 36.6 H-5-vcMMAE 25.9 H-11-vcMMAE 38.1 H-6-vcMMAE 19.1 H-12-vcMMAE 33.8

(158) As shown in Table 3, compared to naked antibody, the binding ability of the ADCs prepared from tetramaleimide linkers to the antigen shows no significant difference.

Test Example 2

(159) Cell Proliferation Inhibition of the ADCs of the Invention

(160) Cell Proliferation Assay

(161) Cell proliferation inhibition of an antibody or ADC is measured by the following method. Mammalian cells expressing tumor-associated antigens or receptor proteins (Her2 expressing breast cancer cell, SK-BR-3, was used in this assay) were seed in 96-well plate at a concentration of 8000 cells/well, and the cells were suspended in DMEM (GIBCO). The initial ADC concentration was 2 μg/mL, which was 3 times gradient diluted with DMEM containing 2% FBS (GIBCO). The initial cell culture media was removed and 200 μL of ADC was added to each well. The cells were incubated for 72 h, and the media was removed. 100 μL of CCK-8 was added, followed by incubation of 30 min. The absorption was read by a microplate reader at 450 nm/630 nm, based on which the curve was drawn and the IC.sub.50 was calculated.

(162) The cell proliferation inhibition result of the ADCs of the invention was listed in table 4.

(163) TABLE-US-00005 TABLE 4 Cell Proliferation Inhibition Result of the ADCs of the Invention ADC IC.sub.50 (ng/mL) ADC IC.sub.50 (ng/mL) H-1-vcMMAE 5.1  H-7-vcMMAE 8.8 H-2-vcMMAE 7.9  H-8-vcMMAE 9.1 H-3-vcMMAE 7.7  H-9-vcMMAE 5.1 H-4-vcMMAE 8.8 H-10-vcMMAE 6.3 H-5-vcMMAE 8.6 H-11-vcMMAE 6.1 H-6-vcMMAE 9.4 H-12-vcMMAE 7.4 P-7-vcMMAE 8.8

(164) Table 4 shows that the ADCs of the invention have excellent cell proliferation inhibition activity

(165) All references mentioned in the present application are incorporated herein by reference to the same extent as if each individual reference is individually incorporated by reference. In addition, it should be understood that after reading the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.