Di-substituted maleic amide linker for antibody drug conjugating and preparation method and use thereof

Abstract

Provided in the present invention are a di-substituted maleic amide linker conjugated to an antibody and a preparation method and use thereof. In particular, the present invention conjugates a strongly cytotoxic active substance to a biomacromolecule through a class of new linkers. The class of linkers can selectively act simultaneously with disulphide chains, so as to greatly improve the substance homogeneity of a conjugate. The conjugate prepared by the linker of the present invention has a high inhibitory activity on a cell strain expressing the corresponding antigen. Also provided is a method for preparing the above-mentioned conjugate and the use.

Claims

1. A substituted maleamide linker as shown in Formula Ia, ##STR00143## wherein, R is X or ArS-, X is selected from halogen; Ar is selected from the group consisting of substituted or unsubstituted C.sub.6-C.sub.10 aryl and substituted or unsubstituted 5-12 membered heteroaryl; Ar′ is selected from the group consisting of substituted or unsubstituted C.sub.6-C.sub.10 arylene, and substituted or unsubstituted 5-12 membered heteroarylene; and L.sub.1 is —O(CH.sub.2CH.sub.2O).sub.n— linked to Ar′, in which n is any integer between 1 and 20.

2. The substituted maleamide linker according to claim 1, wherein Ar is selected from the group consisting of phenyl, halogen-substituted phenyl, C.sub.1-C.sub.4 alkylphenyl, C.sub.1-C.sub.4 alkoxyphenyl, 4-methylphenyl, 4-methoxyphenyl, 2-pyridyl, 2-pyrimidinyl, 1-methylimidazol-2-yl, and ##STR00144## in which W is amido R.sup.1 attached to carbonyl, and R.sup.1 is selected from the group consisting of —NH.sub.2, ##STR00145##

3. The substituted maleamide linker according to claim 1, wherein Ar′ is selected from substituted phenylene and pyridyl, and the substitution means that hydrogen atom on the group is substituted by one or more substituents selected from the group consisting of: halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, trifluoromethyl, cyano, and amide group.

4. The substituted maleamide linker according to claim 1, wherein the linker has a structure selected from the group consisting of: ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151## ##STR00152## ##STR00153##

5. A substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof, the conjugate having a structure as shown in Formula Ib: ##STR00154## wherein R is X or ArS-; X is selected from halogen; Ar is selected from the group consisting of substituted or unsubstituted C.sub.6-C.sub.10 aryl and substituted or unsubstituted 5-12 membered heteroaryl; Ar′ is selected from the group consisting of substituted or unsubstituted C.sub.6-C.sub.10 arylene, and substituted or unsubstituted 5-12 membered heteroarylene; L.sub.1 is —O(CH.sub.2CH.sub.2O).sub.n— linked to Ar′, in which n is any integer between 1 and 20; L.sub.2 is a chemical bond or has a structure of AA-PAB, in which AA is a dipeptide or tripeptide fragment, and PAB is p-aminobenzylcarbamoyl; and CTD is a cytotoxic small molecule drug and/or a drug for treating autoimmune disease and/or inflammation, which is bonded to L.sub.2 via an amide bond.

6. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 5, wherein AA is selected from the group consisting of Val-Cit, Val-Ala, Phe-Lys, Ala-Ala-Asn, and D-Ala-Phe-Lys.

7. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 5, wherein the CTD is selected from the group consisting of tubulin inhibitor, topoisomerase inhibitor and DNA binding agent.

8. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the tubulin inhibitor is selected from the group consisting of maytansine or its derivatives, Monomethyl auristatin E, Monomethylauristatin F, Monomethyl Dolastatin 10, Tubulysin or its derivatives, Cryptophycin or its derivatives, and Taltobulin.

9. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the DNA binding agent is selected from the group consisting of PBD or its derivatives and duocarmycin or its derivatives.

10. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the topoisomerase inhibitor is selected from the group consisting of metabolite PNU-159682 of Doxorubicin or its derivatives, metabolite SN38 of irinotecan or its derivatives, and Exatecan.

11. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 5, wherein the CTD has a structure selected from the group consisting of D1-D13′: ##STR00155## ##STR00156## ##STR00157##

12. The substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 5, wherein the conjugate as shown in Formula Ib is selected from the group consisting of: ##STR00158## ##STR00159## ##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169##

13. An antibody-drug conjugate, formed by coupling an antibody with the substituted maleamide linker-drug conjugate, a pharmaceutically acceptable salt or solvate thereof according to claim 5.

14. The antibody-drug conjugate according to claim 13, wherein the conjugate has a structure as shown in Formula Ic and/or Formula Id: ##STR00170## Ar′, L.sub.1, L.sub.2, and CTD are defined as claim 5; m=1.0-5.0; Ab is selected from the group consisting of protein, enzyme, antibody, antibody fragment, and peptide.

15. The antibody-drug conjugate according to claim 13, wherein the antibody is selected from the group consisting of monoclonal antibody, bispecific antibody, chimeric antibody, humanized antibody and antibody fragment.

16. The antibody-drug conjugate according to claim 13, wherein the antibody is that capable of binding to a tumor-associated antigen selected from the group consisting of HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2.

17. The antibody-drug conjugate according to claim 16, wherein the HER2 antibody is selected from Trastuzumab or Pertuzumab; or the EGFR antibody is selected from Erbitux or Vectibix.

18. A pharmaceutical composition, comprising: (a) the antibody-drug conjugate according to claim 13; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

19. A use of the antibody-drug conjugate according to claim 13 for manufacturing a medicament for the treatment of a tumor.

20. A method for preparing the antibody-drug conjugate according to claim 13, the method including the following steps: (1) reacting an antibody with a reducing reagent in a buffer solution to obtain a reduced antibody; (2) cross-linking a linker-drug conjugate with the reduced antibody obtained in step (1) in a mixture solution of a buffer solution and an organic solvent to obtain the antibody-drug conjugate.

21. The method for preparing the antibody-drug conjugate according to claim 20, wherein synthesis scheme of the method is as follows: ##STR00171## wherein R, Ab, Ar′, L.sub.1, L.sub.2, CTD, and m are defined as claim 14.

22. A method for preparing the substituted maleamide linker according to claim 1, the method including the following steps: performing a cyclization reaction of intermediate C and maleic anhydride dihalide to generate intermediate D, and performing a substitution reaction of intermediate D with aryl thiophenol to generate a linker as shown in Formula E, and synthesis scheme is as follows: ##STR00172## wherein R and n are defined as claim 1; X represents halogen; and U and V are independently N or C.

23. The method for preparing the substituted maleamide linker according to claim 22, wherein intermediate C is obtained by reducing intermediate B, and synthesis scheme is as follows: ##STR00173## wherein R, n, U, and V are defined as claim 22.

24. The method for preparing the substituted maleamide linker according to claim 23, wherein intermediate B is obtained by a substitution reaction of compound A with fluoronitrobenzene, and synthesis scheme is as follows: ##STR00174## wherein R, n, U, and V are defined as claim 22.

25. The method for preparing the substituted maleamide linker according to claim 24, wherein intermediate B is prepared as follows: ##STR00175## wherein R, n, U, and V are defined as claim 22.

26. The method for preparing the substituted maleamide linker according to claim 25, wherein compound A is obtained by reacting n-ethylene glycol with tert-butyl haloacetate, and synthesis scheme is as follows: ##STR00176## wherein n and X are defined as claim 22.

27. A method for preparing the substituted maleamide linker-drug conjugate according to claim 5, the method including: performing a condensation reaction of a substituted maleimide linker and CTD containing a dipeptide/tripeptide-PAB to generate F1 or F′1 respectively; and synthesis schemes are as follows: ##STR00177## wherein R is defined as claim 1, Rx represents halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, trifluoromethyl, cyano, or amide group, and Ry represents H or alkyl.

28. The method for preparing the substituted maleamide linker according to claim 22, wherein X is Br or Cl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows chromatograms of hydrophobic interaction chromatography (HIC) of Pertuzumab and Pertuzumab drug conjugate for comparison;

(2) FIG. 2 shows chromatograms of mass spectrum of Pertuzumab and Pertuzumab drug conjugate for comparison;

(3) FIG. 3 shows experiment results of binding of ADC-2, P-mcVC-MMAE and Pertuzumab to antigen Her 2 on the surface of NCI-N87 cells;

(4) FIG. 4 shows experiment results of proliferation inhibition of ADC-2, P-mcVC-MMAE, Kadcyla and Pertuzumab on human breast cancer cells SK-BR-3;

(5) FIG. 5 shows experiment results of proliferation inhibition of ADC-2, P-mcVC-MMAE, Kadcyla and Pertuzumab on human breast cancer cells BT-474;

(6) FIG. 6 shows experiment results of proliferation inhibition of ADC-2, P-mcVC-MMAE, Kadcyla and Pertuzumab on human gastric cancer cells N87;

(7) FIG. 7 shows experiment results of proliferation inhibition of ADC-2 on SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells and Panc-1 human pancreatic cancer cells;

(8) FIG. 8 shows experiment results of proliferation inhibition of ADC-2 on human breast cancer cells MCF-7, MDA-MB-231 and MDA-MB-468;

(9) FIG. 9-1 shows experiment results of proliferation inhibition of ADC-4 on human gastric cancer cells N87;

(10) FIG. 9-2 shows experiment results of proliferation inhibition of ADC-2, ADC-3 and ADC-4 on Calu-3 human lung cancer cells;

(11) FIG. 10 shows results of study on inhibition activity of ADC-2, ADC-3, P-mcVC-MMAE and Kadcyla on human gastric cancer NCI-N87 subcutaneous xenograft tumor in nude mice;

(12) FIG. 11-1 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab;

(13) FIG. 11-2 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-I;

(14) FIG. 11-3 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-II;

(15) FIG. 11-4 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-III;

(16) FIG. 11-5 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-IV;

(17) FIG. 11-6 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-V;

(18) FIG. 11-7 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-VI;

(19) FIG. 11-8 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Pertuzumab-drug conjugate ADC-VI;

(20) FIG. 12-1 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Trastuzumab;

(21) FIG. 12-2 shows a chromatogram of hydrophobic interaction chromatography (HIC) of Trastuzumab-drug conjugate ADC-VIII;

(22) FIG. 13-1 shows a chromatogram of mass spectrum of Pertuzumab;

(23) FIG. 13-2 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-I;

(24) FIG. 13-3 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-II;

(25) FIG. 13-4 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-III;

(26) FIG. 13-5 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-IV;

(27) FIG. 13-6 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-V;

(28) FIG. 13-7 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-VI;

(29) FIG. 13-8 shows a chromatogram of mass spectrum of Pertuzumab-drug conjugate ADC-VII;

(30) FIG. 14-1 shows a chromatogram of mass spectrum of Trastuzumab;

(31) FIG. 14-2 shows a chromatogram of mass spectrum of Trastuzumab-drug conjugate ADC-VIII;

(32) FIG. 15 shows a trend chart of secondary hydrolyzates produced by ADC control, ADC-I, ADC-II, and ADC-VII, which are detected using LC-MS (Q-TOF) at day 0-7 at room temperature;

(33) FIG. 16-1 shows a HIC chromatogram of ADC control at day 0 at room temperature;

(34) FIG. 16-2 shows a HIC chromatogram of ADC control at day 2 at room temperature;

(35) FIG. 16-3 shows a HIC chromatogram of ADC control at day 4 at room temperature;

(36) FIG. 16-4 shows a HIC chromatogram of ADC control at day 7 at room temperature;

(37) FIG. 17-1 shows a HIC chromatogram of ADC-I at day 0 at room temperature;

(38) FIG. 17-2 shows a HIC chromatogram of ADC-I at day 2 at room temperature;

(39) FIG. 17-3 shows a HIC chromatogram of ADC-I at day 4 at room temperature;

(40) FIG. 17-4 shows a HIC chromatogram of ADC-I at day 7 at room temperature;

(41) FIG. 18-1 shows a HIC chromatogram of ADC-II at day 0 at room temperature;

(42) FIG. 18-2 shows a HIC chromatogram of ADC-II at day 2 at room temperature;

(43) FIG. 18-3 shows a HIC chromatogram of ADC-II at day 4 at room temperature;

(44) FIG. 18-4 shows a HIC chromatogram of ADC-II at day 7 at room temperature;

(45) FIG. 19-1 shows a HIC chromatogram of ADC-VII at day 0 at room temperature;

(46) FIG. 19-2 shows a HIC chromatogram of ADC-VII at day 2 at room temperature;

(47) FIG. 19-3 shows a HIC chromatogram of ADC-VII at day 4 at room temperature;

(48) FIG. 19-4 shows a HIC chromatogram of ADC-VII at day 7 at room temperature;

(49) FIG. 20 shows experiment results of proliferation inhibition of ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and Pertuzumab (Perjeta) on human gastric cancer cells NIC-N87;

(50) FIG. 21 shows experiment results of proliferation inhibition of ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and Pertuzumab (Perjeta) on human breast cancer cells BT-474;

(51) FIG. 22 shows experiment results of proliferation inhibition of ADC-VIII and Trastuzumab (Herceptin) on human gastric cancer cells NIC-N87;

(52) FIG. 23 shows experiment results of proliferation inhibition of ADC-VIII and Trastuzumab (Herceptin) on human breast cancer cells BT-474;

(53) FIG. 24 shows results of study on inhibition activity of P-mcVC-MMAE (1.0 mg/kg), ADC control (0.5, 1.0 mg/kg), ADC-I (1.0 mg/kg), ADC-IV (1.0 mg/kg), ADC-V (1.0 mg/kg), ADC-VI (1.0 mg/kg) and ADC-VII (0.5, 1.0 mg/kg) on human gastric cancer NCI-N87 subcutaneous xenograft tumor in nude mice.

DETAILED DESCRIPTION OF EMBODIMENTS

(54) After having conducted extensive and intensive researches, the inventors found a class of linkers which can cross-couple with light chain-heavy chain and heavy chain-heavy chain of an antibody in whole or in part. Moreover, compared with traditional antibody drug conjugates, the antibody drug conjugates obtained by the above conjugation method have a narrower drug to antibody ratio (DAR) distribution. Based on the above findings, the inventors completed the present invention.

(55) One of Specific Designs:

(56) preparation and use of the substituted maleamide linker as shown in Formula Ia, wherein Ar′ is selected from the group consisting of unsubstituted C.sub.6-C.sub.10 arylene and unsubstituted 5-12 membered heteroarylene.

(57) ##STR00048##

Section 1: Synthesis and Preparation Methods of the Linker Solution 1

(58) The substituted maleamide linker as shown in Formula Ia, which is provided in the first aspect of the present invention, can be synthesized by the method listed in Solution 1. Specifically, intermediate B can be obtained through a substitution reaction between n-ethylene glycol and fluoronitrobenzene, in which the nitro subsequently is reduced to generate amino compound C; intermediate D can be obtained through a substitution reaction between 2,3-dibromomaleimide and aryl thiophenol, and is then reacted with methyl chloroformate to generate intermediate E; alternatively, the 2,3-dibromomaleimide can react with methyl chloroformate directly to generate intermediate E′; and intermediate C is reacted with intermediate E or intermediate E′ to generate linker F. Synthesis scheme and specific examples are as follows:

(59) ##STR00049##

Example 1: Synthesis and Preparation of Compounds as Shown in Formulas 1-12

(60) 1.1 Synthesis of Compound F-1 (Formula 1)

(61) 1.1.1 Synthesis of Intermediate B-1 (Step a)

(62) ##STR00050##

(63) 4-fluoronitrobenzene (10.0 g, 0.071 mol), diethylene glycol (75.2 g, 0.71 mol) and potassium carbonate (14.7 g, 0.11 mol) were weighted and placed in a 250 mL round-bottom flask, and were stirred for 22 hours at 80° C. under the protection of nitrogen. The obtained mixture was then cooled down to room temperature slowly, extracted with dichloromethane, washed successively with 1 mol/L diluted hydrochloric acid, water and saturated salt water, and dried with anhydrous sodium sulfate, and the solvent was rotary evaporated off. The residue was subjected to column chromatography (silica gel, 200-300 mesh, PE/EtOAC 10:1) to obtain a light yellow transparent liquid product (15.1 g, 94% yield). Theoretical value via LC-MS (M+): 227.08, and measured value via LC-MS (ESI, M+H+): 228.12.

(64) 1.1.2 Synthesis of Intermediate C-1 (Step b)

(65) ##STR00051##

(66) Intermediate B-1 (3.0 g, 9.52 mmol) was dissolved in acetone (30 mL), and cooled in ice cold water, and then a freshly prepared Jones reagent (15 mL) was dropped slowly therein. The obtained reaction mixture was stirred at room temperature for 3 hours, cooled in ice water followed by slowly dropping isopropanol therein, and then stirred for 15 minutes in an ice-water bath. Subsequently the organic solvent was rotary evaporated off. The aqueous phase was extracted with diethyl ether for three times, and then the organic phases was combined, washed with saturated salt water, and dried with anhydrous sodium sulfate, and the solvent therein was rotary evaporated off. The obtained yellow oily crude was used in the next step directly without being purified.

(67) The crude obtained was dissolved in tetrahydrofuran (30 mL), and then 10% palladium-carbon (300 mg) was added thereto to allow a hydrogenation at 30° C. for 6 hours. After removing the catalyst by suction filtration, the solvent was rotary evaporated off to give a brownish yellow oily crude, which was used in the next step directly without being purified. Theoretical value via LC-MS (M+): 211.08, and measured value via LC-MS (ESI, M+H+): 212.05.

(68) 1.1.3 Synthesis of Intermediate D-1 (Step c)

(69) ##STR00052##

(70) 2,3-dibromomaleimide (7.0 g, 27.69 mmol) was dissolved in methanol (80 mL), and sodium acetate (4.5 g, 55.4 mmol) and thiophenol (11.3 mL, 110.7 mmol) were added thereto. The obtained reaction mixture was stirred at room temperature for 30 minutes, and then the organic solvent was rotary evaporated off. The residue was extracted with dichloromethane, washed successively with water and saturated salt water, and dried with anhydrous sodium sulfate, and then the solvent was rotary evaporated off. The obtained crude was co-crystallized with petroleum ether/ethyl acetate, suction filtrated and dried to obtain a bright yellow solid D-1 (6.5 g, 75% yield).

(71) 1.4.4 Synthesis of Intermediate E-1 (Step d)

(72) ##STR00053##

(73) Intermediate D-1 (3.0 g, 9.6 mmol) and N-methylmorpholine (1.37 mL, 12.5 mmol) were dissolved in ethyl acetate (40 mL), and cooled in ice water followed by slowly dropping methyl chloroformate (1.11 mL, 14.4 mmol) thereto. The reaction mixture was diluted with ethyl acetate after being stirred at room temperature for 30 minutes, extracted with water added, washed successively with water and saturated salt water, and dried with anhydrous sodium sulfate, and then the solvent was rotary evaporated off. The obtained crude was co-crystallized with petroleum ether/ethyl acetate, suction filtrated and dried to obtain a bright yellow solid E-1 (3.5 g, 98% yield).

(74) 1.1.5 Synthesis of Intermediate F-1 (Step e)

(75) ##STR00054##

(76) Intermediate C-1 (2.0 g, 9.5 mmol) was dissolved in anhydrous dichloromethane (40 ml), and then intermediate E-1 (3.5 g, 9.5 mmol) was added thereto. The obtained reaction mixture was stirred at room temperature for 24 hours under the protection of nitrogen. The reaction mixture was stirred at room temperature overnight after addition of silica gel (12 g, 200-300 mesh). Then the solvent was rotary evaporated off and the residue was subjected to dry column chromatography (silica gel, 200-300 mesh, dichloromethane/methanol 10:1) to obtain F-1, which was an orange oily product (4.1 g, 85% yield). Theoretical value via LC-MS (M+): 507.08, and measured value via LC-MS (ESI, M+H+): 508.11.

(77) 1.2 Synthesis of Compound F-2 (Formula 2)

(78) ##STR00055##

(79) Compound F-2 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to triethylene glycol. Product F-2 obtained after 5 steps of reaction was orange oily product. Theoretical value via LC-MS (M+): 551.11, and measured value via LC-MS (ESI, M+H+): 552.13.

(80) 1.3 Synthesis of Compound F-3 (Formula 3)

(81) ##STR00056##

(82) Compound F-3 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to tetraethylene glycol. Product F-3 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 595.13, and measured value via LC-MS (ESI, M+H+): 596.14.

(83) 1.4 Synthesis of Compound F-4 (Formula 4)

(84) ##STR00057##

(85) Compound F-4 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to pentaethylene glycol. Product F-4 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 639.16, and measured value via LC-MS (ESI, M+H+): 640.18.

(86) 1.5 Synthesis of Compound F-5 (Formula 5)

(87) ##STR00058##

(88) Compound F-5 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to hexaethylene glycol. Product F-5 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 683.19, and measured value via LC-MS (ESI, M+H+): 684.21.

(89) 1.6 Synthesis of Compound F-6 (Formula 6)

(90) ##STR00059##

(91) Compound F-6 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to octaethylene glycol. Product F-6 obtained after 5 steps of reaction was an orange yellow oily product. Theoretical value via LC-MS (M+): 771.24, and measured value via LC-MS (ESI, M+H+): 772.26.

(92) 1.7 Synthesis of Compound F-7 (Formula 7)

(93) ##STR00060##

(94) Compound F-7 was synthetized by the same steps for synthesizing compound F-1 of Example 1.1, with the exception that diethylene glycol in step a was changed to decaethylene glycol. Product F-7 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 895.29, and measured value via LC-MS (ESI, M+H+): 896.31.

(95) 1.8 Synthesis of Compound F-8 (Formula 8)

(96) ##STR00061##

(97) Compound F-8 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to p-methylthiophenol. Product F-8 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 623.16, and measured value via LC-MS (ESI, M+H+): 624.18.

(98) 1.9 Synthesis of Compound F-9 (Formula 9)

(99) ##STR00062##

(100) Compound F-9 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to p-methoxythiophenol. Product F-9 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 655.15, and measured value via LC-MS (ESI, M+H+): 656.17.

(101) 1.10 Synthesis of Compound F-10 (Formula 10)

(102) ##STR00063##

(103) Compound F-10 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 4-(N-methylformamide) thiophenol. Product F-10 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 709.18, and measured value via LC-MS (ESI, M+H+): 710.23.

(104) 1.11 Synthesis of Compound F-11 (Formula 11)

(105) ##STR00064##

(106) Compound F-11 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 2-mercaptopyridine. Product F-11 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 597.12, and measured value via LC-MS (ESI, M+H+): 598.13.

(107) 1.12 Synthesis of Compound F-12 (Formula 12)

(108) ##STR00065##

(109) Compound F-12 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 2-mercaptopyrimidine. Product F-12 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 599.11, and measured value via LC-MS (ESL, M+H+): 600.13.

(110) 1.13 Synthesis of Compound F-13 (Formula 13)

(111) ##STR00066##

(112) Compound F-13 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that 4-fluoronitrobenzene in step a was changed to 3-fluoronitrobenzene. Product F-13 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 595.13, and measured value via LC-MS (ESI, M+H+): 596.15.

(113) 1.14 Synthesis of Compound F-14 (Formula 14)

(114) ##STR00067##

(115) Compound F-14 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that intermediate E-1 in step e was changed to intermediate E′ (substituted with bromine). Product F-14 obtained after 4 steps of reaction was a colorless oily product. Theoretical value via LC-MS (M+): 534.95, and measured value via LC-MS (ESI, M+H+): 536.01.

(116) 1.15 Synthesis of Compound F-15 (Formula 15)

(117) ##STR00068##

(118) Compound F-15 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 2-mercapto-1-methylimidazole. Product F-15 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 603.15, and measured value via LC-MS (ESI, M+H+): 604.14.

(119) 1.16 Synthesis of Compound F-16 (Formula 16)

(120) ##STR00069##

(121) Compound F-16 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 4-(N-morpholineformamide) thiophenol. Product F-16 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 821.23, and measured value via LC-MS (ESI, M+H+): 822.21.

(122) 1.17 Synthesis of Compound F-17 (Formula 17)

(123) ##STR00070##

(124) Compound F-17 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 4-(N-2-methoxyethylformamide) thiophenol. Product F-17 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 797.23, and measured value via LC-MS (ESI, M+H+): 798.31.

(125) 1.18 Synthesis of Compound F-18 (Formula 18)

(126) ##STR00071##

(127) Compound F-18 was synthetized by the same steps for synthesizing compound F-3 of Example 1.3, with the exception that thiophenol in step c was changed to 4-(N-methoxy-N-methylformamide) thiophenol. Product F-18 obtained after 5 steps of reaction was an orange oily product. Theoretical value via LC-MS (M+): 769.20, and measured value via LC-MS (ESI, M+H+): 770.28.

(128) The substituted maleamide linker represented by Formula Ia can also be synthesized by the method shown in the following scheme. Specifically, intermediate B can be obtained through a substitution reaction between n-ethylene glycol and fluoronitrobenzene, and is then reacted with tert-butyl bromoacetate to generate intermediate Z; alternatively, intermediate Z can also be obtained by reacting n-ethylene glycol with tert-butyl bromoacetate following by another substitution reaction with fluoronitrobenzene; intermediate Z was then reduced to generate intermediate Y; intermediate D can be obtained through a substitution reaction between 2,3-dibromomaleimide and aryl thiophenol, and is then reacted with methyl chloroformate to generate intermediate E; Intermediate E is reacted with intermediate Y to obtain intermediate X; and the tert-butyl ester in intermediate X is removed under acidic condition to obtain linker W. An example of synthesis scheme is as follows:

(129) ##STR00072##
Solution 2:

(130) The substituted maleamide linker-drug conjugate (Formula 19-Formula 25) as shown in Formula Ib, which is provided in the second aspect of the present invention, can be synthesized by the synthesis scheme listed in Solution 2. Specifically, compound F is condensed and coupled with a cytotoxic drug CTD (D1-D11, which are commercially available) to obtain a series of molecules represented by Formula G. Synthesis scheme and specific examples are as follows:

(131) ##STR00073##

Example 2: Synthesis and Preparation of Compounds as Shown in Formulas 19-25

(132) 2.1 Synthesis of Compound G-1 (Formula 19)

(133) ##STR00074##

(134) Compound F-3 (200 mg, 0.34 mmol) and compound D1 (220 mg, 0.34 mmol) were dissolved in N,N-dimethylformamide (10 mL) and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) (77 mg, 0.40 mmol) and 1-hydroxybenzotriazole (HOBT) (54 mg, 0.40 mmol) were added thereto. The reaction mixture was stirred overnight at room temperature, and then diluted with ethyl acetate, extracted with addition of water, washed successively with water and saturated salt water, and dried with anhydrous sodium sulfate, and then the solvent was rotary evaporated off. The obtained crude was isolated and purified by using a silica gel chromatographic column (dichloromethane/methanol), suction filtrated and dried to obtain a yellow amorphous solid G-1 (3.5 g, 98% yield). Theoretical value via LC-MS (M+): 1226.40, and measured value via LC-MS (ESI, M+H+): 1227.42.

(135) 2.2 Synthesis of Compound G-2 (Formula 20)

(136) ##STR00075##

(137) Compound G-2 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D2. Product G-2 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1294.63, and measured value via LC-MS (ESI, M+H+): 1295.64.

(138) 2.3 Synthesis of Compound G-3 (Formula 21)

(139) ##STR00076##

(140) Compound G-3 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D3. Product G-3 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1308.61, and measured value via LC-MS (ESI, M+H+): 1309.63.

(141) 2.4 Synthesis of Compound G-4 (Formula 22)

(142) ##STR00077##

(143) Compound G-4 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D9. Product G-4 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1302.41, and measured value via LC-MS (ESI, M+H+): 1303.43.

(144) 2.5 Synthesis of Compound G-5 (Formula 23)

(145) ##STR00078##

(146) Compound G-5 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D6. Product G-5 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1290.51, and measured value via LC-MS (ESI, M+H+): 1291.53.

(147) 2.6 Synthesis of Compound G-6 (Formula 24)

(148) ##STR00079##

(149) Compound G-6 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D7. Product G-6 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1274.44, and measured value via LC-MS (ESI, M+H+): 127545.

(150) 2.7 Synthesis of Compound G-7 (Formula 25)

(151) ##STR00080##

(152) Compound G-7 was synthetized by the same steps for synthesizing compound G-1 of Example 2.1, with the exception that compound D1 was changed to compound D11. Product G-7 obtained was yellow amorphous solid. Theoretical value via LC-MS (M+): 1291.38, and measured value via LC-MS (ESI, M+H+): 1292.40.

(153) Solution 3:

(154) The substituted maleamide linker-drug conjugate (Formula 26-Formula 49) as shown in Formula Ib, which is provided in the second aspect of the present invention, can be synthesized by the synthesis scheme listed in Solution 3. Specifically, compound F is condensed with an amino in dipeptide/tripeptide-PAB linker (commercially available), and then the PAB group is condensed and coupled with a cytotoxic drug CTD (D1-D11) after being activated by bis(p-nitrophenyl) carbonate, thus a series of molecules represented by Formula K are obtained. Synthesis scheme and specific examples are as follows:

(155) ##STR00081##
3.1 Synthesis of Compound K-1 (Formula 26)
3.1.1 Synthesis of Intermediate I-1 (Step f)

(156) ##STR00082##

(157) Compound F-3 (2.0 g, 3.36 mmol) was dissolved in anhydrous N,N-dimethylformamide (25 mL) and then EDCl (963 mg, 5.04 mmol), HOBt (681 mg, 5.04 mmol) and N-methylmorpholine (1.11 ml, 10.08 mmol) were successively added thereto under the protection of nitrogen. The reaction mixture was stirred under the protection of nitrogen at room temperature for 20 minutes, and then compound H-1 (Val-Cit-PAB, 1.91 g, 5.04 mmol) was added thereto. The reaction mixture was stirred under the protection of nitrogen overnight at room temperature. The solvent was rotary evaporated off and the residue was subjected to dry column chromatography (silica gel, 200-300 mesh, DCM/MeOH 10:1) to obtain 1-1, which was an orange oily product (2.0 g, 62.2% yield). Theoretical value via LC-MS (M+): 956.34, and measured value via LC-MS (ESI, M+H+): 957.37.

(158) 3.1.2 Synthesis of Intermediate J-1 (Step g)

(159) ##STR00083##

(160) Compound I-1 (1.5 g, 1.57 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL) and then N,N-diisopropylethylamine (0.52 mL, 3.14 mmol) and bis(p-nitrophenyl) carbonate (717 mg, 2.335 mmol) were successively added thereto under the protection of nitrogen. The reaction mixture was stirred at room temperature for 15 hours and then the solvent was rotary evaporated off. The residue was subjected to dry column chromatography (silica gel, 200-300 mesh, DCM/MeOH 20:1) to obtain J-1, which was an orange oily product (1.4 g, 79.9% yield). Theoretical value via LC-MS (M+): 1121.35, and measured value via LC-MS (ESI, M+H+): 1122.37.

(161) 3.1.3 Synthesis of Intermediate K-1 (Step h)

(162) ##STR00084##

(163) Compound J-1 (0.5 g, 0.52 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 mL) and then N,N-diisopropylethylamine (0.172 mL, 1.04), HOBt (70 mg, 0.52 mmol) and compound D1 (340 mg, 0.52 mmol) were successively added thereto under the protection of nitrogen. The reaction mixture was stirred under the protection of nitrogen overnight at room temperature, and then the solvent was rotary evaporated off. The residue was subjected to dry column chromatography (silica gel, 200-300 mesh, DCM/MeOH 10:1) to obtain K-1, which was yellow amorphous solid (310 mg, 36.33% yield). Theoretical value via LC-MS (M+): 1631.60, and measured value via LC-MS (ESI, M+H+): 1632.62.

(164) 3.2 Synthesis of Compound K-2 (Formula 27)

(165) ##STR00085##

(166) Compound K-2 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to compound D2 (Monomethylauristatin E). Product K-2 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1699.83, and measured value via LC-MS (ESI, M+H+): 1610.85.

(167) 3.3 Synthesis of Compound K-3 (Formula 28)

(168) ##STR00086##

(169) Compound K-3 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound F-3 in step f was changed to F-5 and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-3 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1787.88, and measured value via LC-MS (ESI, M+H+): 1788.90.

(170) 3.4 Synthesis of Compound K-4 (Formula 29)

(171) ##STR00087##

(172) Compound K-4 was synthetized by the same steps for synthesizing compounds K-1 of Example 3.1, with the exception that compound F-3 in step f was changed to F-6 and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-4 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1875.93, and measured value via LC-MS (ESI, M+H+): 1876.95.

(173) 3.5 Synthesis of Compound K-5 (Formula 30)

(174) ##STR00088##

(175) Compound K-4 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound F-3 in step f was changed to F-7 and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-4 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1963.99, and measured value via LC-MS (ESI, M+H+): 18765.01.

(176) 3.6 Synthesis of Compound K-6 (Formula 31)

(177) ##STR00089##

(178) Compound K-6 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound H-1 in step f was changed to H-2 (Val-Ala-PAB) and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-6 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1613.78, and measured value via LC-MS (ESI, M+H+): 1614.80.

(179) 3.7 Synthesis of Compound K-7 (Formula 32)

(180) ##STR00090##

(181) Compound K-7 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound H-1 in step f was changed to H-3 (Phe-Lys-PAB) and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-7 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1718.84, and measured value via LC-MS (ESI, M+H+): 1719.86.

(182) 3.8 Synthesis of Compound K-8 (Formula 33)

(183) ##STR00091##

(184) Compound K-8 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound H-1 in step f was changed to H-4 (Ala-Ala-Asn-PAB) and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-8 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1699.79, and measured value via LC-MS (ESI, M+H+): 1700.80.

(185) 3.9 Synthesis of Compound K-9 (Formula 34)

(186) ##STR00092##

(187) Compound K-9 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound H-1 in step f was changed to H-5 (D-Ala-Phe-Lys-PAB) and compound D1 in step h was changed to D2 (Monomethylauristatin E). Product K-9 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1789.88, and measured value via LC-MS (ESI, M+H+): 179090.

(188) 3.10 Synthesis of Compound K-10 (Formula 35)

(189) ##STR00093##

(190) Compound K-10 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D in step h was changed to D3 (Monomethylauristatin F). Product K-10 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1713.81, and measured value via LC-MS (ESI, M+H+): 1714.83.

(191) 3.11 Synthesis of Compound K-11 (Formula 36)

(192) ##STR00094##

(193) Compound K-12 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D4 (Monomethyl Dolestatin 10, MMAD). Product K-12 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1752.80, 125 and measured value via LC-MS (ESI, M+H+): 1753.82.

(194) 3.12 Synthesis of Compound K-12 (Formula 37)

(195) ##STR00095##

(196) Compound K-12 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D5 (Tubulysin derivative 1). Product K-12 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1506.59, and measured value via LC-MS (ESI, M+H+): 1507.61.

(197) 3.13 Synthesis of Compound K-13 (Formula 38)

(198) ##STR00096##

(199) Compound K-13 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D6 (Tubulysin derivative 2). Product K-13 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1695.71, and measured value via LC-MS (ESI, M+H+): 1696.73.

(200) 3.14 Synthesis of Compound K-14 (Formula 39)

(201) ##STR00097##

(202) Compound K-14 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D6 (Cryptophycin derivative). The product K-14 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1679.64, and measured value via LC-MS (ESI, M+H+): 1680.66.

(203) 3.15 Synthesis of Compound K-15 (Formula 40)

(204) ##STR00098##

(205) Compound K-15 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D in step h was changed to D8 (Taltobulin). Product K-15 obtained after 3 steps of reaction was brownish yellow amorphous solid. Theoretical value via LC-MS (M+): 1455.65, and measured value via LC-MS (ESI, M+H+): 1456.6.

(206) 3.16 Synthesis of Compound K-16 (Formula 41)

(207) ##STR00099##

(208) Compound K-16 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D9 PB dimer) duct K-16 obtained ater 3 steps of reaction was brownish yellow amorphous solid. Theoretical value via LC-MS (M+): 1455.61, and measured value via LC-MS (ESI, M+H+): 145708.63.

(209) 3.17 Synthesis of Compound K-17 (Formula 42)

(210) ##STR00100##

(211) Compound K-17 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D10 (Duocarmycin derivative 1). Product K-17 obtained after 3 steps of reaction was brownish yellow amorphous solid. Theoretical value via LC-MS (M+): 1618.55, and measured value via LC-MS (ESI, M+H+): 1619.57.

(212) 3.18 Synthesis of Compound K-18 (Formula 43)

(213) ##STR00101##

(214) Compound K-18 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D11 (Duocarmycin derivative 2). Product K-18 obtained after 3 steps of reaction was brownish yellow amorphous solid. Theoretical value via LC-MS (M+): 1696.58, and measured value via LC-MS (ESI, M+H+): 1697.60.

(215) 3.19 Synthesis of Compound K-19 (Formula 44)

(216) ##STR00102##

(217) Compound K-19 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that compound D1 in step h was changed to D12 (PNU-159682 derivative). Product K-19 obtained after 3 steps of reaction was brownish yellow amorphous solid. Theoretical value via LC-MS (M+): 1737.61, and measured value via LC-MS (ESI, M+H+): 173868.

(218) 3.20 Synthesis of Compound K-20 (Formula 45)

(219) ##STR00103##

(220) Compound K-20 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that intermediate F-3 in step f was changed to intermediate F-14. Product K-20 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1639.64, and measured value via LC-MS (ESI, M+H+): 1640.61.

(221) 3.21 Synthesis of Compound K-21 (Formula 46)

(222) ##STR00104##

(223) Compound K-21 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that intermediate F-3 in step f was changed to intermediate F-11. Product K-21 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1701.82, and measured value via LC-MS (ESI, M+H+): 1702.86.

(224) 3.22 Synthesis of Compound K-22 (Formula 47)

(225) ##STR00105##

(226) Compound K-22 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that intermediate F-3 in step f was changed to intermediate F-16. Product K-22 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1925.92, and measured value via LC-MS (ESI, M+H+): 1926.88.

(227) 3.23 Synthesis of Compound K-23 (Formula 48)

(228) ##STR00106##

(229) Compound K-23 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that intermediate F-3 in step f was changed to intermediate F-16 and compound D1 in step h was changed to D11 (Duocarmycin derivative 2). Product K-23 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1848.64, and measured value via LC-MS (ESI, M+H+): 1849.58.

(230) 3.24 Synthesis of Compound K-24 (Formula 49)

(231) ##STR00107##

(232) Compound K-24 was synthetized by the same steps for synthesizing compound K-1 of Example 3.1, with the exception that intermediate F-3 in step f was changed to intermediate F-16 and compound D1 in step h was changed to D13 (SN38 derivative). Product K-24 obtained after 3 steps of reaction was yellow amorphous solid. Theoretical value via LC-MS (M+): 1714.64, and measured value via LC-MS (ESI, M+H+): 1715.61.

Section 2: Preparation and Detection of the Antibody Drug Conjugate

Example 1: Preparation of ADC-1

(233) A pertuzumab stock solution was diluted to 5 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 6.0× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 35° C. for 10 hours.

(234) Subsequently, without being purified, the reaction solution was cooled down to 8° C. and an appropriate amount of dimethyl sulfoxide (DMSO) and 6× excess molar ratio of compound G-2 (10 mg/ml, pre-dissolved in DMSO) were added thereto, and DMSO in the reaction system was ensured to be no more than 15% by volume. The obtained reaction solution was stirred at 37° C. for 3 hours for coupling.

(235) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.15 μm, and the obtained product was stored at −60° C.

Example 2: Preparation of ADC-2

(236) A pertuzumab stock solution was diluted to 5 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 10× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 10° C. for 4 hours.

(237) Subsequently, without being purified, the reaction solution was cooled down to 5° C. and an appropriate amount of dimethylacetamide (DMA) and 6× excess molar ratio of compound K-2 (10 mg/ml, pre-dissolved in DMA) were added thereto, and DMA in the reaction system was ensured to be no more than 10% by volume. The obtained reaction solution was stirred at 25° C. for 2.5 hours for coupling.

(238) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.22 μm, and the obtained product was stored at −80° C.

Example 3: Preparation of ADC-3

(239) A pertuzumab stock solution was diluted to 5 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 20× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 15° C. for 2 hours.

(240) Subsequently, without being purified, the reaction solution was cooled down to 10° C. and an appropriate amount of acetonitrile (ACN) and 6× excess molar ratio of compound K-3 (10 mg/ml, pre-dissolved in ACN) were added thereto, and ACN in the reaction system was ensured to be no more than 10% by volume. The obtained reaction solution was stirred at 10° C. for 4 hours for coupling.

(241) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized by filtrating and the obtained product was stored at low temperature. For example, the collected samples were sterilized through a filtration device with a pore size of 0.20 μm, and the obtained product was stored at −90° C.

Example 4: Preparation of ADC-4

(242) A pertuzumab stock solution was diluted to 5 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 25° C. for 48 hours.

(243) Subsequently, without being purified, the reaction solution was cooled down to 0° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound K-4 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 2 hours for coupling.

(244) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.3 μm, and the obtained product was stored at −100° C.

(245) The product obtained from Example 1 and pertuzumab were compared by hydrophobic interaction chromatography (HIC) (FIG. 1) and mass spectrometry (FIG. 2). Results showed that, the conjugates obtained by using the linker provided by the present invention had a very narrow distribution of DAR values, average of which was close to 4. In this regard, the conjugates obtained by present invention have high homogeneity, in which conjugates with a single distribution (with a DAR of 4) account for more than 80%. The value of m was identified to be between 3.0 and 4.2. The same results were obtained when the products obtained from Examples 2-4 were analyzed with the same methods, in which m was between 1.0 and 5.0.

Section 3: Biological Detections of the Antibody Drug Conjugate

(246) 1. Molecular Binding Assay

(247) The principle of Biacore instrument for detecting the intermolecular affinity of proteins is based on Surface Plasmon Resonance (SPR) technology. SPR is an optical physical phenomenon. A surface plasma wave is generated at the interface between a prism and a metal film (Au) when a P-polarized beam is incident on one facet of the prism in a certain angle range. Resonance of free electrons in the metal film is caused when the propagation constant of the incident beam matches the propagation constant of the surface plasma wave. So for analysis, a biomolecular recognition film is firstly fixed on the surface of a sensor chip, and then a sample to be tested is allowed to flow through the surface of the sensor chip. If there is a molecule in the sample that can interact with the biomolecular recognition film on the surface of the sensor chip, refractive index of the surface of the Au film changes accordingly, thereby resulting in changes in SPR angle finally. Information such as concentration, affinity, kinetic constant, and specificity of an analyte can be obtained by detecting the changes in SPR angle.

(248) Binding affinities of three monoclonal antibody samples comprising Pertuzumab, ADC-2 and ADC-4 to Human ErbB2 were detected through binding experiment using Biacore.

(249) TABLE-US-00002 TABLE 1 affinity and kinetic parameters of the three monoclonal antibody samples with Human ErbB2 Sample ka (1/Ms) kd (1/s) K.sub.D (M) Pertuzumab 1.902E+05 1.239E−03 6.517E−10 ADC-2 2.232E+05 1.294E−04 5.799E−10 ADC-4 1.956E+05 1.310E−04 6.969E−10

(250) In this experiment, the binding activities of three monoclonal antibody samples Pertuzumab, ADC-2 and ADC-4 to Human ErbB2 were characterized by using surface plasmon resonance technology, and all the three samples showed binding to Human ErbB2. The experiment results showed that the above three monoclonal antibody samples had similar affinity to Human ErbB2, all in a range of 0.5-0.7 nM.

(251) 2. Cellular Binding Assay of ADC-2 to Her2

(252) Experimental materials used in the following experiment: RPMI1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100× sodium pyruvate and 100× penicillin-streptomycin were purchased from Gibco; Secondary antibody labeled with fluorescein isothiocyanate (FITC) was purchased from Invitrogen; and NCI-N87 gastric cancer cells were obtained from Kunming Cell Bank of Chinese Academy of Sciences. All other reagents used were analytical grade. FACSCalibur flow cytometer (BD) was used.

(253) In this Example, binding affinities of ADC-2, P-mcVC-MMAE and Pertuzumab to Her2 highly expressing cells were investigated.

(254) Her2 highly expressing human gastric cancer NCI-N87 cells were used in this Example. The NCI-N87 cells were incubated in RPMI1640 medium containing 10% fetal bovine serum in a 5% CO2 incubator at 37° C. The cells that subcultured for 4 to 5 days were counted and then collected into a 15 mL centrifuge tube, washed twice with cold PBS, and centrifuged at 1000 rpm for 5 min at 4° C. The obtained cells were resuspended in PBS containing 5% fetal bovine serum, incubated at 37° C. for 30 min, then centrifuged at 1000° C. for 5 min at 4° C., and the supernatant was removed. The obtained cells were resuspended in cold PBS, dispensed into EP tubes at 1×10.sup.6 cells/1.5 mL, centrifuged at 1000 rpm for 5 min at 4° C., and the supernatant was removed. Subsequently, 0.5 mL of ADC-2, P-mcVC-MMAE, Pertuzumab and human IgG in different concentrations were added to the EP tubes, which were then centrifuged at 1000 rpm for 5 min at 4° C. after being placed on ice for 40 min. The obtained residues were washed twice with 1 ml cold PBS, and then 200 μL of FITC-labeled secondary antibody was added to each tube. The EP tubes were placed on ice for 40 minutes in the dark, centrifuged at 1000 rpm for 5 min at 4° C. and the supernatant was removed. The obtained cells were washed twice with 1 ml cold PBS, and then resuspended by addition of 0.5 ml cold PBS, placed on ice in the dark. Mean fluorescence intensity (MFI) of the binding of each of ADC-2, P-mcVC-MMAE and Pertuzumab in different concentrations to the cells was detected using FACSCalibur flow cytometer. The fluorescence intensity of the human IgG binding was of non-specific binding.

(255) As shown in FIG. 3, ADC-2, P-mcVC-MMAE, and Pertuzumab all bound to antigen Her2 on the surface of the NCI-N87 cells, and with the increasing of concentration, the bindings of ADC-2, P-mcVC-MMAE and Pertuzumab to Her2 were increased. What's more, the binding affinities of the monoclonal antibodies to Her2 of NCI-N87 cells were comparable and no significant difference was observed.

(256) 3. Biological Activity Assay of Cell Proliferation In Vitro

(257) Experimental materials used in the following experiment: DMEM, RPMI1640 medium, DMEM/F12K, 0.25% trypsin-EDTA, fetal bovine serum, 100× sodium pyruvate and 100× penicillin-streptomycin were purchased from Gibco; sulforhodamine B (SRB) was purchased from Sigma; BT-474 human breast cancer cells, SK-RB-3 human breast cancer cells, MDA-MB-231 human breast cancer cells and NCI-N87 human gastric cancer cells were obtained from Kunming Cell Bank of Chinese Academy of Sciences; Panc-1 human pancreatic cancer cells, MDA-MB-468 human breast cancer cells and MCF-7 human breast cancer cells were obtained from Cell Bank in Shanghai Institutes for Biological Sciences; and SKOV-3 human ovarian cancer cells and Du-145 human prostate cancer cells were obtained from American Type Culture Collection (ATCC). All other reagents used were analytical grade. 96-well Flat Bottom Polystyrene (Corning, catalog No. 3599) and Synergy 2 Microplate Reader (Bio-Tek) were used.

(258) In this Example, effects of ADC-2, ADC-4, P-mcVC-MMAE, Kadcyla, and Pertuzumab on the proliferation of tumor cell lines were investigated.

(259) Sulforhodamine B (SRB)-based colorimetric method was used in this Example to evaluate the anti-proliferative effect of the drugs. SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids making up proteins in cells under an acidic condition. It provides an absorption peak at 510 nm, and the absorbance is linearly and positively correlated with the amount of cells. In this regard, the method can be used in a quantitative detection of cell number.

(260) Cell lines used in this Example were: BT-474, SK-RB-3, MDA-MB-231, MDA-MB-468, MCF-7 human breast cancer cells, NCI-N87 human gastric cancer cells, SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells, and Panc-1 human pancreatic cancer.

(261) BT-474, SK-BR-3 and NCI-N87 cells in RPMI 1640 medium containing 10% fetal bovine serum, SKOV-3, Du-145, Panc-1, MCF-7 and MDA-MB-231 cells in DMEM containing 10% fetal bovine serum, and MDA-MB-468 cells in DMEM/F12 containing 10% fetal bovine serum were incubated to logarithmic growth phase in a 5% CO2 incubator at 37° C. The above cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 2×10.sup.3 to 9×10.sup.3 cells per well, 100 μL per well, cultured for 24 hours and then different concentrations of drugs were added thereto for 5 days. Specifically, each drug was prepared into nine concentrations by diluting in 3, 4 or 5-fold, each concentration was set in duplicate wells, and corresponding concentration of vehicle control wells and medium control wells without cells were set too. At the end of drug action, culture solutions were decanted, and 100 μl of a pre-cooled trichloroacetic acid solution (30%, w/v) at 4° C. was added to each well and the cells were fixed at 4° C. for 1 hour. Subsequently, the cells were washed with deionized water for 5 times, and dried at room temperature, and then 100 μL of 0.4% (w/v) SRB dye (Sigma, prepared with 1% glacial acetic acid) was added to each well. After being incubated and stained at room temperature for 30 minutes, the cells were washed with 1% glacial acetic acid for 4 times to remove unbound dyes, and then dried at room temperature. Afterwards, 100 μL of 10 mM Tris solution was added per well. After being incubated and stained at room temperature for 15 minutes, the cells was washed with 1% glacial acetic acid for 5 times to remove unbound SRB, and then dried at room temperature. Dyes bound to the proteins in cells were dissolved by addition of 10 mM Tris buffer (pH=10.5) per well, and the absorbance (OD value) was measured at wavelengths of 510 nm and 690 nm using Synergy 2 Microplate Reader (Bio-Tek). A=OD.sub.510−OD.sub.690.
Inhibition rate (%)=(A.sub.control−A.sub.drug)/A.sub.control×100%

(262) In this experiment, effects of ADC-2, ADC-4, P-mcVC-MMAE, Kadcyla, and Pertuzumab on the proliferation of various Her2 highly expressing tumor cell lines in vitro cultured were investigated. Meanwhile, effects of ADC-2 on the proliferation of various tumor cell lines which do not highly express Her2 in vitro cultured were also studied. As shown in FIGS. 4-6, significant inhibition of tumor cell proliferation could be observed with Her2 highly expressing SK-BR-3, BT-474 human breast cancer cells and NCI-N87 human gastric cancer cells which were treated with ADC-2, P-mcVC-MMAE and Kadcyla. Specifically, the inhibitory activity of ADC-2 and P-mcVC-MMAE on tumor cell proliferation was significantly higher than that of Kadcyla and the inhibitory activity of ADC-2 on tumor cell proliferation was essentially comparable to that of P-mcVC-MMAE. However, ADC-2 had a slightly higher inhibitory activity on the proliferation of BT-474 tumor cells than P-mcVC-MMAE. In addition, compared to Pertuzumab which was naked, the antibody drug conjugates, including ADC-2, P-mcVC-MMAE and Kadcyla had significantly higher inhibitory activity on the proliferation of tumor cells. As shown in FIG. 7, ADC-2 also showed good inhibitory effects on the proliferation of SKOV-3 human ovarian cancer cells, Du-145 human prostate cancer cells and Panc-1 human pancreatic cancer cells which did not highly express Her2. In contrast, the inhibitory activities of ADC-2 on the proliferation of human breast cancer MCF-7, MDA-MB-231 and MDA-MB-468 cells which did not express Her2 were significantly lower (FIG. 8). The above results indicate that antibody drug conjugate ADC-2 essentially has no effects on cells not expressing the target antigen, and thus, the toxic and side effects of ADC-2 will be significantly lesser. As shown in FIG. 9-1, ADC-4 showed a potent proliferation inhibition effect on NCI-N87 human gastric cancer cells which highly expressed Her2. And as shown in FIG. 9-1, ADC-2, ADC-3 and ADC-4 showed potent proliferation inhibition effects on Calu-3 human lung cancer cells which expressed Her2 moderately.

(263) 4. In Vivo Anti-Tumor Efficacy Assay

(264) Efficacy of the conjugates of the present invention can be detected in vivo. In brief, an allograft or xenograft of cancer cells can be implanted into rodents and then the implanted tumors are treated with the conjugates. Tested mice can be administered drug treatment or control treatment, monitored for weeks or longer to observe tumor doubling time, log-killing, and tumor suppression.

(265) 1) Experimental Animals

(266) 6-7 weeks BALB/cA-nude mice (♀) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK (Shanghai) 2013-0018; animal certificate number: 2013001815683; and feeding environment: SPF level.

(267) 2) Experimental Steps

(268) Those nude mice were subcutaneously inoculated with human gastric cancer NCI-N87 cells, and then randomly divided into groups (D0) when tumors grew to 100-250 mm.sup.3. Doses and regimen of administration are provided in FIG. 10. Tumor volume was measured 2-3 times a week, the mice were weighed, and corresponding data were recorded. The tumor volume (V) was calculated as: V=½×a×b.sup.2; in which a and b represent length and width respectively. T/C (%)=(T−T0)/(C−C0)×100, in which T and C represent the tumor volumes at the end of the experiment; T0 and C0 represent the tumor volumes at the beginning of the experiment.

(269) 3) Experiment Results

(270) FIG. 10 shows that ADC-2 (0.5, 1, 2 mg/kg, IV, once a week, 2 times in total) can inhibit the growth of human gastric cancer NCI-N87 subcutaneous xenografts in nude mice dose-dependently and significantly, and the tumor inhibition rates are 59%, 94%, and 200% respectively. Specifically, in the group treated with 1 mg/kg, the tumors of 3/6 mice were partially regressed; and in the group treated with 2 mg/kg, the tumors of 6/6 mice were completely regressed. Inhibition rates of ADC3 (0.5, 1, 2 mg/kg, IV, once a week, 2 times in total) on NCI-N87 are 65%, 69% and 185% respectively. Specifically, in the group treated with 2 mg/kg, the tumor of 1/6 mouse was partially regressed and the tumors of 5/6 mice were completely regressed. Inhibition rate of P-vcMMAE (1 mg/kg, IV, once a week, 2 times in total) on NCI-N87 is 94%. Specifically, the tumors of 2/6 mice were partially regressed. Inhibition rates of reference drug Kadcyla (2 mg/kg, IV, once a week, 2 times in total) is 77%. What's more, those tumor-bearing mice had good tolerance to the drugs, and no symptom such as weight loss occurred.

(271) Another One of Specific Designs:

(272) preparation and use of the substituted maleamide linker as shown in Formula Ia, wherein Ar′ is selected from the group consisting of substituted C.sub.6-C.sub.10 arylene and substituted 5-12 membered heteroarylene.

The First Group of Examples: Synthesis and Preparation Methods of the Compound

(273) 1.1 Synthesis of Compound E-1 (Formula Ia-1)

(274) 1.1.1 Synthesis of Intermediate A-1 (Step a)

(275) ##STR00108##

(276) Triethylene glycol (92 g, 613 mmol) was dissolved in tBuOH (200 ml). The obtained solution was placed in ice bath, KOtBu (22.91 g, 204 mmol) was added thereto, and stirred for 30 minutes. Subsequently, tert-butyl bromoacetate (39.8 g, 204 mmol) dissolved in tBuOH (40 ml) was added dropwise under the protection of argon, and the obtained mixture was stirred overnight at room temperature. TLC on the next day indicated that the reaction was completed. Tert-butanol was rotary evaporated off. The residue was added to 400 ml of dichloromethane, and the organic phase was washed with 400 ml of water. The obtained aqueous phase was extracted once with 300 ml of dichloromethane. The organic phases were combined and washed once with saturated salt water, and dried over anhydrous sodium sulfate and then the solvent was rotary evaporated off. The obtained crude was subjected to column chromatography (petroleum ether: ethyl acetate=3:1->1:1) to obtain intermediate A-1 (24 g, 44.5% yield), which was a yellow oily product.

(277) 1.1.2 Synthesis of Intermediate B-1 (Step b)

(278) ##STR00109##

(279) Intermediate A-1 (7.8 g, 29.5 mmol), 5-fluoro-2-nitrobenzotrifluoride (9.26 g, 44.3 mmol), and K.sub.2CO.sub.3 powder (6.12 g, 44.3 mmol) were placed in a 250 mL round bottom reaction flask. Under the protection of nitrogen, the obtained mixture was heated to 80° C. and stirred for 48 hours. TLC indicated that only a small amount of starting materials was remained.

(280) It was then cooled down to room temperature, extracted with 500 ml dichloromethane. The organic phase was washed once with 400 ml of 1 N diluted hydrochloric acid, once with 400 ml water and once with 400 ml saturated salt water, and dried with anhydrous sodium sulfate, and the solvent was rotary evaporated off. The residue was purified by column chromatography (silica gel, 200-300 mesh), rinsed with petroleum ether: ethyl acetate 30:1-10:1, to obtain intermediate B-1 (7.5 g, 56.1% yield), which was a yellow oily product.

(281) 1.1.3 Intermediate C-1

(282) ##STR00110##

(283) Intermediate B-1 (6 g, 13.23 mmol) was dissolved in 100 mL of anhydrous ethanol and then the obtained solution was added to a reaction flask containing 1.2 g of 10% Pd—C. A hydrogenation reaction was carried out for 6 hours (1 atm, 38° C.), and TLC indicated that the reaction was completed. The reaction mixture was filtered through diatomaceous earth, and the filter cake was rinsed with ethanol, and the filtrate was rotary evaporated off to obtain intermediate C-1 (5 g, 89% yield), which was a yellow oily product.

(284) 1.1.4 Compound E-1

(285) ##STR00111##

(286) Intermediate C-1 (0.8 g, 1.889 mmol) was weighed and placed into a parallel reaction tube, then AcOH (3 ml) was added thereto under the protection of nitrogen, and stirred to dissolve. Then, 3,4-dibromomaleic anhydride (0.483 g, 1.889 mmol) was added to the tube, and the obtained reaction mixture was heated to 110° C. and stirred overnight under the protection of nitrogen. The reaction was detected by TLC. Afterwards, the reaction mixture was cooled to room temperature and the solvent was rotary evaporated off. The residue was further rotary evaporated twice by addition of toluene, thereby obtaining compound E-1, which was a brown oily product and was used in the next step directly without being purified.

(287) 1.2 Synthesis of Compound E-2 (Formula Ia-2) (Step e)

(288) ##STR00112##

(289) Compound E-1 (2.0 g, 1.35 mmol) was weighed and placed into a 100 ml round bottom flask, then 30 ml of anhydrous dichloromethane was added thereto under the protection of nitrogen and stirred to dissolve. Then, 297 mg of thiophenol was weighed and added thereto under the protection of nitrogen. After thiophenol dissolved, DIPEA (0.44 ml, 2.70 mmol) was slowly added dropwise to the flask which was in an ice bath. Afterwards, the reaction mixture was stirred for 5 minutes, and then the ice bath was removed. After stirring at room temperature for 2 hours under the protection of nitrogen, TLC indicated that the reaction was completed.

(290) The solvent was evaporated under reduced pressure, the residue was isolated and purified by column chromatography (200-300 mesh silica gel). The column was loaded and rinsed with dichloromethane, and then rinsed with methanol which polarity slowly increased from 2% to 10%. The eluent was collected and the solvent was evaporated to obtain E-2 (0.92 g, 79% yield), which was an orange oily product. Theoretical value via LC-MS (M+): 595.13, and measured value: 596.15 (ESI, M+H+).

(291) 1.3 Synthesis of Compound E-3 (Formula Ia-3)

(292) Compound E-3 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that thiophenol in step e was changed to 2-mercaptopyridine. Product E-3 obtained was an orange oily product.

(293) 1.4 Synthesis of Compound E-4 (Formula Ia-4)

(294) Compound E-4 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 4-fluoro-2-methoxy-1-nitrobenzene and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-4 obtained was an orange oily product.

(295) 1.5 Synthesis of Compound E-5 (Formula Ia-5)

(296) Compound E-5 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 1-fluoro-2-methoxy-4-nitrobenzene and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-5 obtained was an orange oily product.

(297) 1.6 Synthesis of Compound E-6 (Formula Ia-6)

(298) Compound E-6 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 5-fluoro-2-nitrobenzonitrile and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-6 obtained was an orange oily product.

(299) 1.7 Synthesis of Compound E-7 (Formula Ia-7)

(300) Compound E-7 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 2-fluoro-5-nitrobenzonitrile and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-7 obtained was an orange oily product.

(301) 1.8 Synthesis of Compound E-8 (Formula Ia-8)

(302) Compound E-8 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 5-fluoro-2-nitrobenzamide and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-8 obtained was an orange oily product.

(303) 1.9 Synthesis of Compound E-9 (Formula Ia-9)

(304) Compound E-9 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 4-fluoro-1-nitro-2-(trifluoromethyl)benzene and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-9 obtained was an orange oily product.

(305) 1.10 Synthesis of Compound E-10 (Formula Ia-10)

(306) Compound E-10 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 1-fluoro-4-nitro-2-(trifluoromethyl)benzene and the thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-10 obtained was an orange oily product.

(307) 1.11 Synthesis of Compound E-11 (Formula Ia-11)

(308) 1.11.1 Intermediate F-11 (Step f)

(309) ##STR00113##

(310) In a 250 ml round bottom flask, intermediate A-1 (4 g, 15.13 mmol), triethylamine (2.53 ml, 18.16 mmol) and dimethylaminopyridine (0.370 g, 3.03 mmol) were dissolved in 100 ml dichloromethane dried with molecular sieve and then stirred. Subsequently, 4-toluene sulfonyl chloride (3.17 g, 16.65 mmol) was added in batches in an ice bath, and the obtained reaction system was stirred under the protection of argon at room temperature overnight.

(311) To the reaction system, 100 ml of dichloromethane was added for extraction, and then the organic phase was washed once with 200 ml of 1 N diluted hydrochloric acid, twice with 200 ml water and once with 200 ml saturated brine, and dried with anhydrous sodium sulfate, and the solvent was rotary evaporated off. The residue was isolated by column chromatography, in which the column was packed with 200-300 mesh silica gel and eluted with PE:EA=5:1-2:1. The collected eluent was rotary evaporated off to obtain intermediate F-11 (2.8 g, 44.2% yield).

(312) 1.11.2 Intermediate B-11 (Step g)

(313) ##STR00114##

(314) Intermediate F-11 (1 g, 2.389 mmol) and 2,6-difluoro-4-nitrophenol (0.315 g, 1.797 mmol) were dissolved in 20 ml DMF, and then K.sub.2CO.sub.3 (0.497 g, 3.59 mmol) was added thereto. The obtained mixture was heated to 100° C. and stirred for 5 hours. The solvent was rotary evaporated off and then the residue was dissolved in 200 ml dichloromethane for extraction. The organic phase was then washed once with 200 ml of 1 N diluted hydrochloric acid, once with 200 ml water and once with 200 ml saturated salt water, and dried with anhydrous sodium sulfate, and the solvent was rotary evaporated off. The residue was purified by column chromatography, in which the column was packed with 200-300 mesh silica gel and eluted with PE:EA=5:1-3:1. The collected eluent was rotary evaporated off to obtain intermediate B-11 (600 mg, 79% yield).

(315) 1.11.3 Intermediate C-11

(316) ##STR00115##

(317) Intermediate B-11 (600 mg, 1.42 mmol) was dissolved in 100 mL of anhydrous ethanol and then the obtained solution was added to a reaction flask containing 120 mg of 10% Pd—C. A hydrogenation reaction was carried out for 6 hours (1 atm, 38° C.), and TLC indicated that the reaction was completed. The reaction mixture was filtered through diatomaceous earth, and the filter cake was rinsed with ethanol, and the filtrate was rotary evaporated off to obtain intermediate C-11 (450 mg, 81% yield), which was a yellow oily product.

(318) 1.11.4 Intermediate D-11

(319) ##STR00116##

(320) Intermediate C-11 (0.40 g, 1.02 mmol) was weighed and placed into a parallel reaction tube, then AcOH (3 ml) was added thereto under the protection of nitrogen, and stirred to dissolve. Then, 3,4-dibromomaleic anhydride (0.261 g, 1.02 mmol) was added to the tube, and the obtained reaction mixture was heated to 110° C. and stirred overnight under the protection of nitrogen. The reaction was detected by TLC. Afterwards, the reaction mixture was cooled to room temperature and the solvent was rotary evaporated off. The residue was further rotary evaporated twice by addition of toluene, thereby obtaining intermediate D-11, which was a brown oily product and was used in the next step directly without being purified.

(321) 1.11.5 Intermediate E-11

(322) ##STR00117##

(323) Compound D-11 (600 mg, 0.95 mmol) was weighed and placed into a 100 ml round bottom flask, then 30 ml of anhydrous dichloromethane was added thereto under the protection of nitrogen and stirred to dissolve. Then, 425 mg (1.91 mmol) of 4-(N-morpholineformamide) thiophenol was weighed and added thereto under the protection of nitrogen. After 4-(N-morpholineformamide) thiophenol dissolved, DIPEA (0.36 ml, 1.91 mmol) was slowly added dropwise to the flask which was in an ice bath. Afterwards, the reaction mixture was stirred for 5 minutes, and then the ice bath was removed. After stirring at room temperature for 2 hours under the protection of nitrogen, TLC indicated that the reaction was completed.

(324) The solvent was evaporated under reduced pressure, the residue was isolated and purified by column chromatography (200-300 mesh silica gel). The column was loaded and rinsed with dichloromethane, and then rinsed with methanol which polarity slowly increased from 2% to 10%. The eluent was collected and the solvent was evaporated to obtain E-11 (0.62 g, 76% yield), which was an orange oily product. Theoretical value via LC-MS (M+): 857.21, and measured value: 858.23 (ESL, M+H+).

(325) 1.12 Synthesis of Compound E-12 (Formula Ia-12)

(326) Compound E-12 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that 2,6-difluoro-4-nitrophenol in step g was changed to 3-fluoro-4-nitrophenol. Product E-12 obtained was an orange oily product.

(327) 1.13 Synthesis of Compound E-13 (Formula Ia-13)

(328) Compound E-13 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that 2,6-difluoro-4-nitrophenol in step g was changed to 2,5-difluoro-4-nitrophenol. Product E-13 obtained was an orange oily product.

(329) 1.14 Synthesis of Compound E-14 (Formula Ia-14)

(330) Compound E-14 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that triethylene glycol in step a was changed to diethylene glycol. Product E-14 obtained was an orange oily product.

(331) 1.15 Synthesis of Compound E-15 (Formula Ia-15)

(332) Compound E-15 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that triethylene glycol in step a was changed to tetraethylene glycol. Product E-15 obtained was an orange oily product.

(333) 1.16 Synthesis of Compound E-16 (Formula Ia-16)

(334) Compound E-16 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that triethylene glycol in step a was changed to pentaethylene glycol. Product E-16 obtained was an orange oily product.

(335) 1.17 Synthesis of Compound E-17 (Formula Ia-17)

(336) Compound E-17 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that triethylene glycol in step a was changed to hexaethylene glycol. Product E-17 obtained was an orange oily product.

(337) 1.18 Synthesis of Compound E-18 (Formula Ia-18)

(338) Compound E-18 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that triethylene glycol in step a was changed to dodecaethylene glycol. Product E-18 obtained was an orange oily product.

(339) 1.19 Synthesis of Compound E-19 (Formula Ia-19)

(340) Compound E-19 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that 4-(N-morpholineformamide) thiophenol in step e was changed to thiomorpholine-1,1-dioxide. Product E-19 obtained was an orange oily product.

(341) 1.20 Synthesis of Compound E-20 (Formula Ia-20)

(342) Compound E-20 was synthetized by the same steps for synthesizing compound E-11 of Example 1.11, with the exception that 4-(N-morpholineformamide) thiophenol in step e was changed to 4-(N-methylformamide) thiophenol. Product E-20 obtained was an orange oily product.

(343) 1.21 Synthesis of Compound E-21 (Formula Ia-21)

(344) Compound E-21 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 5-fluoro-2-nitropyridine and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-21 obtained was an orange oily product.

(345) 1.22 Synthesis of Compound E-22 (Formula Ia-22)

(346) Compound E-22 was synthetized by the same steps for synthesizing compound E-2 of Example 1.2, with the exception that 5-fluoro-2-nitrobenzotrifluoride in step b was changed to 2-fluoro-5-nitropyridine and thiophenol in step e was changed to 4-(N-morpholineformamide) thiophenol. Product E-21 obtained was an orange oily product.

The Second Group of Examples: Synthesis and Preparation of Compounds as Shown in Formulas Ib-1-Ib-24

(347) 2.1 Synthesis of Compound F1-1 (Formula Ib-1)

(348) ##STR00118##

(349) Compound E1-9 (300 mg, 0.337 mmol) was weighted and placed into a 100 ml round-bottom flask, and stirred to dissolve by addition of anhydrous DMF (20 mL) under the protection of nitrogen. Afterwards, HATU (154 mg, 0.404 mmol) and DIEA (0.11 ml, 0.674 mmol) were weighted and added into the flask successively. The obtained mixture was stirred for 15 minutes at room temperature followed by adding compound D1-1 (219 mg, 0.337 mmol) thereto. The obtained reaction mixture was stirred overnight at room temperature at the protection of nitrogen. TLC and HPLC indicated that raw material E9 disappeared with overnight reaction. The solvent was evaporated off under reduced pressure and the residue was quantitatively analyzed, and then isolated and purified by reverse-phase HPLC to obtain a yellow amorphous powder product F1-1 (0.350 g, 0.230 mmol, 68.2% yield). Theoretical value via LC-MS (M+): 1520.48, and measured value: 1521.51 (ESI, M+H+).

(350) 2.2 Synthesis of Compound F1-2 (Formula Ib-2)

(351) ##STR00119##

(352) Compound F1-2 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-2. Product F1-2 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1993.91, and measured value: 1994.93 (ESI, M+H+).

(353) 2.3 Synthesis of Compound F1-3 (Formula Ib-3)

(354) ##STR00120##

(355) Compound F1-3 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-3. Product F1-3 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 2007.89, and measured value: 2008.91 (ESI, M+H+).

(356) 2.4 Synthesis of Compound F1-4 (Formula Ib-4)

(357) ##STR00121##

(358) Compound F1-4 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-4. Product F1-4 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 2046.88, and measured value: 2047.86 (ESI, M+H+).

(359) 2.5 Synthesis of Compound F1-5 (Formula Ib-5)

(360) ##STR00122##

(361) Compound F1-5 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-5. Product F1-5 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1800.67, and measured value: 1801.65 (ESI, M+H+).

(362) 2.6 Synthesis of Compound F1-6 (Formula Ib-6)

(363) ##STR00123##

(364) Compound F1-6 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-6. Product F1-6 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1989.79, and measured value: 1990.80 (ESI, M+H+).

(365) 2.7 Synthesis of Compound F1-7 (Formula Ib-7)

(366) ##STR00124##

(367) Compound F1-7 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-7. Product F1-7 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1973.72, and measured value: 1974.72 (ESI, M+H+).

(368) 2.8 Synthesis of Compound F1-8 (Formula Ib-8)

(369) ##STR00125##

(370) Compound F1-8 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-8. Product F1-8 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1749.73, and measured value: 1750.75 (ESI, M+H+).

(371) 2.9 Synthesis of Compound F1-9 (Formula Ib-9)

(372) ##STR00126##

(373) Compound F1-9 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-17 and D1-9 respectively. Product F1-9 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 2015.72, and measured value: 2016.73 (ESI, M+H+).

(374) 2.10 Synthesis of Compound F-10 (Formula Ib-10)

(375) ##STR00127##

(376) Compound F1-10 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-10. Product F1-10 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1912.63, and measured value: 1913.65 (ESI, M+H+).

(377) 2.11 Synthesis of Compound F1-11 (Formula Ib-11)

(378) ##STR00128##

(379) Compound F1-1 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-11. Product F1-11 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1916.63, and measured value: 1917.61 (ESI, M+H+).

(380) 2.12 Synthesis of Compound F1-12 (Formula Ib-12)

(381) ##STR00129##

(382) Compound F1-12 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-12. Product F1-12 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 2031.70, and measured value: 2032.71 (ESI, M+H+).

(383) 2.13 Synthesis of Compound F1-13 (Formula Ib-13)

(384) ##STR00130##

(385) Compound F1-13 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compound D1-1 was changed to compound D1-13. Product F1-13 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1711.57, and measured value: 1712.55 (ESI, M+H+).

(386) 2.14 Synthesis of Compound F1-14 (Formula Ib-14)

(387) ##STR00131##

(388) Compound F1-14 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-2 and D1-2 respectively. Product F1-14 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1767.82, and measured value: 1768.83 (ESI, M+H+).

(389) 2.15 Synthesis of Compound F1-15 (Formula Ib-15)

(390) ##STR00132##

(391) Compound F1-15 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-19 and D1-2 respectively. Product F1-15 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 2057.84, and measured value: 2058.87 (ESI, M+H+).

(392) 2.16 Synthesis of Compound F1-16 (Formula Ib-16)

(393) ##STR00133##

(394) Compound F1-16 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-20 and D1-2 respectively. Product F1-16 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1849.85, and measured value: 1850.83 (ESI, M+H+).

(395) 2.17 Synthesis of Compound F1-17 (Formula Ib-17)

(396) ##STR00134##

(397) Compound F1-17 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-10 and D1-2 respectively. Product F1-17 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1993.91, and measured value: 1994.90 (ESI, M+H+).

(398) 2.18 Synthesis of Compound F1-18 (Formula Ib-18)

(399) ##STR00135##

(400) Compound F1-18 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-11 and D1-2 respectively. Product F1-18 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1961.90, and measured value: 1962.91 (ESI, M+H+).

(401) 2.19 Synthesis of Compound F1-19 (Formula Ib-19)

(402) ##STR00136##

(403) Compound F1-19 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-5 and D1-2 respectively. Product F1-19 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1955.93, and measured value: 1956.95 (ESI, M+H+).

(404) 2.20 Synthesis of Compound F1-20 (Formula Ib-20)

(405) ##STR00137##

(406) Compound F1-20 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-4 and D1-2 respectively. Product F1-20 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1955.93, and measured value: 1956.95 (ESI, M+H+).

(407) 2.21 Synthesis of Compound F1-21 (Formula Ib-21)

(408) ##STR00138##

(409) Compound F1-21 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-12 and D1-2 respectively. Product F1-21 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1943.91, and measured value: 1944.90 (ESI, M+H+).

(410) 2.22 Synthesis of Compound F1-22 (Formula Ib-22)

(411) ##STR00139##

(412) Compound F1-22 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-6 and D1-2 respectively. Product F1-22 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1950.92, and measured value: 1951.93 (ESI, M+H+).

(413) 2.23 Synthesis of Compound F1-23 (Formula Ib-23)

(414) ##STR00140##

(415) Compound F1-23 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-21 and D1-2 respectively. Product F1-23 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1926.92, and measured value: 1927.93 (ESI, M+H+).

(416) 2.24 Synthesis of Compound F1-24 (Formula Ib-24)

(417) ##STR00141##

(418) Compound F1-24 was synthetized by the same steps for synthesizing compound F1-1 of Example 2.1, with the exception that compounds E1-9 and D1-1 were changed to compounds E1-22 and D1-2 respectively. Product F1-24 obtained was a yellow amorphous powder. Theoretical value via LC-MS (M+): 1926.92, and measured value: 1927.93 (ESI, M+H+).

The Third Group of Examples: Preparation of the Antibody Drug Conjugate

(419) 1. Preparation of ADC-I

(420) A pertuzumab stock solution was diluted to 2 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7, and then 6.0× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 35° C. for 2.5 hours.

(421) Subsequently, without being purified, the reaction solution was cooled down to 8° C. and an appropriate amount of dimethyl sulfoxide (DMSO) and 6× excess molar ratio of compound F1-17 (10 mg/ml, pre-dissolved in DMSO) were added thereto, and DMSO in the reaction system was ensured to be no more than 15% by volume. The obtained reaction solution was stirred at 37° C. for 3 hours for coupling.

(422) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.15 μm, and the obtained product was stored at −60° C.

(423) 2. Preparation of ADC-II

(424) A pertuzumab stock solution was diluted to 5 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 6, and then 10× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 10° C. for 40 hours.

(425) Subsequently, without being purified, the reaction solution was cooled down to 5° C. and an appropriate amount of dimethylacetamide (DMA) and 6× excess molar ratio of compound F1-2 (10 mg/ml, pre-dissolved in DMA) were added thereto, and DMA in the reaction system was ensured to be no more than 10% by volume. The obtained reaction solution was stirred at 25° C. for 2.5 hours for coupling.

(426) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.22 μm, and the obtained product was stored at −80° C.

(427) 3. Preparation of ADC-III

(428) A pertuzumab stock solution was diluted to 5 mg/mL with PBS//1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 20× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 15° C. for 2 hours.

(429) Subsequently, without being purified, the reaction solution was cooled down to 10° C. and an appropriate amount of acetonitrile (ACN) and 6× excess molar ratio of compound F1-20 (10 mg/ml, pre-dissolved in ACN) were added thereto, and ACN in the reaction system was ensured to be no more than 10% by volume. The obtained reaction solution was stirred at 10° C. for 4 hours for coupling.

(430) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 8.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.20 μm, and the obtained product was stored at −90° C.

(431) 4. Preparation of ADC-IV

(432) A pertuzumab stock solution was diluted to 8 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7, and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 25° C. for 25 hours.

(433) Subsequently, without being purified, the reaction solution was cooled down to 5° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound F1-19 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 2 hours for coupling.

(434) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.3 μm, and the obtained product was stored at −80° C.

(435) 5. Preparation of ADC-V

(436) A pertuzumab stock solution was diluted to 6 mg/mL with 50 mM histidine-sodium hydroxide/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 35° C. for 15 hours.

(437) Subsequently, without being purified, the reaction solution was cooled down to 10° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound F1-22 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 5 hours for coupling.

(438) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.15 μm, and the obtained product was stored at −100° C.

(439) 6. Preparation of ADC-VI

(440) A pertuzumab stock solution was diluted to 10 mg/mL with 50 mM boric acid-borax/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 9; and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 25° C. for 10 hours.

(441) Subsequently, without being purified, the reaction solution was cooled down to 10° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound F1-21 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 4 hours for coupling.

(442) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.2 μm, and the obtained product was stored at −60° C.

(443) 7. Preparation of ADC-VII

(444) A pertuzumab stock solution was diluted to 3 mg/mL with 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH.sub.2PO.sub.4—NaOH)/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 8, and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 15° C. for 48 hours.

(445) Subsequently, without being purified, the reaction solution was cooled down to 0° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound F1-18 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 3 hours for coupling.

(446) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.3 μm, and the obtained product was stored at −70° C.

(447) 8. Preparation of ADC-VIII

(448) A trastuzumab stock solution was diluted to 5 mg/mL with 50 mM disodium hydrogen phosphate-citric acid/150 mM sodium chloride (NaCl)/1 mM diethylene triamine pentacetate acid (DTPA) reaction buffer solution with a pH of 7.4, and then 8× excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added thereto. The obtained reaction solution was stirred at 25° C. for 5 hours.

(449) Subsequently, without being purified, the reaction solution was cooled down to 0° C. and an appropriate amount of dimethylformamide (DMF) and 6× excess molar ratio of compound F1-2 (10 mg/ml, pre-dissolved in DMF) were added thereto, and DMF in the reaction system was ensured to be no more than 8% by volume. The obtained reaction solution was stirred at 0° C. for 2 hours for coupling.

(450) The reaction solution after coupling reaction was purified by filtration with a desalting column using histidine-acetic acid/sucrose gel (pH 6.0), and then sample at absorption peak was collected according to UV280 ultraviolet absorption value. The collected sample was sterilized through a filtration device with a pore size of 0.3 μm, and the obtained product was stored at −80° C.

The Fourth Group of Examples: Detection and Stability Study of the Antibody Drug Conjugate

(451) By hydrophobic interaction chromatography (HIC) analysis on antibody conjugated drugs, important information such as the number and position of coupling sites and the drug to antibody ratio (DAR) can be obtained. In this regard, the inventors performed HIC analysis on the above ADC products based on the following conditions, and the obtained chromatograms were shown in FIGS. 11-1 to 11-8 and FIGS. 12-1 to 12-2.

(452) Agilent 1290 Infinity

(453) Chromatographic column: Waters Protein-Pak Hi Res HIC (4.6*100 mm, 2.5 μm)

(454) Mobile phase: 2.5 M ammonium sulfate (containing 125 mM phosphate buffer): 125 mM phosphate buffer: isopropanol;

(455) Flow rate: 0.7 mL/min, column temperature: 25° C.

(456) In addition, LC-MS technology has been used to analyze structure and composition of ADCs, evaluate stability of the linkers in the ADCs, and analyze and determine relative proportions between components having different DARs. In this regard, the inventors performed LC-MS analysis on the above ADC products based on the following conditions, and the obtained chromatograms were shown in FIGS. 13-1 to 13-8 and FIGS. 14-1 to 14-2.

(457) Instrument: Agilent 6520 Q-TOF

(458) Chromatographic column: Polyhydroxyethyl-A (PHEA) (PolyLC, Columbia, Md.) 2.1 mm*200 mm; 5 μm particles with 300 Å pores

(459) Mobile phase: 200 mM ammonium acetate

(460) Flow rate: 0.1 mL/min:

(461) Column temperature: 25° C.

(462) The maleamide-based disulfide bond bridging has a better stability, which is less prone to sulfhydryl-ether exchange in the body. In order to further prove that the introduction of a substituent to Ar′ can greatly slow down the secondary hydrolysis reaction occurred after the ring of the maleamide is opened, and also enhance the stability of the antibody-drug conjugate compared to the unsubstituted phenyl, the inventors had prepared a control ADC in this experiment. The control ADC was obtained by coupling pertuzumab and a compound in which Ar′ is just a 1,4-substituted phenyl (as shown in following formula) through the same coupling method as that for preparing ADC-I.

(463) ##STR00142##

(464) Antibody drug conjugates ADC-1, ADC-II and ADC-VII were selected to compare with the control ADC. Specifically, ADC samples which had the same antibody concentration and were stored in buffers, were placed at 25° C. and then sampled on day 0, 2, 4, and 7 respectively.

(465) Corresponding amount of secondary hydrolyzate formed in each antibody drug conjugate (ADC) sample was determined by LC-MS (Q-TOF), and characteristic peak in mass spectrum of the secondary hydrolyzate was extracted and the peak area was calculated. Changing trend in the amounts of the secondary hydrolyzates in each ADC sample were obtained by comparing the change in the peak areas from day 0 to 7, and see the following data and FIG. 15 for details. From the following data it can be seen that the amounts of the secondary hydrolyzates formed in the control ADC sample are significantly higher than those formed in the ADC-I, ADC-II, and ADC-VII samples.

(466) TABLE-US-00003 Days Control ADC ADC-I ADC-VII ADC-II Day 0 61779.22 12978.85 24559.1 0 Day 2 1335141 513786.8 679699.3 11842.82 Day 4 2876314 1204614 1561592 34018.8 Day 7 4835396 1933476 3061651 79192.73

(467) In addition, the changes of each ADC sample on day 0, 2, 4, and 7 were also determined by HIC. As can be seen from FIGS. 16-1 to 16-4, in the chromatogram of the control ADC sampled on day 7, an impurity peak appeared at the position where the retention time was 6.904. However, as shown in FIGS. 17-1 to 17-4, FIGS. 18-1 to 18-4, and FIGS. 19-1 to 19-4, the chromatograms of the ADC-I, ADC-II, and ADC-VII samples that sampled from day 0 to 7 showed no substantial changes.

The Fifth Group of Examples: Biological Detections of the Antibody Drug Conjugate

(468) 1. Biological Activity Assay of Cell Proliferation In Vitro

(469) Experimental materials used in the following experiment: DMEM, DMEM/F12K, RPMI 1640 medium, 0.25% trypsin-EDTA, fetal bovine serum, 100× sodium pyruvate and 100× penicillin-streptomycin were purchased from Gibco; sulforhodamine B (SRB) was purchased from Sigma; and NCI-N87 human gastric cancer cells and BT-474 human breast cancer cells were purchased from Kunming Cell Bank of Chinese Academy of Sciences. All other reagents used were analytical grade. 96-well Flat Bottom Polystyrene (Corning, catalog No. 3599) and Synergy 2 Microplate Reader (Bio-Tek) were used.

(470) In this Example, effects of ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII and ADC-VIII on the proliferation of tumor cell lines were investigated.

(471) Sulforhodamine B (SRB)-based colorimetric method was used in this Example to evaluate the anti-proliferative effect of the drugs. SRB is a pink anionic dye which is easily soluble in water and can specifically bind to basic amino acids of proteins in cells under an acidic condition. It provides an absorption peak at 510 nm, and the absorbance is linearly and positively correlated with the amount of cells. In this regard, the method can be used in a quantitative detection of cell number.

(472) Cell lines used in this Example are: BT-474 human breast cancer cells and NCI-N87 human gastric cancer cells.

(473) BT-474 and NCI-N87 cells in RPMI 1640 medium containing 10% fetal bovine serum were incubated to logarithmic growth phase in a 5% CO2 incubator at 37° C. The above cells in the logarithmic growth phase were inoculated into 96-well plates at a density of 2×10.sup.3 to 9×10.sup.3 cells per well, 100 μL per well, cultured for 24 hours and then different concentrations of drugs were added thereto for 5 days. Specifically, each drug was prepared into nine concentrations by diluting in 3, 4 or 5-fold, each concentration was set in duplicate wells, and corresponding concentration of vehicle control wells and control wells without cells were set too. At the end of drug action, culture solutions were decanted, and 100 μl of a pre-cooled trichloroacetic acid solution (30%, w/v) at 4° C. was added to each well and the cells were fixed at 4° C. for 1 hour. Subsequently, the cells were washed with deionized water for 5 times, and dried at room temperature, and then 100 μL of 0.4% (w/v) SRB dye (Sigma, prepared with 1% glacial acetic acid) was added per well. After being incubated and stained at room temperature for 30 minutes, the cells were washed with 1% glacial acetic acid for 4 times to remove unbound dyes, and then dried at room temperature. Afterwards, 100 μL of 10 mM Tris solution was added per well. After being incubated and stained at room temperature for 15 minutes, the cells was washed with 1% glacial acetic acid for 5 times to remove unbound SRB, and then dried at room temperature. Dyes bound to the proteins in cells were dissolved by addition of 10 mM Tris buffer (pH=10.5) per well, and the absorbance (OD value) was measured at wavelengths of 510 nm and 690 nm using Synergy 2 Microplate Reader (Bio-Tek). A=OD.sub.510−OD.sub.690.
Inhibition rate (%)=(A.sub.control−A.sub.drug)/A.sub.control×100%

(474) In this experiment, effects of ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, and ADC-VIII on the proliferation of various Her2 highly expressing tumor cell lines in vitro cultured were investigated. As shown in following Table, proliferation of Her2 highly expressing NCI-N87 human gastric cancer cells and BT-474 human breast cancer cells were significantly inhibited after being treated with ADC-I, ADC-II, ADC-III, ADC-IV, ADC-V, ADC-VI, ADC-VII, and ADC-VIII compared to that treated with naked antibodies Perjeta and Herceptin. Corresponding proliferation inhibition curves are shown in FIG. 20-23.

(475) TABLE-US-00004 IC.sub.50, μg/mL, 120 h Sample NCI-N87 BT-474 Perjeta >10 >10 Herceptin 0.440 0.0986 ADC-I 0.000140 0.000183 ADC-II 0.000690 0.000469 ADC-III 0.001562 0.000905 ADC-IV 0.000623 0.000364 ADC-V 0.001645 0.001255 ADC-VI 0.000612 0.000607 ADC-VII 0.000534 0.000421 ADC-VIII 0.00159 0.000431
2. In Vivo Anti-Tumor Efficacy Assay

(476) Efficacy of the conjugates of the present invention can be detected in vivo. In brief, an allograft or xenograft of cancer cells can be implanted into rodents and then the implanted tumors are treated with the conjugates. Tested mice can be administered drug treatment or control treatment, monitored for weeks or longer to observe tumor doubling time, log-killing, and tumor suppression.

(477) 1) Experimental Animals

(478) 6-7 weeks BALB/cA-nude mice (♀) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Production license number: SCXK (Shanghai) 2013-0018; animal certificate number: 2013001815683; and feeding environment: SPF level.

(479) 2) Experimental Steps

(480) Those nude mice were subcutaneously inoculated with 6×10.sup.6 human gastric cancer NCI-N87 cells, and after the tumors grew to 100-250 mm.sup.3, the mice were divided into groups (D0) according to tumor volumes. The mice were injected intravenously (IV), each with an administration volume 10 mL/kg; mice in vehicle control group was administered with the same volume of “vehicle” (physiological saline containing 0.1% BSA). Doses and regimen of administration are provided in FIG. 24. Tumor volume was measured 2 times a week, the mice were weighed, and corresponding data were recorded.

(481) The purpose of this experiment was to investigate the effect of the drugs on tumor growth using specific indicators T/C % or tumor growth inhibition value (TGI) (%).

(482) Tumor diameters were measured twice a week with a vernier caliper. The tumor volume (V) was calculated as:
V=½×a×b.sup.2;
in which a and b represent length and width respectively.
T/C(%)=(T−T0)/(C−C0)×100,
in which T and C represent the tumor volumes at the end of the experiment; T0 and C0 represent the tumor volumes at the beginning of the experiment.
The tumor growth inhibition value (TGI) (%)=100−T/C (%).
When tumor regresses, the tumor growth inhibition value (TGI) (%)=100−(T−−T0)/T0×100.

(483) If the tumor had a smaller volume than it initially had, i.e. T<T0 or C<C0, it was defined partially regressed (PR); whereas if the tumor completely disappeared, it was defined completely regressed (CR).

(484) When the experiment was finished (D21), when the end of the experiment was reached, or when the volume of a tumor reached 15000 mm.sup.3, the corresponding mouse was sacrificed with carbon dioxide narcosis, and dissected, and the tumor was removed and photographed.

(485) 3) Experiment Results

(486) Efficacies of the drugs on the human gastric cancer NCI-N87 subcutaneous xenografts in nude mice were shown in the following Table and FIG. 24. What's more, those tumor-bearing mice had good tolerance to the drugs, and no symptom such as weight loss occurred.

(487) TABLE-US-00005 number of mice per number of Mean Mean group mice per tumor tumor com- at the group volume volume % % Partial plete beginning at the end Adminis- (mm3) (mm3) T/C TGI p value regres- regres- of the of the Group tration Route D 0 SEM D 23 SEM D 23 D 23 D 23 sion sion experiment experiment vehicle D 0 IV 102.3 ±1.1 643.6 ±57.1 — — — 0 0 12 12 control P-mcVC- 1.0 mg/kg D 0 IV 103.5 ±3.1 194.6 ±77.7 17 83 0.000 3 0 6 6 MMAE Control 0.5 mg/kg D 0 IV 105.4 ±3.9 330.5 ±48.5 42 58 0.003 0 0 6 6 ADC Control 1.0 mg/kg D 0 IV 107.6 ±4.6 107.8 ±40.5 0 100 0.000 3 1 6 6 ADC ADC-I 1.0 mg/kg D 0 IV 103.6 ±3.8 74.6 ±32.8 −28 128 0.000 3 2 6 6 ADC-IV 1.0 mg/kg D 0 IV 109.5 ±4.0 180.1 ±42.6 13 87 0.000 1 0 6 6 ADC-V 1.0 mg/kg D 0 IV 105.8 ±1.0 78.7 ±17.3 −26 126 0.000 3 1 6 6 ADC-VI 1.0 mg/kg D 0 IV 107.9 ±3.7 116.0 ±22.5 1 99 0.000 3 0 6 6 ADC-VII 0.5 mg/kg D 0 IV 104.2 ±2.2 233.2 ±58.1 24 76 0.000 2 0 6 6 ADC-VII 1.0 mg/kg D 0 IV 107.0 ±3.6 46.8 ±20.8 −56 156 0.000 3 2 6 6

(488) All the documents mentioned in the present invention are incorporated by reference in the present application, as if each document is incorporated by reference alone. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art would be able to make various modifications or amendments to the present invention, and these equivalents likewise fall within the scope defined by the appended claims of the present application.