LIGAND-CYTOTOXIC DRUG CONJUGATE, PREPARATION METHOD THEREOF, AND USES THEREOF

Abstract

Ligand-cytotoxic drug conjugates, pharmaceutical compositions, preparation methods, and pharmaceutical uses thereof are provided. More specifically, a ligand-cytotoxic drug conjugate of general formula Pc-(X-Y-D).sub.n is provided. The ligand-cytotoxic drug conjugate can be used to treat cancer via receptor modulation.

Claims

1. A ligand-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-cytotoxic drug conjugate comprises a connecting unit X having the following structure: ##STR00092## wherein X.sub.1 is selected from the group consisting of H, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more groups selected from the group consisting of halogen, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, X.sub.2 is selected from the group consisting of alkyl, cycloalkyl, alkyl-cycloalkyl, cycloalkyl-alkyl, alkyl-cycloalkyl-alkyl, heterocyclyl, alkyl-heterocyclyl, heterocyclyl-alkyl, alkyl-heterocyclyl-alkyl, aryl, alkyl-aryl, aryl-alkyl, alkyl-aryl-alkyl, heteroaryl, alkyl-heteroaryl, heteroaryl-alkyl, alkyl-heteroaryl-alkyl, (CH.sub.2).sub.p(OCH.sub.2CH.sub.2).sub.p, and (CH.sub.2CH.sub.2O).sub.p(CH.sub.2).sub.p, each p is an integer independently selected from 1 to 10, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, or when X.sub.1 is not H, X.sub.1 and X.sub.2 with the carbon atom joining X.sub.1 and X.sub.2 are taken together to form a cycloalkyl group, wherein the cycloalkyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; and S is a sulfur atom.

2. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein X.sub.1 is H or alkyl.

3. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein X.sub.2 is alkyl or aryl.

4. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, which has a structure of formula (I)
Pc(-X-Y-D).sub.n(I) wherein: Pc is a ligand; X is as defined in claim 1; Y is an interval unit; D is a cytotoxic drug; and n is selected from 1 to 8.

5. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein the connecting unit X is linked to at least one of an N-terminal amino group of the Pc polypeptide chain and a -amino group of a lysine residue of the Pc polypeptide chain, and n is selected from 1 to 4.

6. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein the ligand is an antibody.

7. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 6, wherein the antigen of said antibody is a cell surface antigen expressed on at least one of a target cell and tissue of a proliferative disease.

8. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 7, wherein the cell surface receptor is selected from the group consisting of: 1) HER2 (ErbB2), 2) HERS (ErbB3), 3) HER4 (ErbB4), 4) CD20, 5) CD22, 6) CD30, 7) CD33, 8) CD44, 9) Lewis Y, 10) CD56, 11) CD105, 12) VEGFR, and 13) GPNMB.

9. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 8, wherein the cell surface receptor is selected from the group consisting of: 1) HER2 (ErbB2), 2) CD 22, 3) CD30 4) CD33, 5) CD44 6) CD56, 7) Lewis Y, and 8) GPNMB.

10. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 6, wherein the antibody is selected from the group consisting of: 1) Trastuzumab (HER2), 2) Inotuzumab (CD22), 3) Pinatuzumab (CD22), 4) Brentuximab (CD30), 5) Gemtuzumab (CD33), 6) Bivatuzumab (CD44), 7) Lorvotuzumab (CD56), 8) cBR96 (Lewis Y), 9) Glematumamab (GPNMB) and 10) Pertuzumab.

11. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 10, wherein the antibody is capable of binding to a HER2 protein, wherein the antibody comprises: 1) a light chain comprising at least one CDR selected from the group consisting of CDR-L1, CDR-L2 and CDR-L3 defined according to Kabat numbering system, wherein i) CDR-L1 is a CDR of SEQ ID NO: 1, or of at least one sequence having at least 80% identity to SEQ ID NO: 1 after optimal alignment; ii) CDR-L2 is a CDR of SEQ ID NO: 2, or of at least one sequence having at least 80% identity to SEQ ID NO: 2 after optimal alignment; iii) CDR-L3 is a CDR of SEQ ID NO: 3, or of at least one sequence having at least 80% identity to SEQ ID NO: 3 after optimal alignment; and 2) a heavy chain comprising at least one CDR selected from the group consisting of CDR-H1, CDR-H2 and CDR-H3 defined according to Kabat numbering system, wherein iv) CDR-H1 is a CDR of SEQ ID NO: 4, or of at least one sequence having at least 80% identity to SEQ ID NO: 4 after optimal alignment; v) CDR-H2 is a CDR of SEQ ID NO: 5, or of at least one sequence having at least 80% identity to SEQ ID NO: 5 after optimal alignment; vi) CDR-H3 is a CDR of SEQ ID NO: 6, or of at least one sequence having at least 80% identity to SEQ ID NO: 6 after optimal alignment.

12. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 11, wherein the antibody capable of binding to the HER2 protein comprises at least one of a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 7, and the heavy chain comprises the amino acid sequence of SEQ ID NO: 8.

13. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, wherein the cytotoxic drug is selected from the group consisting of tubulin inhibitors, topoisomerase inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, and DNA synthesis inhibitors.

14. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 13, wherein the cytotoxic drug tubulin inhibitor is selected from the group consisting of maytansinoids, calicheamicin, taxanes, vincristine, colchicine, and Dolastatins/Auristatins.

15. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein D is selected from the group consisting of Dolastatins/Auristatins having a structure of formula (D.sub.1): ##STR00093## wherein: R.sub.1 is a bond, H, alkyl or cycloalkyl; R.sub.2 is H or alkyl; or R.sub.1 and R.sub.2 with the joined N atom are taken together to form a heterocyclyl, wherein the heterocyclyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; or form a structure of (CR.sub.aR.sub.b).sub.e, R.sub.a and R.sub.b are independently selected from the group consisting of H, alkyl, and heterocyclyl, e is an integer selected from 2 to 6; R.sub.3 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.4 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.5 is H or methyl; R.sub.6 is H or alkyl; R.sub.7 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.8 is selected from the group consisting of H, hydroxy, alkyl, cycloalkyl, and alkoxy; R.sub.9 is H or alkyl; when R.sub.1 is alkyl or cycloalkyl, or R.sub.1 and R.sub.2 with the joined N atom are taken together to form a heterocyclyl, wherein the heterocyclyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl, R.sub.10 is selected from the following structures: ##STR00094## when R.sub.1 is H, R.sub.10 is selected from the following structures: ##STR00095## when R.sub.1 is a bond, it is connected to the interval unit Y, wherein R.sub.10 is selected from the following structures: ##STR00096## Z is selected from the group consisting of O, S, NH and N(R.sub.14); R.sub.11 is selected from the group consisting of H, hydroxy, amino, NHR.sub.14, N(R.sub.14).sub.2 alkoxy, alkyl, cycloalkyl, aryl, heterocyclyl, alkyl-aryl, alkyl-cycloalkyl, and alkyl-heterocyclyl; or when R.sub.11 is O, it can replace H attached on the joined carbon atom, and form a carbonyl group (CO) with this carbon atom; R.sub.12 is selected from the group consisting of aryl and heterocyclyl, the aryl or heterocyclyl is optionally substituted by one or more groups selected from the group consisting of hydroxy, alkoxy, alkyl, and halogen; R.sub.13 is selected from the group consisting of H, hydroxy, amino, NHR.sub.14, N(R.sub.14).sub.2, COOR.sub.14, alkoxy, alkyl, cycloalkyl, aryl, heterocyclyl, alkyl-aryl, alkyl-cycloalkyl, alkyl-heterocyclyl and alkoxy-alkoxy-alkoxy; R.sub.14 is H or alkyl; R.sub.15 is selected from the group consisting of H, alkyl, aryl, heterocyclic, (R.sub.16O).sub.mR.sub.14 and (R.sub.16O).sub.mCH(R.sub.17).sub.2; m is an integer selected from 1 to 1000; R.sub.16 is C.sub.2-C.sub.8 alkyl; R.sub.17 is selected from the group consisting of H, carboxyl, (CH.sub.2).sub.tN(R-.sub.18).sub.2 and (CH.sub.2).sub.tSO.sub.3R.sub.14; R.sub.18 is selected from the group consisting of H, alkyl, and (CH.sub.2).sub.tCOOH; t is an integer selected from 0 to 6; and R.sub.19 is selected from the group consisting of aryl, cycloalkyl and heterocyclyl.

16. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein D is maytansine having a structure of formula (DM): ##STR00097## wherein: R.sub.20 is O or S; R.sub.21 is selected from the group consisting of H, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are independently and optionally substituted by one or more groups selected from the group consisting of halogen, hydroxy, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

17. The ligand-cytotoxic drug conjugate or pharmaceutically acceptable salt or solvate thereof according to claim 4, wherein the interval unit Y has a structure of the following formula: ##STR00098## wherein: YL is selected from the group consisting of alkyl, cycloalkyl, O-alkyl, O-alkoxy, aryl, alkyl-cycloalkyl, cycloalkyl-alkyl, alkyl-aryl, alkyl-cycloalkyl-alkyl, heterocyclyl, alkyl-heterocyclyl, heterocyclyl-alkyl, alkyl-heterocyclyl-alkyl, aryl, alkyl-aryl, aryl-alkyl, alkyl-aryl-alkyl, heteroaryl, alkyl-heteroaryl, heteroaryl-alkyl, alkyl-heteroaryl-alkyl, CH.sub.2(OCH.sub.2CH.sub.2).sub.t, (CH.sub.2CH.sub.2O).sub.tCH.sub.2, and (CH.sub.2CH.sub.2O).sub.t, and t is an integer selected from 1 to 10; K.sub.k is an amino acid unit, wherein K is an amino acid, and k is an integer selected from 0 to 10; and Qq is an extended unit, wherein q is 0, 1 or 2.

18. A compound of formula (II):
Pc(-X-T).sub.n(II) wherein: Pc is a ligand; T is selected from the group consisting of H, t-butyl, acetyl, n-propionyl, isopropionyl, triphenylmethyl, methoxymethyl, and 2-(trimethylsilyl)ethoxymethyl; n is selected from 1 to 8; and X is a connecting unit having the following structure: ##STR00099## wherein X.sub.1 is selected from the group consisting of H, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more groups selected from the group consisting of halogen, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, X.sub.2 is selected from the group consisting of alkyl, cycloalkyl, alkyl-cycloalkyl, cycloalkyl-alkyl, alkyl-cycloalkyl-alkyl, heterocyclyl, alkyl-heterocyclyl, heterocyclyl-alkyl, alkyl-heterocyclyl-alkyl, aryl, alkyl-aryl, aryl-alkyl, alkyl-aryl-alkyl, heteroaryl, alkyl-heteroaryl, heteroaryl-alkyl, alkyl-heteroaryl-alkyl, (CH.sub.2).sub.p(OCH.sub.2CH.sub.2).sub.p, and (CH.sub.2CH.sub.2O).sub.p(CH.sub.2).sub.p, each p is an integer independently selected from 1 to 10, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl, or when X.sub.1 is not H, X.sub.1 and X.sub.2 with the carbon atom joining X.sub.1 and X.sub.2 are taken together to form a cycloalkyl group, wherein the cycloalkyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; and S is a sulfur atom.

19. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, having a structure of formula (III): ##STR00100## wherein: Pc is an antibody; X is as defined in claim 1; YL is selected from the group consisting of alkyl, cycloalkyl, O-alkyl, O-alkoxy, aryl, alkyl-cyclo alkyl, cycloalkyl-alkyl, alkyl-aryl, alkyl-cycloalkyl-alkyl, heterocyclyl, alkyl-heterocyclyl, heterocyclyl-alkyl, alkyl-heterocyclyl-alkyl, aryl, alkyl-aryl, aryl-alkyl, alkyl-aryl-alkyl, heteroaryl, alkyl-heteroaryl, heteroaryl-alkyl, alkyl-heteroaryl-alkyl, CH.sub.2(OCH.sub.2CH.sub.2).sub.t, (CH.sub.2CH.sub.2O).sub.tCH.sub.2, and (CH.sub.2CH.sub.2O).sub.t, and t is an integer selected from 1 to 10; K.sub.k is an amino acid unit, wherein K is an amino acid, and k is an integer selected from 0 to 10; Qq is an extended unit, wherein q is 0, 1 or 2; n is selected from 1 to 4; and D is a cytotoxic drug.

20. A ligand-cytotoxic drug conjugate or a pharmaceutically acceptable salt or solvate thereof selected from the group consisting of: ##STR00101## ##STR00102## ##STR00103## wherein Pc is a ligand, and n is 1 to 8.

21. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 20, wherein Pc is selected from the group consisting of Trastuzumab, Inotuzumab and Brentuximab.

22. The ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 4, selected from the group consisting of: ##STR00104## ##STR00105## ##STR00106## ##STR00107## wherein n is selected from 1 to 8.

23. A process of preparing an antibody-cytotoxic drug conjugate of formula (III) according to claim 19: ##STR00108## wherein the process comprises the steps of: 1) adding a reducing agent RA to a compound of formula IA and a compound of formula IB, and reaction the reducing agent RA, the compound of formula IA and the compound of formula IB at a pH of 3 to 6 and a temperature of 0 C. to 40 C., thereby obtaining a compound of formula IC; ##STR00109## 2) adding a deprotecting agent to the compound of formula IC and reacting at a temperature of 0 C. to 40 C. to remove the protective group T of the thiol group to obtain a compound of formula ID; ##STR00110## and 3) performing a Michael addition reaction between the compound of formula ID and a compound of formula IE at a temperature of 0 C. to 40 C., thereby obtaining the compound of formula (III); ##STR00111## wherein X.sub.1 and X.sub.2 are as defined in claim 1; Pc is a ligand; T is selected from the group consisting of H, t-butyl, acetyl, n-propionyl, isopropionyl, triphenylmethyl, methoxymethyl, and 2-(trimethylsilyl)ethoxymethyl; n is 1 to 8; K.sub.k is an amino acid unit, wherein K is an amino acid, and k is an integer selected from 0 to 10; Qq is an extended unit, wherein q is 0, 1 or 2; and D is a cytotoxic drug selected from the group consisting of Dolstatins/Auristatins having a structure of formula (D.sub.1) and maytansine having a structure of formula (DM): ##STR00112## wherein: R.sub.1 is a bond, H, alkyl or cycloalkyl; R.sub.2 is H or alkyl; or R.sub.1 and R.sub.2 with the joined N atom are taken together to form a heterocyclyl, wherein the heterocyclyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl; or form a structure of (CR.sub.aR.sub.b).sub.e, R.sub.a and R.sub.b are independently selected from the group consisting of H, alkyl, and heterocyclyl, e is an integer selected from 2 to 6; R.sub.3 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.4 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.5 is H or methyl; R.sub.6 is H or alkyl; R.sub.7 is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkyl-aryl, alkyl-cycloalkyl, heterocyclyl and alkyl-heterocyclyl; R.sub.8 is selected from the group consisting of H, hydroxy, alkyl, cycloalkyl, and alkoxy; R.sub.9 is H or alkyl; when R.sub.1 is alkyl or cycloalkyl, or R.sub.1 and R.sub.2 with the joined N atom are taken together to form a heterocyclyl, wherein the heterocyclyl is independently and optionally substituted with one or more groups selected from the group consisting of halo, hydroxy, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl, R.sub.10 is selected from the following structures: ##STR00113## when R.sub.1 is H, R.sub.10 is selected from the following structures: ##STR00114## when R.sub.1 is a bond, it is connected to the interval unit Y, wherein R.sub.10 is selected from the following structures: ##STR00115## Z is selected from the group consisting of O, S, NH and N(R.sub.14); R.sub.11 is selected from the group consisting of H, hydroxy, amino, NHR.sub.14, N(R.sub.14).sub.2 alkoxy, alkyl, cycloalkyl, aryl, heterocyclyl, alkyl-aryl, alkyl-cycloalkyl, and alkyl-heterocyclyl; or when R.sub.11 is O, it can replace H attached on the joined carbon atom, and form a carbonyl group (CO) with this carbon atom; R.sub.12 is selected from the group consisting of aryl and heterocyclyl, the aryl or heterocyclyl is optionally substituted by one or more groups selected from the group consisting of hydroxy, alkoxy, alkyl, and halogen; R.sub.13 is selected from the group consisting of H, hydroxy, amino, NHR.sub.14, N(R.sub.14).sub.2, COOR.sub.14, alkoxy, alkyl, cycloalkyl, aryl, heterocyclyl, alkyl-aryl, alkyl-cycloalkyl, alkyl-heterocyclyl and alkoxy-alkoxy-alkoxy; R.sub.14 is H or alkyl; R.sub.15 is selected from the group consisting of H, alkyl, aryl, heterocyclic, (R.sub.16O).sub.mR.sub.14 and (R.sub.16O).sub.mCH(R.sub.17).sub.2; m is an integer selected from 1 to 1000; R.sub.16 is C.sub.2-C.sub.8 alkyl; R.sub.17 is selected from the group consisting of H, carboxyl, (CH.sub.2).sub.tN(R-.sub.18).sub.2 and (CH.sub.2).sub.tSO.sub.3R.sub.14; R.sub.18 is selected from the group consisting of H, alkyl, and (CH.sub.2).sub.tCOOH; t is an integer selected from 0 to 6; R.sub.19 is selected from the group consisting of aryl, cycloalkyl and heterocyclyl; R.sub.20 is O or S; and R.sub.21 is selected from the group consisting of H, alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are independently and optionally substituted by one or more groups selected from the group consisting of halogen, hydroxy, cyano, nitro, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl.

24. A pharmaceutical composition, wherein the pharmaceutical composition comprises a therapeutically effective amount of the ligand-cytotoxic drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to claim 1, and one or more pharmaceutically acceptable carriers, diluents or excipients.

25. (canceled)

26. A method for modulating a receptor in vitro, the method comprising administering to an object to be tested an effective amount of the pharmaceutical composition according to claim 24, wherein the receptor is selected from the group consisting of: 1) HER2(ErbB2), 2) CD22, 3) CD30, 4) CD33, 5) CD44, 6) CD56, 7) Lewis Y, and 8) GPNMB.

27. A method of treating cancer in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the pharmaceutical composition according to claim 24, wherein the mammal is a human, and the cancer is selected from the group consisting of breast cancer, ovarian cancer, stomach cancer, endometrial cancer, salivary gland cancer, lung cancer, colon cancer, renal cancer, rectal cancer, thyroid cancer, pancreatic cancer, prostate cancer, bladder cancer, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma and relapsed anaplastic large cell lymphoma.

28. (canceled)

29. A method of treating cancer in a subject in the need thereof, the method comprising administering to the subject the pharmaceutical composition according to claim 24, wherein the cancer is a tumor-associated receptor overexpressed cancer, wherein the tumor-associated receptor is one or more selected from the group consisting of (1)-(8): 1) HER2(ErbB2), 2) CD22, 3) CD30, 4) CD33, 5) CD44, 6) CD56, 7) Lewis Y, and 8) GPNMB.

30. The process according to claim 23, wherein the compound of formula (IE) is a compound of formula (IV): ##STR00116##

Description

BRIEF DESCRIPTION OF FIGURES

[0279] FIG. 1 shows the efficacy of Compound 16, Compound 17, Compound 18, and the positive control Compound 35 on NCI-N87 human gastric cancer xenografts in nude mice; and

[0280] FIG. 2 shows the influence of Compound 16, Compound 17, Compound 18, and the positive control Compound 35 on the weight of tumor-bearing nude mice.

PREFERRED EMBODIMENTS

[0281] The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are merely intended to demonstrate the invention without limiting the scope of the invention.

[0282] Compound structures were identified by Nuclear Magnetic Resonance (NMR) and/or Mass Spectrometry (MS). NMR was determined by a Bruker AVANCE-400 machine. The solvents were deuterated-dimethyl sulfoxide (DMSO-d.sub.6), deuterated-chloroform (CDCl.sub.3), and deuterated-methanol (CD.sub.3OD), with tetramethylsilane (TMS) as an internal standard. NMR chemical shifts () are given in 10.sup.6 (ppm).

[0283] MS was determined by a FINNIGAN LCQAd (ESI) Mass Spectrometer (manufacturer: Thermo, type: Finnigan LCQ advantage MAX).

[0284] High performance liquid chromatography (HPLC) was determined on an Agilent 1200DAD high pressure liquid chromatography spectrometer (Sunfire C18 1504.6 mm chromatographic column) and a Waters 2695-2996 high pressure liquid chromatography spectrometer (Gimini C18 1504.6 mm chromatographic column).

[0285] For thin-layer silica gel chromatography (TLC), Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates were used. The dimension of the plates used in TLC was 0.15 mm to 0.2 mm, and the dimension of the plates used in product purification was 0.4 mm to 0.5 mm.

[0286] For Column chromatography, generally Yantai Huanghai 200 to 300 mesh silica gel was used as carrier.

[0287] The known starting materials of the invention can be prepared by conventional synthesis methods in the prior art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., or Dari Chemical Company, etc.

[0288] Unless otherwise stated, the following reactions were performed under nitrogen atmosphere or argon atmosphere.

[0289] The term argon atmosphere or nitrogen atmosphere means that a reaction flask is equipped with a 1 L argon or nitrogen balloon.

[0290] Unless otherwise stated, the solution used in the examples refers to an aqueous solution.

[0291] Unless otherwise stated, the reaction temperature in the examples was room temperature in the range of 20 C. to 30 C.

[0292] The reaction process was monitored by thin layer chromatography (TLC), and the system of developing solvent included A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: acetone. The ratio of the volume of the solvent was adjusted according to the polarity of the compounds.

[0293] The elution system for purification of the compounds by column chromatography and the developing solvent by thin layer chromatography included: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: n-hexane and acetone system, D: n-hexane, E: ethyl acetate. The volume of the solvent was adjusted according to the polarity of the compounds, and sometimes a little triethylamine and acidic or alkaline reagent was also dded.

[0294] The structures of the compounds of the present invention were determined by Q-TOF LC/MS. For Q-TOF LC/MS, Agilent 6530 Accurate-Mass QuadrupoleTime of Flight Mass Spectrometer and Agilent 1290-Infinity UHPLC (Agilent Poroshell 300SB-C8 5 m, 2.175 mm Column) were used.

[0295] Known starting materials of the present invention were synthesized by adopting or using the methods known in the art, and the experimental methods in the following examples for which the specific conditions are not indicated were carried out according to conventional conditions or the conditions recommended by the product manufacturers. The experimental reagents for which the specific sources are not indicated were the conventional reagents generally purchased from market.

Example 1

Preparation of Intermediates

[0296] Preparation of Intermediates as Drugs

[0297] 1. The following intermediate compounds 1-6 were prepared by a method disclosed in PCT Patent Application Publication WO2004010957.

TABLE-US-00001 No. molecular structure formula 1 [00041] 2 [00042] 3 [00043] 4 [00044] 5 [00045] 6 [00046]

[0298] 2. The following intermediate compounds 7-11 were prepared by a method disclosed in PCT Patent Application publication WO2005081711.

TABLE-US-00002 No. molecular structural formula 7 [00047] 8 [00048] 9 [00049] 10 [00050] 11 [00051]

[0299] 3. The following intermediate compounds 12-14 were prepared by a method disclosed in U.S. Pat. No. 7,750,116.

TABLE-US-00003 No molecular structural formula 12 [00052] 13 [00053] 14 [00054]

[0300] 4. Preparation of intermediate compound 15

##STR00055##

[0301] Specific synthetic route was as follows:

##STR00056## ##STR00057## ##STR00058##

Step 1

3-(maleimide) propionic acid

[0302] The -alanine 1a (2.29 g, 25.8 mmol) and maleic anhydride (2.52 g, 25.8 mmol) were dissolved in 20 mL of acetic acid, heated to reflux, and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, the residue was treated with toluene for azeotropic distillation, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was recrystallized with ethyl acetate, filtered and dried to obtain the entitled product 3-(maleimide) propionic acid 1b (2.80 g, yield 64.3%), as a colorless crystal.

[0303] MS m/z (ESI):170.04M+11.

Step 2

3-(maleimide) propionate succinimide ester

[0304] 3-(maleimide) propionic acid 1b (2.80 g, 16.6 mmol) and N-hydroxysuccinimide (2.25 g, 19.9 mmol) were dissolved in 30 mL of DMF in an ice bath and cooled to 0 C., stirred for 10 minutes with addition of N, N-dicyclohexyl carbodiimide (6.85 g, 33.2 mmol), and then the reaction was warmed to room temperature and stirred overnight. After filtration, the filtrate was mixed with 80 mL of dichloromethane, washed with water (60 mL3), 5% sodium bicarbonate aqueous solution (60 mL3), and saturated saline solution (50 mL3) respectively. The organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 3-(maleimide) propionate succinimide ester 1c (2.76 g, yield 62.5%) as a white solid.

[0305] MS m/z (ESI):267.06 [M+1].

Step 3

(1R, 4R)-4-(t-butoxycarbonyl-aminomethyl) cyclohexyl methanoic acid

[0306] (1R, 4R)-4-(aminomethyl) cyclohexyl methanoic acid 1d (4.72 g, 30.0 mmol) and sodium hydroxide (1.28 g, 32.0 mmol) were dissolved in a solvent mixture of 20 mL of water and 44 mL of tert-butanol, followed by addition of tert-butyl dicarbonate (6.99 g, 32.0 mmol), and the reaction was stirred for 18 hours at room temperature. The reaction solution was added with 100 mL of water, washed with n-hexane (100 mL3), the aqueous layer was cooled to 4 C., the pH was adjusted to 3 with saturated citric acid aqueous solution, the acidified solution was extracted with ethyl acetate (50 mL3), and the organic layers were pooled, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain (1R, 4R)-4-(t-butoxycarbonyl-aminomethyl) cyclohexyl methanoic acid 1e (7.33 g, yield 95%) as a colorless crystal.

[0307] MS m/z (ESI):258.17 [M+1].

Step 4

(1R, 4R)-4-((tert-butoxycarbonylamino) methyl) cyclohexyl methanoic acid succinimide ester

[0308] (1R, 4R)-4-(t-butoxycarbonyl-aminomethyl) cyclohexyl methanoic acid 1e (7.33 g, 28.5 mmol) and N-hydroxysuccinimide (3.87 g, 34.2 mmol) were dissolved in 35 mL of DMF in an ice bath and cooled to 0 C., stirred for 10 minutes with addition of N, N-dicyclohexyl carbodiimide (11.76 g, 57.0 mmol), and then the reaction was warmed to room temperature and stirred overnight. After filtration, the filtrate was mixed with 90 mL of dichloromethane, washed with water (60 mL3), 5% NaHCO.sub.3 aqueous solution (60 mL3), saturated saline solution (50 mL3), respectively. The organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain (1R, 4R)-4-((tert-butoxycarbonyl amino methyl) cyclohexyl methanoic acid succinimide ester if (6.75 g, yield 66.8%) as an almost white solid.

[0309] MS m/z (ESI):355.18 [M+1].

Step 5

S-2-((1R, 4S)-4-((tert-butoxycarbonylamino) methyl)-N-methyl-N-cyclohexylformyl) propanoic acid

[0310] (1R, 4R)-4-((tert-butoxycarbonylaminomethyl) cyclohexyl methanoic acid succinimide ester if (6.75 g, 19.0 mmol) and N-methyl-L-alanine 1g (1.96 g, 19.0 mmol) were dissolved in 90 mL of solvent mixture of ethylene glycol dimethyl ether/water with a volume ratio of 1:1, followed by addition of triethylamine (4.05 g, 40 mmol). The mixture was reacted for 6 hours at room temperature, concentrated under reduced pressure, and the residue was dissolved with 100 mL of ethyl acetate, washed with saturated saline solution (80 mL3), and the organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography with developing solvent dichloromethane/methanol (50:1) to obtain S-2-((1R, 4R)-4-((tert-butoxycarbonylamino) methyl)-N-methyl-N-cyclohexylformyl) propanoic acid 1h (4.78 g, yield 73.5%) as an almost white solid.

[0311] MS m/z (ESI):343.22 [M+1].

Step 6

[0312] Maytansinol 1i (565.5 mg, 1.0 mmol, prepared by a well-known synthetic method published by Wayne C W, Sharon D W, Emily E C, et al., J. Med. Chem, 2006, 49, 4392-4408) and S-2-((1R, 4S)-4-((tert-butoxycarbonylamino)methyl)-N-methyl-N-cyclohexylformyl) propanoic acid 1h (2.05 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane. N, N-dicyclohexyl carbodiimide (1.30 g, 6.3 mmol) was dissolved in 5 mL of dichloromethane and added into the above reaction solution, then the mixture was dropwise slowly titrated with 1M zinc chloride in diethyl ether solution (1.2 mL, 1.2 mmol), and stirred at room temperature for 2 hours. After that the reaction solution was dissolved in 30 mL of ethyl acetate, filtered, and the filtrate was washed with saturated sodium bicarbonate (15 mL2) and saturated saline solution (10 mL2) respectively, and the organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent dichloromethane/methanol (50:1) to obtain the compound 1j (201.8 mg, yield 22.7%) as an almost white solid.

[0313] MS m/z (ESI):889.43 [M+1].

Step 7

[0314] Compound 1j (88.9 mg, 0.1 mmol) was dissolved in 8 mL of dichloromethane, followed by addition of trifluoroacetic acid (12.6 mg, 0.11 mmol). The reaction mixture was stirred at room temperature for 1 hour, concentrated under reduced pressure, and the residue was dissolved in 20 mL of ethyl acetate, washed with 5% sodium carbonate aqueous solution (6 mL3), and the organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the compound 1m (76.8 mg, yield 97.3%) as an almost white solid.

[0315] MS m/z (ESI): 789.38 [M+1].

Step 8

[0316] Compound 1m (76.8 mg, 0.097 mmol) and 3-(maleimide) propionate succinimide ester 1c (29.3 mg, 0.11 mmol) were dissolved in 10.0 ml of N, N-dimethyl formamide, followed by addition of triethylamine (30.6 mg, 0.3 mmol). The mixture was reacted at room temperature for 8 hours, concentrated under reduced pressure, and the residue was dissolved with 20 mL of ethyl acetate, washed with saturated saline solution (10 mL3), the organic layer was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography with developing solvent dichloromethane/methanol (40:1) to obtain the compound 15 (40.6 mg, yield 44.5%) as an almost white solid.

[0317] MS m/z (ESI):940.41 [M+1].

[0318] Preparation of Intermediates as Antibodies

[0319] The following antibodies were prepared according to conventional methods: for instance, vector construction, HEK293 cell transfection (Life Technologies Cat. No. 11625019), purification and expression.

1. Antibody sequences
(1) Trastuzumab, capable of specifically binding to target HER2:

TABLE-US-00004 Sequenceoflightchain: (SEQIDNO:1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Sequenceofheavychain: (SEQIDNO:2) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
(2) Inotuzumab, capable of specifically binding to target CD22:

TABLE-US-00005 Sequenceoflightchain: (SEQIDNO:3) DVQVTQSPSSLSASVGDRVTITCRSSQSLANSYGNTFLSWYLHKPGKAPQ LLIYGISNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQGTHQP YTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC Sequenceofheavychain: (SEQIDNO:4) EVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGG INPGNNYATYRRKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCTREG YGNYGAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
(3) Brentuximab, capable of specifically binding to target CD30:

TABLE-US-00006 Sequenceoflightchain: (SEQIDNO:5) DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKV LIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPW TFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Sequenceofheavychain: (SEQIDNO:6) QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGW IYPGSGNTKYNEKFKGKATLTVDTSSSTAFMQLSSLTSEDTAVYFCANYG NYWFAYWGQGTQVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSLSL SPGK
(4) Pertuzumab, capable of specifically binding to target HER2:

TABLE-US-00007 SequenceoflightchainCDR-L1: (SEQIDNO:7) KASQDVSIGVA SequenceoflightchainCDR-L2: (SEQIDNO:8) SASYRYT SequenceoflightchainCDR-L3: (SEQIDNO:9) QQYYIYPYT SequenceofheavychainCDR-H1: (SEQIDNO:10) DYTMD SequenceofheavychainCDR-H2: (SEQIDNO:11) DVNPNSGGSIYNQRFKG SequenceofheavychainCDR-H3: (SEQIDNO:12) NLGPSFYFDY Sequenceoflightchain: (SEQIDNO:13) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYS ASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Sequenceofheavychain: (SEQIDNO:14) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVAD VNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNL GPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 2

Preparation of Antibody Drug Conjugate Compound 16

[0320] ##STR00059##

[0321] The synthetic route is as follows:

##STR00060##

Step 1

Trastuzumab-propanethiol ethyl ester

[0322] 32.0 mL (1.49 mol) of trastuzumab stock solution (6.9 mg/ml, pH=6.3 in PBS solution) was replaced with an equal volume of 0.1 M acetic acid/sodium acetate buffer with pH 5.0. 3-acetyl-mercapto-propionaldehyde (0.79 mg, 5.98 mol) was dissolved in 3.0 mL of acetonitrile and then dropwise titrated into the above solution buffer, dropwise added with sodium cyanoborohydride (0.92 mg, 14.6 mol) dissolved in 1.0 mL water, and stirred for 3 hours at 25 C. After the reaction stopped, desalting and purification was performed by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.2) to obtain 45.1 mL of trastuzumab-propanethiol ethyl ester (16a) solution at a concentration of 4.8 mg/mL.

Step 2

Trastuzumab-propanethiol

[0323] Trastuzumab-propanethiol ethyl ester (16a) solution (prepared in step 1) was mixed with 73 L of 2 M hydroxylamine hydrochloride, stirred at 25 C. for 1 hour, and then desalting and purification were performed by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.2) to obtain 71.3 mL of trastuzumab-propanethiol (16b) solution at a concentration of 3.0 mg/mL.

Step 3

Trastuzumab-propyl-1-sulfur-MC-Val-Cit-PAB-MMAE (compound 16)

[0324] Compound 1 (MC-Val-Cit-PAB-MMAE, 19.1 mg, 14.5 mol) was dissolved in 7 mL of acetonitrile, followed by addition of trastuzumab-propanethiol (16b) solution (prepared in step 2). The reaction solution was stirred at 25 C. for 4 hours, the reaction was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M of PBS solution containing 2 mM EDTA, pH 6.2) and filtrated under a sterile condition through a 0.2 m filter to obtain 101.0 mL of Trastuzumab-propyl-1-sulfur-MC-Val-Cit-PAB-MMAE (16) solution at a concentration of 2.03 mg/mL. The solution was filtrated under a sterile condition through a 0.2 m filter and stored at 20 C. frozen storage.

[0325] Q-TOF LC/MS: 148381.5(M.sub.Ab+OD), 149613.2(M.sub.Ab+1D), 151169.8(M.sub.Ab+2D), 152587.7(M.sub.Ab+3D), 153868.1(M.sub.Ab+4D), 155484.4(M.sub.Ab+5D).

[0326] n=1.9.

Example 3

Preparation of Antibody Drug Conjugate Compound 22

[0327] ##STR00061##

[0328] The synthetic route is as follows: (consistent with those described above)

##STR00062##

Step 1

Inotuzumab-1-methyl propanethiol ethyl ester

[0329] S-(3-oxobutyl) thio-ethyl acetate (0.70 mg, 4.32 mol) was dissolved in 3.0 mL of acetonitrile, then dropwise titrated into 20.0 mL (1.08 mol) of Inotuzumab stock solution (8.1 mg/mL, pH=6.0 in PBS solution). Trimethoxy sodium borohydride (2.29 mg, 10.8 mol) was dissolved in 2.0 mL of water and dropwise titrated into the above reaction solution, and stirred for 48 hours at 30 C. After that, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.2) to obtain 29.2 mL of Inotuzumab-1-methyl propanethiol ethyl ester (22a) solution at a concentration of 5.4 mg/mL.

[0330] Compound 22 was prepared according to the procedure of steps 2 and 3 in Example 2, and in addition, compound 2 was used in step 3.

[0331] Q-TOF LC/MS: 149611.1(M.sub.Ab+0D), 151039.9(M.sub.Ab+1D), 152470.6(M.sub.Ab+2D), 153904.4(M.sub.Ab+3D), 155317.0(M.sub.Ab+4D), 156757.9(M.sub.Ab+5D).

[0332] n=1.8.

[0333] Referring to the preparation procedures of the antibody drug conjugate 16 described in Example 2 as a reference, the antibody drug conjugates 17-21, 24-27, 29, and 30 were prepared by using the corresponding antibodies (Trastuzumab, Inotuzumab, Brentuximab) and cytotoxic drugs.

TABLE-US-00008 Interval Unit- cytotoxic antibody drug conjugate drug Q-TOF LC/MS [00063] Compound 1 148381.5(M.sub.Ab + 0D), 149613.2(M.sub.Ab + 1D), 151169.8(M.sub.Ab + 2D), 152587.7(M.sub.Ab + 3D), 153868.1(M.sub.Ab + 4D), 155484.4(M.sub.Ab + 5D) [00064] Compound 12 148220.4(M.sub.Ab + 0D), 149212.0(M.sub.Ab + 1D), 150212.8(M.sub.Ab + 2D), 151272.8(M.sub.Ab + 3D), 152205.0(M.sub.Ab + 4D), 153236.5(M.sub.Ab + 5D) [00065] Compound 11 148223.9(M.sub.Ab + 0D), 149227.6(M.sub.Ab + 1D), 150264.5(M.sub.Ab + 2D), 151302.8(M.sub.Ab + 3D), 152363.8(M.sub.Ab + 4D), 153412.8(M.sub.Ab + 5D) [00066] Compound 7 148229.9(M.sub.Ab + 0D), 149639.7(M.sub.Ab + 1D), 151050.5(M.sub.Ab + 2D), 152449.3(M.sub.Ab + 3D), 153900.1(M.sub.Ab + 4D), 155317.2(M.sub.Ab + 5D). [00067] Compound 15 148233.1(M.sub.Ab + 0D), 149248.5(M.sub.Ab + 1D), 150271.9(M.sub.Ab + 2D), 151286.2(M.sub.Ab + 3D), 152294.1(M.sub.Ab + 4D), 153294.3(M.sub.Ab + 5D) [00068] Compound 3 148241.8(M.sub.Ab + 0D), 149605.6(M.sub.Ab + 1D), 150972.4(M.sub.Ab + 2D), 152323.1(M.sub.Ab + 3D), 153693.6(M.sub.Ab + 4D), 155059.8(M.sub.Ab + 5D) [00069] Compound 5 149617.8(M.sub.Ab + 0D), 151106.6(M.sub.Ab + 1D), 152599.5(M.sub.Ab + 2D), 154098.6(M.sub.Ab + 3D), 155577.1(M.sub.Ab + 4D), 157051.7(M.sub.Ab + 5D) [00070] Compound 8 149623.1(M.sub.Ab + 0D), 151056.9(M.sub.Ab + 1D), 152479.8(M.sub.Ab + 2D), 153901.1(M.sub.Ab + 3D), 155343.2(M.sub.Ab + 4D), 156787.4(M.sub.Ab + 5D) [00071] Compound 6 148268.0(M.sub.Ab + 0D), 149718.1(M.sub.Ab + 1D), 151151.3(M.sub.Ab + 2D), 152595.5(M.sub.Ab + 3D), 154042.4(M.sub.Ab + 4D), 155501.0(M.sub.Ab + 5D) [00072] Compound 9 148240.7(M.sub.Ab + 0D), 149771.5(M.sub.Ab + 1D), 151316.2(M.sub.Ab + 2D), 152852.4(M.sub.Ab + 3D), 154403.2(M.sub.Ab + 4D), 155964.3(M.sub.Ab + 5D) [00073] Compound 13 148230.2(M.sub.Ab + 0D), 149485.9(M.sub.Ab + 1D), 150754.2(M.sub.Ab + 2D), 152009.5(M.sub.Ab + 3D), 153276.2(M.sub.Ab + 4D), 154536.9(M.sub.Ab + 5D) [00074] Compound 14 148210.1(M.sub.Ab + 0D), 149360.3(M.sub.Ab + 1D), 150515.5(M.sub.Ab + 2D), 151677.0(M.sub.Ab + 3D), 152821.9(M.sub.Ab + 4D), 153980.7(M.sub.Ab + 5D)

[0334] Referring to the preparation procedures of antibody drug conjugate 22 described in Example 3, the antibody drug conjugates 23 and 28 were prepared by using the corresponding antibodies (Inotuzumab, Brentuximab) and drugs.

TABLE-US-00009 Interval Unit- cytotoxic antibody drug conjugate drug Q-TOF LC/MS [00075] Com- pound 2 149611.1(M.sub.Ab + 0D), 151039.9(M.sub.Ab + 1D), 152470.6(M.sub.Ab + 2D), 153904.4(M.sub.Ab + 3D), 155317.0(M.sub.Ab + 4D), 156757.9(M.sub.Ab + 5D) [00076] Com- pound 4 149607.2(M.sub.Ab + 0D), 150971.0(M.sub.Ab + 1D), 152340.4(M.sub.Ab + 2D), 153712.0(M.sub.Ab + 3D), 155067.2(M.sub.Ab + 4D), 156414.5(M.sub.Ab + 5D) [00077] Com- pound 10 148255.2(M.sub.Ab + 0D), 149786.1(M.sub.Ab + 1D), 151315.0(M.sub.Ab + 2D), 152870.4(M.sub.Ab + 3D), 154431.3(M.sub.Ab + 4D), 155954.1(M.sub.Ab + 5D)

[0335] The antibody drug conjugates 31, 32, 33 and 34 were prepared by using the antibody pertuzumab and the corresponding drugs according to Example 4, Example 5, Example 6, and Example 7.

TABLE-US-00010 Interval Unit- cytotoxic antibody drug conjugate drug Q-TOF LC/MS [00078] Com- pound 12 148096.2(M.sub.Ab + 0D), 149096.1(M.sub.Ab + 1D), 150095.7(M.sub.Ab + 2D), 151096.5(M.sub.Ab + 3D), 152097.0(M.sub.Ab + 4D), 153097.1(M.sub.Ab + 5D) [00079] Com- pound 11 148094.6(M.sub.Ab + 0D), 149081.2(M.sub.Ab + 1D), 150066.9(M.sub.Ab + 2D), 151053.3(M.sub.Ab + 3D), 152040.0(M.sub.Ab + 4D), 153025.7(M.sub.Ab + 5D) [00080] Com- pound 7 148094.8(M.sub.Ab + 0D), 149501.3(M.sub.Ab + 1D), 150908.5(M.sub.Ab + 2D), 152315.0(M.sub.Ab + 3D), 153721.4(M.sub.Ab + 4D), 155128.1(M.sub.Ab + 5D) [00081] Com- pound 1 148095.3(M.sub.Ab + 0D), 149488.2(M.sub.Ab + 1D), 150880.7(M.sub.Ab + 2D), 152273.6(M.sub.Ab + 3D), 153666.2(M.sub.Ab + 4D), 155059.1(M.sub.Ab + 5D)

Example 4

Preparation of Antibody Drug Conjugate Compound 31

[0336] ##STR00082##

The synthetic route is as follows:

##STR00083##

Step 1

Pertuzumab-propanethiol ethyl ester

[0337] Pertuzumab stock solution (preserved in buffer system with 20 mM L-histidine acetate, 120 mM sucrose, pH=5.7) was exchanged to 100 mM acetic acid-sodium acetate buffer (pH=4.3-4.5) by using G-25 size exclusion column, and concentrated to a concentration of about 10.0 mg/mL to obtain 200 mL of P-mAb acetic acid-sodium acetate buffer (13.5 mmol). 3-acetyl-mercapto-propionaldehyde (14.3 mg, 0.108 mmol) was dissolved in 20 mL of acetonitrile and then dropwise titrated into the above buffer. Then, sodium cyanoborohydride (173 mg, 2.7 mmol) was dissolved in 10 mL of water and dropwise titrated into the above reaction solution, and stirred at 25 C. for 3 hours. After that, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 300 mL of pertuzumab-propanethiol ethyl ester (31a) solution at a concentration of 6.5 mg/mL.

Step 2

Pertuzumab-propanethiol

[0338] 6.0 mL of 2M hydroxylamine hydrochloride was added into Pertuzumab-propanethiol ethyl ester (31a) solution (prepared in step 1), and stirred at 25 C. for 1 hour. Then, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 450 mL of pertuzumab-propanethiol (31b) solution at a concentration of 4.3 mg/mL.

Step 3

Pertuzumab-propyl-1-sulfur-MC-MMAF

[0339] Compound 12 (MC-MMAF, 125 mg, 13.5 mmol) was dissolved in 45 mL of acetonitrile and added into pertuzumab-propanethiol (31b) solution (prepared in step 2). After stirring at 25 C. for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3), and concentrated and filtrated under a sterile condition through a 0.2 m filter to obtain 240 mL of pertuzumab-propyl-1-sulfur-MC-MMAF (compound 31) solution at a concentration of 8.02 mg/mL, and then stored at 20 C. frozen storage.

Example 5

Preparation of Antibody Drug Conjugate Compound 32

[0340] ##STR00084##

The synthetic route is as follows:

##STR00085##

Step 1

Pertuzumab-propanethiol ethyl ester

[0341] Pertuzumab stock solution (preserved in buffer system with 20 mM L-histidine acetate, 120 mM sucrose, pH=5.7) was exchanged to 100 mM acetic acid-sodium acetate buffer (pH=4.3-4.5) with G-25 size exclusion column, and concentrated to a concentration of about 10.0 mg/mL to obtain 2.0 mL of P-mAb acetic acid-sodium acetate buffer (0.135 mmol). 3-acetyl-mercapto-propionaldehyde (0.15 mg, 1.1 mol) was dissolved in 0.2 mL of acetonitrile and then dropwise titrated into the above solution buffer. Sodium cyanoborohydride (173 mg, 2.7 mmol) was dissolved in 0.2 mL of water and dropwise titrated into the above reaction solution, and stirred at 25 C. for 3 hours. After that, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 3.0 mL of pertuzumab-propanethiol ethyl ester (31a) solution at a concentration of 6.5 mg/mL.

Step 2

Pertuzumab-propanethiol

[0342] 0.06 mL of 2 M hydroxylamine hydrochloride was added into Pertuzumab-propanethiol ethyl ester (31a) solution (prepared in step 1), and stirred at 25 C. for 1 hour. Then, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 5.0 mL of pertuzumab-propanethiol (31b) solution at a concentration of 3.8 mg/mL.

Step 3

Pertuzumab-propyl-1-sulfur-MC-MMAE

[0343] Compound 11 (MC-MMAE, 1.3 mg, 1.4 mol) was dissolved in 0.55 ml of acetonitrile and was added into pertuzumab-propanethiol (31b) solution (prepared in step 2). After stirring at 25 C. for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3), filtrated under a sterile condition through a 0.2 m filter to obtain 8.0 mL of pertuzumab-propyl-1-sulfur-MC-MMAE (compound 32) solution at a concentration of 2.4 mg/mL, and then stored at 20 C. frozen storage.

Example 6

Preparation of Antibody Drug Conjugate Compound 33

[0344] ##STR00086##

The synthetic route is as follows:

##STR00087##

Step 1

Pertuzumab-propanethiol ethyl ester

[0345] Pertuzumab stock solution (preserved in buffer system with 20 mM L-histidine acetate, 120 mM sucrose, pH=5.7) was exchanged to 100 mM acetic acid-sodium acetate buffer (pH=4.3-4.5) with a G-25 size exclusion column, and concentrated to a concentration of about 10.0 mg/mL to obtain 200 mL of P-mAb acetic acid-sodium acetate buffer (13.5 mmol). 3-acetyl-mercapto-propionaldehyde (14.3 mg, 0.108 mmol) was dissolved in 20 mL of acetonitrile and then dropwise titrated into the above buffer. Sodium cyanoborohydride (173 mg, 2.7 mmol) was dissolved in 10 mL of water and dropwise titrated into the reaction solution, and stirred at 25 C. for 3 hours. After that, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 300 mL of pertuzumab-propanethiol ethyl ester (31a) solution at a concentration of 6.5 mg/mL.

Step 2

Pertuzumab-propanethiol

[0346] 6.0 mL of 2 M hydroxylamine hydrochloride was added into Pertuzumab-propanethiol ethyl ester (31a) solution (prepared in step 1), and stirred at 25 C. for 1 hour. Then, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 450 mL of pertuzumab-propanethiol (31b) solution at the concentration of 4.3 mg/mL.

Step 3

Pertuzumab-propyl-1-sulfur-MC-VC-PAB-MMAF

[0347] Compound 7 (MC-VC-PAB-MMAF, 180 mg, 13.5 mmol) was dissolved in 45 mL of acetonitrile and added into pertuzumab-propanethiol (31b) solution (prepared in step 2). After stirring at 25 C. for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3), concentrated and filtrated under a sterile condition through a 0.2 m filter to obtain 240 mL of pertuzumab-propyl-1-sulfur-MC-VC-PAB-MMAF (compound 33) solution at a concentration of 8.0 mg/mL, and then stored at 20 C. frozen storage.

Example 7

Preparation of Antibody Drug Conjugate Compound 34

[0348] ##STR00088##

The synthetic route is as follows:

##STR00089##

Step 1

Pertuzumab-propanethiol ethyl ester

[0349] Pertuzumab stock solution (preserved in buffer system with 20 mM L-histidine acetate, 120 mM sucrose, pH=5.7) was exchanged to 100 mM acetic acid-sodium acetate buffer (pH=4.3-4.5) with a G-25 size exclusion column, and concentrated to a concentration of about 10.0 mg/mL to obtain 2.0 mL of P-mAb acetic acid-sodium acetate buffer (0.135 mmol). 3-acetyl-mercapto-propionaldehyde (0.15 mg, 1.1 mol) was dissolved in 0.2 mL of acetonitrile and then dropwise titrated into the above buffer solution. Sodium cyanoborohydride (173 mg, 2.7 mmol) was dissolved in 0.2 mL of water and was dropwise titrated into the above reaction solution, and then stirred at 25 C. for 3 hours. After that, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 3.0 mL of pertuzumab-propanethiol ethyl ester (31a) solution at a concentration of 6.5 mg/mL.

Step 2

Pertuzumab-propanethiol

[0350] 0.06 mL of 2 M hydroxylamine hydrochloride was added into the Pertuzumab-propanethiol ethyl ester (31a) solution (prepared in step 1), and stirred at 25 C. for 1 hour. Then, the reaction solution was desalted and purified by Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3) to obtain 5.0 mL of pertuzumab-propanethiol (31b) solution at a concentration of 3.8 mg/mL.

Step 3

Pertuzumab-propyl-1-sulfur-MC-VC-PAB-MMAE

[0351] Compound 1 (MC-VC-PAB-MMAE, 1.7 mg, 1.4 mol) was dissolved in 0.55 mL of acetonitrile and was added into pertuzumab-propanethiol (31b) solution (prepared in step 2). After stirring at 25 C. for 4 hours, the reaction solution was desalted and purified by a Sephadex G25 gel column (eluting phase: 0.05 M PBS solution containing 2 mM EDTA, pH 6.3), concentrated and filtrated under a sterile condition through a 0.2 m filter to obtain 8.0 mL of pertuzumab-propyl-1-sulfur-MC-VC-PAB-MMAE (compound 34) at a concentration of 2.4 mg/mL, and then stored at 20 C. frozen storage.

Example 8

Preparation of Antibody Drug Conjugate Compound 35 as Positive Control

[0352] ##STR00090##

[0353] The synthetic route is as follows:

##STR00091##

[0354] The antibody drug conjugate compound 35 was prepared by a method disclosed in U.S. Patent Application Publication No. 20050169933.

[0355] The following test examples serve to illustrate the invention, but the examples should not be considered as limiting the scope of the invention.

TEST EXAMPLES

[0356] Biological Evaluation

Test Example 1

BT474 Cell Proliferation Assay

[0357] Purpose

[0358] To test the inhibitory effects of the samples of the present invention on the proliferation of BT474 cells.

[0359] Materials:

[0360] Samples of the present invention: antibody drug conjugate compound 16 (hereinafter referred to as compound), compound 17, compound 18, and compounds 31-34;

[0361] Positive control drug: compound 35;

[0362] BT474 cell: purchased from Chinese Academy of Sciences Cell Bank, Catalog No: TCHu 143;

[0363] CCK-8: Cell Counting Kit-8, available from Dojindo, Catalog No: CK04;

[0364] FBS: Fetal Bovine Serum, available from Gibco, Catalog No: 10099-141;

[0365] RPMI1640: available from Hyclone, Catalog No: SH30809.01B;

[0366] NOVOSTAR Multifunctional Microplate Reader (BMG).

[0367] Process:

[0368] 1. 100 L of RPMI1640 medium containing 10% FBS and 15,000 BT474 cells were added into each well of a 96-well plate, and cultured in the incubator at 37 C., 5% CO.sub.2.

[0369] 2. The sample was two-fold gradient diluted with RPMI1640 medium-containing 10% FBS, 9 dilutions total, and the initial concentration was 5 g/mL.

[0370] 3. The diluted drugs were transferred to a 96-well plate, which was pre-plated with BT474 cells, 50 L/well. Each concentration was added in triplicate. Meanwhile, wells without any drug were set as control in triplicate. Thereafter, the cells were continuously cultured under conditions of 37 C., 5% CO.sub.2.

[0371] 4. 96 hours later, each well was added with 10 L of CCK-8 solution for color development, and placed in the incubator at 37 C., 5% CO.sub.2. After 4 hours of color development, the OD.sub.450 value was read on an ELISA microplate reader, and the IC.sub.50 value was obtained using Graphpad Prism 5 software.

[0372] Results:

[0373] Biological activity of the compounds of the present invention was obtained by the above procedures; the calculated IC.sub.50 values are listed in Table 1 below:

TABLE-US-00011 TABLE 1 IC.sub.50 of the compounds of the present invention for inhibiting BT474 cell proliferation No. of compound IC.sub.50 (BT474)/nM 35 1.867 16 0.358 17 3.38 18 0.152 19 0.124

[0374] Conclusion: All of the preferred compounds of the present invention have significant inhibitory activity on BT474 cell proliferation.

Test Example 2

Daudi Cell Proliferation Assay

[0375] Purpose:

[0376] To test the inhibitory effects of the samples of the present invention on the proliferation of Daudi cells.

[0377] Materials and equipment:

[0378] Samples of the present invention: Compound 22, Compound 23, Compound 24, Compound 25;

[0379] RPMI1640: available from Hyclone, Catalog No.: SH30809.01B;

[0380] Pen Strep (P/S), purchased from Gibco, Catalog No. 15140;

[0381] CCK-8: Cell Counting Kit-8, available from Dojindo, Catalog No.: CK04;

[0382] 75 cm TC-Treated Culture Flask, available from Corning Incorporated, Item: 430641;

[0383] PBS: purchased from Gibco, Catalog No.: 20012-027;

[0384] Daudi human Burkitt's lymphoma cells, purchased from Chinese Academy of Sciences Cell Bank, Catalog No.: TcHu140;

[0385] NOVOSTAR Multifunctional Microplate Reader (BMG);

[0386] Antibody Inotuzumab: positive control.

[0387] Process:

[0388] 1. Daudi human Burkitt's lymphoma cells were incubated in RPMI-1640 medium containing 10% FBS and 1% P/S. On the day of the experiment, the cell density was adjusted to 510.sup.4 cells/mL, and 90 l of medium was added into each well of a 96-well plate.

[0389] 2. The sample was four-fold gradient diluted with PBS, 9 dilutions total; and the initial concentration was 2.5 g/ml.

[0390] 3. The diluted drugs were transferred to a 96-well plate which was pre-plated with Daudi human Burkitt's lymphoma cells, 10 L/well. Control wells were added with 10 l of PBS. Thereafter, the cells were continuously cultured in an incubator at 37 C., 5% CO.sub.2.

[0391] 4. 72 hours later, each well was added with 10 L of CCK-8 developing solution, and placed in the incubator at 37 C., 5% CO.sub.2. After 4 hours of development, the OD.sub.450 value was read on an ELISA microplate reader, and the IC.sub.50 value was obtained using Graphpad Prism 5 software.

TABLE-US-00012 TABLE 2 IC.sub.50 of the compounds of the present invention for inhibiting proliferation of Daudi human Burkitt's lymphoma cells Compound No. IC.sub.50 (Daudi)/nM Inotuzumab 95.6 22 1.43 23 22.71 24 0.511 25 5.94

[0392] Conclusion: All the preferred compounds of the present invention have a significant effect in inhibiting proliferation of the Daudi human Burkitt's lymphoma cells.

Test Example 3

Test of Inhibition Rate of NCI-N87

[0393] Purpose

[0394] To evaluate and compare the efficacy of antibody cytotoxic conjugates of the present invention on inhibiting the growth of human gastric cancer NCI-N87 cell (ATCC, CRL-5822) xenografts in nude mice.

[0395] Test Drugs

[0396] Samples of the present invention: Compound 16; Compound 17; Compound 18; and Compound 31;

[0397] Positive control drug: Compound 35;

[0398] Preparation method: formulated with saline.

[0399] Animals

[0400] BALB/cA-nude mice, 6-7 weeks, female, purchased from Shanghai SLAC laboratory Animal Co., Ltd. Certificate No.: SCXK (Shanghai) 2012-0002. Housing environment: SPF level.

[0401] Process:

[0402] Nude mice were inoculated subcutaneously with NCI-N87 human gastric cancer cells. When the tumor volume reached 100-200 mm.sup.3, the animals were grouped randomly (D0). The dosage and schedule are shown in Table 1. The tumor volume and weight were measured 2-3 times per week, and the data was recorded. Tumor volume (V) was calculated as follows:

V=1/2ab.sup.2

[0403] Wherein: a and b represent length and width, respectively.

[0404] T/C (%)=(T-T.sub.0)/(C-C.sub.0)100%, wherein T and C were measured at the end of the experiment; T.sub.0 and C.sub.0 were measured at the beginning of the experiment.

TABLE-US-00013 TABLE 3 Efficacy of compounds (16, 17, 18, 35) on NCI-N87 human gastric cancer xenografts in nude mice Mean tumor % volume Mean tumor Inhibition P Animals Compound Dosing (mm.sup.3) volume (mm.sup.3) % T/C rate value Partial per group dosing route D0 SD D17 SD D17 D17 D17 regression group solvent D0,7,14 IV 135.4 11.5 1054.8 170.9 0 10 16 D0,7,14 IV 136.1 9.8 273.3 183.5 15 85 0.000 1 6 (3 mg/kg) 16 D0,7,14 IV 134.3 9.8 59.7 8.7 56 156 0.000 6 6 (10 mg/kg) 17 D0,7,14 IV 128.4 9.5 888.3 169.3 83 17 0.085 0 6 (3 mg/kg) 17 D0,7,14 IV 139.6 12.4 602.7 130.3 50 50 0.000 0 6 (10 mg/kg) 18 D0,7,14 IV 131.6 13.5 366.7 100.3 26 74 0.000 0 6 (3 mg/kg) 18 D0,7,14 IV 133.4 17.2 66.3 8.3 50 150 0.000 6 6 (10 mg/kg) 35 D0,7,14 IV 137.1 10.5 446.9 69.6 34 66 0.000 0 6 (3 mg/kg) 35 D0,7,14 IV 136.2 5.2 74.5 18.2 45 145 0.000 6 6 (10 mg/kg) D0: time of first administration. P value, versus control group; the number of mice at the beginning of the experiment: control group, n = 10, treatment group, n = 6.

TABLE-US-00014 TABLE 4 Efficacy of compound (31, 35) on NCI-N87 human gastric cancer xenografts in nude mice Mean Mean tumor tumor % Animals volume volume Inhibition P per Compound Dosing (mm.sup.3) (mm.sup.3) % T/C rate value Partial Complete group at group dosing approach D0 SEM D21 SEM D21 D21 D21 regression regression the end solvent D0,7 IV 117.3 3.2 1247.9 144.8 0 0 10 31 D0,7 IV 116.0 5.4 708.2 77.3 52 48 0.017 0 0 6 (1 mg/kg) 31 D0,7 IV 120.3 6.1 99.3 11.4 18 118 0.000** 4 0 6 (3 mg/kg) 31 D0,7 IV 116.4 3.3 0.0 0.0 100 200 0.000 0 6 6 (10 mg/kg) 35 D0,7 IV 115.3 5.5 277.1 45.2 14 86 0.000 0 0 6 (3 mg/kg) 35 D0,7 IV 109.8 4.3 23.9 15.1 78 178 0.000 2 4 6 (10 mg/kg) D0: Time of first administration; P value versus solvent group, **p < 0.01, versus group of 3 mg/kg of compound 35; For all, Student's t test was used. Number of Mice at the beginning of the experiment: control group n = 10, treatment group n = 6.

[0405] Results

[0406] In the first experimental group, the compound 16 (3, 10 mg/kg, IV, D0, 7, 14) significantly inhibited the growth of HER2-highly-expressing gastric cancer NCI-N87 subcutaneously transplanted into nude mice, the inhibition rate was 85% and 156% respectively, and partial tumor regression was caused in 1/6 and 6/6 mice. For the same dosage and schedule of compound 18, the inhibition rate on NCI-N87 was 74% and 150%, respectively, wherein the higher dose caused partial tumor regression in 6/6 mice; The inhibition rate of the control compound 35 on NCI-N87 was 66% and 145%, respectively, wherein the higher dose caused partial tumor regression in 6/6 mice. The inhibition rate of the control compound 17 on NCI-N87 was 17% and 50%, respectively. The tumor-bearing mice were well tolerated to the drugs indicated above. The inhibitory effects of test drugs on tumor growth are shown in Table 1 and FIG. 1. No deaths occurred during the administration, and the body weight of each group of mice was not significantly decreased during the administration, as shown in FIG. 2, suggesting that the current dose had no significant side effects.

[0407] In the second experimental group, compound 31 (1, 3, 10 mg/kg, IV, once per week, totally twice) dose-dependently inhibited the growth of HER2-highly-expressing gastric cancer NCI-N87 subcutaneously transplanted into nude mice, and the inhibition rate was 48%, 118% and 200%, respectively. For the 3 mg/kg group, partial tumor regression was shown in 4/6, and for the 10 mg/kg group, complete tumor regression was shown in 6/6. For control compound 35 (3, 10 mg/kg, IV, once per week, total twice), the inhibition rate on NCI-N87 was 86% and 178%, respectively. For the 10 mg/kg group, partial tumor regression was displayed in 2/6, and complete tumor regression was seen in 4/6. Tumor bearing mice tolerated these drugs well. The efficacy of compound 31 on NCI-N87 was stronger than that of positive control compound 35 (P<0.01, compared with 3 mg/kg group) (Table 4).

Test Example 4

SK-BR-3 Cell Proliferation Assay

[0408] Purpose:

[0409] Test the inhibitory effect of samples on the proliferation of Daudi cells by using CCK method, and evaluate in vitro activity of samples according to IC.sub.50.

[0410] Materials:

[0411] SK-BR-3 cells: ATCC, Catalog No: HTB-30;

[0412] McCoy's 5A medium: purchased from Gibco, Catalog No: 16600-108;

[0413] CCK-8: Cell Counting Kit-8, available from Dojindo, Catalog No: CK04;

[0414] PBS: purchased from Gibco, Catalog No: 20012-027;

[0415] Process:

[0416] 1. SK-BR-3 cells were cultured in McCoy's 5A medium containing 10% FBS, passaged twice to three times per week at a passage ratio of 1:3 or 1:6. For cell passage, the medium was aspirated, the cell adherence layer was washed with 5 mL of 0.25% trypsin, then the trypsin was aspirated, the cells were digested for 3 to 5 minutes in the incubator, and then resuspended with the addition of fresh medium.

[0417] 2. 100 L cell suspension was added into each well of a 96-well plate at a cell density of 510.sup.4 cells/mL with culture medium of McCoy's 5A medium containing 10% FBS, and the periphery of 96-well plate was only added with 100 L of McCoy's 5A medium containing 10% FBS.

[0418] 3. After 24 hours of cell adherence, the medium was removed, and 90 L of McCoy's 5A medium containing 2% FBS was added into each well.

[0419] 4. The sample was gradient diluted to different concentrations with PBS, and 10 L of sample with different concentrations was added into each well of a 96-well plate. Each concentration was repeated in duplicate.

[0420] 5. The plate was incubated for 3 days in the incubator (37 C., 5% CO.sub.2).

[0421] 6. 10 L of CCK-8 solution was added into each well (being careful not to create bubbles in the wells, since it may affect the reading of OD values)

[0422] 7. After 3 hours of incubation in the incubator, the absorbance value was read on an ELISA microplate reader at 450 nm.

[0423] Results:

TABLE-US-00015 Compound No SK-BR-3 IC50 (ng/mL) 31 5.94 35 54.75

[0424] Conclusion: the preferred compounds of the present invention have a significant inhibitory effect on the proliferation of SK-BR-3 cells.