NOVEL USE OF MULTIKINASE INHIBITOR

Abstract

The present invention belongs to the technical field of medicines, relates to novel use of a multi-kinase inhibitor, and particularly relates to a compound of general formula (I) or a pharmaceutically acceptable salt, a stereoisomer and a crystal form thereof for use in the treatment of biliary tract cancer, a pharmaceutical composition comprising the compound, a method for treating biliary tract cancer by using the compound, use of the compound in the treatment of biliary tract cancer, and use of the compound in the preparation of a medicament for treating biliary tract cancer. The variables in the general formula are defined in the specification. Research shows that the multi-kinase inhibitor compound of general formula (I) or the pharmaceutically acceptable salt, the stereoisomer and the crystal form thereof have a treatment effect on biliary tract cancer, and especially on cholangiocarcinoma, so that the compound of the present invention has huge clinical application potential.

##STR00001##

Claims

1. A method of treating a patient suffering from biliary tract cancer; comprising administering to the patient an effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt, a stereoisomer and a crystal form thereof: ##STR00080## wherein Ar is phenyl optionally substituted with 1-3 R.sub.6, each R.sub.6 is independently selected from hydrogen, amino, cyano, halogen, C.sub.1-4 alkyl and trifluoromethyl; Y is CR.sub.3; P is CR.sub.4; W is N; R.sub.3 is hydrogen or C.sub.1-4 alkyl; R.sub.4 is —(CH.sub.2).sub.n−(5-11) membered heterocyclyl, wherein n=0-6, a ring-forming S atom in the heterocyclyl is optionally oxidized to S(O) or S(O).sub.2, a ring-forming C atom in the heterocyclyl is optionally oxidized to C(O), and the heterocyclyl is optionally substituted with one to more substituents independently selected from C.sub.1-3 alkyl and C.sub.3-6 cycloalkyl.

2. The method according to claim 1, wherein Ar is phenyl optionally substituted with 1-3 R.sub.6, and each R.sub.6 is independently selected from hydrogen and halogen; Y is CR.sub.3; P is CR.sub.4; W is N; R.sub.3 is hydrogen; R.sub.4 is selected from —(CH.sub.2).sub.n−(5-6) membered monocyclic heterocyclyl and —(CH.sub.2).sub.n−(7-11) membered fused heterocyclyl, wherein n=0-6, a ring-forming S atom in the heterocyclyl is optionally oxidized to S(O) or S(O).sub.2, a ring-forming C atom is optionally oxidized to C(O), and the heterocyclyl is optionally substituted with one to more substituents independently selected from C.sub.1-3 alkyl and C.sub.3-6 cycloalkyl.

3. The method according to claim 2, wherein R.sub.4 is ##STR00081## and n=0-3, wherein the heterocyclyl is optionally substituted with one to more substituents independently selected from C.sub.1-3 alkyl and C.sub.3-6 cycloalkyl.

4. The method according to claim 3, wherein the compound is selected from compounds of the following structures, and a pharmaceutically acceptable salt, a stereoisomer and a crystal form thereof: ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086##

5. The method according to claim 4, wherein the compound is ##STR00087## or a pharmaceutically acceptable salt, a stereoisomer, and a crystal form thereof.

6. The method according to claim 1, wherein the biliary tract cancer is cholangiocarcinoma.

7. The method according to claim 6, wherein the cholangiocarcinoma is a cholangiocarcinoma with non-FGFR aberration.

8. The method according to claim 6, wherein the cholangiocarcinoma is a cholangiocarcinoma with FGFR aberration.

9. The method according to claim 8, wherein the cholangiocarcinoma is cholangiocarcinoma with any one of FGFR fusion, FGFR mutation and FGFR overexpression or any combination thereof.

10. The method according to claim 9, wherein the cholangiocarcinoma is cholangiocarcinoma with any one of FGFR2 fusion, FGFR2 and/or FGFR3 mutation and FGFR overexpression or any combination thereof.

11. The method according to claim 6, wherein the cholangiocarcinoma refers to drug-resistant cholangiocarcinoma that is positive for FGFR aberration but not responsive to an FGFR inhibitor, or drug-resistant cholangiocarcinoma after administration of an FGFR inhibitor.

12. The method according to claim 6, wherein the cholangiocarcinoma is any one of intrahepatic cholangiocarcinoma, perihilar cholangiocarcinoma and distal cholangiocarcinoma or any combination thereof.

13. (canceled)

14. The method according to claim 1, wherein the patient is further administered one or more second therapeutically active agents in addition to the compound of general formula (I) or the pharmaceutically acceptable salt, the stereoisomer and the crystal form thereof, wherein the one or more second therapeutically active agents are any one of an antimetabolite, a growth factor inhibitor, a mitotic inhibitor, an anti-tumor hormone, an alkylating agent, metallic platinum, a topoisomerase inhibitor, a hormonal agent, an immunomodulator, a tumor suppressor gene, a cancer vaccine and an immune checkpoint inhibitor or any combination thereof.

15. The method according to claim 14, wherein the compound of general formula (I) or the pharmaceutically acceptable salt, the stereoisomer and the crystal form thereof and the second therapeutically active agents are administered to a patient in need of treatment sequentially, simultaneously or in a combined formulation.

16. The method according to claim 15, wherein the patient is a mammal.

17. The method according to claim 16, wherein the patient is a human.

18. The method according to claim 1, wherein the biliary tract cancer is an FGFR-mediated cholangiocarcinoma.

19. The method according to claim 1, wherein the biliary tract cancer is cholangiocarcinoma mediated by any one of FGFR1, FGFR2 and FGFR3 or any combination thereof.

20. The method according to claim 6, wherein the cholangiocarcinoma is a cholangiocarcinoma with FGFR2 aberration and/or a cholangiocarcinoma with FGFR3 aberration.

Description

DETAILED DESCRIPTION

[0192] In order to make the objective, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below. It should be apparent that the examples described herein are only some examples of the present invention, but not all examples. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0193] The abbreviations and English expressions used in the present invention have the following meanings:

TABLE-US-00002 TABLE 2 Abbreviations and English expressions Abbreviation/English Meaning DMSO Dimethyl sulfoxide MOPS 3-morpholinopropanesulfonic acid EDTA Ethylenediaminetetraacetic acid MnCl.sub.2 Manganese chloride MC Methylcellulose ATP Adenosine triphosphate Glu Glutamic acid Tyr Tyrosine PEG Polyethylene glycol Qd Administration once daily Qw Administration once a week BiW Administration twice a week

Experimental Example 1: Assay on Inhibitory Activity of Compounds of the Present Invention Against FGFR Wild-Type Enzyme

[0194] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1.

Experiment Method

[0195] (1) Preparation of the Compound Plate

[0196] The compounds were each dissolved in DMSO to prepare stock solutions with a maximum concentration of 500 μM. The compound stock solutions were diluted with DMSO to final concentrations of 500, 150, 50, 15, 5, 1.5, 0.5, 0.15, 0.05 μM to obtain compound working solutions (50×).

(2) Experimental Procedures

[0197] a) Preparation of Different Reaction Systems

[0198] FGFR1(h) was dissolved in 8 mM MOPS (pH 7.0), 0.2 mM EDTA and 250 μM KKKSPGEYVNIEFG;

[0199] FGFR2(h) was dissolved in 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 2.5 mM MnCl.sub.2 and 0.1 mg/mL poly (Glu, Tyr) (4:1);

[0200] FGFR3(h) was dissolved in 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 10 mM MnCl.sub.2 and 0.1 mg/mL poly (Glu, Tyr) (4:1);

[0201] b) Enzymatic Reaction

[0202] The reaction was initiated by the addition of 10 mM magnesium acetate and 10μM [γ−.sup.33P]−ATP. After incubation at room temperature for 40 min, the reaction was stopped by using a 3% (v/v) phosphoric acid solution. 10 μL of the reaction solution was pipetted onto P30 filter paper, washed 3 times with 75 mM phosphoric acid solution for 5 min each time, dried and counted by scintillation. 2% (v/v) DMSO was taken as a positive control (Max) instead of the compound solutions; high concentration positive control inhibitor (10 μM Staurosporine) was taken as a negative control (Min) instead of the compound solutions.

[0203] Test Results:

TABLE-US-00003 TABLE 3 Inhibitory activity of compounds of the present invention against wild-type FGFRs (IC.sub.50) FGFR1 FGFR2 FGFR3 Test samples (nM) (nM) (nM) Compound 29 2 3 5

[0204] It can be seen from the experimental results in Table 3 that the compounds of the present invention can target FGFR1-3, have a good inhibitory activity against FGFR1-3, and have good clinical application potential in the aspect of treating diseases mediated by FGFR1-3.

Experimental Example 2: Assay on Inhibitory Activity of Compounds of the Present Invention Against Wild-Type and Mutant FGFR

[0205] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1.

TABLE-US-00004 TABLE 4 Names and sources of enzymes/reagents Abbreviations Name Source FGFR2 WT Wild-type FGFR2 enzyme Invitrogen FGFR2 N549H gene mutant Signalchem N549H FGFR2 enzyme FGFR2 V564F gene mutant Signalchem V564F FGFR2 enzyme FGFR3 WT Wild-type FGFR3 enzyme Invitrogen FGFR3 K650E gene mutant Signalchem K650E FGFR3 enzyme HTRF KinEASE-TK Homogeneous time-resolved Cisbio kit fluorescence method kit BIBF 1120 Nintedanib ChemExpress

Experiment Method

[0206] (1) Preparation of the Compound Plate

[0207] The compounds were dissolved in DMSO and 3-fold diluted with DMSO to obtain 10 serially diluted stock solutions. The stock solutions were added into a 384-well plate to obtain a 3-fold diluted series of 10 concentrations of the compound starting from 10 μm.

(2) Experimental Procedures

[0208] Different enzyme solutions (2×) were prepared, transferred into the 384-well plate, incubated with the compound solutions with different concentrations for 10 min at room temperature, and a mixed solution (2×) of biotinylated tyrosine kinase substrate/ATP was added to activate the reaction. After incubation for 50 min at room temperature, an HTRF detection reagent and a corresponding kinase antibody cryptate were added, and after incubation for 1 h at room temperature, fluorescence readings at 615 nm (cryptate) and 665 nm (HTRF detection reagent) were detected by using an Envision 2104 multifunctional microplate reader. 10 μM BIBF-1120 was taken as a compound positive control (PC) instead of the compound solutions, and 0.1% (v/v) DMSO was taken as a solvent negative control (VC) instead of the compound solutions.

[0209] (3) Data Processing

[0210] The fluorescence ratio of 665/615 nm was calculated, and the enzyme activity inhibition rate (%) was calculated according to the following formula:

[00001]$\mathrm{Inhibition}\mathrm{rate}\%=100-\frac{\mathrm{Fluorescence}\mathrm{ratio}\mathrm{of}\mathrm{compounds}-\mathrm{Mean}\mathrm{fluorescence}\mathrm{ratio}\mathrm{of}\mathrm{PC}}{\mathrm{Mean}\mathrm{fluorescence}\mathrm{ratio}\mathrm{of}\mathrm{VC}-\mathrm{Mean}\mathrm{fluorescence}\mathrm{ratio}\mathrm{of}\mathrm{PC}}\times 100$

[0211] IC.sub.50 values were fitted using GraphPad 6.0 based on inhibition rates of compounds with different concentrations.

[0212] Test Results:

TABLE-US-00005 TABLE 5 Inhibitory Activity of Compounds of the Present Invention Against FGFR enzyme (IC.sub.50) Test samples Enzymes IC.sub.50 (nM) Compound 29 FGFR2 WT 0.5 FGFR2 N549H 1.9 FGFR2 V564F 0.7 Compound 29 FGFR3 WT 1.0 FGFR3 K650E 1.7

[0213] It can be seen from the experimental results in Table 5 that the compounds of the present invention have an obvious inhibitory effect on both wild-type and mutant FGFR2 and FGFR3, which indicates that the compounds of the present invention have better clinical application potential in treating diseases mediated by wild-type and/or mutant FGFR, such as cholangiocarcinoma.

Experimental Example 3: Cholangiocarcinoma Organoid Experiment of Compounds of the Present Invention

[0214] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1.

[0215] Organoid information: Human distal cholangiocarcinoma (dCC) from K2 Oncology Co. Ltd., Beijing, code KOBD-002

TABLE-US-00006 TABLE 6 Names and sources of reagents and instruments Name Source/model No. 96-channel high-throughput full- NAYO N96 automatic liquid workstation Microplate reader BMG FLUOstar Fetal bovine serum Gibco Penicillin/streptomycin double-antibody Gibco 15140122 Pancreatin Gibco 12604013 Cell Viability fluorescence Promega G7573 detection kit CellTiter-Glo ® Luminescent Cell Viability Assay GAS-Ad-BD medium K2 Oncology, K2O-CML-01801 BEZ235 MCE Gemcitabine Cisplatin MCE 5-fluorouracil MCE Matrigel BD 356231

Experiment Method

[0216] (1) Preparation of the Compound Plate

[0217] The compounds were dissolved in DMSO, then diluted 3-fold to obtain stock solutions (1000×) with 10-concentration gradients, and diluted 100-fold using GAS-Ad-BD medium to obtain working solutions (10×).

(2) Experimental Procedures

[0218] After tumor organoids could be passaged, matrigel was melted at 4° C. for later use. Cultured tumor organoids were collected using a Pasteur dropper and added with pancreatin to form a single cell suspension. After cell counting, the cell concentration was adjusted to 8×10.sup.4 cell/mL by using GAS-Ad-BD medium, and 2 mL of the cell suspension was placed on ice for later use. A matrigel mixed solution was prepared by mixing the cell suspension and the matrigel and then placed on ice for later use, 50 uL of the mixed solution was added to a 96-well plate, incubated at 37° C. for 30 min and added with GAS-Ad-BD medium, and after 2 days of incubation in a 37° C. cell incubator, the formation and growth of tumor organoids were observed.

[0219] After the formation and growth of the tumor organoids were observed, 10 μL of working solutions (10×) with 10-concentration gradients prepared on the same day were sequentially added. The solutions were incubated at 37° C. for 96 h with 5% carbon dioxide. The starting concentrations of each compound were as follows: compound 29 at 10 μM, gemcitabine at 40 μM, cisplatin at 60 μM, and 5-fluorouracil at 30 μM. A solvent negative control group (DMSO, 100% survival) and a positive control group (2.5 μM BEZ235, 0% survival) were set.

[0220] After the cultivation was completed, 70 μL of CellTiter-Glo® solution was added, and the fluorescence value was determined after the operation was performed according to the specification.

[0221] (3) Data Processing

[0222] The corresponding compound concentration at 50% survival was calculated by using GraphPad Prism 5 software, that is the IC.sub.50 value (absolute IC.sub.50 value) for the compounds on these cells.

[0223] Test results:

TABLE-US-00007 TABLE 7 Inhibitory Activity of Compounds of the Present Invention Against KOBD-002 Distal Cholangiocarcinoma Organoids Test samples IC.sub.50(μM) Compound 29 1.27 Gemcitabine 17.97 Cisplatin 18.77 5-fluorouracil >30

[0224] It can be seen from the experimental results in Table 7 that the compounds of the present invention have a good inhibitory activity against KOBD-002 distal cholangiocarcinoma organoids, and are superior to gemcitabine, cisplatin and 5-fluorouracil, which indicates that the compounds have good clinical application potential in treating cholangiocarcinoma, and especially distal cholangiocarcinoma.

Experimental Example 4: In Vivo Efficacy Test of Compounds of the Present Invention on HuPrime® Human Cholangiocarcinoma CC6204 Subcutaneous Xenograft Tumor Model

[0225] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1; gemcitabine hydrochloride, commercially available.

[0226] The source of tumor mass: CC6204 was a HuPrime® xenograft model established by a female cholangiocarcinoma patient. The pathological diagnosis was intrahepatic cholangiocarcinoma, with FGFR2-BICC1 fusion aberration.

[0227] Animals: Balb/c nude female mice at 5-6 weeks (weeks of age at time of mice inoculation).

[0228] Test Method:

[0229] (1) Construction and Grouping of Tumor-Bearing Mice

[0230] Tumor tissues were collected from tumor-bearing mice of HuPrime® cholangiocarcinoma xenograft model CC6204, cut into tumor masses of a diameter of 2-3 mm and then subcutaneously inoculated into the right anterior scapula of the Balb/c nude mice, and the mice were grouped and administered when an average volume of tumors reached 133 mm.sup.3. Grouping method: animals were weighed before administration and tumor volumes were measured. Grouping was designed in blocks according to the tumor volume, with 6 mice per group.

[0231] (2) Administration Regimen

TABLE-US-00008 TABLE 8 Administration regimen Dosing Route Frequency Dosage volume of of Administration Group Vehicle (mg/kg) (mL/kg) administration administration period Vehicle 0.5% (v/v) MC 0 10 Oral Qd 68 days control containing 0.5% intragastric (v/v) Tween 80 administration Compound 29 0.5% (w/v) MC 15 10 Oral Qd 68 days intragastric administration Gemcitabine Normal saline 120 10 Intraperitoneal QW .sup.a 68 days hydrochloride Note: .sup.a gemcitabine hydrochloride was administered once a week for a total of 10.

[0232] (3) Experimental Observation Index

[0233] Animals were monitored daily for health and mortality, body weight and tumor volume were measured twice a week, and samples were collected after the last administration. The therapeutic effect of tumor volume was evaluated by TGI %, wherein the relative tumor inhibition rate TGI (%): TGI=1-T/C (%). T/C % is the relative tumor proliferation rate, i.e., the percentage of the relative tumor volume in the treatment and control groups at a given time point. T and C are relative tumor volumes (RTVs) of the treatment and control groups at a given time point, respectively.

[0234] The calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV×100% (T.sub.RTV: mean RTV for treatment group; C.sub.RTV: mean RTV for vehicle control group; RTV=V.sub.t/V.sub.0, wherein V.sub.0 is the tumor volume of the animal at the time of grouping, and V.sub.t is the tumor volume of the animal after treatment). The drug is considered to be effective according to the National Institute of Health (NIH) Guidelines that TGI is ≥58%.

[0235] Test Results:

TABLE-US-00009 TABLE 9 Effect of Compounds of the Present Invention on Tumor Growth in Mice of HuPrime ® Human Cholangiocarcinoma CC6204 subcutaneous xenograft tumor model Number of Tumor volume .sup.a TGI Group animals (mm.sup.3) (%).sup.b P.sup.c Vehicle control 6 696.52 — — Compound 29 6 147.8 80 <0.001 Gemcitabine 6 397.22 43 0.973 hydrochloride Note: .sup.a tumor volumes were statistic data for 21 days of administration. .sup.bTGI: relative tumor inhibition rate, statistic data for 21 days of administration. .sup.cP < 0.05 indicates that there was a statistical difference, P < 0.01 indicates that there was a significant statistical difference, and P < 0.001 indicates that there was an extremely significant statistical difference.

TABLE-US-00010 TABLE 10 Effect of Compounds of the Present Invention on Survival Rate of Mice of HuPrime ® Human Cholangiocarcinoma CC6204 subcutaneous xenograft tumor model Number Number of of surviving Survival Group animals animals .sup.a rate (%) P.sup.b Vehicle control 6 0 0 — Compound 29 6 6 100 <0.001 Gemcitabine 6 1 16.7 0.467 hydrochloride Note: .sup.a surviving animals were data statistics after the last administration (day 67). .sup.bP < 0.05 indicates that there was a statistical difference, P < 0.01 indicates that there was a significant statistical difference, and P < 0.001 indicates that there was an extremely significant statistical difference.

[0236] It can be seen from the experimental results in Table 9 and Table 10 that the compounds of the present invention have a remarkable inhibitory effect on HuPrime® human cholangiocarcinoma CC6204 subcutaneous xenograft tumor model, effectively prolong the life cycle of tumor-bearing animals, and are significantly superior to clinical standard treatment of gemcitabine, which indicates that the compounds of the present invention can be used in clinical treatment of intrahepatic cholangiocarcinoma tumors with FGFR2 aberration, and have good clinical application potential.

Experimental Example 5: In Vivo Efficacy Test of Compounds of the Present Invention on HuPrime® Human Cholangiocarcinoma CC6639 Subcutaneous Xenograft Tumor Model

[0237] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1.

[0238] The source of tumor mass: CC6639 was a HuPrime® xenograft model established by a male cholangiocarcinoma patient. The pathological diagnosis was intrahepatic cholangiocarcinoma, with no FGFR2 aberration.

[0239] Animals: Balb/c nude female mice at 4-5 weeks (weeks of age at time of mice inoculation).

[0240] Experiment Method

[0241] (1) Construction and Grouping of Tumor-Bearing Mice

[0242] Tumor tissues were collected from tumor-bearing mice of HuPrime® cholangiocarcinoma xenograft model CC6639, cut into tumor masses of a diameter of 2-3 mm and then subcutaneously inoculated into the right anterior scapula of the Balb/c nude mice, and the mice were grouped and administered when an average volume of tumors reached 202 mm.sup.3. Grouping method: animals were weighed before administration and tumor volumes were measured. Grouping was designed in blocks according to the tumor volume, with 5 mice per group.

[0243] (2) Administration Regimen Shown in Table 11

TABLE-US-00011 TABLE 11 Administration regimen Dosing Dosage volume Route of Frequency of Administration Group Vehicle (mg/kg) (mL/kg) administration administration period Vehicle 0.5% 0 10 Oral intragastric Qd 21 days control (w/v)MC administration Compound 0.5% 15 10 Oral intragastric Qd 21 days 29 (w/v)MC administration

[0244] (3) Experimental Observation Index

[0245] Animals were monitored daily for health and mortality, body weight and tumor volume were measured twice a week, and samples were collected after the last administration. The therapeutic effect of tumor volume was evaluated by TGI %, wherein the relative tumor inhibition rate TGI (%): TGI=1-T/C (%). T/C % is the relative tumor proliferation rate, i.e., the percentage of the relative tumor volume in the treatment and control groups at a given time point. T and C are relative tumor volumes (RTVs) of the treatment and control groups at a given time point, respectively. The calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV×100% (T.sub.RTV: mean RTV for treatment group; C.sub.RTV: mean RTV for vehicle control group; RTV=V.sub.t/V.sub.0, wherein V.sub.0 is the tumor volume of the animal at the time of grouping, and V.sub.t is the tumor volume of the animal after treatment). The drug is considered to be effective according to the NIH Guidelines that TGI is ≥58%.

[0246] Test Results:

TABLE-US-00012 TABLE 12 Effect of Compound 29 of the present invention on tumor growth in mice of HuPrime ® human cholangiocarcinoma CC6639 subcutaneous xenograft tumor model Tumor volume TGI Group Number of animals (mm.sup.3) (%).sup.a P.sup.b Vehicle control 5 2195.88 — — Compound 29 5 623.72 72 0.0045 Note: .sup.astatistical data after the last administration; TGI: relative tumor inhibition rate. .sup.bP < 0.05 indicates that there was a statistical difference, P < 0.01 indicates that there was a significant statistical difference, and P < 0.001 indicates that there was an extremely significant statistical difference.

[0247] It can be seen from the experimental results in Table 12 that Compound 29 has a remarkable inhibitory effect on HuPrime® human cholangiocarcinoma CC6639 subcutaneous xenograft tumor model, which indicates that the compound can be used in clinical treatment of intrahepatic cholangiocarcinoma tumors with non-FGFR2 aberration and has good clinical application potential.

Experimental Example 6: In Vivo Efficacy Test of Compounds of the Present Invention on Human Perihilar Cholangiocarcinoma PDTX Subcutaneous Xenograft Tumor Model

[0248] Test samples: the compounds of the present invention, which have structures shown in Table 1 and are prepared as described in the embodiments of WO2018108079A1.

[0249] The source of tumor mass: the human perihilar cholangiocarcinoma tumor sample was derived from a female patient.

[0250] Animals: NCG Male Mice at 5-8 Weeks.

[0251] Test Method:

[0252] (1) Construction and Grouping of Tumor-Bearing Mice

[0253] The tumor samples excised in the surgery were inoculated into mice as P0 generation and then passaged as P1 generation for drug efficacy evaluation. The tumor masses were inoculated into the right back of the mice, and the mice were grouped and administered when an average volume of tumors reached approximately 100 mm.sup.3. Grouping method: animals were weighed before administration and tumor volumes were measured. Grouping was designed in blocks according to the tumor volume, with 6 mice per group.

[0254] (2) Administration According to Administration Regimen of Table 13

TABLE-US-00013 TABLE 13 Administration regimen Dosing Dosage volume Route of Frequency of Administration Group Vehicle (mg/kg) (mL/kg) administration administration period Vehicle 0.5%(w/v)MC 0 10 Oral Qd 25 days control intragastric administration Compound 29 0.5%(w/v)MC 15  10 Oral Qd 25 days intragastric administration Gemcitabine Normal saline 30 + 10 + Intraperitoneal BiW 25 days hydrochloride + 3 10 Qw Cisplatin

[0255] (3) Experimental Observation Index

[0256] Animals were monitored daily for health and mortality, body weight and tumor volume were measured twice a week, and samples were collected after the last administration. The therapeutic effect of tumor volume was evaluated by TGI %, wherein the relative tumor inhibition rate TGI (%): TGI=1-T/C (%). T/C % is the relative tumor proliferation rate, i.e., the percentage of the relative tumor volume in the treatment and control groups at a given time point. T and C are relative tumor volumes (RTVs) of the treatment and control groups at a given time point, respectively. The calculation formula is as follows: T/C %=T.sub.RTV/C.sub.RTV×100% (T.sub.RTV: mean RTV for treatment group; C.sub.RTV: mean RTV for vehicle control group; RTV=V.sub.t/V.sub.0, wherein V.sub.0 is the tumor volume of the animal at the time of grouping, and V.sub.t is the tumor volume of the animal after treatment). The drug is considered to be effective according to the NIH Guidelines that TGI is ≥58%.

[0257] Test Results:

TABLE-US-00014 TABLE 14 Effect of Compound 29 of the Present Invention on Tumor Growth in Mice of HuPrime ® Human Cholangiocarcinoma CC6204 subcutaneous xenograft tumor model Number of Tumor volume .sup.a TGI Group animals (mm.sup.3) (%).sup.b P.sup.c Vehicle control 6 233.9 — — Compound 29 6 70.9 70 0.0039 Gemcitabine 6 157.3 35 0.247 hydrochloride + Cisplatin Note: .sup.a tumor volumes were statistical data for 21 days of administration; .sup.bTGI: relative tumor inhibition rate, statistic data for 21 days of administration. .sup.cP < 0.05 indicates that there was a statistical difference, P < 0.01 indicates that there was a significant statistical difference, and P < 0.001 indicates that there was an extremely significant statistical difference.

[0258] It can be seen from the experimental results in Table 14 that Compound 29 has a remarkable inhibitory effect on human perihilar cholangiocarcinoma PDTX subcutaneous xenograft tumor model, and is significantly superior to the clinical standard treatment of gemcitabine+cisplatin, which indicates that the compound of the present invention can be used in clinical treatment of perihilar cholangiocarcinoma tumors and has good clinical application potential.

[0259] The above description is only for the purpose of illustrating the preferred example of the present invention, and is not intended to limit the scope of the present invention. Any modifications, equivalents, improvements and the like made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.