Isochroman compound

Abstract

Provided is an isochroman compound represented by formula (I), or a pharmaceutically acceptable salt thereof, which is used as an aldo-keto reductase (AKR1C3) inhibitor for the treatment of liver cancer. ##STR00001##

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, ##STR00020## wherein T is N or CH; R.sub.1 and R.sub.2 are each independently H, F, Cl, Br, I, or C.sub.1-3 alkyl, wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2, or 3 R.sub.a; each R.sub.a is independently F, Cl, Br, I, CN, OH or NH.sub.2; R.sub.3 and R.sub.4 are each independently H, F, Cl, Br, I, CN, C.sub.1-3 alkyl, C.sub.1-3 alkoxy, ##STR00021## wherein the C.sub.1-3 alkyl is optionally substituted by 1, 2 or 3 R.sub.e; R.sub.b and R.sub.c are each independently H, CH.sub.3, CH.sub.2CH.sub.3, (CH.sub.2).sub.2CH.sub.3 or CH(CH.sub.3).sub.2; R.sub.d is CH.sub.3, CH.sub.2CH.sub.3, (CH.sub.2).sub.2CH.sub.3 or CH(CH.sub.3).sub.2; each R.sub.e is independently F, Cl, Br, I, CN, OH or NH.sub.2.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has a structure represented by formula (I-1): ##STR00022##

3. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has a structure represented by formula (I-3) or (I-4): ##STR00023## wherein the carbon atom with * is a chiral carbon atom, which exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.1 and R.sub.2 are each independently H, F, Cl, Br, I or CH.sub.3, wherein the CH.sub.3 is optionally substituted by 1, 2 or 3 R.sub.a.

5. The compound or the pharmaceutically acceptable salt thereof according to claim 4, wherein R.sub.1 is H, F, Cl, Br, I or CH.sub.3; R.sub.2 is H.

6. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein R.sub.3 and R.sub.4 are each independently H, F, Cl, Br, I, CN, CH.sub.3, OCH.sub.3, ##STR00024## wherein the CH.sub.3 is optionally substituted by 1, 2 or 3 R.sub.e.

7. The compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein R.sub.3 and R.sub.4 are each independently H, F, CH.sub.3, CHF.sub.2, OCH.sub.3, ##STR00025##

8. The compound or the pharmaceutically acceptable salt thereof according to claim 7, wherein R.sub.3 and R.sub.4 are each independently H.

9. A compound selected from the group consisting of: ##STR00026## or a pharmaceutically acceptable salt thereof.

10. A compound selected from the group consisting of: ##STR00027## or a pharmaceutically acceptable salt thereof.

11. A method for treating liver cancer in a subject in need thereof, comprising: administering an effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 1 to the subject.

12. A method for treating liver cancer in a subject in need thereof, comprising: administering an effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 9 to the subject.

13. A method for treating liver cancer in a subject in need thereof, comprising: administering an effective amount of the compound or the pharmaceutically acceptable salt thereof according to claim 10 to the subject.

14. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound has a structure represented by formula (I-2): ##STR00028##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the tumor volume growth curve of each group during the administration period.

(2) FIG. 2 is the relative weight growth curve of animal of each group during the administration period.

(3) FIG. 3 is the tumor growth signal-time curve.

(4) FIG. 4 is the schematic diagram of the tumor weight at the end point of the experiment.

(5) FIG. 5 is the weight of animaltime curve.

(6) FIG. 6 is the changes of animal weighttime curve.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(7) The following embodiments further illustrate the present disclosure in detail, but it does not mean that there are any adverse restrictions on the present disclosure. The present disclosure has been described in detail herein, and specific embodiments thereof have also been disclosed. For one skilled in the art, it is obvious to make various modifications and improvements to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

Embodiment 1

(8) ##STR00017##

(9) Step A: Compound 1-1 (5 g, 44.21 mmol) was dissolved in DMF (50 mL), and potassium carbonate (18.33 g, 132.64 mmol) and Compound 1-2 (9 g, 48.63 mmol) were added. The reaction solution was stirred at 50 C. for 12 hours. The reaction solution was concentrated under reduced pressure, water (50 mL) was added to the residue, and the pH was adjusted to 1 with dilute hydrochloric acid (1 mol/L). After filtration, the filter cake was vacuum dried to obtain Compound 1-3. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 13.88-13.37 (m, 1H), 8.55 (d, J=2.0 Hz, 1H), 8.21-8.16 (m, 2H), 7.97 (ddd, J=1.6, 8.1, 9.9 Hz, 1H), 7.50 (dd, J=5.0, 7.8 Hz, 1H), 7.29 (d, J=8.8 Hz, 1H).

(10) Step B: Compound 1-3 (6 g, 21.57 mmol) were dissolved in dioxane (150 ml), and dichloro(p-cymene)ruthenium(II) dimer (1.32 g, 2.16 mmol), guanidine carbonate (1.94 g, 10.78 mmol), Compound 1-4 (3.91 g, 29.55 mmol) and AcOH (1.30 g, 21.57 mmol) were added. Under nitrogen protection, the reaction solution was stirred at 105 C. for 16 hours. The reaction solution was concentrated under reduced pressure. The crude product was purified by column chromatography (SiO.sub.2, PE:EtOAc=1:0-5:1) to obtain Compound 1-5.

(11) .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.64 (s, 1H), 8.14 (td, J=1.5, 4.8 Hz, 1H), 7.92 (ddd, J=1.7, 8.1, 10.0 Hz, 1H), 7.80 (s, 1H), 7.60 (s, 1H), 7.52-7.42 (m, 4H), 7.39-7.32 (m, 2H), 5.42 (d, J=1.2 Hz, 2H).

(12) Step C: Compound 1-5 (2.5 g, 6.37 mmol) was added to toluene (50 ml), and cooled to minus 60 degrees Celsius, then diisobutylaluminum hydride (1 mol/L, 12.11 ml) was added. The reaction solution was stirred at minus 60 degrees Celsius for 2 hours. The reaction solution was quenched by adding water (1 ml) at minus 60 degrees Celsius, and sodium tartrate aqueous solution (4.5 g dissolved in 100 ml) was added, and the mixture was stirred for 1.5 hours, then extracted with EtOAc (100 ml2), then the organic phases were combined and washed with brine (50 mL1), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude Compound 1-6 was obtained.

(13) Step D: Compound 1-6 (2.4 g, 1.06 mmol) and triethylsilyl hydride (2.12 g, 18.26 mmol) were added to DCM (50 mL), and TFA (2.08 g, 18.26 mmol) was added at 0 degrees Celsius. The mixture was slowly heated to 25 C. and stirred for 2 hours. DCM (100 mL) was added to the reaction solution, and the solution was washed with sodium bicarbonate (50 mL1), then the combined organic phase was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO.sub.2, PE/DCM/EtOAc=20:1:1-5:1:1) to obtain Compound 1-7. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.07 (s, 1H), 8.04 (s, 1H), 7.99 (br d, J=4.9 Hz, 1H), 7.57 (ddd, J=1.4, 8.2, 10.1 Hz, 1H), 7.52 (s, 1H), 7.47-7.40 (m, 2H), 7.38-7.30 (m, 4H), 4.84 (s, 2H), 4.75 (s, 2H).

(14) Step E: Compound 1-7 (400 mg, 1.06 mmol) were dissolved in DCM (50 ml), cooled to minus 70 degrees Celsius, and ozone was bubbled into the solution for about 15 minutes, then excess ozone was blown away with nitrogen flow. A mixture of sodium borohydride (131.09 mg, 3.47 mmol) and methanol (5 mL) was added at minus 20 degrees Celsius, and the reaction solution was stirred at 0-20 C. for 1 hour. The reaction solution was quenched by adding water (10 mL) at 20 degrees Celsius and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO.sub.2, PE:EtOAc=100:0-1:1) to obtain Compound 1-8. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) 8.10 (td, J=1.5, 4.8 Hz, 1H), 7.94 (s, 1H), 7.77 (ddd, J=1.5, 8.1, 10.1 Hz, 1H), 7.44 (ddd, J=0.6, 4.8, 7.9 Hz, 1H), 7.25 (s, 1H), 5.76 (d, J=6.2 Hz, 1H), 4.83-4.67 (m, 2H), 4.55 (q, J=6.1 Hz, 1H), 3.94 (dd, J=4.9, 11.4 Hz, 1H), 3.58 (dd, J=6.8, 11.4 Hz, 1H).

(15) Step F: Compound 1-8 (190 mg, 620.42 mol) was dissolved in THE (8 mL), lithium hexamethyldisilazide (1 mol/L, 930.63 L) was added at minus 60 degrees Celsius, and the mixture solution was stirred at minus 60 degrees Celsius for 15 minutes under nitrogen protection. Phosphorus oxychloride (190.26 mg, 1.24 mmol) was added at minus 60 degrees Celsius, and the mixture was stirred at minus 60 degrees Celsius for 15 minutes under nitrogen protection. 2-bromoethylamine hydrobromide (1.02 g, 4.96 mmol) and diisopropylethylamine (641.46 mg, 4.96 mmol) were added. The reaction solution was stirred at 0 C. for 1 hour under nitrogen protection. Water (10 ml) was added to the reaction solution, and the solution was extracted with EtOAc (50 mL3), then the combined organic phase was washed with brine (10 mL1) and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (SiO.sub.2, PE:EtOAc=100:0-1:1) to obtain Compound 1-9.

(16) Step G: Compound 1-9 (360 mg, 601.85 mmol) was dissolved in THE (18 mL), and silver oxide (4.18 g, 18.06 mmol) was added. The mixture was stirred at 63 C. for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to p-HPLC (separation column: Welch Ultimate XBSiOH (Spec: 250 mm50 mm, particle size: 10 m); mobile phase: [n-hexane-isopropanol]; elution gradient: isopropanol 20%-60%, 15 minutes) to obtain Compound 1. .sup.1H NMR (CDCl.sub.3, 400 MHz) 8.00 (td, J=1.6, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J=1.7, 7.8, 9.6 Hz, 1H), 7.20-7.16 (m, 2H), 5.42-5.29 (m, 1H), 4.87-4.59 (m, 2H), 4.00 (d, J=4.4 Hz, 2H), 2.13-1.96 (m, 8H).

(17) Step H:

(18) ##STR00018##

(19) Compound 1 was chirally separated (separation column: DAICEL CHIRALPAK AD (Spec: 250 mm30 mm, particle size: 10 m); mobile phase: [neutral-isopropanol]; elution gradient: isopropanol 35%-35%) to obtain Compound 1A (retention time=1.671 mins) and Compound 1B (retention time=1.870 mins).

(20) Compound 1A: .sup.1H NMR (CDCl.sub.3,400 MHz) 8.00 (td, J=1.6, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J=1.7, 7.9, 9.6 Hz, 1H), 7.21-7.15 (m, 2H), 5.35 (dt, J=8.76, 4.25 Hz, 1H), 4.87-4.57 (m, 2H), 4.00 (d, J=4.4 Hz, 2H), 2.14-1.96 (m, 8H). ee value (enantiomeric excess): 100%.

(21) Compound 1B: .sup.1H NMR (CDCl.sub.3,400 MHz) 8.00 (td, J=1.5, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J=1.7, 7.9, 9.6 Hz, 1H), 7.19-7.16 (m, 2H), 5.35 (dt, J=8.63, 4.32 Hz, 1H), 4.84-4.63 (m, 2H), 4.00 (d, J=4.4 Hz, 2H), 2.13-1.95 (m, 8H). ee value (enantiomeric excess): 98%.

Embodiment 2

(22) ##STR00019##

(23) As regards the synthesis of Compound 2, one may refer to step A to G of the synthesis method of Compound 1. In the synthetic route of Compound 2, the starting material 1-1 was substituted by 2-fluorophenol.

(24) Compound 2: .sup.1H NMR (400 MHz, CDCl.sub.3) 7.62 (s, 1H), 7.18-7.10 (m, 4H), 7.08 (s, 1H), 5.31 (td, J=4.2, 8.7 Hz, 1H), 4.82-4.73 (m, 1H), 4.68-4.58 (m, 1H), 3.99 (d, J=4.4 Hz, 2H), 2.08-1.92 (m, 8H).

BIOLOGICAL ACTIVITY

Experimental Embodiment 1: Antiproliferative Activity of the Compound of the Present Disclosure on NCI-H460 Cell Line

(25) Experimental Materials:

(26) RPMI-1640 medium and penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega. The NCI-H460 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo Multimode Plate Reader (PerkinElmer).

(27) Experimental Method:

(28) NCI-H460 cells (lung cancer) were seeded in a 96-well plate, 80 L of cell suspension per well, which contained 4000 NCI-H460 cells. The cell plate was incubated overnight in a carbon dioxide incubator. The compound to be tested was diluted 5-fold to the ninth concentration with a multi-channel pipette, that is, the compound to be tested was diluted from 2 mM to 5.2 nM, and an experiment for the sample assayed in duplicate was set up. 78 L of medium was added to the middle plate, and 2 L of the gradient-diluted compound per well was transferred to the middle plate according to the corresponding position, then 20 L per well was transferred to the cell plate after mixing. The concentration of the compound transferred to the cell plate ranged from 10 M to 0.026 nM. The cell plate was incubated in the carbon dioxide incubator for 2 hours, and then the drug-containing medium was removed. The cell plate was rinsed once with fresh medium, and 100 L of fresh medium without drug was added to each well to continue the incubation for 70 hours. Another cell plate was prepared, and the signal value was read as the maximum value (Max value in the following equation) on the day of drug addition to participate in data analysis. 25 L of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings. 25 L of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings.

(29) Data Analysis:

(30) The equation (SampleMin)/(MaxMin)*100% is used to convert the raw data into an inhibition rate, and the value of IC.sub.50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. responseVariable slope mode in GraphPad Prism). Table 1 provides the inhibitory activity of the compounds of the present disclosure on the proliferation of NCI-H460 cells.

(31) TABLE-US-00002 TABLE 1 Data of antiproliferative activity of the compounds of the present disclosure on NCI-H460 cell line Compound NO. IC.sub.50 (nM) Compound 1 3.8 Compound 1A 10.1 Compound 1B 16.6 Compound 2 21.6
Conclusion: the compound of the present disclosure has excellent antiproliferative activity against NCI-H460 with high expression of AKR1C3.

Experimental Embodiment 2: Antiproliferative Activity of the Compound of the Present Disclosure on HepG2 Cell Line

(32) Experimental Materials:

(33) DMEM medium and penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) Reagent was purchased from Promega. The HepG2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multimode Plate Reader (PerkinElmer).

(34) Experimental Method:

(35) HepG2 cells (liver cancer) were seeded in a white 384-well plate, 25 M of cell suspension per well, which contained 1000 HepG2 cells. The cell plate was incubated overnight in a carbon dioxide incubator. The compound to be tested was diluted 3-fold to the ninth concentration with a multi-channel pipette, that is, the compound to be tested was diluted from 200 mM to 30 nM, and an experiment for the sample assayed in duplicate was set up. 99 M of medium was added to the middle plate, and 1 L of the gradient-diluted compound per well was transferred to the middle plate according to the corresponding position, then 25 M per well was transferred to the cell plate after mixing. The concentration of the compound transferred to the cell plate ranged from 1 M to 0.15 nM. The cell plate was incubated in the carbon dioxide incubator for 5 days. Another cell plate was prepared, and the signal value as the maximum value (Max value in the following equation) was read on the day of drug addition to participate in data analysis. 20 M of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings. 20 M of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings.

(36) Data Analysis:

(37) The equation (SampleMin)/(MaxMin)*100% was used to convert the raw data into an inhibition rate, and the value of IC.sub.50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. responseVariable slope mode in GraphPad Prism). Table 2 provides the inhibitory activity of the compounds of the present disclosure on the proliferation of HepG2 cells.

(38) TABLE-US-00003 TABLE 2 Data of antiproliferative activity of the compound of the present disclosure on HepG2 cell line Compound NO. IC.sub.50 (nM) Compound 1A 9.3 Compound 1B 7.4

(39) Conclusion: the compounds of the present disclosure have excellent antiproliferative activity against HepG2 with high expression of AKR1C3.

Experimental Embodiment 3: Antiproliferative Activity of the Compound of the Present Disclosure on Hep3B Cell Line

(40) Experimental Materials:

(41) EMEM medium and penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) Reagent was purchased from Promega. The Hep3B cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo Multimode Plate Reader (PerkinElmer).

(42) Experimental Method:

(43) Hep3B cells (liver cancer) were seeded in a white 96-well plate, 80 L of cell suspension per well, which contained 3000 Hep3B cells. The cell plate was incubated overnight in a carbon dioxide incubator. The compound to be tested was diluted 5-fold to the ninth concentration with a multichannel pipette, that is, the compound to be tested was diluted from 2 mM to 5.12 nM, and an experiment for the sample assayed in duplicate was set up. 78 L of medium was added to the middle plate, and 2 L of each well of the gradient dilution compound was transferred to the middle plate according to the corresponding position, then 20 L per well was transferred to the cell plate after mixing. The concentration of the compound transferred to the cell plate ranged from 10 M to 0.0256 nM. The cell plate was incubated in the carbon dioxide incubator for 3 days. Another cell plate was prepared, and the signal value as the maximum value (Max value in the following equation) was read on the day of drug addition to participate in data analysis. 25 L of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings. 25 L of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescent signal. The multimode plate reader was used for readings.

(44) Data Analysis:

(45) The equation (SampleMin)/(MaxMin)*100% was used to convert the raw data into an inhibition rate, and the value of IC.sub.50 can be obtained by curve fitting with four parameters (log(inhibitor) vs. responseVariable slope mode in GraphPad Prism). Table 3 provides the inhibitory activity of the compounds of the present disclosure on the proliferation of Hep3B cells.

(46) TABLE-US-00004 TABLE 3 Data of antiproliferative activity of the compound of the present disclosure on Hep3B cell line Compound NO. IC.sub.50 (nM) Compound 1A >10,000 Compound 1B >10,000

(47) Conclusion: the compound of the present disclosure has no antiproliferative activity on Hep3B with low expression of AKR1C3.

Experimental Embodiment 4: In Vivo Pharmacodynamics Study of the Compound of the Present Disclosure on Transplanted Tumor Model of Human Liver Cancer HepG2 Cells in Nude Mice

(48) Experimental Purpose:

(49) The inhibition of tumor growth in vivo for transplanted tumor model of human liver cancer HepG2 cells in nude mice was used in this experiment.

(50) Experimental Materials:

(51) Female NU/NU nude mice (number: 90; week-age: 6-8 weeks), human liver cancer cell HepG2, MEM medium, fetal bovine serum (FBS), trypsin, Penicillin-Streptomycin, PBS, Matrigel, etc.

(52) Experimental Method and Steps:

(53) 1. Cell Culture

(54) Routine cell culture was carried out in MEM medium containing 10% fetal bovine serum at 37 C. in 5% CO.sub.2; 0.25% trypsin was used for digestion and passage; according to the cell growth, the cell was passaged 2 to 3 times a week, and the ratio of passage was 1:3 to 1:6.

(55) 2. Preparation of Animal Model

(56) Hep G2 cells in the logarithmic growth phase were collected, counted and resuspended in 50% serum-free MEM medium and 50% Matrigel, the cell concentration was adjusted to 2.510.sup.7 cells/mL; the cells were blown with a pipette to disperse evenly and were put into a 50 mL centrifuge tube, and the centrifuge tube was put in an ice box; the cell suspension was drawn up with a 1 mL syringe and injected subcutaneously into the armpit of the right fore limb of nude mice, and each animal was inoculated 200 L (510.sup.6 cells/mouse) and the transplanted tumor model of HepG2 cells in nude mice was established. After inoculation, the state of the animals and the growth of the tumor were observed regularly, and the diameter of the tumor was measured with an electronic vernier caliper. The data was directly input into an Excel spreadsheet to calculate the tumor volume. When the tumor volume reached 100-300 mm.sup.3, 48 animals with good health and similar tumor volume were selected, and randomized block method was used to divide the animals into 8 groups (n=6) according to the tumor volume, and the average weight of each group was kept consistent as much as possible. The day of grouping was regarded as the first day of the experiment (D1). After the experiment started, the tumor diameter was measured twice a week, and the tumor volume was calculated. At the same time, the weight of the animals was weighed and recorded.

(57) The formula for calculating tumor volume (TV) is as follows:
TV (mm.sup.3)=lw.sup.2/2

(58) Wherein, l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

(59) 3. Animal Grouping and Administration:

(60) The animal grouping and dosage regimen are shown in Table 4. The administration was started on the day of grouping, and the experiment was ended 3 weeks later (or the tumor volume in the solvent control group reached 2000 mm.sup.3 or above, whichever came first), and the administration volume was 10 mL.Math.kg.sup.1. As the solvent control group, Group 1 was given DMSO & 30% HP--CD (10:90, v:v) by intravenous injection once a week for 3 consecutive weeks; Groups 2 and 3 were given Compound 1A and 1B by intravenous injection with the dose of 1 mg.Math.kg.sup.1.

(61) TABLE-US-00005 TABLE 4 Dosage regimen of pharmacodynamic experiment for transplanted tumor model of HepG2 cells in nude mice Administration Number of Dose volume Route of Administration Group Sample animals (mg .Math. kg.sup.1) (mL .Math. kg.sup.1) administration frequency 1 DMSO & 6 10 intravenous Day 0, Day 7, 30% HP-B-CD injection (IV) Day 14 (10:90, V:V) 2 Compound 6 1 10 intravenous Day 0, Day 7, 1A injection (IV) Day 14 3 Compound 6 1 10 intravenous Day 0, Day 7, 1B injection (IV) Day 14

(62) 4. Experimental Indicators:

(63) The calculation formula of tumor growth inhibition rate (GI) is:
TGI=100%[1(TV.sub.t(T)TV.sub.initial(T))/(TV.sub.t(c)TV.sub.initial(C))]

(64) where TV.sub.t(T) represents the tumor volume measured each time in the treatment group; TV.sub.initial(T) represents the tumor volume in the treatment group at the time of grouping and administration; TV.sub.t(C) represents the tumor volume measured each time in the solvent control group; TV.sub.initial(C) represents the tumor volume of the solvent control group at the time of grouping and administration. The formula of the relative animal weight is calculated as:
Relative animal weight=BW.sub.t/BW.sub.initial*100

(65) wherein, BW.sub.t represents the animal weight measured each time during the administration period; B.sub.Initial represents the animal weight at the time of grouping and administration.

(66) 5. The Inhibition of the Compounds on the Growth of Subcutaneously Transplanted Tumors of HepG2 Liver Cancer in Nude Mice:

(67) In this experiment, the efficacy of Compounds 1A and 1B in the transplanted tumor model of HepG2 liver cancer was evaluated. After 21 days of administration, Compound 1B had a significant effect of inhibiting tumor growth at a dose of 1 mg/kg, all p<0.05 compared with the vehicle control group. The tumor volume and inhibition rate are shown in Table 5, and the tumor growth curve is shown in FIG. 1.

(68) TABLE-US-00006 TABLE 5 Tumor volume and tumor growth inhibition rate of animals in each group during administration Mean tumor volume SEM (mm.sup.3) or tumor growth inhibition rate (TGI) Group D1 D5 D8 D12 D15 D19 D22 solvent control 140 7 370 31 648 74 946 112 1568 186 2198 145 2545 199 group Compound 1A, 140 7 245 13 313 22 474 60 853 112 1158 186 1415 220 1.0 mg/kg TGI 54% 66% 58% 50% 51% 47% Compound 1B, 140 6 185 24 215 35 276 58 412 100 602 157 681 189 1.0 mg/kg TGI 80% 85% 83% 81% 78% 77%

(69) 6. Changes in Weight:

(70) In this model, the weight of animals in all treatment groups did not fluctuate greatly, and the whole average weight loss of animals did not exceed 10%, as shown in FIG. 2 for details.

(71) Conclusion: the compound of the present disclosure exhibits good antitumor efficacy.

Experimental Embodiment 5: In Vivo Pharmacodynamics Study of the Compound of the Present Disclosure on Orthotopic Xenograft Tumor Model of Human Liver Cancer HepG2

(72) Experimental Purpose:

(73) In this experiment orthotopic xenograft tumor model of HepG2 in nude mouse was used to evaluate the antitumor effect of the compound.

(74) Experimental Materials:

(75) Female Balb/C nude mice, the week age is 6-8 weeks, the weight is 18-22 g, fetal bovine serum (PBS), EMEM medium (Cat. No. 30-2003), phosphate buffer, Antibiotic-Antimycotic (Cat. No. 15240-062), Matrigel and Pancreatin.

(76) Experimental Method and Steps:

(77) 1. Preparation of Cell Culture: HepG2-Luc Cells were Cultured in Monolayer In Vitro.

(78) The culture conditions were that 10% heat-inactivated fetal bovine serum was added into EMEM medium and the cells were cultured in an incubator containing 5% CO.sub.2 at 37 C.

(79) Digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation was 80%-90%, the cells were digested with trypsin-EDTA, counted and resuspended in PBS and Matrigel (PBS:Matrigel=1:1), and the density was 166.6710.sup.6 cells/mL.

(80) Tumor cell inoculation and grouping: Animals were anesthetized by intramuscular injection of 60 mg/kg Zytex 50 and 1.5 mg/kg xylazine. When the animals were under deep anesthesia, the animals were properly fixed, and the abdomen skin was cleaned with 75% alcohol cotton balls. An incision of about 10 mm was cut with surgery, and 0.03 mL (PBS:Matrigel=1:1) of HepG2-luc cells were orthotopically inoculated on the left lobe of the liver of each mouse, and then the incision on the muscle layer was sutured with absorbable catgut, and the incision on the skin was sutured with a stapler. After surgery, the animals were kept warm on a warming blanket until they woke up. To relieve the pain of the animals, 2 mg/kg of meloxicam (administered subcutaneously once a day) will be administered for 3 days in a row after surgery. 15 animals were randomly selected to detect the growth of the signal. When the signal began to rise, they were randomly divided into groups according to the value of the bioluminescence signal, and drug treatment was started. The detailed treatment plan is shown in Table 6.

(81) TABLE-US-00007 TABLE 6 Grouping and dosage regimen of experimental animals Administration Number of Dose volume Route of Administration Group animals Drug (mg .Math. kg.sup.1) (L/g).sup.2 administration frequency 1 6 blank 10 intravenous Day 0, Day 7, group injection (IV) Day 14 2 6 Compound 1 10 intravenous Day 0, Day 7, 1B injection (IV) Day 14 3 6 Compound 3 10 intravenous Day 0, Day 7, 1B injection (IV) Day 14

(82) 3. Experimental Indicator:

(83) The experimental indicator is whether the tumor growth can be delayed or whether the tumor can be cured. After tumor inoculation, the bioluminescence signal and animal weight were detected once a week, which continued until the end of the observation period. The bioluminescent signal value can be used to calculate T/C (wherein T is the administration group, and C is the average intensity value of bioluminescence of the blank control group at the set time). Calculation formula of Tumor Growth Inhibition Rate (TGI): TGI (%)=[1(T.sub.iT.sub.0)/(V.sub.iV.sub.0)]100, wherein T.sub.i is the average intensity of bioluminescence of the treatment group at the set time; T.sub.0 is the average intensity of bioluminescence at the starting point of administration. V.sub.i is the average intensity of bioluminescence of the blank control group at the set time; V.sub.0 is the average intensity of bioluminescence at the starting point of administration.

(84) 4. Inhibitory of Compounds on the Growth of Subcutaneously Transplanted Tumors of HepG2 Liver Cancer in Nude Mice:

(85) In this experiment, the efficacy of Compound 1B in the orthotopic xenograft tumor model of HepG2 liver cancer was evaluated. After 21 days of administration, Compound 1B had a significant effect of inhibiting tumor growth at a dose of 1 mg/kg, all p<0.05 compared with the vehicle control group. With the dose of compound 1B increased to 3 mg/kg, the anti-tumor effect is significantly enhanced.

(86) Experimental results: See FIG. 3 and Tables 7, 8 and 9. The tumor weight at the end point of the experiment is shown in FIG. 4 and Table 10.

(87) TABLE-US-00008 TABLE 7 Antitumor effect of the compound of the present disclosure on orthotopic xenograft tumor model of HepG2 Bioluminescence Bioluminescence (photons/sec).sup.a (photons/sec).sup.a RBL TGI (%) T/C (%) Group (Day 1) (Day 21) (Day 21) (Day 21) (Day 21) 1 3.51E+09 7.27E+08 3.09E+10 6.17E+09 9.78 1.67 2 3.51E+09 7.41E+08 4.35E+09 2.19E+09 0.98 0.31 96.97 10.04 3 3.51E+09 7.96E+08 1.81E+09 7.52E+08 0.41 0.12 106.21 4.22 Note: .sup.aMean SEM, n = 6.

(88) TABLE-US-00009 TABLE 8 The p value of the comparison of the relative tumor signal growth value (RBL) between groups in the xenograft tumor model of HepG2 Group Group 1 Group 2 Group 3 Group 4 1 N/A 0.038 0.029 0.024 2 0.029 0.921 N/A 0.682 3 0.024 0.384 0.682 N/A Note: The p value was obtained by using one-way ANOVA to analyze the relative value of tumor volume (RBL). There was a significant difference in the F value between groups (p < 0.001), and the Games-Howell test was used.

(89) TABLE-US-00010 TABLE 9 Bioluminescence signal values of tumor tissue at different time points in each group Bioluminescence (photons/sec).sup.a Days after administration group 0 7 14 21 1 1.47E+09 3.54E+08 4.86E+09 9.42E+08 1.24E+10 3.98E+09 1.80E+10 3.52E+09 2 1.47E+09 3.20E+08 4.34E+09 1.74E+09 4.76E+09 2.40E+09 4.52E+09 2.29E+09 3 1.47E+09 2.84E+08 2.77E+09 5.16E+08 2.59E+09 3.58E+08 2.16E+09 3.23E+08 Note: .sup.aMean SEM, n = 6

(90) TABLE-US-00011 TABLE 10 Tumor weight in each group Tumor weight (g).sup.a T/C.sub.weight.sup.b Group (Day 21) (%) p value.sup.c 1 0.569 0.115 2 0.090 0.036 15.80 0.072 3 0.058 0.013 10.23 0.059 Note: .sup.aMean SEM, n = 6. .sup.bTumor growth inhibition was calculated as T/C.sub.weight = TW.sub.treatment/TW.sub.vehicle. .sup.cp value was obtained by one-way ANOVA and vehicle treatment group to analyze tumor weight, F value was significantly different (p < 0.001), and the Games-Howell test was used for analysis.

(91) 5. Changes in Weight

(92) In this model, the weight of animals in all treatment groups did not fluctuate greatly, and the decrease of average weight of all animals did not exceed 5%, as shown in FIG. 5 and FIG. 6.

(93) Conclusion: The compound of the present disclosure has the effect of significantly inhibiting tumor growth, and the animal weight in the administration group does not decrease significantly, showing good safety.