MALEATE OF NICOTINYL ALCOHOL ETHER DERIVATIVE, CRYSTAL FORM THEREOF, AND APPLICATION THEREOF

Abstract

The present invention relates to the technical field of medicine, and disclosed a maleate of a nicotinyl alcohol ether derivative, a crystal form thereof, and an application thereof, i.e., (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serine isopropyl ester maleate and a stereoisomer and a crystal form thereof, a preparation method therefor, a pharmaceutical composition, and a use thereof. Specifically, the present invention relates to the (S)-N-(2-(pyridin-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serine isopropyl ester maleate represented by formula I, and a crystal form thereof, a stereoisomer thereof, a preparation method therefor, a composition containing said compound or the crystal form thereof, and a use of said compound or the crystal form thereof in the preparation of a medicine for treating diseases related to a PD-1/PD-L1 signaling pathway, such as cancer, infectious diseases and autoimmune diseases.

##STR00001##

Claims

1. The isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate as shown in formula (I) and a stereoisomer thereof, ##STR00005##

2. A crystal form A solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate, characterized in that, when powder X-ray diffraction analysis is used and Cu Target Radiation experimental conditions are adopted, the position of diffraction peak: 2-theta value () or d value () and relative intensity (%) of diffraction peak have the following characteristics: TABLE-US-00017 Peak 2-Theta 0.2 d() 0.2 intensity % 5% 1 4.5 19.6 42.3 2 5.6 15.9 50.1 3 6.8 13.0 14.0 4 8.5 10.4 16.2 5 9.1 9.8 100 6 10.2 8.7 28.1 7 12.4 7.1 11.1 8 13.3 6.7 18.6 9 13.6 6.5 35.6 10 14.6 6.1 12.1 11 15.5 5.7 15 12 16.0 5.5 11 13 16.6 5.3 13.6 14 17.1 5.2 14.2 15 17.4 5.1 13.8 16 17.9 4.9 17.4 17 18.7 4.7 17.6 18 19.0 4.7 20.1 19 19.7 4.5 35.5 20 20.0 4.4 46.6 21 20.7 4.3 58.3 22 21.6 4.1 35.5 23 22.2 4.0 15 24 22.7 3.9 33.6 25 23.1 3.9 50.5 26 23.9 3.7 42 27 25.0 3.6 16.4 28 25.4 3.5 19.2 29 26.6 3.4 28 30 28.0 3.2 13.9 31 28.4 3.1 15.9 32 29.3 3.1 19.1 33 29.9 3.0 14.2 34 30.4 2.9 14.4 35 31.0 2.9 15.7 36 31.6 2.8 15.6 37 32.7 2.7 12.2 38 34.0 2.6 12.7 39 34.9 2.6 11.9 40 35.7 2.5 13.4 41 39.0 2.3 11.2

3. The crystal form A solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 2, characterized in that, when analyzed by infrared spectrum, the absorption peaks at 3059, 2984, 2841, 2761, 2519, 2170, 1988, 1968, 1807, 1741, 1716, 1623, 1602, 1580, 1505, 1481, 1460, 1446, 1425, 1401, 1389, 1368, 1309, 1262, 1242, 1205, 1171, 1111, 1095, 1069, 1040, 1004, 972, 953, 924, 884, 870, 864, 854, 824, 788, 761, 721, 703, and 662 cm.sup.12 cm.sup.1 are the characteristic peak positions of infrared spectrum presented by the crystal form A solid substance.

4. The crystal form A solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 2, characterized in that, when analyzed by differential scanning calorimetry, there is an endothermic peak at 175 C.3 C. in the DSC spectrum with a heating rate of 10 C. per minute.

5. A mixed crystal solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate, characterized in that, it comprises any non-zero proportion of the crystal form A solid substance of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 2.

6. A preparation method of the isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 1, characterized in that, the preparation method is as follows: reacting isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate with maleic acid in a solvent to form a salt, preferably, the solvent is isopropyl alcohol, tetrahydrofuran, or acetone; crystallizing the obtained isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate in a mixed solvent of acetone and water, the ratio of acetone to water ranges from 200:1 to 1:1, preferably, the ratio ranges from 50:1 to 5:1, more preferably, the ratio ranges from 25:1 to 10:1.

7. A pharmaceutical composition, wherein the pharmaceutical composition comprises the isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 1 and a stereoisomer thereof as an active ingredient, and a pharmaceutically acceptable carrier or excipient thereof.

8. A method for preventing and/or treating diseases related to PD-1/PD-L1 signal pathway, which comprises administering the isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate according to claim 1 and a stereoisomer thereof to a subject in need thereof.

9. The method according to claim 8, wherein the diseases related to PD-1/PD-L1 signal pathway are selected from cancer, infectious diseases and autoimmune diseases.

10. The method according to claim 9, wherein the cancer is selected from skin cancer, lung cancer, urological tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious diseases are selected from bacterial infection and viral infection; the autoimmune diseases are selected from organ specific autoimmune diseases and systemic autoimmune diseases, wherein, the organ specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis, the systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease and autoimmune hemolytic anemia.

11. A preparation method of the crystal form A solid substance according to claim 2, wherein the preparation method is as follows: reacting isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate with maleic acid in a solvent to form a salt, preferably, the solvent is isopropyl alcohol, tetrahydrofuran, or acetone; crystallizing the obtained isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate in a mixed solvent of acetone and water, the ratio of acetone to water ranges from 200:1 to 1:1, preferably, the ratio ranges from 50:1 to 5:1, more preferably, the ratio ranges from 25:1 to 10:1.

12. A pharmaceutical composition, wherein the pharmaceutical composition comprises the crystal form A solid substance according to claim 2 as an active ingredient, and a pharmaceutically acceptable carrier or excipient thereof.

13. A pharmaceutical composition, wherein the pharmaceutical composition comprises the crystal form A solid substance according to claim 3 as an active ingredient, and a pharmaceutically acceptable carrier or excipient thereof.

14. A pharmaceutical composition, wherein the pharmaceutical composition comprises the crystal form A solid substance according to claim 4 as an active ingredient, and a pharmaceutically acceptable carrier or excipient thereof.

15. A pharmaceutical composition, wherein the pharmaceutical composition comprises the mixed crystal solid substance according to claim 5 as an active ingredient, and a pharmaceutically acceptable carrier or excipient thereof.

16. A method for preventing and/or treating diseases related to PD-1/PD-L1 signal pathway, which comprises administering the crystal form A solid substance according to claim 2 to a subject in need thereof.

17. The method according to claim 16, wherein the diseases related to PD-1/PD-L1 signal pathway are selected from cancer, infectious diseases and autoimmune diseases.

18. The method according to claim 17, wherein the cancer is selected from skin cancer, lung cancer, urological tumor, blood tumor, breast cancer, glioma, digestive system tumor, reproductive system tumor, lymphoma, nervous system tumor, brain tumor, head and neck cancer; the infectious diseases are selected from bacterial infection and viral infection; the autoimmune diseases are selected from organ specific autoimmune diseases and systemic autoimmune diseases, wherein, the organ specific autoimmune diseases include chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pulmonary hemorrhage nephritis syndrome, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis, the systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease and autoimmune hemolytic anemia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1. Differential scanning calorimetry/thermogravimetric analysis spectrum of compound of Example 1.

[0034] FIG. 2. Differential scanning calorimetry/thermogravimetric analysis spectrum of compound of Example 2.

[0035] FIG. 3. Powder X-ray diffraction pattern of compound of Example 2.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

[0036] The present invention will be further described below in combination with specific examples, but the scope of the present invention is not limited.

[0037] Test instruments: Bruker AVANCE III 500 high resolution superconducting nuclear magnetic resonance spectrometer is used for nuclear magnetic resonance spectroscopy. QSTAR Elite LC/MS/MS System is used for mass spectrometry. FLASH1112 trace element analyzer and MX-5 millionth Balance instrument are used for elemental analysis. Shimadzu UV-2700 UV-Vis spectrophotometer was used for UV analysis. American PE Model 343 polarimeter is used for specific rotation. D8-Advance X-ray diffractometer is used for powder X-ray diffraction analysis. Swiss Mettler TGA/DSC3.sup.+ thermal analyzer is used for differential scanning calorimetry/thermogravimetric analysis (DSC/TG).

1. Preparation of Salt:

[0038] Example 1: isopropyl (S)-N-[2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate hydrochloride (the Example is a comparative example, in which the compound is a known compound, and its preparation method is exactly the same as the Example 6 of the international application PCT/CN2017/085418)

##STR00004##

[0039] 598 mg of (S)-N-[2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serine and 60 ml of anhydrous isopropanol were placed in a 100 ml round bottom flask, 6 ml of sulfoxide chloride and 2 drops of DMF were added under stirring in an ice water bath. The mixture was stirred at room temperature for 2 hours, heated and refluxed until the reaction was complete. The solvent was removed under reduced pressure to obtain isopropyl (S)-N-[2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate hydrochloride, as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6) 9.62 (s, 1H, HCl), 9.40 (s, 1H, HCl), 9.10 (s, 1H, ArH), 8.86 (d, 1H, ArH), 8.59 (d, J=7.6 Hz, 1H, ArH), 8.01-7.91 (m, 1H, ArH), 7.73-7.64 (m, 2H, ArH), 7.57-7.46 (m, 3H, ArH), 7.46-7.37 (m, 4H, ArH), 7.16 (s, 1H, ArH), 5.47 (s, 2H, CH.sub.2), 5.36 (s, 2H, CH.sub.2), 4.92 (m1H, CH), 4.30-4.15 (m, 2H, CH.sub.2), 4.05 (s, 1H, CH), 3.97 (dd, J=12.0, 3.0 Hz, 1H, CH.sub.2), 3.84 (dd, J=12.0, 3.8 Hz, 1H, CH.sub.2), 1.20 (d, J=6.4 Hz, 3H, CH.sub.3), 1.18 (d, J=6.4 Hz, 3H, CH.sub.3). MS (FAB): 640 (M).

[0040] Example 2: isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate (IMMH-010)

[0041] At room temperature, isopropyl (S)-N-[2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-(phenyl)benzyloxy)-5-chlorobenzyl]serinate (2.6 g) and isopropanol (9 ml) were added into a 50 ml reaction flask. The mixture was heated to 40 C., stirred for 0.5 h, and maleic acid (0.594 g) -isopropanol (4 ml) solution was added dropwise. The temperature was controlled at 3545 C., and a solid was precipitated, then the mixture was stirred at the temperature for 0.5 h, naturally cooled down to room temperature, and stirred overnight. On the next day, the mixture was filtered by suction, and the filter cake was washed with 0.5 ml of isopropanol and 0.5 ml of acetone successively to obtain a light yellow solid, which is the crude product of isopropyl (S)-N-(2-(pyridine-3-yl-methoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl) serinate maleate. At room temperature, the crude product and acetone (26 ml) were added into a 50 ml reaction flask, heated to reflux, and 1.4 ml of purified water was added dropwise. After being fully dissolved, the mixture was subjected to thermal filtration. After filtration, the filter cake was transferred to a 50 ml reaction flask, naturally cooled down to room temperature, and stirred for crystallization overnight. On the next day, the mixture was cooled down to 515 C., stirred for 2 h, and filtered by suction. The filter cake was washed with 0.5 ml of acetone, and subjected to forced air drying to constant weight at 45 C. to obtain a pure product (0.73 g), as a white solid.

2. Structural Confirmation of the Compound of Example 2:

1) Elemental analysis: [0042] (1) Test instrument: trace element analyzer FLASH1112 and millionth balance MX-5. [0043] (2) Test method: carbon, hydrogen and nitrogen were measured twice in parallel. [0044] (3) Measured results:
Elemental analysis data table of the compound of Example 2

TABLE-US-00002 Content (%) C H N Theoretical 57.19 4.80 3.71 Value (%) Measured value 57.10 4.80 3.63 (%) 57.21 4.78 3.59 Average value 57.16 4.79 3.61 (%) Absolute error 0.03 0.01 0.1 (%)

2) High Resolution Mass Spectrometry:

[0045] (1) Test instrument: QSTAR Elite LC/MS/MS System [0046] (2) Test condition: ESI source [0047] (3) Measured data: High resolution mass spectrometry data of the compound of Example 2

High Resolution Mass Spectrometry Data of the Compound of Example 2

TABLE-US-00003 Measured value (m/z) of [M + H].sup.+ 639.1259 Theoretical value (m/z) of [M + H].sup.+ 639.1256 Error (10.sup.6) 0.49 Molecular constitute of molecular ion peak C.sub.32H.sub.33N.sub.2O.sub.5ClBr Molecular formula C.sub.32H.sub.32N.sub.2O.sub.5ClBr

3) UV Absorption Spectrum:

[0048] (1) Test instrument: Shimadzu UV-2700 UV-Vis spectrophotometer [0049] (2) Test method: the sample was prepared into a solution in a certain concentration, the same batch of solvent was used as the blank control, and 1 cm absorption cell was used to measure the absorption value in the range of 190400 nm. [0050] Solvent: watermethanol (1:1), 0.1 mol/L of hydrochloric acidmethanol (1:1), 0.1 mol/L of sodium hydroxidemethanol (1:1) [0051] Test solution concentration: 20 l/ml. [0052] (3) Measured data:

UV Data Table of the Compound of Example 2

TABLE-US-00004 .sub.max Absorption Spectral band IMMH-010 (nm) value assignment Methanol-water (1:1) 206.50 2.567 K-band 237.50 0.622 absorption 285.30 0.116 0.1 mol/L of hydrochloric 206.60 2.464 acid - methnol (1:1) 237.50 0.621 285.40 0.147 0.1 mol/L of sodium 227.60 0.550 hydroxide - methanol (1:1) 285.70 0.042

4) Infrared Absorption Spectrum:

[0053] (1) Test instrument: British PE (Spectrum 400) infrared spectrometer [0054] (2) Test conditions: attenuated total reflection (ATR) infrared spectroscopy, powder direct injection

IR Data Table of the Compound of Example 2

TABLE-US-00005 absorption peak (cm.sup.1) Vibration type Group Intensity 3059 CH stretching Aromatic ring, w vibration alkene 2984, 2840 CH stretching Methyl, w vibration methylene 1716 CO stretching Carboxyl w vibration (maleate) 1741 CO stretching Ester bond m vibration 1623 CC stretching Alkene bond m vibration (maleate) 1600, 1580, CC stretching Aromatic ring s 1505, 1446 vibration 1368, 1389, CH deformation Isopropyl s 1460 vibration 703, 761, 788 CH out of plane Aromatic ring s deviational vibration

5) Nuclear Magnetic Resonance Hydrogen Spectrum and Carbon Spectrum:

[0055] (1) Test instrument: Bruker AVANCE III 500 high resolution superconducting nuclear magnetic resonance spectrometer [0056] (2) Test conditions: the solvent was DMSO-d6 and the internal standard was TMS

.SUP.1.H-NMR Data of the Compound of Example 2

TABLE-US-00006 chemical shift Spectral line Proton No (ppm) multiplicity J (Hz) number 1 8.72 d 1.7 1 2 8.56 dd 4.8, 1.7 1 3 7.92 m \ 1 4 7.64 dd 7.7, 1.7 1 5 7.55 s \ 1 6 7.51 t 7.6 1 7 7.49-7.46 m \ 1 8 7.46-7.44 m, \ 1 9 7.44-7.42 m \ 1 10 7.42-7.40 m \ 1 11 7.40-7.38 m \ 2 12 7.38-7.36 m \ 1 13 7.14 s \ 1 14 6.05 s \ 2 15 5.48 brs \ 1 17 5.34 s \ 2 18 5.29 s \ 2 19 4.86 p 6.3 1 20 4.10 s \ 2 21 3.87-3.73 m \ 3 22 1.16 dd \ 6

.SUP.13.C-NMR Data of the Compound of Example 2

TABLE-US-00007 Number chemical shift of carbon Carbon functional No (ppm) atoms type group 1 167.69 1 4 Ester group 2 167.12 2 4 carboxyl 3 156.60 1 4 Aromatic ring carbon 4 154.76 1 4 Aromatic ring carbon 5 149.33 1 3 Aromatic ring carbon 6 148.97 1 3 Aromatic ring carbon 7 143.10 1 4 Aromatic ring carbon 8 140.76 1 4 Aromatic ring carbon 9 136.16 1 4 Aromatic ring carbon 10 135.68 1 3 Aromatic ring carbon 11 135.24 2 3 Alkene bond 12 132.51 1 3 Aromatic ring carbon 13 132.00 1 4 Aromatic ring carbon 14 131.27 1 3 Aromatic ring carbon 15 129.25 2 3 Aromatic ring carbon 16 129.10 1 3 Aromatic ring carbon 17 128.20 2 3 Aromatic ring carbon 18 127.84 1 3 Aromatic ring carbon 19 127.78 1 3 Aromatic ring carbon 20 123.63 1 3 Aromatic ring carbon 21 123.06 1 4 Aromatic ring carbon 22 112.90 1 4 Aromatic ring carbon 23 100.19 1 3 Aromatic ring carbon 24 71.08 1 2 methylene 25 69.66 1 3 Methyne 26 68.16 1 2 methylene 27 60.47 1 3 Methyne 28 59.50 1 2 methylene 29 43.34 1 2 methylene 30 21.40 1 1 methyl 31 21.34 1 1 methyl

6) Specific Rotation:

[0057] (1) Test instrument: American PE Model 343 polarimeter. [0058] (2) Test method: the product of the present invention was weighed accurately, dissolved in DMSO, and diluted quantitatively to obtain a solution containing about 50 mg per 1 ml, and the specific rotation was determined. [0059] (3) Test temperature: 20 C. [0060] (4) Results

[0061] With DMSO as the solvent and the determination concentration of 50 mg/ml, the specific rotation of the compound of Example 2 was +5.5. [].sub.589.sup.20=+5.5 (C=5, DMSO)

3. Crystal Form Analysis of the Compound of Example 2

1) Powder X-Ray Diffraction Analysis:

[0062] (1) Test instrument: D8-Advance X-ray diffractometer [0063] (2) Test conditions: working voltage: 40 kV, working current: 40 mA, Cu target. Scanning speed: 0.02 degrees/step, dwell time: 0.1 seconds/step. [0064] (3) Test results (see FIG. 3):

Powder X-Ray Diffraction Data Table of the Compound of Example 2

TABLE-US-00008 Intensity Angle Intensity d value % 2-theta Cont Angstrom 42.3 4.509 1707 19.58167 50.1 5.565 2020 15.86828 14.0 6.785 567 13.01670 16.2 8.481 653 10.41733 100.0 9.054 4033 9.75969 28.1 10.206 1133 8.66043 11.1 12.392 449 7.13700 18.6 13.285 749 6.65934 35.6 13.620 1435 6.49629 12.1 14.550 486 6.08317 15.0 15.497 603 5.71323 11.0 15.984 442 5.54049 13.6 16.574 550 5.34433 14.2 17.119 573 5.17560 13.8 17.401 558 5.09221 17.4 17.929 703 4.94346 17.6 18.697 711 4.74221 20.1 18.963 809 4.67625 35.5 19.650 1432 4.51412 46.6 20.037 1880 4.42785 58.3 20.655 2351 4.29674 35.5 21.556 1433 4.11923 15.0 22.174 604 4.00580 33.6 22.706 1356 3.91306 50.5 23.110 2036 3.84561 42.0 23.863 1692 3.72585 16.4 24.974 660 3.56261 19.2 25.356 774 3.50974 28.0 26.594 1127 3.34912 13.9 27.986 560 3.18567 15.9 28.414 641 3.13867 19.1 29.261 772 3.04971 14.2 29.872 574 2.98871 14.4 30.398 581 2.93813 15.7 30.948 634 2.88721 15.6 31.580 631 2.83082 12.2 32.673 493 2.73853 12.7 33.988 512 2.63555 11.9 34.858 480 2.57176 13.4 35.712 539 2.51218 11.2 39.004 453 2.30741 [0065] (4) Analysis: powder X-ray diffraction analysis showed that the compound of Example 2 was a crystalline substance.

2) Differential Scanning Calorimetry/Thermogravimetric Analysis (DSC/TG):

[0066] (1) Test instrument: Swiss Mettler TGA/DSC3+thermal analyzer [0067] (2) Parameter setting: starting temperature: 35 C.; termination temperature: 250 C.; heating rate: 10 C./min. [0068] (3) Measured data: DSC: peak temperature: 174.68 C. (endothermic). [0069] TGA: weight loss started at about 170 C., and the weight loss was obvious at 175 C. [0070] (4) Analysis: DSC showed that there was an endothermic peak at 174.68 C., which should be caused by the heat absorbed from the melting of the compound of Example 2. TGA showed that the thermogravimetric curve was basically unchanged before 160 C., and there was no weight loss, indicating that the compound of Example 2 did not contain crystallization solvent. When the temperature rose to the top of DSC endothermic peak (about 175 C.), the weight loss was obvious, which was the weight loss caused by the melting decomposition of the sample. The DSC endothermic peak was the decomposition point of the compound of Example 2.

4. Comparison of Stability Between the Compound of Example 1 and the Compound of Example 2

1) Stability of the Compound of Example 1

[0071] (1) Physical and chemical properties [0072] Appearance: off white powder [0073] Melting point: 119.36 C. (DSC method) (see FIG. 1) [0074] Purity: 99.5% (HPLC normalization method) [0075] log P=2.4 [0076] (2) Influencing factor test

TABLE-US-00009 Melting point Related (DSC method, Purity substances Appearance C.) (%) (%) Initial off white powder 119.36 99.5 0.5 60 C., 5 days off white powder 118.69 99.5 0.5 RH 92.5%, colorless viscous 99.5 0.5 5 days liquid Illumination, transparent bulk 96.0 4.0 5 days solid

[0077] The compound of Example 1 showed no obvious change in appearance, melting point and impurity content after being placed at a high temperature of 60 C. for 5 days, indicating that it was stable under high temperature conditions. After being placed at a high humidity of RH92.5% for 5 days, the compound showed serious moisture absorption, colorless viscous liquid in appearance, and no change in related substances. The compound showed transparent bulk in appearance under illumination conditions, and impurity content thereof increased to 4.0%, indicating that it was unstable under illumination conditions.

2) Stability of the Compound of Example 2

[0078] (1) Physical and chemical properties [0079] Appearance: white solid [0080] Melting point: 174.68 C. (DSC method) (see FIG. 2) [0081] Purity: 98.7% (HPLC normalization method) [0082] log P=3.2 [0083] (2) Influencing factor test

TABLE-US-00010 Melting point (DSC Chiral Related method, impurities Purity substances Appearance C.) (%) (%) (%) Initial white 174.68 0.2 98.7 1.3 powder 60 C., white 174.64 0.2 98.7 1.3 10 days powder RH 92.5%, white 174.57 0.2 98.7 1.3 10 days powder Illumination, white 174.60 0.2 98.6 1.4 10 days powder

[0084] The compound of Example 2 was stable under the conditions of illumination, high temperature and high humidity.

5. Comparison of Antitumor Effects of the Compound of Example 1 and the Compound of Example 2 on Mouse Melanoma B16F10 In Vivo

Purpose of the Experiment:

[0085] The in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors on mouse melanoma B16F10 were evaluated on the subcutaneous transplanted tumor model of mice.

Experimental solution:

[0086] Animal grouping: the experiment animals were divided into solvent control group, cyclophosphamide 80 mg/kg group (CTX), 5 mg/kg group and 10 mg/kg group of the compound of Example 1 and the compound of Example 2 respectively.

[0087] Experimental steps: the subcultured B16F10 tumor strain was ground with a homogenizer, washed twice with sterile normal saline and counted, and the cell concentration of tumor cell suspension was adjusted with normal saline to 910.sup.6/ml. 0.2 ml of the cell suspension was inoculated into the right armpit of C57BL/6J mice and the day was recorded as day 0. The next day after inoculation, the animals were randomly divided into groups, weighed and administered. The mice in the solvent control group were administrated with 0.5% CMC orally every day. The cyclophosphamide was administrated by intraperitoneal administration. The compounds to be tested were administered orally once a day. The animals were weighed and killed during treatment. The tumor tissues were stripped, weighed and photographed. Finally, the tumor inhibition rates were calculated to evaluate the intensity of antitumor effect.

[0088] Calculation and statistical method: tumor proliferation inhibition rate TGI(%): TGI=(1T/C)100. (T: tumor weight in the treatment group; C: tumor weight in the negative control group).

[0089] Statistical method: Graphpad was used for data statistical analysis, and One-way ANOVA test was used,* P<0.05, * * P<0.01, P<0.001.

Experimental Results

[0090] After administration, the animals were executed and the tumors were weighed. The antitumor effects of the compound of Example 1 and the compound of Example 2 on mouse melanoma B16F10 were shown in the table below.

TABLE-US-00011 Dosage Number Body weight (g) X SD Tumor weight (g) Grouping (mg/kg) (Start/End) Start End X SD TGI % Solvent 10/10 17.32 0.46 21.0 0.7 2.32 0.85 NA control group CTX group 80 1 10/10 16.94 0.43 19.2 0.8 0.23 0.18*** 90 Example 2 5 14 10/10 17.09 0.81 20.0 1.2 1.39 0.84* 40 compound 10 14 10/10 17.15 0.50 20.1 1.0 1.04 0.66** 55 group Note: compared with the solvent control group, *P < 0.05, **P < 0.01, ***P < 0.001, One way ANOVA NA: not applicable
In vivo antitumor effect of the compound of Example 2 on mouse B16F10

TABLE-US-00012 Dosage Number Body weight (g) X SD Tumor weight (g) Grouping (mg/kg) (Start/End) Start End X SD TGI % Solvent 7/7 17.1 0.9 18.8 1.1 3.49 0.91 NA control group CTX group 80 1 7/7 16.9 0.8 17.5 0.9 1.00 0.34*** 70 Example 1 5 14 7/7 17.7 1.1 20.1 1.5 3.11 0.98* 11 compound 10 14 7/7 18.8 1.4 21.0 1.8 3.04 0.94** 13 group Note: compared with the solvent control group, *P < 0.05, **P < 0.01, ***P < 0.001, One way ANOVA NA: not applicable

6. Comparison of Antitumor Effects of the Compound of Example 1 and the Compound of Example 2 on Mouse Colon Cancer MC38 In Vivo

Purpose of the Experiment:

[0091] The in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors on mouse colon cancer MC38 were evaluated on the subcutaneous transplanted tumor model of mice.

Experimental Solution:

[0092] Animal grouping: the experiment animals were divided into solvent control group, cyclophosphamide 80 mg/kg group (CTX), 5 mg/kg group and 10 mg/kg group of the compound of Example 1 and the compound of Example 2 respectively.

[0093] Experimental steps: the subcultured MC38 tumor strain was ground with a homogenizer, washed twice with sterile normal saline and counted, and the cell concentration of tumor cell suspension was adjusted with normal saline to 910.sup.6/ml, 0.2 ml of the cell suspension was inoculated into the right armpit of C57BL/6J mice and the day was recorded as day 0. The next day after inoculation, the animals were randomly divided into groups, weighed and administered. The mice in the solvent control group were administrated with 0.5% CMC orally every day. The cyclophosphamide was administrated by intraperitoneal administration. The compounds to be tested were administered orally once a day. The animals were weighed and killed during treatment. The tumor tissues were stripped, weighed and photographed. Finally, the tumor inhibition rates were calculated to evaluate the intensity of antitumor effect.

[0094] Calculation and statistical method: tumor proliferation inhibition rate TGI (%): TGI=(1-T/C)100. (T: tumor weight in the treatment group; C: tumor weight in the negative control group).

[0095] Statistical method: Graphpad was used for data statistical analysis, and One-way ANOVA test was used, *P<0.05, * *P<0.01, * * *P<0.001.

Experimental Results:

[0096] After administration, the animals were executed and the tumors were weighed. The antitumor effects of the compound of Example 1 and the compound of Example 2 on mouse colon cancer MC38 were shown in the table below.

Antitumor Effect of the Compound of Example 2 on Mouse Colon Cancer MC38

TABLE-US-00013 Dosage Number Body weight (g) X SD Tumor weight (g) Grouping (mg/kg) (Start/End) Start End X SD TGI % Solvent 10/10 22.0 0.4 26.1 1.3 1.70 0.75 NA control group CTX group 40 2 10/10 22.0 0.8 24.5 0.9 0.17 0.10** 90 Example 2 2.5 14 10/10 22.0 0.6 25.3 2.3 1.13 0.78 34 compound 5 14 10/10 21.9 0.8 23.7 1.8 0.42 0.39*** 75 group 10 14 10/10 21.9 0.7 25.0 1.7 0.73 0.54** 57 Note: compared with the solvent control group, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA NA: not applicable

Antitumor Effect of Compound of Example 1 on Mouse Colon Cancer MC38

TABLE-US-00014 Dosage Number Body weight (g) X SD Tumor weight (g) Grouping (mg/kg) (Start/End) Start End X SD TGI % Solvent 7/7 18.6 0.3 24.1 0.8 3.94 0.78 NA control group CTX group 40 1 6/6 18.8 0.9 21.7 1.4 1.30 1.44** 67 Example 1 5 14 5/6 18.7 0.4 23.4 1.2 3.21 0.28*** 18 compound 10 14 5/6 18.5 0.6 23.8 1.4 2.86 1.36** 27 group Note: compared with the solvent control group, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA NA: not applicable

7. Comparison of Antitumor Effects of the Compound of Example 1 and the Compound of Example 2 on Human Lung Cancer NCI-H460 Model

Purpose of the Experiment:

[0097] The in vivo antitumor effects of the compound of Example 1 and the compound of Example 2 as PD-L1 inhibitors were evaluated on the NCI-H460 model of human lung cancer in NSG tumor-bearing mice reconstructed by human immune system.

Experimental Solution:

[0098] Animal grouping: the experiment animals were divided into solvent control group, cyclophosphamide 80 mg/kg group (CTX), 5 mg/kg group and 10 mg/kg group of the compound of Example 1 and the compound of Example 2 respectively.

[0099] Experimental steps: PBMCs were obtained by isolation of fresh human leukocytes, then inoculated into NSG mice through caudal vein, and each mouse was inoculated with 110.sup.7. On the third day, NCI-H460 tumor cells were inoculated into the armpit of mice, and each mouse was inoculated with 110.sup.6. After the tumors grew to 100300 mm.sup.3, the mice were administered in groups. The mice in the solvent control group were administrated with 0.5% CMC orally every day. The cyclophosphamide was administrated by intraperitoneal administration. The compounds to be tested were administered orally once a day. The animals were weighed and killed during treatment. The tumor tissues were stripped, weighed and photographed. Finally, the tumor inhibition rates were calculated to evaluate the intensity of antitumor effect.

[0100] Calculation and statistical method: tumor proliferation inhibition rate TGI (%): TGI=(1T/C)100. (T: tumor weight in the treatment group; C: tumor weight in the negative control group).

[0101] Statistical method: Graphpad was used for data statistical analysis, and One-way ANOVA test was used, *P<0.05, * *P<0.01, * * *P<0.001.

Experimental Results:

[0102] After administration, the animals were executed and the tumors were weighed. The antitumor effects of the compound of Example 1 and the compound of Example 2 on NCI-H460 were shown in the table below.

Antitumor Effect of the Compound of Example 1 and the Compound of Example 2 on NCI-H460

TABLE-US-00015 Dosage Number Tumor volume X SD Tumor weight (g) Grouping (mg/kg) (Start/End) Day 1 Day 20 X SD TGI % Solvent 5/5 180.4 13.0 2129.9 362.7 2.73 0.60 NA control group CTX group 100 1 5/5 165.4 22.4 556.5 81.1 0.90 0.15*** 66.9 Example 2 15 14 compound 5/5 166.5 24.5 1405.2 442.9 1.90 0.0.37 30.6 group Example 1 15 14 compound 5/5 161.9 21.0 1441.4 337.6 2.06 0.22 24.7 group Note: compared with the solvent control group, *p < 0.05, **p < 0.01, ***p < 0.001, One-way ANOVA NA: not applicable

[0103] To sum up, the experimental results showed that:

[0104] In the subcutaneous transplanted tumor model of mouse melanoma highly metastatic strain B16F10, the tumor inhibition rates of the compound of Example 2 at the daily oral dosages of 5 mg/kg and 10 mg/kg were 40% and 55% respectively, while the tumor inhibition rates of the compound of Example 1 at the same dosages were 11% and 13% respectively.

[0105] In the subcutaneous transplanted tumor model of mouse colon cancer MC38, the tumor inhibition rates of the compound of Example 2 at the daily oral dosages of 5 mg/kg and 10 mg/kg were 75% and 57% respectively, while the tumor inhibition rates of the compound of Example 1 at the same dosages were 18% and 27% respectively.

[0106] On NSG tumor-bearing mice (NCI-H460) with reconstructed human immune system, the tumor inhibition rate of the compound of Example 2 at the daily oral dosage of 15 mg/kg was better than that of the compound of Example 1 (tumor inhibition rate: 30.6% vs 24.7%).

TABLE-US-00016 Tumor model Comparison of tumor inhibition rate at the same dosage B16F10 Compound of the Example 2 (maleate) > Compound of the Example 1 (hydrochloride) MC38 Compound of the Example 2 (maleate) > Compound of the Example 1 (hydrochloride) NSG Compound of the Example 2 (maleate) > Compound NCI-H460 of the Example 1 (hydrochloride)