SALT FORM AND CRYSTAL FORM OF A2A RECEPTOR ANTAGONIST AND PREPARATION METHOD THEREFOR

Abstract

Provided are a salt form and a crystal form of adenosine A.sub.2A receptor antagonist, and preparation method therefor. Also provided is an application of the salt form or crystal form in the preparation of a medicine for A.sub.2A receptor-related diseases, the maleate salt in the salt form has a structure of formula (I).

##STR00001##

Claims

1. A compound of formula (I): ##STR00014##

2. A crystal form A of the compound of formula (I) according to claim 1, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 7.16±0.2°, 9.66±0.2°, 19.66±0.2°.

3. The crystal form A of the compound of formula (I) according to claim 2, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 7.16±0.2°, 9.66±0.2°, 13.59±0.2°, 14.30±0.2°, 15.87±0.2°, 17.73±0.2°, 19.66±0.2°, 20.88±0.2%; or, wherein the differential scanning calorimetry curve thereof has an onset of an endothermic peak at 205.67±3° C.; or, wherein the thermogravimetric analysis curve thereof has a weight loss of 0.1846% at 120.00° C.±3° C.

4. The crystal form A of the compound of formula (I) according to claim 3, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 7.16±0.2°, 9.66±0.2°, 13.59±0.2°, 14.30±0.2°, 15.87±0.2°, 17.73±0.2°, 19.66±0.2°, 20.88±0.2°, 26.01±0.2°, 26.76±0.2°, 27.21±0.2°.

5. The crystal form A of the compound of formula (I) according to claim 4, wherein the XRPD pattern thereof is shown in FIG. 1; or, the DSC pattern thereof is shown in FIG. 2; or, the TGA pattern thereof is shown in FIG. 3.

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. A method for treating A.sub.2A receptor-related diseases in a subject in need thereof, comprising administering an effective amount of the compound according to claim 1 to the subject.

11. A crystal form B of a compound of formula (II), wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 12.84±0.2°, 15.84±0.2°, 23.55±0.2°; ##STR00015## n is selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 and 2.0.

12. The crystal form B of the compound of formula (II) according to claim 11, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.43±0.2°, 11.22±0.2°, 12.84±0.2°, 15.84±0.2°, 19.78±0.2°, 21.61±0.2°, 23.55±0.2°, 27.02±0.2°; or, the differential scanning calorimetry curve thereof has an onset of an endothermic peak at 256.37±3° C.

13. The crystal form B of the compound of formula (II) according to claim 12, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ angles: 6.43±0.2°, 11.22±0.2°, 12.41±0.2°, 12.84±0.2°, 15.84±0.2°, 16.36±0.2°, 19.27±0.2°, 19.78±0.2°, 21.61±0.2°, 23.55±0.2°, 27.02±0.2°, 28.62±0.2°.

14. The crystal form B of the compound of formula (II) according to claim 13, wherein the XRPD pattern thereof is shown in FIG. 4; or, the DSC pattern thereof is shown in FIG. 5.

15. (canceled)

16. (canceled)

17. The crystal form B of the compound of formula (II) according to claim 11, wherein the thermogravimetric analysis curve thereof has a weight loss of 0.8860% at 159.53° C.±3° C.

18. The crystal form B of the compound of formula (II) according to claim 17, wherein the TGA pattern thereof is shown in FIG. 6.

19. A crystal form C of a compound of formula (III) or a crystal form D of a compound of formula (IV), wherein the X-ray powder diffraction pattern of a crystal form C of a compound of formula (III) has characteristic diffraction peaks at the following 2θ angles: 8.89±0.2°, 14.10±0.2°, 15.56±0.2°, 15.99±0.2°, 16.48±0.2°, 17.49±0.2°, 20.23±0.2°, 21.63±0.2°, 23.47±0.2°, 24.51±0.2°, 26.33±0.2°, 28.18±0.2°; the X-ray powder diffraction pattern of a crystal form D of a compound of formula (IV) has characteristic diffraction peaks at the following 2θ angles: 4.433±0.2°, 12.38±0.2°, 13.31±0.2°, 16.66±0.2°, 17.08±0.2°, 17.75±0.2°, 18.65±0.2°, 22.58±0.2°, 23.63±0.2°, 24.95±0.2°, 25.40±0.2°, 26.80±0.2°, 27.65±0.2°; ##STR00016##

20. The crystal form C of the compound of formula (III) according to claim 19, wherein the XRPD pattern thereof is shown in FIG. 7.

21. (canceled)

22. The crystal form D of the compound of formula (IV) according to claim 19, wherein the XRPD pattern thereof is shown in FIG. 10.

23. A method for treating A.sub.2A receptor-related diseases in a subject in need thereof, comprising administering an effective amount of the crystal form B of the compound of formula (II) according to claim 11 to the subject.

24. A method for treating A.sub.2A receptor-related diseases in a subject in need thereof, comprising administering an effective amount of the crystal form A according to claim 2 to the subject.

25. A method for treating A.sub.2A receptor-related diseases in a subject in need thereof, comprising administering an effective amount of the crystal form C of the compound of formula (III) according to claim 19 to the subject.

26. A method for treating A.sub.2A receptor-related diseases in a subject in need thereof, comprising administering an effective amount of the crystal form D of the compound of formula (IV) according to claim 19 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0098] FIG. 1 is an XRPD pattern of the crystal form A of the compound of formula (I) measured by Cu-Kα radiation;

[0099] FIG. 2 is a DSC pattern of the crystal form A of the compound of formula (I);

[0100] FIG. 3 is a TGA pattern of the crystal form A of the compound of formula (I);

[0101] FIG. 4 is an XRPD pattern of the crystal form B of the compound of formula (II) measured by Cu-Kα radiation;

[0102] FIG. 5 is a DSC pattern of the crystal form B of the compound of formula (II);

[0103] FIG. 6 is a TGA pattern of the crystal form B of the compound of formula (II);

[0104] FIG. 7 is an XRPD pattern of the crystal form C of the compound of formula (III) measured by Cu-Kα radiation;

[0105] FIG. 8 is a DSC pattern of the crystal form C of the compound of formula (III);

[0106] FIG. 9 is a TGA pattern of the crystal form C of the compound of formula (III);

[0107] FIG. 10 is an XRPD pattern of the crystal form D of the compound of formula (IV) measured by Cu-Kα radiation;

[0108] FIG. 11 is a DSC pattern of the crystal form D of the compound of formula (IV);

[0109] FIG. 12 is a TGA pattern of the crystal form D of the compound of formula (IV).

[0110] FIG. 13 is an XRPD pattern of the crystal form E of the compound of formula (V) measured by Cu-Kα radiation;

[0111] FIG. 14 is a DSC pattern of the crystal form E of the compound of formula (V);

[0112] FIG. 15 is a TGA pattern of the crystal form E of the compound of formula (V).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0113] In order to better understand the content of the present disclosure, a further description will be given below in conjunction with specific embodiments, but the specific embodiments are not a limitation on the content of the present disclosure.

Embodiment 1: Preparation of Compound D

[0114] ##STR00007##

Step 1

[0115] To 50 L of kettle R1 were added 20 L of isopropanol, the material A (10 Kg. 1.0 eq) and N, N-dimethylformamide dimethyl acetal (8.53 Kg, 1.2 eq), The reaction solution was heated to 70-80° C. and stirred for 3 h. A sample were taken for detection, and the material A was disappeared. The reaction mixture was cooled to 70° C. and hydroxylamine hydrochloride (4.8 Kg, 1.2 eq) was added in batches while the internal temperature was controlled ≤80° C. After the addition was completed, the internal temperature was kept at 70-80° C. and stirred for 1 h. The sample was taken for detection and the reaction was completed. Heating was stopped, the reaction solution was cooled to room temperature and filtered. The filter cake was washed with 2 L of isopropanol and dried under reduced pressure and vacuum to obtain compound B.

[0116] LCMS: m/z: 217.7 [M+1].sup.+;

[0117] .sup.1H NMR (400 MHz, MeOD-d.sub.4): δ8.24 (d, J=2.4 Hz, 1H), 7.90 (s, 2H), 7.76 (dd, J=8.8, 2.4 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H).

Step 2

[0118] To 50 L of kettle R1 were added 22.4 L of isopropyl acetate and the compound B (4.48 Kg, 1.0 eq). The reaction system was cooled to 0-10° C. and trifluoroacetic anhydride (6.2 Kg, 1.5 eq) was added dropwise while the internal temperature was controlled at 0-10° C. After the addition was completed, refrigeration was turned off, the reaction solution was slowly warmed to room temperature and stirred for 16 h. A sample was taken for detection and the reaction was completed. 2.44 kg of sodium hydroxide was weighed and mixed with 30 L of ice evenly. The reaction solution was poured into the above solution in batches. The mixture was stirred for 0.2 h and filtered. The filter cake was washed with 1 L of isopropyl acetate. The filtrate was separated and the aqueous phase was extracted twice with 5 L of isopropyl acetate each time. The organic phases were combined and washed with 10 L of water. The organic phase was separated and concentrated to obtain the crude product. The crude product and the filter cake were combined, and the combined solid was slurried at 16-22° C. for 2 h. The suspension was filtered, and the filter cake was washed with 2 L of the above mixed solvents. The solid was collected and dried under reduced pressure and vacuum to obtain compound C.

[0119] LCMS: m/z: 199.7 [M+1].sup.+;

[0120] .sup.1H NMR (400 MHz, deuterated chloroform): δ8.77 (s, 1H), 8.34 (s, 1H), 7.70-7.60 (m, 2H).

Step 3

[0121] To 50 L of kettle R1 were added 30 L of dioxane, compound C (3.0 Kg, 1.0 eq), bis(pinacolato)diboron (3.85 Kg, 1.0 eq), potassium acetate (2.3 Kg, 1.5 eq) and Pd(dppf)Cl.sub.2 (537 g, 0.05 eq). The kettle R1 was evacuated and purged with nitrogen four times. The temperature was controlled at 85-105° C. and the mixture was stirred for 18 h. A sample was Taken for detection and the mixture was cooled to room temperature, filtered through diatomite and the filter cake was washed with dioxane. The combined organic phase was concentrated under reduced pressure to obtain the residue. 2.5 L of isopropyl acetate was added to R1, the residue was transferred to kettle R1, diatomite was added thereto, then n-heptane was added thereto, and the mixture was heated to 100° C. and stirred for 16 h. The mixture was cooled to room temperature, filtered through diatomite. The filter cake was washed with n-heptane, and the combined organic phase was spin-dried to obtain the crude product. The crude product was slurried with n-heptane at room temperature for 16 h. The suspension was filtered and the filter cake was washed with n-heptane. The filter cake was dried to obtain compound D.

[0122] LCMS: m/z: 246.2[M+1].sup.+;

[0123] .sup.1H NMR (400 MHz, deuterated chloroform): δ 8.90 (s, 1H), 8.29 (s, 1H), 7.76-7.66 (m, 2H), 1.30 (s, 12H).

Embodiment 2: Preparation of the Compound of Formula (I)

[0124] ##STR00008## ##STR00009##

Step 1

[0125] To 50 L of kettle was added 24 L of DMF and stirring was started. Compound E (5.0 Kg, 1.0 eq), compound F (4304.14 g, 1.0 eq), cuprous iodide (66.88 g, 0.01 eq) and triethylamine (5330.00 g, 1.5 eq) were added to the kettle, and then the mouth of the kettle was washed with 0.95 kg DMF. Pd(PPh.sub.3).sub.2Cl.sub.2(123.24 g, 0.005 eq) was added to the kettle without replacing nitrogen. The internal temperature was kept below 50° C., and the mixture was stirred for 16 h. A sample was taken for detection and the reaction mixture was directly filtered. Every ⅕ of the filtrate was slowly added to 25 L of water and kept stirring for 0.5 h. The operation was repeated five times to process all the filtrate. The mixed solution was filtered to obtain 9.82 kg of crude product. The crude product was slurried with 14 L of ethanol at room temperature and then the slurry was filtered. The filter cake was dried under reduced pressure in a vacuum drying oven to obtain compound G.

[0126] LCMS: m/z: 223.6 [M+1].sup.+;

[0127] .sup.1H NMR (400 MHz, deuterated chloroform): δ 8.76 (d, J=1.6 Hz, 1H), 8.60 (d, J=2.4 Hz, 1H), 7.73-7.65 (m, 2H), 7.13 (t, J=8.4 Hz, 2H).

Step 2

[0128] To 50 L of kettle was added 23 L of DMF and stirring was started. Compound G (7700.00 g, 1.0 eq), ammonium acetate (4945.45 g, 2.0 eq) and copper trifluoromethanesulfonate (232.07 g, 0.02 eq) were added, and then the mouth of the kettle was washed with 1 L of DMF. Then heating was started and the internal temperature was kept at 100° C., and the mixture was stirred for 16 h. A sample was taken for detection and the reaction solution was directly filtered. The solid was collected as a crude product with a crude product of 6.98 kg. The crude product was slurried at room temperature with 14 L of water and then the slurry was filtered. Then the crude product was slurried with 14 L of ethanol at reflux, and then the slurry was filtered. The filter cake was dried under reduced pressure in a vacuum drying oven to obtain compound H.

[0129] LCMS: m/z: 241.0 [M+1].sup.+;

[0130] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.44 (d, J=2.0 Hz, 1H), 9.19 (d, J=2.0 Hz, 1H), 8.73 (dd, J=8.8, 3.2 Hz, 2H), 8.06 (s, 1H), 7.71 (t, J=8.8 Hz, 2H), 7.29 (br, 2H).

Step 3

[0131] To 50 L of kettle was added 10.6 L of DMF and stirring was started. Compound H (5.30 kg, 1.0 eq) and N-Bromosuccinimide (4.66 kg, 1.2 eq) were added in batches while the internal temperature was kept <50° C. during addition. Then heating was started and the internal temperature was kept at 50° C., and the mixture was stirred for 2 h. A sample was taken for detection and 20 L of EtOAc was added to the reaction solution. The internal temperature was kept at 50° C., and the mixture was stirred for 0.5 h. The reaction mixture was cooled to room temperature and then filtered. The crude product was slurried with 16 L of THF at reflux. The slurry was filtered, and the filter cake was dried under reduced pressure in a vacuum drying oven to obtain compound J.

[0132] LCMS: m/z: 320.9 [M+1].sup.+;

[0133] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.16 (d, J=1.6 Hz, 1H), 8.89 (d, J=2.0 Hz, 1H), 7.72 (dd, J=8.8, 6.0 Hz, 2H), 7.64 (br, 2H), 7.32 (t, J=8.8 Hz, 2H).

Step 4

[0134] To 50 L of kettle A were added 22 L of THF, compound J (4.4 kg, 1.0 eq), P.sub.3KO.sub.4 (7991.97 g, 3.0 eq), compound D (6.15 kg, 1.38 eq), Pd(dtbpf).sub.2Cl.sub.2 (408.97 g, 0.05 eq) and 4.4 L of H.sub.2O. Stirring was started and the kettle was evacuated and purged with nitrogen three times. The temperature was controlled at 70° C. and stirred for 16 h. A sample was taken for detection and the reaction solution was concentrated under reduced pressure. 33 L of DCM and 10 L of EtOH were added to the kettle. Then the mixture was stirred at 30° C. for 0.5 h, the concentrate was added with 4.4 L of EtOAc, the temperature was controlled at 60° C., and the mixture was stirred for 1 h, cooled to room temperature and filtered. The filter cake was diluted with 8.8 L of water and then stirred at 25° C. for 1 h. The suspension was filtered and the filter cake was diluted with 4.4 L of DMF, the temperature was controlled at 50° C., and the mixture was stirred for 0.5 h, 22 L of ethyl acetate was added, then the temperature was controlled at 50° C., and the mixture was stirred for 1 h. Then the mixture was cooled to room temperature, filtered, the filter cake was diluted with 36.7 L of DCM and 9.7 L of EtOH. The mixture was warmed to 40° C. and 1,3,5-triazine-2,4,6-trimercaptan trisodium (0.88 kg) was added. The temperature was controlled at 40° C. and the mixture was stirred for 15 h and then filtered. The operation was repeated three times, filtered and the filtrate was spin-dried to obtain compound L.

[0135] LCMS: m/z: 358.1 [M+1]+.

Step 5

[0136] To 50 L kettle A were added 24.2 L of THF, then compound L (490 g, 1.0 eq) was added to kettle C. The temperature was controlled at 50° C. and the mixture was stirred for 1 h and then filtered. To 50 L kettle A was added the filtrate, and then a solution of maleic acid (165.98 g, 1.05 eq) dissolved in 29 L of THF was added to kettle C, the temperature was controlled at 50° C. and the mixture was stirred for 14 h and then cooled to room temperature 20-25° C. and directly filtered. The filter cake was slurried with 1.32 L of ethanol at room temperature for 16 h. The slurry was filtered and the filter cake was dried under reduced pressure in a vacuum drying oven to obtain the crystal form A of the compound of formula (I).

[0137] LCMS: m/z: 358.1 [M+1].sup.+;

[0138] .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 9.02 (d, J=2.0 Hz, 1H), 8.88 (d, J=2.0 Hz, 1H), 8.79 (d, J=1.6 Hz, 1H), 8.48 (s, 1H), 7.77 (d, J=0.8 Hz, 1H), 7.75 (br, 2H), 7.47-7.43 (m, 3H), 7.14 (t, J=4.6 Hz, 2H), 6.27 (t, J=10.8 Hz, 2H).

Embodiment 3

[0139] According to Table 7, the crystal form A of the compound of formula (I) was weighed into liquid phase vials, and then solvents or mixed solvents in Table 7 were added to form a suspension, respectively. The above suspension samples were placed on a constant temperature mixer at 40° C. and shaken at 700 rpm for 2 days. Then the solid was separated by centrifugation (centrifuge 8000 rpm, for three minutes) and dried overnight in a vacuum drying oven at 30° C. to obtain the crystal form A of the compound of formula (I).

TABLE-US-00007 TABLE 7 Solution suspension crystallization method for screening polycrystalline forms of maleates Amount Volume (mg) (mL) Number Solvent added added 1 methanol 47.978 1.0 2 Ethanol 48.198 1.0 3 Acetonitrile 49.372 1.0 4 Acetone 48.064 1.0 5 Ethyl acetate 47.560 1.0 6 Tert-butyl methyl ether 51.680 1.0 7 Tetrahydrofuran 52.992 1.0 8 Water 48.718 1.0 9 Methanol-water (1:1) 49.108 1.0 10 Acetonitrile-water (1:1) 52.343 1.0

Embodiment 4: Preparation of the Compound of Formula (II-1)

[0140] ##STR00010##

[0141] At 25° C., to the reaction bottle were added 200 mL of acetonitrile, 200 mL of water and compound L (6 g), the mixture was adjusted to pH=3-5 with 1M dilute HCl. And then the reaction solution was stirred at 25° C. for 0.5 h to obtain the compound of formula (II-1).

[0142] LCMS m/z: 358.2 [M+H].sup.+

[0143] .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ=9.24 (t, J=3.6 Hz, 1H), 9.12 (d, J=2.0 Hz, 1H), 8.87 (s, 1H), 8.59 (t, J=5.2 Hz, 1H), 7.84 (t, J=8.4 Hz, 1H), 7.58-7.55 (m, 2H), 7.50 (d, J=15.6 Hz, 1H), 7.27 (t, J=9.2 Hz, 2H).

Embodiment 5: Preparation of the Crystal Form B of the Compound of Formula (II)

[0144] About 100 mg of compound L was weighed at room temperature and added to a 40 mL of flask, and 30 mL of acetone was added to dissolve compound L, and then 24.17 μL of HCl (the molar ratio of compound L: hydrochloric acid was 1:1.05) was slowly added. The sample was placed on a magnetic stirrer (40° C.) and stirred for one day to obtain the crystal form B of the compound of formula (II).

Embodiment 6: Preparation of the Crystal Form C of the Compound of Formula (III)

[0145] ##STR00011##

[0146] About 100 mg of compound L was weighed at room temperature and added to a 40 mL of flask, and 30 mL of acetone was added to dissolve compound L, and then 19.08 μL of methanesulfonic acid (the molar ratio of compound L: methanesulfonic acid was 1:1.05) was slowly added. The sample was placed on a magnetic stirrer (40° C.) and stirred for one day to obtain the crystal form C of the compound of formula (III).

Embodiment 7: Preparation of the Crystal Form D of the Compound of Formula (IV)

[0147] ##STR00012##

[0148] About 100 mg of compound L was weighed at room temperature and added to a 40 mL of flask, and 30 mL of acetone was added to dissolve compound L, and then 200.57 μL of p-toluenesulfonic acid (the molar ratio of compound L: p-toluenesulfonic acid was 1:1.05) was slowly added. The sample was placed on a magnetic stirrer (40° C.) and stirred for one day to obtain the crystal form D of the compound of formula (IV).

Embodiment 8: Preparation of the Crystal Form E of the Compound of Formula (V)

[0149] ##STR00013##

[0150] About 100 mg of compound L was weighed at room temperature and added to a 40 mL of flask, and 30 mL of acetone was added to dissolve compound L, and then 15.99 μL of sulfuric acid (the molar ratio of compound L: sulfuric acid was 1:1.05) was slowly added. The sample was placed on a magnetic stirrer (40° C.) and stirred for one day to obtain the crystal form E of the compound of formula (V).

Embodiment 9: Stability Experiments of Crystal Forms

[0151] A certain amount (approximately 10 mg) of two parts of the crystal form A of the compound of formula (I), the crystal form B of the compound of formula (II) and the crystal form C of the compound of formula (III) were weighed in parallel, and added to a 40 mL of sample bottle, which were placed in a constant temperature and humidity box at 60° C./75% humidity for 1 week. Another sample (about 10 mg) of the crystal form A of the compound of formula (I), the crystal form B of the compound of formula (II) and the crystal form C of the compound of formula (III) were placed in a refrigerator at −20° C. as control samples. At the time point of investigation, the corresponding test sample was taken out of the stability box, the aluminum foil paper was removed from the sample bottle of 60° C./75% RH, and the sample bottle was covered with a bottle cap. The 0-day samples were taken out of the refrigerator and analyzed after the samples were restored to room temperature. The experimental results were shown in Table 8.

TABLE-US-00008 TABLE 8 Experimental results of stability of crystal forms Relative Total retention time 0.36 0.45 0.49 0.67 0.70 1.18 impurities % Crystal form 0 day 0.06 0.09 0.09 0.05 0.11 0.29 0.69 A of the 60 C./75% 0.05 0.09 0.09 0.05 0.12 0.29 0.69 compound of RH_one week formula (I) Crystal form 0 day 0.09 0.10 0.09 0.05 0.15 0.40 0.87 B of the 60 C./75% 0.09 0.10 0.09 0.05 0.15 0.40 0.87 compound of RH_one week formula (II) Crystal form 0 day 0.05 0.09 0.08 0.04 0.09 0.50 0.96 C of the 60 C./75% 0.05 0.09 0.08 0.04 0.09 0.52 1.16 compound of RH_one week formula (III)

[0152] Conclusion: The crystal form A of the compound of formula (I), the crystal form B of the compound of formula (II) and the crystal form C of the compound of formula (III) have good stability under high temperature and high humidity conditions.

Experimental Embodiment 1: In Vitro Activity Test Experiment of the Compound of the Present Disclosure

[0153] Test Experiment of Calcium Current of Human Adenosine A.sub.2A Receptor

[0154] Cell source:

[0155] The A.sub.2A stable cell line was constructed by Shanghai WuXi AppTec, the host cell was CHO.

[0156] Detection kit:

[0157] Fluo-4 Direct kit, (Invitrogen, catalog number F10471). After the fluorescence detection reagent in the kit (which specifically binds to calcium ions and causes an increase in fluorescence signal) was incubated with the cells for an appropriate period of time, the compound was added to stimulate the cells to cause changes in intracellular calcium flow, thereby causing changes in fluorescence signals, which can reflect the strength of the agonistic or inhibitory activity of the compound.

[0158] Cell culture medium:

[0159] F12+10% Fetal Bovine Serum+Geneticin 300 μg/mL+Blasticidin 2 μg/mL

[0160] Compound dilution buffer:

[0161] Hanks balanced salt buffer (Invitrogen)+20 mM HEPES, configure before each use

[0162] Agonist:

[0163] NECA (Sigma-E2387)

[0164] Reference compound (antagonist):

[0165] CGS-15943 (Sigma-C199)

[0166] Compound dilution:

[0167] The test compound was dissolved in DMSO to prepare a 10 mM stock solution. The test compound was diluted with DMSO to 0.2 mM, and the reference compound CGS-15943 was diluted with DMSO to 0.015 mM. Then 10-point 3-fold serial dilution was performed with ECHO. Then the dilution was transferred 900 nL to the compound plate (Greiner-781280), and 30 μL of compound dilution buffer was added thereto. The final initial concentration of the test compound was 1 μM, and the CGS-15943 was 0.075 μM.

[0168] Test methods:

[0169] Cell preparation:

[0170] The cryopreserved A.sub.2A cells were resuscitated and were resuspend in culture medium to 1×10.sup.6 cells/mL, and inoculated 384-well polylysine-coated cell plate (Greiner-781946) with 20 μL/well, and incubated overnight at 37° C. in a 5% CO.sub.2 incubator.

[0171] The cell plate prepared the day before was taken out from the incubator, 20 μL of 2×Fluo-4Direct™ buffer was added to each well, and incubated for 50 minutes at 37° C. in a 5% CO.sub.2 incubator, and placed at room temperature for 10 minutes.

[0172] EC.sub.80 determination of agonist NECA:

[0173] Dilution of agonist NECA: 10-point 3-fold serial dilution of NECA with an initial concentration of 0.15 mM was performed with ECHO, then the dilution was transferred 900 nL to the corresponding compound plate; then 30 μL of compound dilution buffer was added to the corresponding compound plate. The final initial concentration was 750 nM. The FLIPR instrument software was run, according to the set procedure, 10 μL of compound dilution buffer was added to the cell plate, and the fluorescence signal was read. Then 10 μL of the agonist reference compound of a predetermined concentration was added to the cell plate, and the fluorescence signal was read. After reading, the data was exported through the “Max-Min” and “Read 90 to Maximum allowed” methods in the software, the EC80 of the A.sub.2A cell line was calculated, and an agonist with a concentration of 6× EC.sub.K) was prepared. The reference compound agonist with the corresponding cell concentration of 6× EC.sub.K) with buffer salt solution was prepared, and was added 30 μL/well to the corresponding compound plate for later use.

[0174] IC.sub.50 determination of the test compound:

[0175] The FLIPR instrument software was run, according to the set procedure, 10 μL of the test compound and reference compound of a predetermined concentration were added to the cell plate, and the fluorescence signal was read. Then 10 μL of the reference compound agonist at a concentration of 6×EC.sub.80 was added to the cell plate, and the fluorescence signal was read. For agonist detection of compounds, the data was exported through the “Max-Min” and “Read 1 to 90” methods in the software. For antagonist detection of compounds, the data was exported through the “Max-Min” and “Read 90 to Maximum allowed” methods in the software. The data was analyzed with GraphPad Prism 5.0 to calculate the IC.sub.50 value of the test compounds. The test results were shown in Table 9.

TABLE-US-00009 TABLE 9 Results of in vitro screening experiments for the compound of the present disclosure Test sample IC.sub.50 vaue (nM) Compound of formula (II-1) 1.14

[0176] Conclusion: The compound of the present disclosure exhibits excellent adenosine A.sub.2A receptor antagonistic activity.

Experimental Embodiment 2: Pharmacokinetic Experiment of the Compound of the Present Disclosure

[0177] Experimental materials: Balb/c mice (female, 15-30 g, 7-9 weeks old, Shanghai Lingchang) were used.

[0178] Experimental method: The rodent pharmacokinetic characteristics of the compound after intravenous injection and oral administration were tested by a standard protocol. In the experiment, the candidate compound was formulated into a clear solution and given to mice by a single intravenous injection and a single oral administration. The solvent for intravenous injection (IV) was a mixed solvent of 5% DMSO/5% polyethylene glycol hydroxystearate/90% water, and the solvent for oral (PO) was a mixed solvents of 1% tween80, 9% PEG400, 90% H.sub.2O (pH=3). Whole blood samples within 48 hours were collected, centrifuged at 3000 g for 15 minutes at 4 degrees, separated the supernatant to obtain a plasma sample. Then 20 times the volume of acetonitrile solution containing internal standard was added to precipitate the protein, the mixture was centrifuged to take the supernatant, equal volume of water was added and centrifuge to take the supernatant again for injection. The blood drug concentration was quantitatively analyzed by LC-MS/MS analysis method, and the pharmacokinetic parameters were calculated, such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, bioavailability, etc. The test results were shown in Table 10.

TABLE-US-00010 TABLE 10 PK parameters in plasma of embodiment compounds Concentration Clearance rate Half life integral AUC Bioavailability Test sample (mL/min/kg) T.sub.1/2 (h) (nM .Math. hr) F (%) Compound of 5.87 0.82 24050 30.4 formula (II-1)

[0179] Conclusion: The compound of the present disclosure can significantly improve the pharmacokinetic index of mice.

Experimental Embodiment 3: In Vivo Drug Efficacy Experiment of the Compound of the Present Disclosure

[0180] Experimental materials: BALB/c mice (female); mouse colon cancer CT26 cells (cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences), were cultured in a monolayer in vitro, and the culture condition was RPMI-1640 medium containing 10% fetal bovine serum, and cultured in 5% CO.sub.2 incubator at 37° C. Trypsin-EDTA was used for conventional digestion and passage. When the cells were in the exponential growth phase and the saturation was 80%-90%, the cells were collected and counted.

[0181] Compound preparation: The compound of formula (II-1) was weighed and added to a solvent (10% PEG400+90% (10% Cremophor aqueous solution)) to prepare samples of 2.5 mg/mL, 5 mg/mL, and 10 mg/mL, respectively. 72 μL of CS1003 (PD-1 antibody) solution (25 mg/mL) was taken and added with 1.728 mL of Dulbecco's Phosphate Buffer Saline (DPBS) to prepare a solution of 1 mg/mL, then added with 16.2 ml DPBS to prepare a clear solution of 0.1 mg/mL.

[0182] Experimental operation: The cells in Dulbecco's phosphate buffer were resuspended at a density of 3×10.sup.6 cells/mL. 0.1 mL of DPBS (containing 3×10.sup.5 CT26 cells) was subcutaneously inoculated on the right back of each mouse. On the day of inoculation, the mice were randomly divided into groups according to their body weight with 9 mice in each group, and the administration was started for 20 days. During the entire experiment, the animals were weighed and monitored daily. If there were special circumstances, the relevant project leader should be notified in time and corresponding records should be made. The tumor diameter was measured with vernier calipers twice a week. The dosage regimen was shown in Table 11. The calculation formula of tumor volume was: V=0.5×a× b.sup.2, where a and b represent the long diameter and short diameter of the tumor, respectively.

TABLE-US-00011 TABLE 11 Dosage regimen for in vivo drug efficacy experiments of the compounds of the present disclosure Administration Administration Dosage volume Dosage Group Test sample route (mg/kg) (mL/kg) regimen 1 Solvent control Gavage — 10 Once a day administration 2 C51003 Intraperitoneal 1 10 Day injection 7.10, 13, 16 3 Compound of Gavage 100 10 Once a day formula (II-1) administration 4 Compound of Intraperitoneal 25 Compound of 10 + 10 Once a day + formula (II-1) + injection + formula (II-1) + 1 Day7.10, 13, (C51003) Gavage (C51003) 16 administration 5 Compound of Intraperitoneal 50 Compound of 10 + 10 Once a day + formula (II-1) + injection + formula (II-1) + Day7.10, 13, (CS1003) Gavage 1 (CS1003) 16 administration 6 Compound of Intraperitoneal 100 Compound 10 + 10 Once a day + formula (II-1) + injection + of formula (II-1) + Day7.10, 13, (CS1003) Gavage 1 (CS1003) 16 administration 7 Compound of Gavage 50 10 Twice a day formula (II-1) administration 8 Compound of Intraperitoneal 50 Compound of 10 + 10 Twice a day + formula (II-1) + injection + formula (II-1) + Day7.10, 13, (CS1003) Gavage 1 (CS1003) 16 administration

[0183] The anti-tumor efficacy of the compound was evaluated by GI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%)=VtNc×100% (Vt: average tumor volume in the treatment group; Vc: average tumor volume in the negative control group). Vt and Vc are taken from the same day data.

[0184] GI (%), tumor inhibition rate. GI(%)=1−VtNc×100%.

[0185] The statistical analysis was based on the relative tumor volume and tumor weight at the end of the experiment using SPSS software. The comparison between the two groups was analyzed by t-test, and the comparison between three or more groups was analyzed by one-way ANOVA. If the variance was uniform (F values were not significantly different), the analysis should be performed by Tukey's method. If the variance was not uniform (F Values were significantly different), Games-Howell method was used to test. P<0.05 was considered a significant difference.

[0186] On the 20th day after the start of administration, the tumor volume in the solvent group reached 847.09±79.65 mm.sup.3, and the tumor volume in the CS1003 (1 mg/kg) group was 487.34±109.07 mm.sup.3, and its tumor inhibition rate was 42.47% (no significant difference with the solvent control group). Compared with the solvent control group, each group of combination administration could significantly inhibit the growth of transplanted tumors in vivo, and the efficacy of the compound of formula (II-1) combined with CS1003 was positively correlated with its dosage and frequency of administration. The tumor volumes of 25 mg/kg, 50 mg/kg and 100 mg/kg compound of formula (II-1) combined with 1 mg/kg CS1003 at the experimental end point were 312.06±80.17 mm.sup.3, 246.48±62.57 mm.sup.3, and 233.10±59.55 mm.sup.3, respectively; tumor inhibition rates were 63.16%, 70.90%, and 72.48% (P<0.001). The compound of formula (II-1) (50 mg/kg) administered twice a day combined with CS1003 showed a stronger anti-tumor effect. The average tumor volume of this group at the end of the experiment was 142.17±40.30 mm.sup.3, and the tumor suppression rate was 83.22%. (P<0.001). It could be seen that when the compound of formula (II-1) was combined with CS1003 for preventive administration, it could significantly inhibit the growth of in vivo allograft tumors of mouse colon cancer cell CT26.

[0187] Conclusion: The combination of the compound of the present disclosure and CS1003 has a good anti-tumor effect, and the combination of the compound of the present disclosure and CS1003 has a synergistic effect. The combination of compound of formula (II-1) and CS1003 could enhance the anti-tumor effect. Wherein, the compound of formula (II-1) administered at 25 mg/kg, 50 mg/kg, 100 mg/kg once a day combined with CS1003 group, the tumor inhibition rates were 63%, 71% and 72%, respectively; the compound of formula (II-1) administered at 50 mg/kg twice a day combined with CS1003 group, the tumor inhibition rate of was 83% (P<0.001), which was significantly different from the compound of formula (II-1) (50 mg/kg, BID) or CS1003 alone (p values were 0.002, 0.003, respectively).

Experimental Embodiment 4: In Vivo PK Experiment of the Compound of the Present Disclosure

[0188] Blood was collected and tissues were collected from each group of the experiment at different time points (0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h) on day 20 of the administration of experimental embodiment 3. The pharmacokinetic parameters of each experimental group were shown in Table 12, and the biological ratios of the tumor tissue drug concentration and the corresponding blood sampling point tumor tissue drug concentration and plasma drug concentration of each experimental group were shown in Table 13.

TABLE-US-00012 TABLE 12 Pharmacokinetic parameters of each experimental group Compound Compound Compound Compound Compound Compound Test of formula of formula of formula of formula of formula of formula sample (II-1) (II-1) (II-1) (II-1) (II-1) (II-1) PK Group 4 5 6 3 7 8 PO Cmax (nM) 32200 57200 132000 114000 30100 47100 Tmax (h) 0.500 0.250 0.250 0.250 0.500 0.500 T½ (h) 1.09 1.05 0.655 0.81 NR 1.59 AUC.sub.0-last 23500 70600 125000 145000 42000 77900 (nM .Math. h) Note: NR means not obtained.

TABLE-US-00013 TABLE 13 The biological ratio of the tumor tissue drug concentration and the corresponding blood sampling point tumor tissue drug concentration and plasma drug concentration of each experimental group measured in the experiment Compound Compound Compound Compound Compound Compound Test of formula of formula of formula of formula of formula of formula sample (II-1) (II-1) (II-1) (II-1) (II-1) (II-1) Group 25 mpk 50 mpk 100 mpk 100 mpk 50 mpk 50 mpk 4 5 6 3 7 8 Time 1 4 1 4 1 4 1 4 1 4 1 4 point(h) Tumor drug 3480 925 9960 1660 18700 3030 17500 10600 3690 901 ND 3020 concentration (nM) T/P* Ratio 0.7 0.9 0.4 1.8 0.5 4.2 0.6 0.9 0.7 9.8 ND 36 Note: ND means not detected. T/P*Ratio means the ratio of the compound exposure in tumor tissue and plasma.

[0189] Conclusion: The compound of the present disclosure has sufficient exposure in plasma and tumor tissues.