ADENOSINE COMPOUND, ITS PHARMACEUTICALLY ACCEPTABLE SALT OR ITS STEREOISOMER AND USE THEREOF

Abstract

The present invention relates to a compound represented by Formula I, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester, wherein R is selected from heteroaryl, substituted heteroaryl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, alkyl, and substituted alkyl, wherein the substituted heteroaryl, the substituted aryl, the substituted cycloalkyl and the substituted alkyl are each independently substituted with one or more hydroxyalkylene groups. The compound or its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, its pharmaceutically acceptable ester of the present invention has agonistic activity on A.sub.2A adenosine receptor and can improve the permeability of blood-brain barrier to promote the delivery of drug across blood-brain barrier, and can also prevent or treat a disease associated with A.sub.2A adenosine receptor agonistic activity.

##STR00001##

Claims

1. A compound represented by Formula I, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester, ##STR00021## wherein, R is selected from the group consisting of heteroaryl, substituted heteroaryl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, alkyl, and substituted alkyl, wherein the substituted heteroaryl, the substituted aryl, the substituted cycloalkyl and the substituted alkyl are each independently substituted with one or more hydroxyalkylene groups.

2. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from the group consisting of heteroaryl, substituted heteroaryl, substituted aryl, and cycloalkyl, wherein the substituted heteroaryl and the substituted aryl are each independently substituted with one or more hydroxyalkylene groups.

3. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, which is selected from the following compounds, their pharmaceutically acceptable salts or their stereoisomers: ##STR00022##

4. A pharmaceutical composition, which comprises the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, and optionally a pharmaceutical excipient.

5. A method for agonizing A.sub.2A adenosine receptor, comprising administering an effective amount of the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, or an effective amount of the pharmaceutical composition according to claim 4 on an A.sub.2A adenosine receptor.

6. A method for improving the permeability of blood-brain barrier, comprising administering an effective amount of the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, or an effective amount of the pharmaceutical composition according to claim 4 to a subject.

7. A method for promoting the delivery of a drug across blood-brain barrier, comprising improving the permeability of blood-brain barrier by the method according to claim 6, and then administering the drug to the subject; or, administering the drug together with an effective amount of the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, or the drug together with an effective amount of the pharmaceutical composition according to claim 4 to the subject.

8. A method for preventing or treating a disease associated with A.sub.2A adenosine receptor agonistic activity, comprising administering an effective amount of the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, or an effective amount of the pharmaceutical composition according to claim 4 to a subject in need.

9. (canceled)

10. (canceled)

11. (canceled)

12. A method for diagnosing myocardial perfusion abnormality disease, comprising administering a diagnostically effective amount of the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, or a diagnostically effective amount of the pharmaceutical composition according to claim 4 to a subject in need.

13. (canceled)

14. (canceled)

15. (canceled)

16. A pharmaceutical composition, which comprises the compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, and a drug suitable for delivery across blood-brain barrier and optionally a pharmaceutical excipient.

17. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from the group consisting of 5- to 8-membered heteroaryl, substituted 5- to 8-membered heteroaryl, 5- to 8-membered aryl, substituted 5- to 8-membered aryl, 3- to 8-membered cycloalkyl, substituted 3- to 8-membered cycloalkyl, C.sub.1-10 alkyl, and substituted C.sub.1-10 alkyl, wherein the substituted 5- to 8-membered heteroaryl, the substituted 5- to 8-membered aryl, the substituted 3- to 8-membered cycloalkyl and the substituted C.sub.1-10 alkyl are each independently substituted with one or more hydroxyl-C.sub.1-10 alkylene groups.

18. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from the group consisting of 5- to 8-membered heteroaryl, substituted 5- to 8-membered heteroaryl, substituted 5- to 8-membered aryl, and 3- to 8-membered cycloalkyl, wherein the substituted 5- to 8-membered heteroaryl and the substituted 5- to 8-membered aryl are each independently substituted with one or more hydroxyl-C.sub.1-10 alkylene groups.

19. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from the group consisting of pyrrolyl, substituted pyrrolyl, imidazolyl, substituted imidazolyl, thiazolyl, substituted thiazolyl, furyl, substituted furyl, pyridyl, substituted pyridyl, substituted phenyl, and 3- to 6-membered cycloalkyl, wherein the substituted pyrrolyl, the substituted imidazolyl, the substituted thiazolyl, the substituted furyl, the substituted pyridyl and the substituted phenyl are each independently substituted with one or more hydroxyl-C.sub.1-6 alkylene groups.

20. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from the group consisting of pyridin-1-yl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, p-hydroxymethylphenyl, o-hydroxymethylphenyl, m-hydroxymethylphenyl, p-hydroxyethylphenyl, o-hydroxyethylphenyl, m-hydroxyethylphenyl, p-hydroxypropylphenyl, cyclopropyl and cyclobutyl.

21. The compound, its pharmaceutically acceptable salt, its stereoisomer, its pharmaceutically acceptable hydrate or solvate, or its pharmaceutically acceptable ester according to claim 1, wherein R is selected from ##STR00023##

22. The method for preventing or treating a disease associated with A.sub.2A adenosine receptor agonistic activity according to claim 8, wherein the disease associated with A.sub.2A adenosine receptor agonistic activity is selected from the group consisting of autoimmune irritation, inflammation, allergic disease, skin disease, infectious disease, wasting disease, neuropathic pain, open trauma, adverse reaction caused by drug therapy, cardiovascular disease, ischemia-reperfusion injury, gout, chemical trauma, thermal trauma, diabetic nephropathy, sickle cell disease, laminitis, foundrymen's disease, glaucoma, ocular hypertension, spinal cord injury, myocardial infarction, and acute myocardial infarction.

Description

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

[0119] The embodiments of the present invention will be clearly and completely described below in conjunction with examples. Obviously, the described examples are only a part of the examples of the present invention, rather than all of the examples. The following description of at least one exemplary embodiment is actually only illustrative, and in no way serves as any limitation to the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0120] Unless otherwise stated: all temperatures were expressed in ° C. (degrees Celsius); the structure of the compound was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS); the melting point m.p. of the compound was determined by RY-1 melting point instrument, and the thermometer had not been corrected. The m.p. was given in ° C.; 1H NMR was measured by JEOL JNM-ECA-400 nuclear magnetic resonance instrument; mass spectrum was measured by API3000 (ESI) instrument; all reaction solvents that were not specified were subject to standardized pretreatment.

Example 1: Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-(pyridin-3-yl)urea (Compound 1)

[0121] ##STR00016##

[0122] Compound 1 was prepared according to the following reaction scheme, wherein R was pyridin-3-yl.

##STR00017## ##STR00018##

(1) Synthesis of 1-(4-bromophenyl)-3-(pyridin-3-yl) urea (II)

[0123] 0.6 g (3.03 mmol) of 4-bromophenylisocyanate was added to 100 ml of dichloromethane and stirred for dissolution, 10 ml of dichloromethane solution containing 0.34 g (3.63 mmol) of 3-aminopyridine was gradually added dropwise, after the formation of precipitate, the reaction was continued for 2 hours, and filtration was carried out to obtain 0.85 g of white solid 1-(4-bromophenyl)-3-(pyridin-3-yl)urea, which was directly used in the next reaction.

(2) Synthesis of 1-(pyridin-3-yl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea (III)

[0124] 0.75 g (2.56 mmol) of 1-(4-bromophenyl)-3-(pyridin-3-yl)urea, 1.3 g (5.1 mmol) of bis(pinacolato)diboron, 0.9 g (1.78 mmol) of PdCl.sub.2 (PPh.sub.3).sub.2, 0.7 g (7.7 mmol) of potassium acetate were added to 50 ml of DMF, and reacted at 80° C. for 4 hours under nitrogen protection. After the reaction, it was extracted with dichloromethane/water, the organic layer was collected and washed with water and saturated brine, dried with anhydrous Na.sub.2SO.sub.4, filtrated, and purified by column chromatography to obtain 0.7 g of white solid 1-(R group)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea, which was directly used in the next reaction.

(3) Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl) tetrahydrofuran-3,4-diyl diacetate (VI)

[0125] 21 g (0.066 mol) of (2S,3R,4R,5R)-5-(acetoxymethyl)tetrahydrofuran-2,3,4-triethyl triacetate (IV) was heated to 90° C. until it became clear, and at this time 12 g (0.063 mol) of 2,6-dichloropurine (V) and 0.3 g of tin tetrachloride were added and stirred, and then heated to 120° C. and stirred for 15 minutes. The solvent was evaporated in vacuo and the residue was cooled. Methanol (50 ml) was added to the residue, and the crude solid product was separated by filtration. The crude product was recrystallized in ethanol to obtain 12 g of pale yellow powder product (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl) tetrahydrofuran-3,4-diyl diacetate, which was directly used in the next reaction.

(4) Synthesis of (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxymethyl) tetrahydrofuran-3,4-diol (VII)

[0126] 10 g (0.022 mol) of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(2,6-dichloro-9H-purin-9-yl) tetrahydrofuran-3,4-diyl diacetate (VI) was heated to 100° C. in 200 ml of methanol solution containing ammonia and kept in an autoclave for 24 hours. The solution was further stirred for 24 hours until room temperature, and then the solution was evaporated to dryness under reduced pressure to remove ammonia. The residue was purified by flash chromatography, in which the mixed solvent of CHCl.sub.3 and MeOH was used as the eluent. The product was dried below 50° C. to obtain 4.5 g of light yellow powder (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol, which was directly used in the next reaction.

(5) Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-(pyridin-3-yl)urea (VIII, i.e., Compound 1)

[0127] 0.5 g (0.0017 mol) of (2R,3R,4S,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (VII) was added to 0.6 g (0.0018 mol) of 1-(pyridin-3-yl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea (III, 1.1 equivalent), then 3 ml of ethylene glycol dimethyl ether, 10 ml of methanol, 5 ml of 2M Na.sub.2CO.sub.3 aqueous solution were added and stirred, 1.0 g (0.0009 mol) of Pd(PPh.sub.3).sub.4 (0.5 equivalent) was added and then the air in the reaction flask was evacuated by introducing nitrogen gas, it was heated to 100° C. and refluxed overnight. Then the heating was stopped and it was cooled to room temperature, 300 ml of methanol was added and stirred for dissolution, after filtration a crude product was separated by silica gel column chromatography from the filtrate mixture. The crude product was further purified by medium pressure preparative chromatography using C18 reverse phase column to obtain 116 mg of white solid (Compound 1). m.p. 230° C.; 1H NMR (DMSO-d6): δ (ppm) 9.05 (s, 1H), 8.94 (s, 1H), 8.63 (s, 1H), 8.36 (s, 1H), 8.29 (d, 2H, J=8.0 Hz), 8.21 (d, 2H, J=4.8 Hz), 7.98 (d, 1H, J=9.6 Hz), 7.57 (d, 2H, J=8.0 Hz), 7.35-7.32 (m, 2H), 5.99 (d, 1H, J=5.2 Hz), 5.51 (s, 1H), 5.27 (s, 1H), 5.07 (s, 1H), 4.75 (s, 1H), 4.23 (s, 1H), 3.97 (s, 1H), 3.72-3.57 (m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C.sub.22H.sub.22N.sub.8O.sub.5: 479.1786; found: 479.1786.

[0128] Compounds 2 to 3 as follows were prepared with reference to the method of Example 1, in which step (1) and step (2) were used to synthesize different 1-(R group)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea (III) that was used to replace the side chain compound (III) in step (5) for preparation.

Example 2: Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-[4-(hydroxymethyl)phenyl]urea (Compound 2)

[0129] ##STR00019##

(1) Synthesis of 1-(4-bromophenyl)-3-[4-(hydroxymethyl)phenyl]urea (II)

[0130] By referring to the method of step (1) in Example 1, 3-aminopyridine was replaced with 4-aminobenzyl alcohol, and the remaining operations were the same as those in step (1) of Example 1 to obtain 0.91 g of white solid 1-(4-bromophenyl)-3-[4-(hydroxymethyl)phenyl]urea, which was directly used in the next reaction.

(2) Synthesis of 1-[4-(hydroxymethyl)phenyl]-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea (III)

[0131] By referring to the step (2) of Example 1, 1-(4-bromophenyl)-3-(pyridin-3-yl)urea was replaced with 1-(4-bromophenyl)-3-[4-(hydroxymethyl) phenyl]urea, and the remaining operations were the same as those in step (2) of Example 1 to obtain 0.76 g of white solid 1-[4-(hydroxymethyl)phenyl]-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea, which was directly used in the next reaction.

(3) Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-[4-(hydroxymethyl)phenyl]urea (Compound 2)

[0132] By referring to the method of step (5) in Example 1, 1-(pyridin-3-yl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea was replaced with 1-[4-(hydroxymethyl)phenyl]-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea, and the remaining operations were the same as those in step (5) of Example 1 to obtain 138 mg of white solid (Compound 2). m.p. 174° C.; 1H NMR (DMSO-d6): δ (ppm) 8.89 (s, 1H), 8.73 (s, 1H), 8.35 (s, 1H), 8.27 (d, 2H, J=8.8 Hz), 7.55 (d, 2H, J=8.4 Hz), 7.43 (d, 2H, J=8.4 Hz), 7.30 (s, 2H), 7.24 (d, 2H, J=8.8 Hz), 5.98 (d, 1H, J=6.0 Hz), 5.50 (d, 1H, J=6.0 Hz), 5.26 (d, 1H, J=4.8 Hz), 5.11-5.05 (m, 2H), 4.74 (dd, 1H, J=5.6 Hz, 5.6 Hz), 4.43 (d, 2H, J=5.6 Hz), 4.23 (dd, 1H, J=3.6 Hz, 4.4 Hz), 3.96 (d, 1H, J=3.6 Hz), 3.73-3.55 (m, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C.sub.24H.sub.25N.sub.7O.sub.6: 508.1939; found: 508.1938.

Example 3: Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-cyclopropylurea (Compound 3)

[0133] ##STR00020##

(1) Synthesis of 1-(4-bromophenyl)-3-cyclopropylurea (II)

[0134] By referring to the method of step (1) in Example 1, 3-aminopyridine was replaced with cyclopropylamine, and the remaining operations were the same as those in step (1) of Example 1 to obtain 0.72 g of white solid 1-(4-bromophenyl)-3-cyclopropylurea, which was directly used in the next reaction.

(2) Synthesis of 1-cyclopropyl-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea (III)

[0135] By referring to the method of step (2) in Example 1, 1-(4-bromophenyl)-3-(pyridin-3-yl)urea was replaced with 1-(4-bromophenyl)-3-cyclopropylurea, and the remaining operations were the same as those in step (2) of Example 1 to obtain 0.59 g of white solid 1-cyclopropyl-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea, which was directly used in the next reaction.

(3) Synthesis of 1-{4-{6-amino-9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl]-9H-purin-2-yl}phenyl}-3-cyclopropylurea (Compound 3)

[0136] By referring to the method of step (5) in Example 1, 1-(pyridin-3-yl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea was replaced with 1-cyclopropyl-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]urea, and the remaining operations were the same as those in step (5) of Example 1 to obtain 135 mg of white solid (Compound 3). m.p. 174° C.; 1H NMR (DMSO-d6): δ (ppm) 8.51 (s, 1H), 8.33 (s, 1H), 8.21 (d, 2H, J=8.4 Hz), 7.49 (d, 3H, J=8.4 Hz), 7.24 (s, 2H), 6.47 (s, 1H), 5.97 (d, 1H, J=6.0 Hz), 5.46 (d, 1H, J=6.0 Hz), 5.22 (d, 1H, J=4.4 Hz), 5.03 (t, 1H, J=5.6 Hz), 4.72 (d, 1H, J=5.6 Hz), 4.22 (d, 1H, J=4.0 Hz), 3.96 (d, 1H, J=3.6 Hz), 3.71-3.54 (m, 2H), 0.64 (d, 2H, J=5.2 Hz), 0.42 (s, 2H); HRMS (ESI+) m/z [M+H]+ calculated for C.sub.20H.sub.23N.sub.7O.sub.5: 442.1833; found: 442.1833.

Example 4: Radioligand Binding Test

[0137] 1) Experimental Materials:

[0138] [3H]-CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine, [carboxy-1-ethyl-3H(N)]—; 250 μCi): purchased from PerkinElmer Research Products (Boston, Mass.);

[0139] The cell membrane stably transfected with (human) A.sub.2A adenosine receptor was prepared in HEK-293 cells, and the cell membrane was obtained from PerkinElmer Research Products (Boston, Mass.);

[0140] CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine) was purchased from Selleck (Shanghai, CN);

[0141] All other reagents were of analytical grade and obtained from commercial sources.

[0142] 2) Experimental Method:

[0143] The A.sub.2A adenosine receptors used were all expressed in the cell membrane. Each compound was continuously diluted 3 times with DMSO (Solarbio, D8371-250 ml) to generate compound source plates with 10 different concentrations (10 μM, 3.3 μM, 1.1 μM, 0.37 μM, 0.12 μM, 0.0412 μM, 0.0137 μM, 0.0046 μM, 0.0015 μM, 0.0005 μM), 250 nL of compounds were added to 384-well Opti-plate, sealed with parafilm; 1 mL of assay buffer (containing 50 mM Tris-HCl, pH 7.4, 10 mM MgCl.sub.2, 1 mM EDTA, 1 μg/mL adenosine deaminase) was added to 20 U hA.sub.2A HEK-293 cell membrane for dilution, 0.75 μCi [3H]-CGS21680 (final 25 nM) was added to the diluted cell membrane and mixed well; 50 μL of the prepared cell membrane dilution was transferred to the 384-well Opti-plate containing fresh compounds and incubated at 25° C. for 90 minutes; 100 μL of 0.5% polyethyleneimine solution (PEI) was added to UNIFILTER-96 GF/B filter plate, and soaked at 4° C. for 90 minutes, then the UNIFILTER-96 GF/B filter plate was washed twice with 500 μL of washing buffer per well (washing buffer contained 50 mM Tris-HCl pH 7.4, 154 mM NaCl) which was transferred by Cell Harvester; then, the mixture system in the Opti-plate was transferred to the cleaned UNIFILTER-96 GF/B filter plate, and then the UNIFILTER-96 GF/B filter plate was washed 9 times with 500 μL of washing buffer per well (washing buffer contained 50 mM Tris-HCl, pH 7.4, 154 mM NaCl), and incubated for 3 minutes in a 37° C. incubator; 40 μL of ULTIMA GOLD scintillation fluid (Perkin Elmer, Cat #77-16061) was added to each well, MicroBeta liquid scintillation counter (PerkinElmer) was used to read CPM (count per minute) value. The specific binding percentage of [3H]CGS21680 was calculated according to the CPM value, % specific binding of [3H]CGS21680=(CPM.sub.sample−CPM.sub.Low Control)/(CPM.sub.High Control−CPM.sub.Low Control)*100, wherein High Control was 0.5% DMSO, Low Control was 100 μM CGS21680. According to the compound concentration and the specific binding percentage of [3H]CGS21680, curve fitting was performed to calculate IC.sub.50 value.

[0144] 3) Experimental Results:

[0145] The binding inhibition constant (Ki) value was calculated from the IC.sub.50 value according to the Cheng-Prusoff equation, Ki=IC.sub.50/(1+[S]/Km), wherein [S] was radioligand concentration (25 nM), and Km was human A.sub.2AAR dissociation constant of [3H]CGS21680 (22 nM). Table 1 showed the binding inhibition constant Ki values of Compounds 1 to 3 of the present invention binding to A.sub.2A adenosine receptor.

TABLE-US-00001 TABLE 1 Test results of compounds binding to A.sub.2A adenosine receptor Compound IC.sub.50(nM) Ki (nM) Compound 1 1662 778 Compound 2 35.0 16.4 Compound 3 34.0 15.9

Example 5: cAMP Test of A.SUB.2A .Adenosine Receptor

[0146] 1) Experimental Materials:

[0147] DMEM/F12, G418, Penicillin-Streptomycin, Versene solution, HEPES, Hank's buffer saline aqueous solution, PBS (pH 7.4, 1×, sterile), FBS, 7.5% BSA stabilizer, Rolipram, NECA, were purchased from Gibico, Hyclone and Sigma, respectively;

[0148] LANCE® Ultra cAMP kit (containing Eu-cAMP tracer, Ulight-anti-cAMP reagent, cAMP detection buffer) and hADORA.sub.2A-HEK293 cells were purchased from PerkinElmer Research Products (Boston, Mass.);

[0149] All other reagents were of analytical grade and obtained from commercial sources;

[0150] 384-well polypropylene microplate and 384-well white solid plate were purchased from Labcyte and Corning, respectively.

[0151] Experimental instruments: TECAN automated pipetting workstation, Echo ultrasonic pipetting system and EnVison microplate reader were purchased from TECAN, Labcyte and Envision, respectively.

[0152] 2) Experimental Method:

[0153] The cells stably expressing human adenosine receptor A.sub.2A (hADORA.sub.2A-HEK293 cells) were cultured in DMEM/F12 medium containing 10% FBS, 1× Penicillin-Streptomycin and 400 μg/ml G418 in the environment of 37° C. and 5% CO.sub.2. Before the experiment, the cells were digested with Versene solution. The cells were collected by centrifugation at 200 g and room temperature for 5 minutes using a centrifuge, and finally resuspended using the assay buffer (the assay buffer contained Hank's buffer saline aqueous solution, 1M HEPES, 7.5% BSA stabilizer, pH 7.4, 20 mM Rolipram). The compound was continuously diluted 3 times in a 384-well polypropylene microplate with DMSO through TECAN automated pipetting workstation to prepare compound source plates with 11 concentration points (10 mM, 3.33 mM, 1.11 mM, 0.37 mM, 0.12 mM, 0.041 mM, 0.013 mM, 4.57×10.sup.−3 mM, 1.52×10.sup.−3 mM, 5×10.sup.−4 mM and 1.7×10.sup.−4 mM). The test compound was transferred from the compound source plate to assay plate by Echo ultrasonic pipetting system (Labcyte), and the transfer volume of the compound was 10 nl/well. The hADORA.sub.2A-HEK293 cell suspension was diluted to 30,000 cells/ml with assay buffer (the assay buffer contained Hank's buffer saline aqueous solution, 1M HEPES, 20 mM Rolipram, 7.5% BSA stabilizer, pH 7.4), and then the cell suspension was transferred to the assay plate in a volume of 10 μl/well (300 cells/well). The assay plate was centrifuged at 150 g for 1 minute and pre-incubated for 30 minutes at room temperature. Eu-cAMP tracer working solution (including 40 μl of Eu-cAMP tracer, 1.96 ml of cAMP detection buffer) (5 μl/well) was added to the assay plate, and then Ulight-anti-cAMP working solution (13 μl of Ulight-anti-cAMP agent and 1.95 ml of cAMP detection buffer) (5 μl/well) was added to the assay plate. The assay plate was rotated at 150 g for 30 seconds and incubated at room temperature for 30 minutes. EnVison microplate reader (EnVision multimode plate reader, PerkinElmer) was used to determine the level of cyclic adenosine monophosphate in the final solution (λ.sub.ex=320 nm, λ.sub.em=665 nm & 615 nm). The EC.sub.50 (nM) value that stimulated the production level of cyclic adenosine monophosphate when the test compound interacted with the A.sub.2A adenosine receptor was calculated. The A.sub.2A receptor agonist titer of the compound was expressed as the EC.sub.50 (nM) value that stimulated the production level of cyclic adenosine monophosphate when the compound interacted with the A.sub.2A adenosine receptor.

[0154] 3) Experimental Results:

[0155] When the test compound interacted with A.sub.2AAR, the EC.sub.50 (nM) value that stimulated the level of cyclic AMP was shown in Table 2. The results show that the compound of the present invention is shown to be an hA.sub.2AAR agonist. It is generally accepted in this field that cAMP EC.sub.50 value <10 μM indicates significant result, and cAMP EC.sub.50 value <1 μM indicates a strong agonistic activity.

TABLE-US-00002 TABLE 2 EC.sub.50 value test results of compound for A.sub.2A agonist function determination cAMP Compound EC.sub.50 (nM) Compound 2 227.7

[0156] Finally, it should be noted that the above examples are only used to illustrate the technical solution of the present invention but not to limit it; although the present invention has been described in detail with reference to the preferred examples, those of ordinary skill in the art should understand that: the specific implementation of the present invention can be modified or some technical features can be equivalently replaced without departing from the spirit of the technical solution of the present invention, and all of them shall be covered by the scope of the technical solution sought to be protected by the present invention.