Use of triazolotriazine derivatives in treating diseases

Abstract

The present application relates to the use of a triazolotriazine derivative or a pharmaceutically acceptable solvate or salt thereof in the preparation of a drug for treating a disease, and a method for treating a disease.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a triazolotriazine derivative capable of treating diseases. Background Art

[0002] Parkinson's disease occurs as a result of chronic, progressive degeneration of neurons, the causes of which are not yet fully understood. Its main clinical symptoms are resting tremor, rigidity, bradykinesia and postural instability. Current treatments are mainly based on a dopamine replacement therapy that interferes with dopaminergic and / or cholinergic signaling cascades. However, these clinical symptoms are often not satisfactorily controlled by existing therapies, manifesting as non-dopaminergic lesions in the brain, spinal nerves and peripheral autonomic nervous system, and many Parkinson's patients suffer from disability under existing therapies. Therefore, the development of new therapies that can slow down, stop or reverse the progression of the disease is a top priority in Parkinson's research.

[0003] Cancer remains a major global health burden. Despite advances in the treatment of cancer, there is still an unmet medical need for more effective and less toxic treatments, especially for those patients with advanced disease or cancer that is resistant to existing treatments. In recent years, the importance of lymphocytes in tumor immunity has been deeply recognized. The body's immune response to tumors includes immune surveillance, and this cell-mediated immunity-related mechanism can effectively eliminate newly generated tumor cells after recognizing tumor-associated antigens. Although antibody-mediated anticancer effects are also commonly present, they are generally much smaller than those mediated by cell-mediated immunity. Therefore, there is an urgent need to develop promising new anticancer agents to meet medical needs. Summary of the invention

[0004] The present application provides a use of a triazolotriazine derivative or a pharmaceutically acceptable solvate or salt thereof in the preparation of a drug for treating a disease, and a method for treating a disease, wherein the disease includes tumors and Parkinson's disease. The compound of the present application has at least one of the following properties in treating a disease: (1) good pharmacokinetic properties, high in vivo exposure and bioavailability; (2) improving the motor ability of Parkinson's disease subjects; and / or (4) inhibiting tumor growth and reducing tumor volume.

[0005] In one aspect, the present application provides the use of a compound of formula 1 in preparing a drug for treating a disease.

[0006]

[0007] in:

[0008] R is hydrogen or methyl; Ar 1is an optionally substituted furanyl, an optionally substituted phenyl, or an optionally substituted pyridyl; any optionally substituted aromatic ring is substituted by halogen or oxo; Ar 2 is an optionally substituted phenyl, an optionally substituted pyridyl, or an optionally substituted pyrimidyl; any optionally substituted aromatic ring is substituted by halogen, hydroxy, cyano or methoxy; X is oxygen or nitrogen;

[0009] and Y and Z are each independently hydrogen, an optionally substituted C 1-3 Alkyl, optionally substituted C 1-5 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted heterocycloalkylalkyl, optionally substituted aryl, optionally substituted C 1-3 Alkylcarbonyl, optionally substituted C 1-5 cycloalkylcarbonyl, optionally substituted heterocycloalkylcarbonyl, optionally substituted heterocycloalkylalkylcarbonyl, optionally substituted arylcarbonyl, or optionally substituted heteroarylcarbonyl; any of the optionally substituted groups is replaced by halogen, hydroxy, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, alkylamino, cycloalkylamino, heterocyclyl, aryl, heteroaryl, or C 1-3 or Y and Z are linked to form a heterocycloalkyl group having 5 to 7 ring atoms which is optionally substituted; any of the optionally substituted rings is substituted with halogen, oxo, methoxy, ethoxy, trifluoromethoxy, or trifluoroethoxy; or Y or Z is absent, or a pharmaceutically acceptable solvate or salt thereof.

[0010] In certain embodiments, R is a hydrogen radical; Ar 1 is 2-furyl; Ar 2 is an optionally substituted phenyl group; any of the optionally substituted phenyl groups is substituted by halogen; X is oxygen or nitrogen; and Y and Z are each independently hydrogen, an optionally substituted C 2-3 alkyl, optionally substituted heterocycloalkyl, or optionally substituted heterocycloalkylalkyl; any of the optionally substituted groups is methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, or C 1-2 The alkylpolyoxyethylene group is substituted; or Y and Z are linked to form a morpholinyl ring; or Y or Z is absent, or a pharmaceutically acceptable solvate or salt thereof.

[0011] In certain embodiments, R in the compound of Formula 1 is hydrogen; Ar 1 is 2-furyl; Ar 2is phenyl; X is nitrogen; and Y and Z are each independently hydrogen, an ethyl group optionally bearing a substituent, or an oxetanyl group optionally bearing a substituent; any of the optionally substituted groups is substituted by a methoxy group, an ethoxy group, a trifluoromethoxy group, or a trifluoroethoxy group; or Y and Z are connected to form a morpholinyl ring, or a pharmaceutically acceptable solvate or salt thereof.

[0012] In certain embodiments, the compound is selected from the group consisting of:

[0013] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)-3-morpholinepropaneamide;

[0014] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)-2-morpholinepropaneamide;

[0015] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)pivalamide;

[0016] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)-3-hydroxy-2,2-dimethylpropanamide;

[0017] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)-2-hydroxy-2-methylpropanamide;

[0018] N-(4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)phenyl)-4-methyltetrahydro-2H-pyran-4-carboxamide;

[0019] 2-(Furan-2-yl)-N5-(2-(pyrimidin-4-yl)ethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0020] 2-(Furan-2-yl)-N5-(2-(pyridin-3-yl)ethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0021] 2-(Furan-2-yl)-N5-(4-(2-(pyrrolidin-1-yl)ethoxy)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]-triazine-5,7-diamine;

[0022] N5-(4-(2-(azetidin-1-yl)ethoxy)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0023] 2-(Furan-2-yl)-N5-(4-(2-(4-methylpiperazin-1-yl)ethoxy)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0024] 2-(Furan-2-yl)-N5-(4-(3-(4-methylpiperazin-1-yl)propoxy)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0025] N5-(4-(2-(3,3-difluoropyrrolidin-1-yl)ethoxy)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0026] (R)-N5-(4-(2-(3-fluoropyrrolidin-1-yl)ethoxy)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0027] (S)-N5-(4-(2-(3-fluoropyrrolidin-1-yl)ethoxy)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0028] 2-(Furan-2-yl)-N5-(4-((2-morpholinoethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0029] 2-(Furan-2-yl)-N5-(4-((tetrahydro-2H-pyran-4-yl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0030] 2-(Furan-2-yl)-N5-(4-((tetrahydrofuran-3-yl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0031] 2-(Furan-2-yl)-N5-(4-(4-methylpiperazin-1-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0032] N5-(4-(bis(2-methoxyethyl)amino)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0033] 2-(Furan-2-yl)-N5-(4-(oxetan-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0034] 2-(Furan-2-yl)-N5-(4-(oxetan-3-ylmethyl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0035] 2-(Furan-2-yl)-N5-(4-((1-methoxypropan-2-yl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0036] 2-(Furan-2-yl)-N5-(4-((2-methoxyethyl)(methyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0037] N5-(4-(ethyl(2-methoxyethyl)amino)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0038] 2-(Furan-2-yl)-N5-(4-((2-methoxyethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0039] 2-(Furan-2-yl)-N5-(4-((2-(2-methoxyethoxy)ethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0040] 2-(Furan-2-yl)-N5-(4-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0041] N5-(4-((2-ethoxyethyl)amino)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0042] 2-(Furan-2-yl)-N5-(4-((2-(2,2,2-trifluoroethoxy)ethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0043] 2-(Furan-2-yl)-N5-(4-((3-methoxypropyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0044] 2-((4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-yl)amino)ethyl)-phenyl)amino)acetonitrile;

[0045] N5-(4-(1,3-dimethoxypropan-2-ylamino)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0046] 2-(3-fluorophenyl)-N5-(4-((2-methoxyethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine; and

[0047] 2-(3-Fluorophenyl)-N5-(4-(oxetan-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0048] or a pharmaceutically acceptable solvate or salt thereof.

[0049] On the other hand, the present application provides the use of the compound of formula 2 in preparing a drug for treating a disease,

[0050]

[0051] wherein R is hydrogen or methyl; Ar1 ​​is an optionally substituted furanyl group, an optionally substituted phenyl group, or an optionally substituted pyridyl group; any optionally substituted aromatic ring is substituted by halogen or oxo; Ar2 is an optionally substituted phenyl group, an optionally substituted pyridyl group, or an optionally substituted pyrimidyl group; any optionally substituted aromatic ring is substituted by halogen, hydroxyl, cyano or methoxy;

[0052] and Q is a 5-6 membered aromatic ring optionally substituted by X, an aminocarbonyl group optionally substituted on nitrogen by Y and Z, an aminosulfonyl group optionally substituted on nitrogen by Y and Z, a nitro group, or a cyano group; X is halogen or an optionally substituted C1-3 alkyl group; any of the optionally substituted alkyl groups is substituted by halogen, cyano, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, aryl or heteroaryl; Y and Z are each independently hydrogen or an optionally substituted C1-3 alkyl group; any of the optionally substituted alkyl groups is substituted by halogen, hydroxy, methyl, alkylamino or cycloalkylamino; or Y and Z are connected to form an optionally substituted ring having 5 to 7 ring atoms; any of the optionally substituted rings is substituted by halogen, methyl, ethyl, trifluoromethyl or trifluoroethyl, or a pharmaceutically acceptable solvate or salt thereof.

[0053] In certain embodiments, in the compound of Formula 2, R is hydrogen; Ar 1 Ar is 2-furanyl; Ar2 is phenyl or pyridyl; and Q is nitro, cyano, or a 5-6 membered aromatic ring optionally substituted by X; X is an optionally substituted C1-3 alkyl; any of the optionally substituted alkyl is substituted by halogen, cyano, methoxy, aryl or heteroaryl, or a pharmaceutically acceptable solvate or salt thereof

[0054] In certain embodiments, in the compound of Formula 2, R is hydrogen; Ar1 ​​is 2-furyl; Ar2 is phenyl; and Q is a tetrazole ring optionally substituted by X; X is an optionally substituted C1-3 alkyl; any of the optionally substituted alkyl is substituted by halogen, cyano or methoxy, or a pharmaceutically acceptable solvate or salt thereof.

[0055] In certain embodiments, the compound is selected from the group consisting of:

[0056] 4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl)-N,N-dimethylbenzamide;

[0057] N5-(4-(1-methyl-1H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0058] N5-(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0059] N5-(4-(2-ethyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0060] N5-(4-(2-isopropyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0061] N5-(4-(2-propyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0062] 2-(5-(4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl)-phenyl)-2H-tetrazol-2-yl)acetonitrile;

[0063] 4-(2-(7-amino-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-5-ylamino)ethyl)-benzonitrile;

[0064] N5-(4-(2-(2,2,2-trifluoroethyl)-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0065] N5-(4-(2-(2-methoxyethyl)-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0066] 2-(Furan-2-yl)-N5-(4-(pyridin-2-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0067] 2-(Furan-2-yl)-N5-(4-(pyridin-3-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0068] 2-(Furan-2-yl)-N5-(4-(pyridin-4-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0069] 2-(Furan-2-yl)-N5-(4-(pyrimidin-2-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0070] 2-(Furan-2-yl)-N5-(4-(1-methyl-1H-1,2,4-triazol-3-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0071] N5-(4-(2H-tetrazolyl-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0072] 2-(Furan-2-yl)-N-(4-(methylsulfonyl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5-amine;

[0073] 2-(3-fluorophenyl)-N5-(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine; and

[0074] N5-(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-phenyl-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine; and

[0075] 2-(Furan-2-yl)-N5-(2-(6-(2-methyl-2H-tetrazolyl-5-yl)pyridin-3-yl)ethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine,

[0076] or a pharmaceutically acceptable solvate or salt thereof.

[0077] In certain embodiments, the compound is selected from the group consisting of:

[0078] 2-(Furan-2-yl)-N5-(4-(oxetan-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0079] 2-(Furan-2-yl)-N5-(4-((2-methoxyethyl)amino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0080] N5-(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine; and

[0081] N5-(4-(2-ethyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine;

[0082] and pharmaceutically acceptable solvates or salts thereof.

[0083] In certain embodiments, the pharmaceutically acceptable solvate or salt of the compound comprises acetate and / or benzenesulfonate.

[0084] In certain embodiments, pharmaceutically acceptable solvates or salts of the compound include 2-(furan-2-yl)-N5-(4-(oxetan-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine acetate and / or N5-(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine benzenesulfonate.

[0085] In certain embodiments, the disease comprises Parkinson's disease.

[0086] In certain embodiments, the disease comprises a tumor. In certain embodiments, the tumor comprises a solid tumor and a non-solid tumor. In certain embodiments, the solid tumor comprises colon cancer and melanoma. In certain embodiments, the non-solid tumor comprises a lymphoma, for example, a B-cell lymphoma.

[0087] In certain embodiments, the medicament is prepared for oral administration and / or injection.

[0088] In certain embodiments, the medicament further comprises a pharmaceutically acceptable carrier. For example, the pharmaceutically acceptable carrier may include cyclodextrin.

[0089] In another aspect, the present application provides a method for treating a disease, comprising the following step: administering a therapeutically effective dose of the compound described herein to a subject in need thereof.

[0090] In certain embodiments, the disease comprises Parkinson's disease.

[0091] In certain embodiments, the subject exhibits early signs of Parkinson's disease.

[0092] In certain embodiments, the subject has or has not previously received medication to treat Parkinson's disease.

[0093] In certain embodiments, the subject is receiving a drug treating Parkinson's disease prior to, concurrently with, or after administration of the compound.

[0094] In certain embodiments, the subject has developed resistance to a drug used to treat Parkinson's disease.

[0095] In certain embodiments, the Parkinson's disease treating drug comprises dopamine.

[0096] In certain embodiments, said treating comprises reducing the rate of progression of Parkinson's disease in said subject, and / or delaying the clinical progression of Parkinson's disease in said subject.

[0097] In certain embodiments, the subject's Parkinson's disease progression rate is assessed by UPDRS.

[0098] In certain embodiments, the treating comprises delaying the subject's need for symptomatic anti-Parkinson's disease treatment.

[0099] In certain embodiments, the treating comprises reducing the subject's risk of requiring symptomatic treatment for Parkinson's disease.

[0100] In certain embodiments, the disease comprises a tumor. In certain embodiments, the tumor comprises a solid tumor and a non-solid tumor. In certain embodiments, the solid tumor comprises colon cancer and melanoma. In certain embodiments, the non-solid tumor comprises a lymphoma, for example, a B-cell lymphoma.

[0101] In certain embodiments, the compound is administered orally and / or by injection.

[0102] In certain embodiments, the compound is administered once a day or twice a day.

[0103] In certain embodiments, the compound is administered at a dose of about 0.001 mg / kg to about 500 mg / kg.

[0104] In certain embodiments, the compound is administered at a dose of about 1 mg / kg to about 100 mg / kg.

[0105] In certain embodiments, the subject receives immunotherapy to treat the tumor prior to, concurrently with, or after administration of the compound.

[0106] In certain embodiments, the immunotherapy for treating tumors comprises an immune checkpoint inhibitor.

[0107] In certain embodiments, the immunotherapy for treating tumors comprises PD-1 antibodies.

[0108] In another aspect, the present application provides a pharmaceutical combination or kit, which comprises (1) the compound described above, and (2) a drug for treating Parkinson's disease.

[0109] In certain embodiments, the Parkinson's disease treating drug comprises dopamine.

[0110] In another aspect, the present application provides a pharmaceutical combination or kit comprising (1) the compound described above, and (2) an immunotherapy for treating a tumor.

[0111] In certain embodiments, the immunotherapy for treating tumors comprises an immune checkpoint inhibitor.

[0112] In certain embodiments, the immunotherapy for treating tumors comprises PD-1 antibodies.

[0113] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0115] Figure 1 Shown are the XRPD results of the salts of the compounds of the present application.

[0116] Figure 2 Shown are the salts of the compounds of the present application 1 H-NMR spectrum results.

[0117] Figure 3 Shown are the DSC and TGA results of the salts of the compounds of the present application. DETAILED DESCRIPTION

[0118] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0119] Definition of terms

[0120] In this application, the term "alkyl" generally refers to straight and branched saturated aliphatic hydrocarbon groups including a certain number of carbon atoms. For example, C4 alkyl includes n-butyl, isobutyl, sec-butyl and tert-butyl. The term "cycloalkyl" generally refers to monocyclic and bicyclic saturated aliphatic hydrocarbon groups including a certain number of carbon atoms.

[0121] In this application, the term "alkenyl" is generally intended to include straight-chain and branched aliphatic hydrocarbon groups containing at least one carbon-carbon double bond. For example, there is one carbon-carbon double bond.

[0122] Unless otherwise indicated, the term "aryl" generally refers to monocyclic and bicyclic aromatic rings containing 5 to 14 ring atoms, and in some cases, 6 to 10 ring atoms. The aryl group may be optionally substituted with one or more substituents. Unless otherwise indicated, the term "aryl" may also include monocyclic and bicyclic heteroaryl rings containing 5 to 14 ring atoms, and in some cases, 6 to 10 ring atoms.

[0123] In the present application, the term "heterocycloalkyl" generally refers to saturated monocyclic and bicyclic systems including 3 to 14 ring atoms, for example, 4 to 10 ring atoms, wherein one or more of the atoms is a non-carbon element, such as nitrogen, oxygen or sulfur, and these heteroelements may exist alone or with other heteroelements. Examples of heterocycloalkyl include, for example, azetidinyl, hexahydroazapyridinyl, piperazinyl, piperidinyl, pyrrolidinyl, morpholinyl, tetrahydrofuranyl, thiomorpholinyl and tetrahydrothienyl, and N-oxides thereof.

[0124] In the present application, the term "halogen" generally refers to include fluorine, chlorine, bromine and iodine. The term "trifluoromethyl" generally refers to the "-CF3" group, and the term "hydroxyl" or "hydroxy" generally refers to the "-OH" group.

[0125] In the present application, the term "anti-Parkinson's disease symptomatic treatment" generally includes any therapy for treating Parkinson's disease, including but not limited to bromocriptine, benztropine, levodopa, ropinirole, pramipexole, rotigotine, cabergoline, entacapone, tolcapone, amantadine, pergolide, apomorphine, lisuride or selegiline. The term "delaying the need for anti-Parkinson's disease symptomatic treatment" means delaying the need for anti-Parkinson's disease symptomatic treatment in patients with Parkinson's disease who receive the compounds described in the present application compared to patients who do not receive the compounds described in the present application.

[0126] In the present application, the term "reducing the rate of progression of Parkinson's disease" generally refers to reducing the deterioration experienced by a Parkinson's disease patient, for example, such deterioration can be quantified by UPDRS score compared to the deterioration experienced by a Parkinson's disease patient who has not received the compounds described in the present application over a period of time. The rate of progression is quantified by the Total Unified Parkinson's Disease Rating Scale (Total UPDRS) score. An increase in the Total UPDRS score represents the progression of Parkinson's disease symptoms, and the increment in the increase in the UPDRS score over a period of time represents the degree of progression of Parkinson's disease symptoms.

[0127] In this application, the term "functional decline" generally refers to the worsening of symptoms over time in a Parkinson's disease patient as determined by the total UPDRS score.

[0128] As used herein, the term "delaying the need for symptomatic treatment of Parkinson's disease" generally refers to delaying the need for symptomatic treatment of Parkinson's disease in patients receiving the compounds described herein compared to patients who did not receive the compounds described herein.

[0129] In this application, the term "early signs of Parkinson's disease" generally includes one or more of the following signs:

[0130] a) 4-8 Hz pill-rolling tremor in one hand at rest;

[0131] b) Tremor is greatest when at rest, decreases with movement, and disappears during sleep;

[0132] c) stiffness and slowness of movement (bradycardia), decreased activity (hypokinesia), difficulty initiating movement (loss of mobility);

[0133] d) The face becomes mask-like, the mouth opens, and blinking slows, which may be confused with depression;

[0134] e) The body becomes bent;

[0135] f) Difficulty starting to walk; gait is unsteady and takes small steps, arms are bent at the waist and do not swing with the steps;

[0136] g) The pace may occasionally speed up unintentionally, and the patient may suddenly start to jog to avoid falling (panic pace);

[0137] h) tending to lean forward (propulsion) or backward (counterpropulsion) when the center of gravity is shifted due to loss of postural reflexes;

[0138] i) Weakened pronunciation, monotonous characteristics, stuttering, and dysphonia;

[0139] j) Hypokinesia and impaired control of distal musculature, leading to micrographia and increased difficulty in daily activities;

[0140] k) Rare blinking and lack of facial expression;

[0141] l) Decreased exercise;

[0142] m) Postural reflex disorders; and / or

[0143] n) Typical gait abnormalities.

[0144] The stage of Parkinson's disease patients in this application refers to the following 5 distinct stages described by Hoehn and Yahr depending on symptoms (Hoehn MM, Yahr MD, Parkinsonism: onset, progression and mortality. Neurology 1967, 17: 427-42).

[0145] Stage I: (mild or early disease): Symptoms affect one side of the body.

[0146] Stage II: Both sides of the body are affected, but posture remains normal.

[0147] Stage III: (Moderate disease): Both sides of the body are affected, and slight imbalance is present in standing and walking; but the patient is able to remain independent.

[0148] Stage IV (advanced disease): Both sides of the body are affected, and impaired instability is present in standing and walking; patients at this stage require considerable assistance.

[0149] Stage V: Presents with severe, full-blown illness; patient is confined to bed or chair.

[0150] In this application, "early Parkinson's disease patients" refer to patients in Parkinson's disease stage I or II as defined by Hoehn and Yahr, who do not require anti-Parkinson's disease symptomatic treatment. For example, those patients do not require anti-Parkinson's disease symptomatic treatment for at least the next 9 months. Those patients in early Parkinson's disease can be confirmed by performing relevant tests.

[0151] In the present application, the term "delaying the clinical progression of Parkinson's disease" generally refers to a lower rate of increase in the total UPDRS score in the control group, for example, 12 weeks after receiving the compound, after the compound has fully taken effect, compared to a subject who did not receive the compound described in the present application. Or a control group, for example, a subject who delayed receiving the compound treatment, showed a lower deterioration in the total UPDRS score after a sufficient period of time to eliminate the changes caused by the delayed start of the compound treatment. The sufficient time is necessarily more than 52 weeks (for example, at least more than 72 weeks) after the initial treatment with the compound. Or a control group, for example, a subject who delayed receiving the compound treatment, showed a substantially similar rate of deterioration in the total UPDRS score during the period after the delayed start of the compound treatment. The total UPDRS score is within 0.15 units per week.

[0152] In this application, the terms "cancer" and "tumor" are used interchangeably and generally refer to cells that have undergone malignant transformation or cellular changes that have resulted in abnormal or unregulated growth or excessive proliferation. Such changes or malignant transformations generally render such cells pathogenic to the host organism, and thus are also intended to include precancerous or precancerous cells that will or may become pathogenic and require or benefit from intervention. The tumor may include solid tumors and non-solid tumors. DETAILED DESCRIPTION OF THE INVENTION

[0154] On the one hand, the present application provides a compound represented by Formula 1, and its hydrate, solvate, pharmaceutically acceptable salt, prodrug and complex thereof, and use thereof in drugs for treating diseases:

[0155]

[0156] Wherein, R can be hydrogen or optionally substituted C 1-5 Alkyl; any optionally substituted alkyl group is substituted by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy; Ar 1 Ar2 may be a 5-6 membered aromatic ring, which may be optionally substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy; Ar2 may be a monocyclic or bicyclic aromatic ring, which may be optionally substituted by halogen, hydroxyl, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy; X is selected from the group consisting of oxygen, sulfur, carbon, and nitrogen;

[0157] and Y and Z may each independently be hydrogen, an optionally substituted C 1-9 Alkyl, optionally substituted monocyclic or bicyclic C 1-9 Cycloalkyl, optionally substituted C1-9 Alkenyl, optionally substituted monocyclic or bicyclic C 1-9 cycloalkenyl, aryl optionally substituted, heteroaryl optionally substituted, aralkyl optionally substituted, heteroaralkyl optionally substituted, heterocycloalkyl optionally substituted, heterocycloalkenyl optionally substituted, heterocycloalkylalkyl optionally substituted, C 1-9 Alkylcarbonyl, optionally substituted C 1-9 cycloalkylcarbonyl, optionally substituted heterocycloalkylcarbonyl, optionally substituted heterocycloalkylalkylcarbonyl, optionally substituted arylcarbonyl, or optionally substituted heteroarylcarbonyl; the optionally substituted group may be substituted by a substituent selected from the group consisting of halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, heterocyclyl, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, aryl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 alkylpolyoxypropylene; or, Y and Z may be linked to form an optionally substituted ring having 3 to 10 ring atoms; any of the optionally substituted rings may be substituted with a substituent selected from the group consisting of halogen, cyano, hydroxy, oxo, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 Alkyl polyoxypropylene; alternatively, Y or Z may not exist in the structure

[0158] In some cases, R in the compound of Formula 1 may be hydrogen, methyl or trifluoromethyl. In some specific cases, R in the compound of Formula 1 may be hydrogen or methyl. For example, R in the compound of Formula 1 may be hydrogen.

[0159] In some cases, Ar in the compound of Formula 1 1 It can be an imidazolyl, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl group which may optionally have a substituent; and the aromatic ring which may optionally have a substituent is substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0160] In some cases, Ar in the compound of Formula 1 1The aromatic groups may be selected from the aromatic groups in Table (1).

[0161] Table 1 Ar in Formula 1 1 Structure

[0162]

[0163] In some cases, Ar in the compound of Formula 1 1 It may be 2-furyl.

[0164] In some cases, Ar in the compound of Formula 1 2 It may be imidazolyl, triazolyl, tetrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindolyl, azaindazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, benzisoxazolyl, benzisothiazole, benzoxazolyl, benzothiadiazole, quinolyl, isoquinolyl, quinoxalinyl, phthalazinyl, quinazolinyl, cinnolinyl, naphthyridinyl, pyridopyrimidinyl, pyridopyrazine, or pteridinyl which may optionally have a substituent; the aromatic ring which may optionally have a substituent may be substituted by halogen, hydroxyl, cyano, nitro, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0165] In some cases, Ar in the compound of Formula 1 2 It may be phenyl, pyridyl, pyridazinyl or pyrimidinyl which may be optionally substituted; the aromatic ring which may be optionally substituted may be substituted by halogen, hydroxy, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0166] In some cases, Ar in the compound of Formula 1 2 It may be phenyl or pyridyl, which may be optionally substituted by halogen or hydroxy.

[0167] In some cases, X in the compound of Formula 1 is oxygen or nitrogen. For example, X in the compound of Formula 1 is nitrogen.

[0168] In some cases, Y and Z in the compound of Formula 1 can be independently hydrogen, optionally substituted C 2-5 Alkyl, optionally substituted C 3-5 Cycloalkyl, optionally substituted C 2-5 Alkenyl, optionally substituted C 3-5cycloalkenyl, aryl optionally substituted, heteroaryl optionally substituted, aralkyl optionally substituted, heteroaralkyl optionally substituted, heterocycloalkyl optionally substituted, or heterocycloalkylalkyl optionally substituted; any of the optionally substituted groups may be substituted by halogen, cyano, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy or C 1-3 Alkyl polyoxyethylene substitution;

[0169] In some cases, Y and Z in the compound of Formula 1 can be independently hydrogen, optionally substituted C 2-3 Alkyl, or optionally substituted heterocycloalkyl; any of the optionally substituted groups may be substituted by methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy;

[0170] In some cases, Y and Z in the compound of Formula 1 may each independently be hydrogen, an ethyl group optionally bearing a substituent, or an oxetane group optionally bearing a substituent; any of the optionally substituted groups may be substituted by a methoxy group, an ethoxy group, a trifluoromethoxy group, or a trifluoroethoxy group;

[0171] In some cases, Y and Z in the compound of Formula 1 may be linked to form a ring having 4 to 8 ring atoms which may be optionally substituted; the substituent in any of the optionally substituted groups may be halogen, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy;

[0172] In some cases, Y and Z in the compound of Formula 1 may be linked to form a heterocycloalkane having 5 to 7 ring atoms which may be optionally substituted; any of the optionally substituted groups may be substituted with halogen, oxo, methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy;

[0173] In some cases, Y and Z in the compound of Formula 1 may be linked to form an optionally substituted morpholinyl ring; any of the optionally substituted groups may be substituted by halogen, methoxy, ethoxy, trifluoromethoxy or trifluoroethoxy.

[0174] On the other hand, the present application provides a triazolotriazine compound represented by Formula 2, and its hydrate, solvate, pharmaceutically acceptable salt, prodrug and complex thereof, and use thereof in drugs for treating diseases:

[0175]

[0176] Wherein, R can be hydrogen or a C1-5 alkyl group optionally bearing a substituent; any of the alkyl groups optionally bearing a substituent can be substituted by halogen, cyano, hydroxyl, nitro, amino, alkylamino, cycloalkylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy or trifluoroethoxy;

[0177] Ar 1 It can be a 5-6 membered aromatic ring, which can be optionally substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy; Ar 2 It may be a monocyclic or bicyclic aromatic ring, which may be optionally substituted by halogen, hydroxy, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy;

[0178] Q can be a monocyclic or bicyclic aromatic ring optionally substituted by X, an aminocarbonyl optionally substituted by Y and Z on the nitrogen atom, an aminosulfonyl optionally substituted by Y and Z on the nitrogen atom, a nitro group, or a cyano group. X can be halogen, cyano, hydroxy, nitro, amino, alkylamino, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, optionally substituted C 1-9 Alkyl, optionally substituted C 1-9 Cycloalkyl, optionally substituted C 1-9 Alkenyl, optionally substituted C 1-9 cycloalkenyl, aryl optionally substituted, heteroaryl optionally substituted, aralkyl optionally substituted, heteroaralkyl optionally substituted, heterocycloalkyl optionally substituted, or heterocycloalkenyl optionally substituted; the aforementioned optionally substituted groups may be substituted by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, heterocycloalkyl, aryl, heteroaryl, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 Y and Z can each independently be hydrogen, optionally substituted C 1-9 Alkyl, optionally substituted monocyclic or bicyclic C 1-9 Cycloalkyl, optionally substituted C 1-9 Alkenyl, optionally substituted monocyclic or bicyclic C 1-9cycloalkenyl, aryl optionally substituted, heteroaryl optionally substituted, aralkyl optionally substituted, heteroaralkyl optionally substituted, heterocycloalkyl optionally substituted, or heterocycloalkenyl optionally substituted; any of the optionally substituted groups may be substituted by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 or Y and Z may be linked to form a ring having 3 to 10 ring atoms optionally with a substituent; the optionally substituted group may be substituted by halogen, cyano, hydroxyl, oxo, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 Alkyl polyoxypropylene substitution.

[0179] In some cases, R in the compound of Formula 2 may be hydrogen, methyl or trifluoromethyl. In some specific cases, R in the compound of Formula 2 may be hydrogen or methyl. For example, R in the compound of Formula 2 is hydrogen.

[0180] In other cases, Ar in the triazolotriazine compound of Formula 2 is 1 It can be an imidazolyl, triazolyl, tetrazolyl, furyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, or triazinyl group which may have a substituent; any of the substituents may be substituted by halogen, oxo, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0181] In other cases, Ar in the compound of Formula 2 1 is selected from the aromatic groups in Table 2. For example, Ar in the compound shown in Formula 2 1 It is 2-furyl.

[0182] Table 2 Structure of Ar1 in Formula 2

[0183]

[0184]

[0185] In some cases, Ar in the compound of Formula 22 It may be imidazolyl, triazolyl, tetrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, phenyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindolyl, azaindazolyl, purinyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, benzisoxazolyl, benzisothiazole, benzoxazolyl, benzothiadiazole, quinolyl, isoquinolyl, quinoxalinyl, phthalazine, quinazolinyl, cinnolinyl, naphthyridinyl, pyridopyrimidinyl, pyridopyrazine, or pteridinyl which may have a substituent; and the aromatic ring which may have a substituent may be substituted by halogen, hydroxy, cyano, nitro, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0186] In some cases, Ar in the compound of Formula 2 2 It may be phenyl, pyridyl, pyridazinyl or pyrimidinyl which may be optionally substituted; any of the aromatic rings which may be optionally substituted may be substituted by halogen, hydroxy, cyano, methyl, methoxy, trifluoromethyl or trifluoromethoxy.

[0187] In some cases, Ar in the compound of Formula 2 2 It may be phenyl or pyridyl which may be optionally substituted by halogen or hydroxy.

[0188] In some cases, Q in the compound of Formula 2 can be a monocyclic or bicyclic aromatic ring optionally substituted by X; X can be halogen, cyano, hydroxyl, nitro, amino, alkylamino, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, C optionally substituted 1-9 Alkyl, optionally substituted C 1-9 Cycloalkyl, optionally substituted C 1-9 Alkenyl, optionally substituted C 1-9 cycloalkenyl, aryl optionally substituted, heteroaryl optionally substituted, aralkyl optionally substituted, heteroaralkyl optionally substituted, heterocycloalkyl optionally substituted, or heterocycloalkenyl optionally substituted; the optionally substituted group may be halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, heterocycloalkyl, aryl, heteroaryl, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene, or C 1-3 Alkyl polyoxypropylene substitution.

[0189] In some cases, Q in the compound of Formula 2 can be a 5-6 membered aromatic ring optionally bearing a substituent X. X can be halogen, cyano, hydroxyl, nitro, amino, alkylamino, methoxy, ethoxy, trifluoromethoxy, trifluoroethoxy, C 1-5 Alkyl, optionally substituted C 1-5 Cycloalkyl, optionally substituted C 1-5 Alkenyl, optionally substituted C 1-5 Cycloalkenyl, aryl which may be substituted, heteroaryl which may be substituted, aralkyl which may be substituted, heteroaralkyl which may be substituted, heterocycloalkyl which may be substituted, or heterocycloalkenyl which may be substituted; the substituted groups are replaced by halogen, cyano, hydroxy, nitro, amino, alkylamino, cycloalkylamino, aminocarbonyl, sulfonyl, aminosulfonyl, carbonylamino, sulfonylamino, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, heterocycloalkyl, aryl, heteroaryl, polyoxyethylene, polyoxypropylene, C 1-3 Alkyl polyoxyethylene or C 1-3 Alkyl polyoxypropylene substitution.

[0190] In some cases, Q in the compound of Formula 2 may be a tetrazole ring optionally substituted by X. X may be a C 1-3 Alkyl or optionally substituted heterocycloalkyl; any of the optionally substituted groups may be substituted by halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, trifluoromethyl, trifluoroethyl, trifluoromethoxy, trifluoroethoxy, heterocycloalkyl, aryl, or heteroaryl.

[0191] The compounds described in the present application may exist in the form of one or more geometric isomers, enantiomers, diastereomers or tautomers. The compounds described in the application may include all these forms of isomers, including racemates and mixtures of other forms.

[0192] In some cases, the compounds described herein may exist in the form of solvates or ansolvates. The term "solvate" is used herein to describe a compound complex comprising a compound of the invention and a pharmaceutically acceptable solvent molecule such as water and ethanol molecule. The compounds described in the present application include all solvates or ansolvates thereof.

[0193] In some cases, the compounds described herein may exist in the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a physiologically or toxicologically tolerable salt, and when appropriate, includes pharmaceutically acceptable base addition salts and acid addition salts thereof. The compounds described herein include all pharmaceutically acceptable salts thereof. For example, the pharmaceutically acceptable salts may include benzenesulfonates and / or acetates.

[0194] In some cases, the compound described herein can exist in the form of pharmaceutically acceptable nanoparticles. The nanoparticles comprising the compound can be designed to improve the pharmacokinetics and biodistribution performance of the drug. For example, the compound can be wrapped in a liposome, which may extend the life of the drug in the body distribution process. Because the nanoparticles will preferentially leak out in the porous blood vessels around the tumor cells, the nanoparticles of appropriate size can also have better safety. Doing so also helps to reduce the effective dose of the drug.

[0195] In some cases, the compounds described in the present application can exist in the form of prodrugs. The term "prodrug" generally refers to a compound that can be converted into the present application compound by a metabolic process in the body (e.g., by hydrolysis, reduction or oxidation). The compounds described in the present application include all forms of prodrugs thereof.

[0196] In some cases, the compounds described herein also include pharmaceutically acceptable isotopic variants, i.e., one or more atoms are replaced by atoms having the same atomic number but different atomic masses. Atoms suitable for such isotopic replacement include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine. Isotopic variants of some of the compounds, such as deuterium-substituted compounds, may have better therapeutic effects in some cases because of better metabolic stability. Those skilled in the art can prepare isotopic variants of the compounds using conventional techniques known in the art.

[0197] In the present application, the compound may include the following compounds, or pharmaceutically acceptable salts thereof:

[0198] Table 3 Exemplary compounds of the present application

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Preparation of the compounds of the present application

[0205] The compounds described in the present application can be prepared by various synthetic methods. As illustrative examples, two synthetic routes for preparing the target compound are listed in synthetic route (1). In the first method, after obtaining intermediate (1A) by a suitable preparation method, the mesyl group on intermediate (1A) is substituted with an alkylamino group to obtain triazolotriazine compound (1C). In the second method, after obtaining intermediate (1B) by a suitable method, the phenoxy group on intermediate (1B) is substituted with an alkylamino group to obtain triazolotriazine compound (1C).

[0206]

[0207] Synthetic route 1

[0208] The intermediates (1A) and (1B) required in the synthetic route (1) can also be prepared by various synthetic methods. As an illustrative example, a synthetic route for the intermediate compound (1A) is shown in the synthetic route (2). In the first step, an appropriate aryl hydrazide (2A) is reacted with S-methylisothiourea (2B) in an aqueous sodium hydroxide solution to obtain an intermediate (2C). In the subsequent steps, (2C) is first fully heated in an aqueous medium to obtain an intermediate (2D), and the intermediate (2D) is reacted with N-cyanodithioimidocarbonate (2E) to obtain a sulfide intermediate (2F). Thereafter, the intermediate (2F) is oxidized with meta-chloroperbenzoic acid to obtain the desired intermediate sulfone (1A). As shown in the synthetic route (1), the target compound (1C) can be obtained by nucleophilically replacing the methylsulfonyl group with a suitable alkylamine (J. Chem. Soc., Perkin Trans. 1 1995, 801-808; Structural Chemistry. 2013, 24, 1241-1251).

[0209]

[0210] Synthetic route 2

[0211] The intermediate (2D) in the synthetic route (2) can also be prepared by various synthetic methods. As an illustrative example, the synthetic route (3) shows three synthetic routes of the intermediate (2D). It is worth emphasizing that among these three methods, the synthetic route starting from the methyl ester or ethyl ester (3F) can generally improve the efficiency and achieve a higher reaction yield.

[0212]

[0213] Synthetic route 3

[0214] The intermediate (1B) in the synthetic route (1) can be prepared by the synthetic route shown in the synthetic route (4). The preparation starts with cyanuric chloride (4A), which is refluxed in phenol to obtain 2,4,6-triphenoxy-1,3,5-triazine (4B). The next step is to react with hydrazine hydrate to obtain 2-hydrazine-4,6-diphenoxy-1,3,5-triazine (4C), which is reacted with a suitable acyl chloride to obtain acyl hydrazide (4D). Then, hydrazide (4D) is subjected to a cyclization reaction under dehydration conditions to obtain 2-substituted 5,7-diphenoxy triazolotriazine (4E). In the last step, (4E) is refluxed in methanolic ammonia to obtain the key intermediate (1B) (J. Chem. Soc., Perkin Trans. 1 1995, 801-808). As shown in the route (1), the target compound (1C) is obtained by reacting (1B) with a suitable alkylamine.

[0215]

[0216] Synthetic route 4

[0217] The compounds described in the present application can bind to adenosine receptors, for example, they can bind to adenosine A2A receptors. The compounds described in the present application can bind to free adenosine receptor proteins, and can also bind to adenosine receptors on the surface of cells. The compounds described in the present application can selectively bind to adenosine receptors, for example, the ability of the compounds of the present application to bind to adenosine A2A receptors is at least 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or more than that of the compounds of the present application to bind to adenosine A1, A2B and A3 receptors. For example, using radioisotope ligand competitive binding technology, the IC of the compounds of the present application binding to adenosine A2A receptors is 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or more. 50 The values ​​are the IC values ​​for binding to adenosine A1, A2B, and A3 receptors 50 The value is at least 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or more.

[0218] The compounds described in the present application can inhibit the activity of adenosine receptors, for example, they can inhibit the activity of adenosine A2A receptors. The compounds described in the present application can inhibit the activity of free adenosine receptor proteins, and can also inhibit the activity of adenosine receptors on the cell surface. The compounds described in the present application can selectively inhibit the activity of adenosine receptors. For example, the ability of the compounds of the present application to inhibit the activity of adenosine A2A receptors is at least 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or more than the ability to inhibit the activity of adenosine A1, A2B and A3 receptors. For example, using the cAMP accumulation assay or the calcium flow assay, the IC of the compounds of the present application for inhibiting the activity of adenosine A2A receptors is 2.5 times, 1.5 times, 2.5 times, 3.5 times, 4.5 times, 5.5 times, 6.5 times, 7.5 times, 8.5 times, 9.5 times, 10.5 times, 11.5 times, 12.5 times, 13.5 times, 14.5 times, 15.5 times, 16.5 times, 17.5 times, 18.5 times, 19.5 times, 20.5 times, 21.5 times, 22.5 times, 23.5 times, 24.5 times, 25.5 times, 26.5 times, 27.5 times, 28.5 times, 29.5 times, 30.5 times, 31.5 times, 32.5 times, 34.5 times, 35.5 times, 36.5 times, 37.5 times, 38.5 times, 39.5 times, 40.5 times, 50The IC values ​​are for inhibition of adenosine A1, A2B, and A3 receptor activity. 50 The value is at least 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 300 times, 500 times or more.

[0219] Uses and methods

[0220] Parkinson's disease

[0221] The compounds described in the present application can be used to prepare drugs for treating diseases, wherein the diseases may include Parkinson's disease.

[0222] In another aspect, the present application provides a method for treating Parkinson's disease, which may include the following step: administering a therapeutically effective dose of the compound described in the present application to a subject in need thereof.

[0223] In certain embodiments, the subject may exhibit early signs of Parkinson's disease, and the method comprises confirming that the patient exhibits early signs of Parkinson's disease, and regularly administering to the patient an amount of the compound or a pharmaceutically acceptable solvate or salt thereof determined thereby, to effectively treat the patient.

[0224] Early signs may be structural or functional changes in the brain, which may be detectable, for example, physically, by PET and SPECT studies, by transcranial sonography (Becker, J Neurol 249, Suppl 3. 2002, III / 40); Prunier C, et al., Neuroimage. 2003 Jul; 19(3): 810-6) or by detecting biochemical markers such as neuromelanin (WO 02 / 31499).

[0225] In certain embodiments, the speed of the Parkinson's disease progression of the subject can be quantified by the Total Unified Parkinson's Disease Rating Scale (Total UPDRS) score. Early signs may also be olfactory disorders, depression, visual disorders, and cognitive impairment or sleep disorders, and a combination of different tests can be used for early diagnosis (Becker, J Neurol 249, Suppl 3, 2002 III / 40; Stern, Annalsof Neurol 56, 2004, 169).

[0226] In certain embodiments, the subject may exhibit functional decline of Parkinson's disease, which generally refers to a worsening of symptoms of Parkinson's disease over time as determined by the total UPDRS score.

[0227] In certain embodiments, the subject may exhibit Parkinson's disease fatigue, and the functional decline is generally referred to as fatigue in Parkinson's disease patients as determined by the total UPDRS score.

[0228] In certain embodiments, the subject may exhibit non-motor symptoms of Parkinson's disease, which are generally non-motor symptoms of Parkinson's disease patients determined by the total UPDRS score. Non-motor symptoms of Parkinson's disease may include autonomic nervous system dysfunction, neuropsychiatric disorders (including mood, cognition, behavior and thought changes), sensory disorders and sleep disorders, etc.

[0229] In certain embodiments, the subject may or may not have previously received therapy for Parkinson's disease, including medication, rehabilitation (such as physical training, improved gait, slow rotation of limbs or trunk, stretching exercises, oral guidance, Lee Silverman speech therapy, stepping exercises, environmental modification, rhythmic activation, abdominal breathing and meditation), palliative care, diet therapy, repetitive transcranial magnetic stimulation, cauterization, deep brain stimulation surgery, and pallidotomy.

[0230] In certain embodiments, the subject may or may not have previously received drugs for the treatment of Parkinson's disease. Drugs for the treatment of Parkinson's disease may include, but are not limited to, L-dopa, dopamine receptor agonists, monoamine oxidase inhibitors, other drugs such as amantadine and anticholinergics. Drugs for the treatment of Parkinson's disease may include: L-dopa, bromocriptine, benztropine, levodopa, ropinirole, pramipexole, rotigotine, cabergoline, safinamide, sildenafil, rasagiline, entacapone, tolcapone, amantadine, pergolide, apomorphine, lisuride or selegiline

[0231] In certain embodiments, the treatment may include reducing the rate of progression of the subject's Parkinson's disease and / or delaying the clinical progression of the subject's Parkinson's disease. After starting to administer the compound described herein or a pharmaceutically acceptable solvate or salt thereof, the rate of disease progression is a decrease in the average increase in the total UPDRS score.

[0232] In certain embodiments, the treatment may include delaying the need for symptomatic treatment of the subject's Parkinson's disease. In certain embodiments, the treatment may include determining that the patient is at a stage of Parkinson's disease and regularly administering to the patient an amount of the compound or a pharmaceutically acceptable solvate or salt thereof determined thereby, effectively delaying the need for symptomatic treatment of Parkinson's disease.

[0233] In certain embodiments, the treatment may include reducing the risk of the subject needing symptomatic treatment for Parkinson's disease. In certain embodiments, the treatment includes regularly administering to a Parkinson's disease patient an amount of the compound described herein or a pharmaceutically acceptable solvate or salt thereof, effectively reducing the risk of needing treatment for Parkinson's disease.

[0234] In certain embodiments, the treatment may include reducing the functional decline of the subject. In certain embodiments, the method includes determining that the patient is in a Parkinson's disease stage, and regularly administering to the patient the compound or a pharmaceutically acceptable solvate or salt thereof in an amount determined thereby, effectively reducing the functional decline. In certain embodiments, the functional decline of the Parkinson's disease patient is quantified by the total Unified Parkinson's Disease Rating Scale (total UPDRS) score, and an increase in the total UPDRS score represents functional decline.

[0235] In certain embodiments, the treatment may include reducing the fatigue of the subject. In certain embodiments, the method includes determining that the patient is at a stage of Parkinson's disease, and regularly administering to the patient an amount of the compound described herein or a pharmaceutically acceptable solvate or salt thereof determined thereby, effectively reducing fatigue.

[0236] In certain embodiments, the treatment may include reducing the severity of the subject's motor symptoms. The motor symptoms may mainly include tremor (such as resting tremor), limb rigidity, slow movement, postural instability, hypokinesia, slow movement, posture, speech and swallowing abnormalities, etc.

[0237] In certain embodiments, the treatment may include reducing the severity of the non-motor symptoms of the subject. In certain embodiments, the method includes determining that the patient is at a stage of Parkinson's disease and periodically administering to the patient an amount of the compound or a pharmaceutically acceptable solvate or salt thereof determined thereby, effective to reduce the severity of the non-motor symptoms. In certain embodiments, the non-motor symptoms are defined by the Unified Parkinson's Disease Rating Scale (UPDRS) Fourth Edition, Part I.

[0238] The total UPDRS (unified Parkinson's disease rating scale) score represents the severity of Parkinson's disease and is used to measure the change from baseline in efficacy parameters during treatment. The UPDRS consists of the following tests:

[0239] The UPDRS consists of the following parts: Part I: Assessment of Psychological, Behavioral, and Emotional, Part II: Self-assessment of Activities of Daily Living (ADL), including speech, swallowing, handwriting, dressing, hygiene, falls, drooling, bed rest, walking, and cutting food, Part III: Clinician-rated Monitoring Motor Assessment, Part IV: Treatment Complications, Part V: Hoehn and Yahr Stages of Parkinson's Disease Severity, Part VI: Schwab and England ADL Scale.

[0240] The MDS-UPDRS published by the Movement Disorders Society can also be used to score Parkinson's disease. It contains four parts with a total of 50 subscales, namely; (1) non-motor experience of daily life (13 items), (2) motor experience of daily life (13 items), (3) physical movement examination (18 items), and (4) motor complications (6 items). Each subscale is scored on a scale of 0-4, where 0 = normal, 1 = mild, 2 = mild, 3 = moderate, and 4 = severe. Completing each item results in a score of 0-4, where 0 represents no impairment and 4 represents the highest degree of impairment.

[0241] The compounds described herein can reduce the subject's score on one or more UPDRS subscale items from 4 points to 3 points, 4 points to 2 points, 4 points to 1 point, 4 points to 0 points, 3 points to 2 points, 3 points to 1 point, 3 points to 0 points, 2 points to 1 point, 2 points to 1 point, or 1 point to 0 points.

[0242] The compound of the present application can prolong the walking distance, walking time, number of upright times and upright time of the subject.

[0243] For example, compared to not administering the compound of the present application, the walking distance of a subject using the compound of the present application can be extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, as tested in a reserpine-induced mouse movement disorder model.

[0244] For example, compared to not administering the compound of the present application, the walking time of a subject using the compound of the present application can be extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, as detected in a reserpine-induced mouse movement disorder model.

[0245] For example, compared to not administering the compound of the present application, the number of upright times of a subject using the compound of the present application can be extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, as tested in a reserpine-induced mouse movement disorder model.

[0246] For example, compared to not administering the compound of the present application, the upright time of a subject using the compound of the present application can be extended by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, as tested in a reserpine-induced mouse movement disorder model.

[0247] The compound of the present application can reduce the time a subject spends on the stick, increase the time a subject spends standing, and effectively improve the symptoms of stiffness in the subject.

[0248] For example, compared to not administering the compound of the present application, the time on the rod of a subject administered with the compound of the present application can be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, as measured in the haloperidol-induced rat spasticity model of Parkinson's disease.

[0249] For example, compared to not administering the compound of the present application, the standing time of a subject using the compound of the present application can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, as detected in the haloperidol-induced rat Parkinson's disease spasticity model or the MPTP-induced mouse Parkinson's disease model.

[0250] For example, compared to not administering the compound of the present application, the walking distance of a subject using the compound of the present application can be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300% or more, as detected in an MPTP-induced mouse Parkinson's model.

[0251] In the present application, in the treatment of Parkinson's disease, the compound can be administered at a dose of about 0.001 to about 500 mg / kg, about 0.001 to about 500 mg / kg, about 0.01 to about 500 mg / kg, about 0.1 to about 500 mg / kg, about 1 to about 500 mg / kg, about 0.001 to about 400 mg / kg, about 0.001 to about 300 mg / kg, about 0.001 to about 200 mg / kg, about 0.001 to about 100 mg / kg, about 0.001 to about 50 mg / kg, about 1 to about 200 mg / kg, about 1 to about 100 mg / kg, about 1 to about 50 mg / kg, about 1 to about 30 mg / kg, about 10 to about 30 mg / kg, about 3 to about 30 mg / kg or about 5 to about 30 mg / kg. The compound may be administered at a dose of about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 30 mg / kg, about 50 mg / kg, or about 100 mg / kg.

[0252] The compounds described in the present application can inhibit the drug resistance of a subject to Parkinson's drugs, for example, can inhibit the drug resistance of a subject to levodopa (L-DOPA) or Benserazide.

[0253] Tumor

[0254] The compounds described in the present application can also be used to prepare drugs for treating or preventing cancer in a host and related diseases related to abnormal cell proliferation in the host, wherein the host can be any multicellular vertebrate, including humans and non-human mammals. The host can be humans. The diseases include tumors. The tumors can include solid tumors and non-solid tumors.

[0255] In the present application, the solid tumor may include colon cancer and melanoma. In the present application, the non-solid tumor may include lymphoma, for example, B-cell lymphoma.

[0256] In the present application, in the treatment of tumors, the compound can be administered at a dose of about 0.001 to about 500 mg / kg, about 0.001 to about 500 mg / kg, about 0.01 to about 500 mg / kg, about 0.1 to about 500 mg / kg, about 1 to about 500 mg / kg, about 0.001 to about 400 mg / kg, about 0.001 to about 300 mg / kg, about 0.001 to about 200 mg / kg, about 0.001 to about 100 mg / kg, about 0.001 to about 50 mg / kg, about 1 to about 200 mg / kg, about 1 to about 100 mg / kg, about 1 to about 50 mg / kg, about 1 to about 30 mg / kg, about 10 to about 30 mg / kg, about 3 to about 30 mg / kg or about 5 to about 30 mg / kg. The compound may be administered at a dose of about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 30 mg / kg, about 50 mg / kg, or about 100 mg / kg.

[0257] The compounds described in the present application can be used to enhance the anti-tumor activity of host immune cells. The compounds can reduce the immune anergy of T cells or the tolerance of T cells to cancer, can make cancer cells more susceptible to immune clearance, can inhibit the proliferation of regulatory T cells, and can contribute to the formation of memory T cells. The compounds can enhance the host's own immune response and also enhance its efficacy in various adaptive immunotherapies.

[0258] For example, the drug of the present application can inhibit or delay the development or progression of the disease, can reduce the size of the tumor (or even substantially eliminate the tumor), and / or can alleviate and / or stabilize the disease state. Examples of inhibiting tumor or tumor cell growth include: the tumor growth volume is reduced relative to the corresponding level before the intervention.

[0259] In another aspect, the present application provides a method for treating tumors, which may include the following step: administering a therapeutically effective dose of the compound described in the present application to a subject in need thereof.

[0260] In order to make it more effective, the compounds described in the present application can also be used in combination with other anti-tumor treatment methods (such as chemotherapy, tumor vaccines and various immune checkpoint inhibitors) to achieve a synergistic effect. The term "combination therapy" refers to the simultaneous administration of active agents, which can also be administered in different orders.

[0261] In certain embodiments, the method for treating or preventing abnormal proliferation of cells in a host may include co-administering or alternating the compound described herein and an immune checkpoint inhibitor to the patient. The immune checkpoint inhibitor here may be selected from the following group: PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, BTLA inhibitors, LAG3 inhibitors, TIM-3 inhibitors, B7-H3 inhibitors, B7-H4 inhibitors, KIR inhibitors, TIGIT inhibitors or VISTA inhibitors, etc. For example, the other anti-tumor treatment methods may be the PD-1 inhibitor.

[0262] In another embodiment, the method for treating or preventing abnormal proliferation of cells in a host may include administering to a patient a combination or alternation of the compounds described herein and a cell vaccine. The cell vaccine is based on cells that match the tumor to be prevented. For example, if a host suffers from prostate cancer, or is at risk of prostate cancer, the cell vaccine will be based on prostate cancer cells. In this case, the cells are usually irradiated with a certain amount of radiation, or otherwise deprived of their replication function. Cells may also be genetically modified to secrete colony stimulating factors.

[0263] In another embodiment, the method for treating or preventing abnormal cell proliferation comprises administering the compound in combination or alternatingly to a patient with chimeric antigen receptor CAR-T cell therapy.

[0264] In another embodiment, the method for treating or preventing abnormal cell proliferation may include administering the compound in combination or alternating administration to a patient with another anticancer drug to treat abnormal cell proliferation. The anticancer drug may be an alkylating agent, an antimetabolite, an anthracycline derivative, a plant alkaloid, a topoisomerase inhibitor, an antitumor antibiotic, a kinase inhibitor, or a monoclonal antibody against a tumor antigen, etc.

[0265] The compounds of the present application have good pharmacokinetic properties, for example, the compounds of the present application have high exposure and bioavailability. In certain embodiments, the compounds of the present application have high oral in vivo exposure and high oral bioavailability. For example, the blood T 1 / 2 It is about 0.1h to about 10h, for example, about 0.2h to about 10h, about 0.3h to about 10h, about 0.4h to about 10h, about 0.5h to about 10h, about 0.5h to about 9h, about 0.5h to about 8h, about 0.5h to about 7h, about 0.5h to about 6h, about 0.5h to about 5h, about 0.5h to about 3h, about 0.5h to about 2h, about 0.5h to about 1h.

[0266] Pharmaceutical composition

[0267] When taking medicine, the compound of the present application can be formulated into a suitable pharmaceutical composition. The specific composition of the pharmaceutical composition is determined by the selected route of administration, which includes oral, parenteral, intravenous, intramuscular, nasal, oral, topical, transdermal or subcutaneous. In the treatment of diseases (such as Parkinson's disease or tumors), the dosage of the compound described in the present application contained in the pharmaceutical composition should be sufficient to play an effective therapeutic role without causing serious toxic side effects to the host. Daily medication or medication every few days can be used, and the duration of medication can last for several days, weeks, months, or even years. A certain specific dose can be divided into multiple interval administrations, such as once a day, twice a day or more, once a week, once every two weeks, once every three weeks, once a month, or once every two months or more. For example, the drug can be applied once a day or twice a day.

[0268] The compound can be administered at intervals before or after other anti-tumor treatment methods. The time of the interval can be 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or longer. In some embodiments, the compound described herein can be administered with the same administration route as other anti-tumor treatment methods or with different administration routes.

[0269] The compound can be administered at intervals before or after other drugs for the treatment of Parkinson's disease. The time of the interval can be 1 minute, 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or longer. In some embodiments, the compound described herein can be administered with the same administration route as other drugs for the treatment of Parkinson's disease or with different administration routes.

[0270] In certain embodiments, the compound can be administered orally. The ingredients of oral drugs generally include an inert diluent or an edible carrier. The drug can be encapsulated in a gelatin capsule or compressed into a tablet. Tablets, pills, capsules, lozenges, and other dosage forms may contain the following ingredients: a binder such as microcrystalline cellulose, tragacanth, or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; a flavoring agent such as mint, methyl salicylate, or orange flavoring; a wetting agent or emulsifier; a preservative; and a pH buffer, such as a phosphate buffer, sodium acetate, or sorbitan monolaurate. When the pharmaceutical composition is placed in a capsule, a liquid carrier such as a fatty oil may also be placed therein. In addition, the pharmaceutical composition may also contain various other materials, such as a sugar coating, shellac, or other enteric solvents.

[0271] The compounds of the present application can also be administered in the form of elixirs, suspensions, syrups, cakes, or chewing gums. In addition to the triazolotriazine derivatives, the syrups may also contain sweeteners such as sucrose, preservatives, colorants, and flavoring agents. Pharmaceutical solutions or suspensions for oral, parenteral, intradermal, subcutaneous, or topical administration may include sterile diluents such as water, saline, Ringer's solution, fixed oils, polyethylene glycols (such as PEG400), glycerol, propylene glycol, fatty acids such as oleic acid and its derivatives, polar solvents such as dimethylacetamide (DMAC) or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid; buffers such as phosphate buffer, acetic acid, citric acid, or phosphates, and tonicity or osmotic pressure regulators such as sodium chloride or glucose, stabilizers such as cyclodextrins, surfactants such as hydroxystearate, and the like. Drug stock solutions can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0272] In order to prevent the drug from being rapidly removed from the body, the compounds described herein may also be placed in a carrier that can provide such protection. Other methods of controlling the rate of drug release may also be used, such as methods using implants and microcapsule delivery systems.

[0273] The compounds described herein can also be administered using a nebulizer, dry powder inhaler, or metered dose spray inhaler. Drugs administered in this manner can be formulated into solutions using saline, to which benzyl alcohol or other suitable preservatives, absorption enhancers, fluorocarbons, and other commonly used solubilizing or dispersing agents are added.

[0274] Generally speaking, the treatment regimen and dosage for a patient will depend on a variety of factors, including the properties of the specific compound used, the patient's age, weight, general health, sex, diet, time of administration, excretion rate, the patient's pathological condition, the goals of treatment and the physician's judgment. If combined therapy is used, the amount of active ingredient used will also depend on which drug is used in the combined therapy.

[0275] In another aspect, the present application provides a pharmaceutical combination and / or kit comprising (1) the compound described herein, and (2) immunotherapy for treating tumors.

[0276] In another aspect, the present application provides a pharmaceutical combination and / or kit comprising (1) the compound, and (2) a drug for treating Parkinson's disease.

[0277] Without intending to be bound by any theory, the following examples are merely intended to illustrate the compounds, preparation methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0278] Example

[0279] In the examples of the present application, the names of the compounds correspond to the following Table 4:

[0280] Table 4 Compounds in Examples of this Application

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] Example 1 Preparation and Characterization of 2-(Furan-2-yl)-N5-(4-(Oxetane-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine Acetate (Compound 22 Acetate)

[0287] A. Preparation of 2-(furan-2-yl)-N5-(4-(oxetane-3-ylamino)phenethyl)-[1,2,4]triazole[1,5-a][1,3,5]triazine-5,7-diamine acetate is as follows:

[0288]

[0289] 500 mg of free base (A05746-093A15) was dispersed in 7.5 mL of ethyl acetate. 115 mg of acetic acid was dissolved in 2.5 mL of ethyl acetate to obtain an acetic acid solution. The acetic acid solution was slowly added dropwise to the suspension of the free base under stirring. Stir overnight, filter to obtain a solid, and dry overnight under vacuum at 40°C. The obtained product was subjected to characterization tests.

[0290] According to the same steps, methyl tert-butyl ether (MTBE), acetone (Acetone), methyl acetate (MAC), and isopropyl alcohol (IPA) were used to obtain the target solid and perform characterization tests.

[0291] B. Analytical Methods

[0292] 1) X-ray powder diffraction (XRPD)

[0293] Solid samples were analyzed using an Empyrean X-ray powder diffractometer (PANalytical) or a D8 Advance X-ray powder diffraction analyzer (Bruker). The Empyrean X-ray powder diffractometer was equipped with a PIXcel1D detector. In the XRPD analysis, the 2θ scanning angle was from 3 to 40°, and the scanning step was 0.013°. The light tube voltage and light tube current were 45KV and 40mA, respectively. The D8 Advance X-ray powder diffraction analyzer was equipped with a LynxEye detector. The 2θ scanning angle was from 3 to 40°, and the scanning step was 0.02°. The light tube voltage and light tube current were 40KV and 40mA, respectively.

[0294] 2) Differential Scanning Calorimetry (DSC)

[0295] The instrument model for differential scanning calorimetry analysis is DSC 250 (TA Instruments, US). The sample is accurately weighed and placed in a DSC perforated sample pan, and the exact mass of the sample is recorded. The sample is heated from 25°C to the final temperature at a heating rate of 10°C / min.

[0296] 3) Thermogravimetric analysis (TGA)

[0297] The model of the thermogravimetric analyzer is TGA 55 (TA Instruments, US). The sample is placed in a balanced open aluminum sample pan, and the mass is automatically weighed in the TGA heating furnace. The sample is heated to the final temperature at a rate of 10°C / min.

[0298] 4) Dynamic Vapor Sorption (DVS)

[0299] project parameter Sample temperature 25℃ Cycle time 10min Minimum time per cycle 50min Maximum time per cycle 120min Weight Limit 100％ Equilibrium conditions 0.01% / 45min Loop #1 Humidity 0% to 0%, temperature 40℃ for 3 hours Loop #2 The temperature is 25℃ and the humidity rises from 0% to 90% Loop #3 Temperature is 25℃, humidity returns from 80% to 0% Adsorption 0，10，20，30，40，50，60，70，80，90 Desorption 80，70，60，50，40，30，20，10，0

[0300] project parameter Step time 60min Sample temperature 25℃ cycle Full cycle Adsorption 0，10，20，30，40，50，60，70，80，90 Desorption 80，70，60，50，40，30，20，10，0 Save data rate 5S Total flow 200sccm Total flow after test 200sccm

[0301] 5) Polarized light microscopy (PLM)

[0302] The instrument used for PLM analysis was ECLIPSE LV100POL polarizing microscope (Nikon, Japan).

[0303] 6) Nuclear Magnetic Resonance (1H-NMR)

[0304] 1 H-NMR was performed using a Bruker AVANCE III HD 300 or 400 equipped with a Sample Xpress 60 autosampler or a Bruker Advance 300 equipped with a B-ACS 120 autosampler.

[0305] 7) High Performance Liquid Chromatography (HPLC)

[0306] The HPLC analysis method uses an Agilent HPLC 1260 series instrument. The HPLC method for solubility and stability testing is as follows:

[0307]

[0308] C. Data and Conclusion

[0309] According to XRPD results ( Figure 1 ) It can be seen that acetate is obtained in all solvents, which has a crystal form different from the free base. The product obtained by the reaction in MTBE still contains a small amount of free base crystal form. The acetate obtained in different solvents has the same crystal form.

[0310] From the 1H-NMR spectrum ( Figure 2 ), combined with XRPD spectra ( Figure 1 ) It can be seen that acetone, ethyl acetate, methyl acetate and isopropanol can all become acetates.

[0311] DSC, TGA results ( Figure 3 ) shows that the sample loses 13.4% of its weight before 210°C, which is presumably due to the removal of acetic acid. A broad endothermic peak is observed at an onset temperature of 148.9°C, which is due to the removal of acetic acid. The endothermic peak at an onset temperature of 226.0°C is presumably due to the dissolution of the free base anhydrous crystalline form.

[0312] 4) Solubility

[0313] The solubility of the free base and acetate salt forms in biological solvents (SGF (Simulated Gastric Fluid), FaSSIF (Fasted State Stimulated Intestinal Fluid) and FeSSIF (Fed State Stimulated Intestinal Fluid)) at 37°C was determined. 3 mg of sample was added to a sample bottle and 1 mL of biologically relevant solvent was added. The suspension was shaken at 500 rpm at 37°C. After 0.5 h and 2 h, 500 μL of the suspension was taken, filtered, and the filtrate was tested by HPLC to obtain the solubility.

[0314] Table 5 Solubility of free base and acetate salt of compound 22

[0315]

[0316] The results in Table 5 show that the solubility of the free base and acetate salt of compound 22 is greatly related to the pH of the solvent. They have the highest solubility in SGF, with the acetate salt being >2.887 mg / mL and the free base being 0.712 mg / mL. The solubility of the acetate salt in the three biologically relevant solvents is greater than that of the free base.

[0317] 5) Stability results and conclusions

[0318] A certain amount of acetate crystals were placed at 60°C closed and 40°C / 75% RH open for up to 7 days. Samples were taken at 0, 3 and 7 days and diluted to ~0.3 mg / mL with diluent for HPLC purity test. Solid samples were subjected to XRPD test to determine the crystal form.

[0319] Table 6 XRPD results of compound 22 acetate

[0320]

[0321] The results in Table 6 show that no significant degradation was found for the acetate salt of compound 22 under the test conditions. The acetate salt crystal form was physically and chemically stable at 60°C. However, under 40°C / 75%RH conditions, part of the acetate salt crystal form was converted into the free base crystal form.

[0322] Example 2 N 5 Preparation of -(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine benzenesulfonate (Compound 40 benzenesulfonate)

[0323] The preparation reaction is as follows:

[0324]

[0325] Benzenesulfonic acid hydrate (4 g, 25.4 mol) was added to methanol (18 mL), and N 5 -(4-(2-methyl-2H-tetrazol-5-yl)phenethyl)-2-(furan-2-yl)-[1,2,4]triazole[1,5-a][1,3,5]triazine-5,7-diamine (2.56 g, 6.35 mol), the solid was dissolved and added in batches. After the addition was completed, a small amount of solid was found to be insoluble, which was completely dissolved after ultrasonication. After stirring for 5 minutes, a large amount of white solid precipitated. 50 mL of ethyl acetate was added to slurry, and the target compound was filtered and dried to obtain a white solid (2.0 g, 56% yield). 1 H NMR (500 MHz, DMSO-d 6 )δ9.04-8.71 (m, 1H), 8.25 (d, J=0.7Hz, 2H), 8.02-7.96 (m, 3H), 7.61 (dd, J=7.7, 1.8Hz, 2H), 7.46 (t, J=9.7H z.2H), 7.39-7.26 (m, 3H), 7.19 (d, J=2.8Hz, 1H), 6.75 (s, 1H), 4.42 (s, 3H), 3.65-3.53 (m, 2H), 2.96 (t, 2H).

[0326] Example 3 The compounds of the present application can inhibit the activity of adenosine receptor A2A

[0327] 1. Experimental Methods

[0328] The inhibitory effect of the test compounds on the adenosine receptor A2A was evaluated by a TR-FRET-based cAMP accumulation assay. The method includes NECA-stimulated cAMP production and its inhibition by A2A receptor antagonists in HEK293 cells expressing A2AR (hADORA2A-HEK293). All cells were cultured in complete medium at 5% C02 and 37°C. After trypsinization, the cells were collected by centrifugation at 200g for 5 minutes. After resuspending the cells in fresh complete medium, the cell viability was counted by trypan blue exclusion. Subsequent cAMP production measurement experiments were performed only when the cell viability was greater than 95%. After diluting the cells with Hank's buffer solution of HEPES (5mM), BSA stabilizer (0.1%) and Rolipram (10μM), the cells were added to a 384-well white non-transparent plate (cells / well, 10μl / well) and incubated with the test compound at appropriate concentrations (11 concentrations) at room temperature for 20 minutes. Then, the A2A receptor agonist NECA (final concentration = EC80, which was determined in the same experiment performed earlier) was added to the sample and incubated again at 37°C for 30 minutes. Finally, Eu-cAMP tracer and Ulight-anti-cAMP were added, and the amount of cAMP produced was determined by measuring the ratio of TR-FRET at 665nm to the fluorescence emission at 615nm. ZM241385 was selected as the reference compound and Istradefylline was selected as the positive control compound. Istradefylline is a selective A2A receptor antagonist that can improve the motor function of PD patients by changing the activity of neurons. It is clinically used to treat PD and improve movement disorders in PD.

[0329] 2. Calculation

[0330] The inhibition rate (%) was calculated according to the following formula, and the IC50 value was calculated from the concentration-inhibition (%) curve after log conversion.

[0331]

[0332] %Inhibitor: % inhibition rate;

[0333] Ratio 665 / 615 high : High fluorescence ratio of reference compound;

[0334] Ratio 665 / 615 low : Low fluorescence ratio of reference compound;

[0335] Ratio 665 / 615 cmpd : Fluorescence ratio of test compounds;

[0336] XLfit software was used to fit the dose response curve and determine the IC 50.

[0337] 3. Results and Conclusions

[0338] Table 7 The activity of the compounds of the present application in inhibiting adenosine receptor A2A

[0339]

[0340] From the data in the above table, it can be seen that the compounds in the present application in the list all exhibit excellent A2A receptor antagonism, IC 50 At a lower level, about 10 nM. The A2A receptor inhibitory activity was higher than that of the positive control compound Istradefylline.

[0341] Example 4 The compounds of the present application selectively inhibit adenosine receptors

[0342] 4.1 cAMP accumulation assay to detect the inhibitory activity of the compounds of the present application on other adenosine receptors

[0343] The inhibitory effects of test compound 22 and Istradefylline (positive control drug) on ​​three other adenosine receptors, A2b, A1, and A3, were evaluated by TR-FRET-based cAMP accumulation assay.

[0344] 1. Experimental Methods

[0345] (1) Gs-coupled adenosine receptor A2b cAMP experiment:

[0346] In this experiment, HEK293 cells stably expressing adenosine receptor A2b were used as the research object (PerkinElmer, ES-013-C). Cells were cultured in EMEM medium (ATCC, 30-2003) containing 10% FBS (Hyclone, SH30406.05), 100 U / mL penicillin-streptomycin mixture (Gibco, 15140-122) and 100 μg / mL G418 (Gibco, 11811031). TrypLE TMThe cells were digested and passaged about 3 times a week using the Cell-Transcriptase Express (Gibco, 12604-013) and maintained at about 70% to 90% confluence. On the day of the experiment, the cells were seeded into a 384-well microplate (Corning, 3570) at a density of 500 cells per well in 10 μL cAMP assay buffer. The cAMP assay buffer was prepared by adding 75 μL 1 M HEPES (Gibco, 15630080), 200 μL 7.5% BSA stabilizer (PerkinElmer, TRF0263) and 7.5 μL 20 mM rolipram (Sigma, R6520) to 14 mL 1×HBSS (Gibco, 14025-076). 1 mM (1000× final concentration) of reference agonist NECA (Sigma, E2387) and reference inhibitor CVT-6883 (Aobious, AOB4675) were prepared and then diluted 3-fold in 100% DMSO (Millipore, 1029312500). Similarly, 10 mM of the test compound was also diluted 3-fold in 100% DMSO. The gradient diluted reference compound and test compound were added to a 384-well compound plate (Labcyte, PP-0200). Use Echo (Labcyte, 550) to transfer 10 nL of the gradient diluted reference agonist NECA per well to the test plate containing the cells, and the test plate was incubated at 37°C for 30 minutes. Add 5 μL of Eu-cAMP tracer working solution per well and 5 μL of ULight-anti-cAMP working solution per well (PerkinElmer, TRF0263) to the test plate, and incubate the test plate at 25°C for 30 minutes. Use EnVision microplate reader (PerkinElmer, 2009-0030) to read the fluorescence signal value (λex=320nm, λem=615nm and 665nm) of each well in the test plate, and calculate its EC50 value and EC80 value using the output fluorescence signal ratio (Ratio 665 / 615) and concentration of the reference agonist NECA. Use 100% DMSO to prepare 1000×EC80 NECA. Use Echo to transfer 10nL of gradient dilutions of reference inhibitor CVT-6883 and test compounds per well to the test plate containing cells. After incubating the test plate at 25°C for 20 minutes, transfer 10 nL of 1000×EC80 NECA per well to the test plate. Incubate the test plate at 37°C for 30 minutes. Add 5 μL of Eu-cAMP tracer working solution per well and 5 μL of ULight-anti-cAMP working solution per well to the test plate, and incubate the test plate at 25°C for 30 minutes.The fluorescence signal ratio of each well in the test plate is read, and the IC50 value is calculated using the output data of the reference inhibitor and the test compound and the concentration of the compound.

[0347] (2) Gi-coupled adenosine receptor A1 cAMP experiment:

[0348] In this experiment, a CHO cell line stably expressing adenosine receptor A1 was used as the research object (PerkinElmer, ES-010-C). The cells were cultured in F12 medium (Gibco, 11765-062) containing 10% FBS (Hyclone, SH30406.05), 100 U / mL penicillin-streptomycin mixture (Gibco, 15140-122) and 400 μg / mL G418 (Gibco, 11811031). TrypLE TMThe cells were digested and passaged about 3 times a week using 1x HBSS (Gibco, 12604-013) and maintained at about 70% to 90% confluence. On the day of the experiment, the cells were seeded into a 384-well microplate (Corning, 3570) at a density of 2000 cells per well. The solution system for cell seeding was 10 μL cAMP assay buffer. The cAMP assay buffer was prepared by adding 75 μL 1M HEPES (Gibco, 15630080), 200 μL 7.5% BSA stabilizer (PerkinElmer, TRF0263) and 7.5 μL 20 mM rolipram (Sigma, R6520) to 14 mL 1×HBSS (Gibco, 14025-076). 1 mM (1000× final concentration) of reference agonist NECA (Sigma, E2387) and reference inhibitor DPCPX (Sigma, C101) were prepared and then diluted 3-fold in 100% DMSO (Millipore, 1029312500). Similarly, 10 mM of the test compound was also diluted 3-fold in 100% DMSO. 1 mM forskolin (Sigma, F3917) was prepared in 100% DMSO. The gradient dilutions of reference compound, test compound and 1 mM forskolin were added to a 384-well compound plate (Labcyte, PP-0200). Use Echo (Labcyte, 550) to transfer 10 nL per well of 1 mM forskolin and 10 nL per well of the gradient dilution of reference agonist NECA to the test plate containing cells, and the test plate was placed at 37°C and incubated for 30 minutes. Add 5 μL of Eu-cAMP tracer working solution per well and 5 μL of ULight-anti-cAMP working solution per well (PerkinElmer, TRF0263) to the test plate, and incubate the test plate at 25°C for 30 minutes. Use EnVision microplate reader (PerkinElmer, 2009-0030) to read the fluorescence signal value (λex=320nm, λem=615nm and 665nm) of each well in the test plate, and calculate its EC50 value and EC80 value using the data and concentration of the output reference agonist NECA. Use 100% DMSO to prepare 1000×EC80 NECA. Use Echo to transfer 10nL of gradient dilutions of reference inhibitor DPCPX and test compounds per well to the test plate containing cells, and incubate the test plate at 25°C for 20 minutes. Transfer 10nL of 1000×EC80 NECA per well and 10nL of 1mM forskolin per well to the test plate. The test plate was incubated at 37°C for 30 minutes.Add 5 μL of Eu-cAMP tracer working solution and 5 μL of ULight-anti-cAMP working solution to the test plate, and incubate the test plate at 25°C for 30 minutes. Read the fluorescence signal ratio of each well in the test plate, and calculate the IC50 value using the output data of the reference inhibitor and the test compound and the concentration of the compound.

[0349] (3) Gi-coupled adenosine receptor A3 cAMP experiment:

[0350] Similarly, experiments were conducted using a CHO cell line stably expressing adenosine receptor A3 (PerkinElmer, ES-012-C). The IC50 values ​​of the compounds were calculated.

[0351] 2. Data Analysis

[0352] 1) Gs-coupled adenosine receptor A2b cAMP experiment:

[0353]

[0354] %Inhibitor: % inhibition rate;

[0355] Ratio High Control : High fluorescence ratio of reference compound;

[0356] Ratio Low Control : Low fluorescence ratio of reference compound;

[0357] Ratio Compound : Fluorescence ratio of test compounds;

[0358] 2) Gi-coupled adenosine receptor A1 and A3 cAMP assay:

[0359]

[0360] %Inhibitor: % inhibition rate;

[0361] Ratio High Control : High fluorescence ratio of reference compound;

[0362] Ratio Low Control : Low fluorescence ratio of reference compound;

[0363] Ratio Compound : Fluorescence ratio of test compounds;

[0364] 3. Experimental results

[0365] Table 8 Inhibitory activity of the compounds of the present application on adenosine receptors

[0366]

[0367] Combined with the data in Example 3, compounds 22, 27, and 40 inhibit adenosine A2A receptor ADORA 2A Active IC 50 The IC for inhibiting adenosine A2b, A1 and A3 receptor activity is about 10 nM. 50 The results showed that under the experimental conditions used in this experiment, compounds 22, 27, 40 and istradefylline all had an inhibitory effect on adenosine ADORA 2A It exhibited selective inhibitory activity superior to the other three adenosine receptors.

[0368] 4.2 Calcium flux assay to determine the selective inhibitory activity of the compounds of the present application on adenosine receptors

[0369] 1. Test principle:

[0370] This experiment used a stably transfected cell line that highly expressed adenosine receptors coupled to human Gq, and evaluated the antagonistic function of compound 22 by testing the changes in intracellular calcium ion concentration. 2A R agonists and A 2A After binding to R, it can activate the downstream pathway and increase the intracellular calcium ion concentration. Antagonists will competitively occupy the agonist binding A 2A R binding site, thereby interrupting the conduction of intracellular signals and causing changes in intracellular calcium ion concentrations. By detecting the strength of intracellular calcium ion signals, the effect of compounds on human ADORA 1 、ADORA 2A 、ADORA 2B and ADORA 3 Adenosine receptor antagonist activity.

[0371] 2. Cell line information:

[0372] Target Reference sequence Host cells Culture conditions ADORA1 NM_000674 HEK293 DMEM+10%FBS; G418 300μg / mL; BS: 2μg / mL ADORA2A NM_000681 CHO F12+10%FBS; G418 300μg / mL; BS: 2μg / mL ADORA2B NM_000676.2 HEK293 DMEM+10%FBS; G418 300μg / mL; BS: 2μg / mL ADORA3 NM_000677 CHO F12+10%FBS; G418 300μg / mL; BS: 2μg / mL

[0373] 3. Reagents and consumables:

[0374]

[0375]

[0376] instrument model factory FLIPR FT-0249 Molecular devices Echo Echo555 Labcyte Cell Counter AN16097 Beckman

[0377] 5. Test methods:

[0378] Preparation of cell plates:

[0379] 1) Take one tube of cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath.

[0380] 2) Add the cell suspension to a centrifuge tube that has been pre-warmed with 20 mL of culture medium.

[0381] 3) Centrifuge at 1000 rpm for 5 minutes at room temperature.

[0382] 4) Slowly pour off the supernatant to avoid disturbing the settled cells.

[0383] 5) Resuspend the cells in 10-30 mL of culture medium and gently pipette the cells.

[0384] 6) Use a cell counter to calculate the cell viability and number.

[0385] 7) Dilute the cells to 1.0×10 6 cells / mL with culture medium, seed 20 μL / well into a 384-well poly-lysine-coated cell plate, and incubate in a 5% CO 2 , 37° C. incubator for 16-20 hours.

[0386] Note: Cell viability must reach more than 90% before use in the experiment.

[0387] Preparation of Fluo4-Direct dye:

[0388] Prepare 250 mM probenecid solution: According to the kit instructions, add 1 mL of FLIPR buffered saline to 77 mg of probenecid and prepare it immediately before use.

[0389] Prepare 2× (8 mM) Fluo-4 DirectTM loading buffer: melt the number of tubes of Fluo-4 DirectTM for the experiment in advance, add 10 mL of FLIPR buffered saline to each tube, add 0.2 mL of 250 mM Probenecid solution, vortex and shake in the dark for > 5 minutes, and prepare it before use.

[0390] Compound preparation and experimental operation:

[0391] 1) During the whole experiment, the compound was diluted 30000 / 900*6=200 times

[0392] Preparation of compounds: The four compounds to be tested were prepared into 10 mM stock solutions with DMSO and stored in a 4°C refrigerator.

[0393] 2) Preparation of agonist reference compounds: The agonist was diluted 1:4 using Echo, 10 points, left and right duplicate wells, and then 900 nL was transferred to the corresponding compound plate; then 30 μL of experimental buffer saline solution was added to the corresponding compound plate.

[0394] 3) Reference and test compound plate preparation: 2mM (10mM stock solution was diluted 5 times to 2mM with DMSO) test compound was used for a 10-point dose response curve with a 1:4 gradient dilution. The reference compound CGS-15943 was used for a 10-point dose response curve with a 1:4 gradient dilution using Echo. The starting concentration of CGS-15943 for ADORA1, ADORA2B and ADORA3 receptors was 200μM, and the starting concentration of CGS-15943 for ADORA2A receptor was 20μM.

[0395] 4) Add 30 μL of FLIPR buffered saline to the corresponding compound plate and set aside.

[0396] 5) Take out the cell plate prepared the day before from the incubator, add 20 μL 2×Fluo-4DirectTM buffer to each well, incubate in a 37°C incubator with 5% CO2 for 50 minutes, and place at room temperature away from light for 10 minutes.

[0397] 6) Place the compound plate, cell plate and pipette tips in the FLIPR instrument.

[0398] 7) Run the FLIPR instrument software, add 10 μL of the experimental buffer solution to the cell plate according to the set program, and read the fluorescence signal. Then add 10 μL of the agonist reference compound of the established concentration to the cell plate and read the fluorescence signal. After reading, export the data through the "Max-Min" and "Read 91 to Maximum allowed" methods in the software, calculate the EC80 of each cell line, and prepare 6×EC80 concentrations of agonists.

[0399] 8) Prepare the reference compound agonist of the corresponding cell at a concentration of 6×EC80 with a buffered saline solution, add 30 μL / well to the corresponding compound plate and set aside.

[0400] 9) Run the FLIPR instrument software, add 10 μL of the test compound and reference compound of the established concentration to the cell plate according to the set program, and read the fluorescence signal. Then add 10 μL of the reference compound agonist at 6×EC80 concentration to the cell plate and read the fluorescence signal. For the antagonist detection of the compound, export the data through the "Max-Min" and "Read 1 to 90" methods in the software.

[0401] 10) Data analysis was performed using Prism statistical software.

[0402] The formula for analyzing data is as follows:

[0403] Antagonist % Inhibition = 100-(RLU-LC) / (DMSO-LC)*100

[0404] RLU: relative light absorbance, reading value from 1 to 90;

[0405] HC: average fluorescence signal of DMSO group;

[0406] LC: average fluorescence signal at the highest antagonist concentration.

[0407] The data were fitted using “log(inhibitor) vs response-variable slope” in GraphPad Prism 5.0 to obtain the IC50.

[0408] 5. Results and Conclusions:

[0409] Table 9 Inhibitory activity of the compounds of the present application on adenosine receptors

[0410]

[0411] The results showed that compounds 22 and 40 had no significant effect on ADORA 2A The inhibitory ability of mediated signal transduction is stronger, and the inhibitory ability of the other three adenosine receptors is weaker, that is, the inhibitory ability of ADORA 2A The inhibition is highly selective.

[0412] Example 5 The compounds of the present application can bind to adenosine receptors

[0413] 1. Test Principle

[0414] This experiment used radioisotope ligand competition binding technology to detect the binding effects of the test compound and the reference compound CGS-15943 on adenosine receptors ADORA1, ADORA2A, ADORA2B, and ADORA3 at the biochemical level.

[0415] Reagents and consumables Part Number supplier Human adenosine ADORA1 receptor membrane protein ES-010-M400UA PerkinElmer Human adenosine ADORA2A receptor membrane protein RBHA2AM400UA PerkinElmer Human adenosine ADORA2B receptor membrane protein ES-013-M400UA PerkinElmer Human adenosine ADORA3 receptor membrane protein ES-012-M400UA PerkinElmer [3H]DPCPX NET974001MC PerkinElmer [3H]CGS-21680 NET1021250UC PerkinElmer [3H]HEMADO ART1456 ARC CGS-15943 C199-25MG Sigma NECA E2387 Sigma PEI P3143 Sigma Microscint 20 scintillation fluid 6013329 PerkinElmer Top Seal-A Sealing Film 6005250 PerkinElmer 96-well conical polypropylene plate 5042-1385 Agilent Unifilter-96 GF / C filter plate 6005174 PerkinElmer

[0416] instrument model factory Harvester C961961 PerkinElmer Microbeta2 2450 Microplate counter PerkinElmer

[0417] 4. Test methods

[0418] 1) Preparation of detection buffer:

[0419] ADORA1: 25mM HEPES pH 7.4, 5mM MgCl 2 , 1 mM CaCl 2 , 100 mM NaCl

[0420] ADORA2A: 50mM Tris-HCl pH 7.4, 10mM MgCl 2 , 1mM EDTA

[0421] ADORA2B: 50mM HEPES pH 7.0, 5mM MgCl 2 , 1mM EDTA

[0422] ADORA3: 25mM HEPES pH 7.4, 10mM MgCl 2 , 1 mM CaCl2, 0.5% BSA

[0423] 2) Preparation of lotion:

[0424] ADORA1: 25mM HEPES pH 7.4, 5mM MgCl 2 , 1 mM CaCl 2 , 100mM NaCl.

[0425] ADORA2A: 50mM Tris-HCl pH 7.4, 154mM NaCl

[0426] ADORA2B: 50mM HEPES pH 6.5, 5mM MgCl 2 , 1 mM EDTA, 0.2% BSA

[0427] ADORA3: 50 mM Tris-HCl pH 7.4

[0428] 3) Reaction system: 100 μL cell membrane, 100 μL isotope, 1 μL compound

[0429] 4) Compound preparation: dilute the test compound and reference compound with 100% DMSO. For the test compound: the starting concentration is 10 μM, double wells, 4-fold gradient dilution, 10 points, and the final working concentration is 0.038nM to 10μM (the second and third detections of the test compound for the ADORA2A target: the starting concentration is 2μM, double wells, 4-fold gradient dilution, 10 points, and the final working concentration is 0.0076nM to 2μM). For ADORA1, ADORA2A, ADORA2B, ADORA3 reference compounds: the starting concentration is 1μM, double wells, 4-fold gradient dilution, 10 points, and the final working concentration is 0.0038nM to 1μM.

[0430] 5) Cell membrane preparation: ADORA receptor cell membrane is prepared into corresponding concentration using detection buffer.

[0431] Target Cell membrane concentration isotope Final isotope concentration ADORA1 2.5 μg / well [3H]DPCPX 1nM ADORA2A 5μg / well [3H]CGS-21680 6nM ADORA2B 20 μg / well [3H]DPCPX 8nM ADORA3 2μg / well [3H]HEMADO 1nM

[0432] 7) IC50 determination of test compounds and reference compounds:

[0433] Add 1 μL of the sample to be tested and the reference compound to the 96-well plate in sequence. The high signal control wells are 0.5% DMSO, and the low signal control wells for ADORA1, ADORA2A, ADORA2B, and ADORA3 are CGS-15943. Add 100 μL of cells and 100 μL of isotope to all experimental wells. Seal the 96-well plate, incubate ADORA1, ADORA2B, and ADORA3 on a shaker (300 rpm) at room temperature (22°C) for 1 hour, and incubate ADORA2A on a shaker (300 rpm) at room temperature (22°C) for 2 hours. At the same time, soak the GF / C filter plate with 0.3% PEI for ADORA1, ADORA2A, ADORA2B, and ADORA3 at room temperature for at least 30 minutes, 50 μL / well. After incubation, the incubated reaction system was collected on the GF / C filter plate with a Harvester, washed 4 times and dried in a 50°C oven for 0.5 hours. The bottom of the dried GF / C filter plate was sealed with a film, 50 μL of scintillation fluid was added to each well, and sealed with a Top-seal-A film. Microbeta was used for reading and GraphPad Prism 5.0 was used for data analysis.

[0434] 5. Data Processing and Analysis

[0435] %Inhibition=100×(1-(Sample_raw value-Low control_Average) / (Highcontrol_Average-Low control_Average))

[0436] %Inhibitor: % inhibition rate;

[0437] Sample_raw value: raw reading of the test compound;

[0438] Low control_Average: low level reading of reference compound;

[0439] High control_Average: high level reading of reference compound;

[0440] The data were fitted using “log(inhibitor) vs response-variable slope” in GraphPad Prism 5.0 to obtain the IC50.

[0441] 6. Test results

[0442] Table 10 Adenosine receptor binding test results (IC50)

[0443]

[0444] Table 11 Adenosine receptor binding test results

[0445]

[0446] The results showed that compounds 22 and 40 had no significant effect on ADORA 2A All of them have extremely high binding effects, and the binding to the other three adenosine receptors is weak, indicating that the compounds of the present application have a strong affinity for ADORA 2A High binding selectivity.

[0447] Example 6 Effects of the compounds of the present application in the reserpine-induced dyskinesia model in mice

[0448] 6.1 Effect of compound 22 in the mouse reserpine-induced dyskinesia model

[0449] 1. Test Principle

[0450] Reserpine is an indole alkaloid drug that can inhibit the reuptake of noradrenergic neuron terminals, leading to slowed movement or inability, resulting in clinical symptoms similar to Parkinson's disease. Its mechanism is that reserpine can block the irreversible transport of monoamines such as DA, 5-hydroxytryptamine (5-HT) and norepinephrine (NA) in vesicles, causing monoamine substances to be degraded intracellularly, thereby consuming central and peripheral monoamines, rapidly reducing monoamine levels, and leading to Parkinson's disease-like symptoms such as movement disorders. A2A receptor antagonists were administered to evaluate whether the test compound could improve movement disorders in mice. Evaluation indicators included walking distance, walking time, number of upright times and upright time.

[0451] 2. Experimental Design

[0452] Group 1: solvent control group (Vehicle 1 + Vehicle 2), Vehicle 1 (0.1% acetic acid, 10 mL / kg, subcutaneous injection) + Vehicle 2 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastrically, administered 60 min before behavioral testing), n = 15;

[0453] Group 2: Model group (Reserpine+Vehicle2), Reserpine (0.6 mg / kg, 10 mL / kg, subcutaneous injection) + Vehicle2 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastric administration, 60 min before behavioral testing), n=15;

[0454] Group 3: Reserpine + Istradefylline group, Reserpine (0.6 mg / kg, 10 mL / kg, subcutaneous injection) + Istradefylline (10 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0455] Group 4: Reserpine + Compound 22 acetate group, Reserpine (0.6 mg / kg, 10 mL / kg, subcutaneous injection) + Compound 22 (3 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0456] Group 5: Reserpine + Compound 22 acetate group, Reserpine (0.6 mg / kg, 10 mL / kg, subcutaneous injection) + Compound 22 (10 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0457] Group 6: Reserpine + Compound 22 acetate group, Reserpine (0.6 mg / kg, 10 mL / kg, subcutaneous injection) + Compound 22 (30 mg / kg, 10 mL / kg, oral administration, 60 min before behavioral testing), n = 15;

[0458] Male CD1 mice weighed 29.5±0.1 g. A total of 90 animals were used. The animals were purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd. (Animal Qualification Certificate No.: 1100112011009984) and kept in the animal room of Shanghai Ruizhi Chemical Research Co., Ltd. (Experimental Animal Use License: SYXK (Shanghai) 2017-0018). The animal room was an SPF-grade environment.

[0459] name supplier model Laboras Animal Spontaneous Activity Platform Metris BV, Netherlands NA Reagents and consumables Article number / Batch number supplier Methylcellulose M0430-100G / SLCB9094 SIGMA Sodium acetate trihydrate G70149B / P1536840 GENERAL-REAGENT reserpine R007 / UQNCN-EH TCI Istradefylline I1100 / 2PJUO Tokyo Chemical Industry Co., Ltd.

[0460] 4. Experimental steps

[0461] 1) Compound preparation

[0462] A reserpine solution with a concentration of 0.06 mg / mL, an istradefylline solution with a concentration of 1 mg / ml, and acetate solutions of compound 22 (concentration 1) with concentrations of 3 mg / ml (concentration 1), 1 mg / ml (concentration 2), and 0.3 mg / ml (concentration 3) were prepared.

[0463] 2) Experiment

[0464] The day before the start of the experiment, the 3-channel timer, syringe, stainless steel workbench and work stool and related record forms required for the experiment should be placed in the test laboratory in advance for standby. After checking the animal status, no animals were found to be underweight, tail-broken, alopecia areata, or in poor physical condition. There were 90 experimental animals in total, and a random grouping sequence was generated before the experiment. According to the random sequence, the experimental animals were randomly divided into a solvent control group, a model group, an Istradefylline group, and a 3, 10, and 30 mg / kg compound 22 acetate group on the day of the experiment.

[0465] On the first day of the experiment, 0.1% acetic acid or 0.6 mg / kg reserpine was subcutaneously injected once according to the pre-set groups and time points. After the injection, the animals were returned to the cages and pushed back to the original feeding room.

[0466] Activity test was conducted on the second day of the experiment. Before the experiment, the Laboras system needed to be calibrated in advance to make it in normal working condition. Then, Vehicle 2 (acetic acid-sodium acetate (pH=4.0) buffer containing 0.5% MC), Istradefylline or Compound 22 acetate were administered orally according to the pre-set time point (17 hours after a single subcutaneous injection of 0.1% acetic acid or 1 mg / kg reserpine). 50 minutes after administration, the mice were placed in an independent Laboras test box, and the Laboras behavioral recording system was started after 10 minutes of adaptation. At this time, the animals were 1 hour after the last administration, and the recording was continuous for 60 minutes.

[0467] After the experiment, the experimental animals were returned to the breeding cages and the Laboras test cages were cleaned.

[0468] After the experiment, the Laboras behavior system automatically analyzed the walking distance (m), walking time (sec), number of upright times and upright time (sec) of the mice during 0-30min, 30-60min, 0-60min and every 10min.

[0469] All quantitative data were expressed as mean ± standard error (mean ± SEM) and analyzed using Prism 8.0 statistical software.

[0470] 5. Results and Conclusions

[0471] Table 12 Effects of the compounds of the present application on the walking distance of mice

[0472]

[0473]

[0474] Table 13 Effects of the compounds of the present application on spontaneous walking time of mice

[0475]

[0476] Table 14 Effects of the compounds of the present application on the number of upright standing times of mice

[0477]

[0478] The results are shown in Tables 12-14, which show that oral administration (OP) of 10 mg / kg and 30 mg / kg of Compound 22 acetate can significantly increase walking distance, walking time, and the number of upright times. The experiment showed that Compound 22 acetate has a therapeutic effect in the reserpine-induced mouse movement disorder model, and the efficacy has a clear dose-effect relationship. In addition, the control drug Istradefylline showed roughly the same efficacy in the experiment. The mouse reserpine-induced movement disorder model experiment supports the application of Compound 22 acetate in the treatment of Parkinson's disease.

[0479] 6.2 Effect of Compound 22 Acetate in the Rat Reserpine-Induced Dyskinesia Model

[0480] 1. Experimental Design

[0481] Group 1: solvent control group (Vehicle 1 + Vehicle 2): Vehicle 1 (0.1% acetic acid, 1 mL / kg, subcutaneous injection) + Vehicle 2 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastrically, administered 60 min before behavioral testing), n = 15;

[0482] Group 2: Model group (Reserpine+Vehicle2), Reserpine (1 mg / kg, 1 mL / kg, subcutaneous injection) + Vehicle2 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastric administration, 60 min before behavioral testing), n=15;

[0483] Group 3: Reserpine + Istradefylline group, Reserpine (1 mg / kg, 1 mL / kg, subcutaneous injection) + Istradefylline (15 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0484] Group 4: Reserpine + Compound 22 acetate group, Reserpine (1 mg / kg, 1 mL / kg, subcutaneous injection) + Compound 22 acetate (7.5 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0485] Group 5: Reserpine + Compound 22 acetate group, Reserpine (1 mg / kg, 1 mL / kg, subcutaneous injection) + Compound 22 acetate (15 mg / kg, 10 mL / kg, oral gavage, 60 min before behavioral testing), n = 15;

[0486] Group 6: Reserpine + Compound 22 acetate group, Reserpine (1 mg / kg, 1 mL / kg, subcutaneous injection) + Compound 22 acetate (30 mg / kg, 10 mL / kg, oral administration, 60 min before behavioral testing), n=15.

[0487] 2. The same method as in 11.1 was used to analyze the walking distance (m), walking time (sec), number of standing up times and standing up time (sec) of mice during 0-30min, 30-60min, 0-60min and every 10min.

[0488] 3. Results and Conclusions

[0489] Table 15 Effects of the compounds of the present application on walking distance of rats

[0490]

[0491] Table 16 Effects of the compounds of the present application on spontaneous walking time of rats

[0492]

[0493] Table 17 Effects of the test compounds on the number of upright times of rats

[0494]

[0495] The test results show that the compound of the present application can significantly increase the walking distance, walking time, upright time and number of animals at a dose of 15 mg / kg and 30 mg / kg. The test shows that compound 22 acetate has a therapeutic effect in the reserpine-induced rat movement disorder model, and there is a dose-effect relationship. In addition, the control drug istradefylline showed roughly the same efficacy in the test. The rat reserpine-induced movement disorder model test supports the application of compound 22 acetate in the treatment of Parkinson's disease.

[0496] Example 7 Effects of the compounds of the present application on the haloperidol-induced Parkinson's disease rigidity model in rats

[0497] Parkinson's disease (PD) is a common neurodegenerative disease. The main pathological changes are the loss of dopaminergic neurons in the substantia nigra and the reduction of dopamine transmitter content, which leads to a variety of clinical symptoms such as resting tremor, muscle rigidity, akinesia and postural reflex disorders in patients. Haloperidol, a dopamine D2 receptor blocker, can block dopamine D2 receptors in the striatum, leading to dystonic posture, i.e. rigidity and decreased movement in mice. This model is a recognized animal model of Parkinson's syndrome. The test substance was evaluated for its improvement on the rigidity symptoms of Parkinson's disease induced by Haloperidol in CD-1 mice.

[0498] 7.1 Effects of Compound 40 Benzenesulfonate, Compound 27 and Compound 22 Acetate on Haloperidol-induced Parkinson's Disease Rigidity Model in Rats

[0499] 1. Experimental Design and Methods

[0500] 1) Experiment 1:

[0501] 57 male CD-1 mice, SPF grade, 4 weeks old when purchased, 8 weeks old when tested, weighing about 40g, were randomly divided into 5 groups, namely Vehicle group (n=11), Compound 40 benzenesulfonate 1mg / kg group (n=11), Compound 40 benzenesulfonate 3mg / kg group (n=12), Compound 40 benzenesulfonate 10mg / kg group (n=12), Compound 40 benzenesulfonate 30mg / kg group (n=11). 1mg / kg Haloperidol was injected subcutaneously, the injection volume was 10ml / kg, and 30min after injection, the compound 40 was gavaged with each dose, the gavage volume was 10ml / kg, and the Rearing and Bar Tests were performed 1h and 4h after the completion of the administration, respectively, and the number of mice standing and the time on the bar were counted to evaluate the improvement effect of compound 40 on the rigidity symptoms of Parkinson's disease induced by Haloperidol in CD-1 mice.

[0502] 2) Experiment 2:

[0503] 40 male CD-1 mice, SPF grade, 4 weeks old when purchased, 5 weeks old when tested, weighing about 25g, were randomly divided into 5 groups, namely Vehicle group (n=8), Compound 22 acetate 3mg / kg group (n=8), Compound 22 acetate 10mg / kg group (n=8), Compound 22 acetate 30mg / kg group (n=8) and Istradefylline 10mg / kg (n=8). First, each dose of Compound 22 acetate was intragastrically administered, with an intragastrically administered volume of 10ml / kg. 30min later, 1mg / kg Haloperidol was subcutaneously injected, with an injection volume of 10ml / kg. Rearing and Bar Tests were performed 1h and 4h after administration, respectively, and the number of mice standing and the time on the bar were counted to evaluate the improvement of Compound 22 acetate on the Parkinson's disease rigidity symptoms induced by Haloperidol in CD-1 mice.

[0504] 3) Experiment 3:

[0505] 40 male CD-1 mice, SPF grade, 4 weeks old when purchased, 5 weeks old during the experiment, weighing about 25-30g, were randomly divided into 5 groups, namely Vehicle group (n=8), Compound 27 3mg / kg group (n=8), Compound 27 10mg / kg group (n=8), Compound 27 30mg / kg group (n=8) and Istradefylline 10mg / kg (n=8). First, each dose of Compound 27 was intragastrically administered with a volume of 10ml / kg. 30min later, 1mg / kg Haloperidol was subcutaneously injected with an injection volume of 10ml / kg. Rearing and Bar Tests were performed 1h and 4h after administration, respectively, and the number of mice standing and the time on the bar were counted to evaluate the improvement of Compound 27 on the Parkinson's disease rigidity symptoms caused by Haloperidol in CD-1 mice.

[0506] 2. Materials, reagents / compound information

[0507] Reagents and consumables Article number / Batch number supplier Methylcellulose P10995 adamas-beta Haloperidol H113852-5g / G1411016 Aladdin Sodium acetate P1246037 GENERAL-REAGENT glacial acetic acid P1480181 adamas-beta 2-Hydroxypropyl-β-cyclodextrin B1922043 Aladdin Istradefylline 2PJUO-EB TCI

[0508] 4. Experimental Procedure

[0509] 1) Drug preparation

[0510] Vehicle solvent control: 40% hydroxypropyl β-cyclodextrin solution

[0511] Weigh 20.00 g of hydroxypropyl β-cyclodextrin, add 50 ml of ultrapure water, vortex and sonicate for 20 min until the solution is clear, and store at room temperature for later use.

[0512] Vehicle solvent control: 0.5% MC in acetic acid-sodium acetate (pH ≈ 4.0) buffer

[0513] Prepare 2M acetic acid solution: Take 11.6mL of acetic acid in a 100mL measuring cylinder, then add ultrapure water to 100mL, which is 2M acetic acid, and store in a 100mL brown bottle.

[0514] Prepare acetic acid-sodium acetate (pH≈4.0) buffer containing 0.5% MC: weigh 0.0904 g of sodium acetate, add 1.95 mL of 2 M acetic acid, add to a 100 mL measuring cylinder, then add ultrapure water to about 80 mL, measure the pH with pH paper, about 3.5-4, then add ultrapure water to 100 mL, then add 0.5013 g of MC, mix until MC is completely dissolved, and store in a 100 mL brown bottle at room temperature.

[0515] Haloperidol preparation (dosing volume 10ml / kg)

[0516] Prepare before use: weigh 0.0037g Haloperidol, add 7.4ml 0.9% saline, vortex, add 20ul 1M HCl, vortex and sonicate at room temperature for 20min, mix well, the concentration is 0.5mg / ml, dilute 5 times to 0.1mg / ml as the working concentration, and store the remaining at room temperature away from light.

[0517] Preparation of Compound 40 benzenesulfonate solution (dosing volume 10 ml / kg)

[0518] Prepare before use, the correction coefficient is 1.415, weigh 0.0199g of the compound, add 4.7ml 40% HP-β-CD, vortex and ultrasonic at room temperature for 10min, mix well, and the working concentration is 3mg / ml; aspirate 1ml of the solution with a concentration of 3mg / ml, add 2ml40% HP-β-CD, and the working concentration is 1mg / ml; aspirate 0.2ml of the solution with a concentration of 3mg / ml, add 1.8ml 40% HP-β-CD, and the working concentration is 0.3mg / ml; aspirate 0.2ml of the solution with a concentration of 1mg / ml, add 1.8ml 40% HP-β-CD, and the working concentration is 0.1mg / ml.

[0519] Preparation of acetate solution of compound 22 (dosing volume 10 ml / kg)

[0520] Prepare before use, the correction coefficient is 1.153, weigh 0.0225g of the compound, add 6.5ml of acetic acid-sodium acetate buffer containing 0.5% MC, vortex and ultrasonicate at room temperature for 10min, mix well, and the working concentration at this time is 3mg / ml; aspirate 1ml of the solution with a concentration of 3mg / ml, add 2ml of acetic acid-sodium acetate buffer containing 0.5% MC, and the working concentration at this time is 1mg / ml; aspirate 0.2ml of the solution with a concentration of 3mg / ml, add 1.8ml of acetic acid-sodium acetate buffer containing 0.5% MC, and the working concentration at this time is 0.3mg / ml.

[0521] Compound 27 preparation (dosing volume 10 ml / kg)

[0522] Prepare before use, with a correction factor of 1.013. Weigh 0.0182 g of the compound, add 6 ml of 40% HP-β-CD, vortex and sonicate at room temperature for 10 minutes, mix well, and the working concentration is 3 mg / ml; aspirate 1 ml of a solution with a concentration of 3 mg / ml, add 2 ml of 40% HP-β-CD, and the working concentration is 1 mg / ml; aspirate 0.2 ml of a solution with a concentration of 3 mg / ml, add 1.8 ml of 40% HP-β-CD, and the working concentration is 0.3 mg / ml.

[0523] Istradefylline preparation (dosing volume 10 ml / kg)

[0524] Prepare it for immediate use, prepare 1 mg / ml, weigh 0.0040 g, add 4 ml of 40% HP-β-CD or acetic acid-sodium acetate buffer containing 0.5% MC, vortex and sonicate at room temperature for 10 minutes, and mix well.

[0525] 2) Haloperidol modeling

[0526] Haloperidol was injected subcutaneously in the abdomen of mice at a dose of 1 mg / kg, a working solution concentration of 0.1 mg / ml, and an injection volume of 10 ml / kg.

[0527] 3) Drug administration

[0528] Follow the experimental design.

[0529] 4) Rearing detection method

[0530] 1h and 4h after drug intervention, the mice were placed in a white transparent plastic bucket with a diameter of 20cm and a height of 21cm, and allowed to adapt to the bucket for 5min before recording. The spontaneous activities of the mice were recorded within 10min, and the number of standing times was counted. The more times the mice stood, the more frequent their spontaneous exploration activities and the lower their degree of rigidity.

[0531] 5) Bar Test

[0532] After the Rearing Test at 1h and 4h, the Bar Test was performed. The mice were placed on a long wooden bar with a diameter of 6cm and a height of 6cm. The mice held the bar with their forelimbs, with only their hips or hindlimbs touching the ground. The timing was stopped when the hindlimbs left the table or the forelimbs left the bar, and the time the mice spent on the bar was recorded. Each mouse was tested 3 times, and the maximum and average time on the bar were counted. The longer the time on the bar, the more severe the stiffness of the mouse.

[0533] 6) Statistical analysis

[0534] Prism GraphPad 7.0 was used to perform independent sample T test for statistical analysis of the data among the groups, and P < 0.05 was considered to be a significant difference.

[0535] 5. Experimental results and conclusions

[0536] Table 18 Effects of different doses of compound 40 benzenesulfonate on the behavioral test results of mice

[0537]

[0538]

[0539] Note: Compared with the Vehicle group, *P<0.05, **P<0.01, ***P<0.001

[0540] Table 18 shows that after the injection of Haloperidol, the mice showed hypoactivity, decreased spontaneous activity, and prolonged rigidity time; the behavior of the mice in the compound 40 benzenesulfonate group was significantly improved, the time on the rod was reduced, and the number of standing times increased, showing a certain dose-effect relationship.

[0541] Table 19 Effects of different doses of compound 22 acetate on the behavioral test results of mice

[0542]

[0543] Note: Compared with the Vehicle group, *P<0.05, **P<0.01, ***P<0.001

[0544] Table 19 shows that in the Bar Test, compound 22 acetate can reduce the time on the bar and prolong the standing time of mice, and the results show a good dose-effect dependence. As the dose of compound 22 acetate increases, the rigidity time decreases and the time on the bar shortens. The efficacy of the 10 mg / kg group and the 30 mg / kg group is equivalent to that of the positive drug.

[0545] Table 20 Effects of different doses of compound 27 on the behavioral test results of mice

[0546]

[0547]

[0548] Note: Compared with the Vehicle group, *P<0.05, **P<0.01, ***P<0.001

[0549] Table 20 shows that in the Bar Test, compound 27 can reduce the time on the bar and prolong the standing time of mice, and the results show a good dose-dependent relationship. As the dose of the compound increases, the rigidity time decreases and the time on the bar shortens. The efficacy of the 10 mg / kg group is equivalent to that of the positive drug, and the efficacy of the 30 mg / kg group is slightly better than that of the positive drug group.

[0550] The test results showed that in mice with haloperidol-induced rigidity, after a single oral administration of the three test compounds, the test mice were able to significantly reduce the rigidity time and shorten the time on the rod, indicating that the test compounds can counteract or prevent the rigidity symptoms induced by haloperidol, and there is a dose-effect relationship. In addition, the control drug istradefylline showed roughly the same efficacy in this test. The haloperidol-induced mouse Parkinson's disease rigidity model test supports the application of the test compounds in the treatment of Parkinson's disease.

[0551] 7.2 Effect of Compound 22 Acetate on Haloperidol-Induced Parkinson's Disease Rigidity Model in Rats

[0552] The effects of the acetate salt of the compound in the following dosing regimen were examined in the haloperidol-induced spasticity model of Parkinson's disease in rats.

[0553] 1. Experimental Design

[0554] Group 1: blank control group (Vehicle 1 + Vehicle 2), Vehicle 1 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastrically, administered 30 min before haloperidol injection) + Vehicle 2 (acetic acid aqueous solution, 5 mL / kg, intraperitoneally, administered before the test), n = 10;

[0555] Group 2: Model group (Vehicle 1 + Haloperidol), Vehicle 1 (acetic acid-sodium acetate containing 0.5% MC, 10 mL / kg, intragastric administration, 30 min before haloperidol injection) + Haloperidol (1 mg / kg, 5 mL / kg, intraperitoneal administration, administration before test), n = 10;

[0556] Group 3: Istradefylline + Haloperidol group: Istradefylline (10 mg / kg, 10 mL / kg, intragastrically, administered 30 min before Haloperidol injection) + Haloperidol (1 mg / kg, 5 mL / kg, intraperitoneally, administered before the test), n = 10;

[0557] Group 4: Compound 22 acetate + Haloperidol group: Compound 22 acetate (3 mg / kg, 10 mL / kg, intragastrically, administered 30 min before Haloperidol injection) + Haloperidol (1 mg / kg, 5 mL / kg, intraperitoneally, administered before the test), n = 10;

[0558] Group 5: Compound 22 acetate + Haloperidol group: Compound 22 acetate (10 mg / kg, 10 mL / kg, intragastrically, administered 30 min before Haloperidol injection) + Haloperidol (1 mg / kg, 5 mL / kg, intraperitoneally, administered before the test), n = 10;

[0559] Group 6: Compound 22 acetate + Haloperidol group: Compound 22 acetate (30 mg / kg, 10 mL / kg, oral administration, administered 30 min before Haloperidol injection) + Haloperidol (1 mg / kg, 5 mL / kg, intraperitoneal administration, administered before the test), n=10.

[0560] 2. Experimental Methods

[0561] The animals needed for the day were transferred from the feeding room to the operating room in advance, and the experiment was started after acclimatization for at least 30 minutes. According to the pre-set grouping, the experimental animals were divided into a blank control group, a model group, an Istradefylline group, and a 3, 10, and 30 mg / kg compound 22AC group. The experimental animals were orally administered with Vehicle 1 (acetic acid-sodium acetate (pH=4.0) buffer containing 0.5% MC), Istradefylline, or Compound 22 acetate, and then the animals were returned to the original feeding cages. After 30 minutes, the experimental animals were intraperitoneally injected with Haloperidol or Vehicle 2 without Haloperidol, and returned to the original feeding cages.

[0562] At 60 and 120 min after the injection of Haloperidol or Vehicle 2, the rat's forelimbs were gently placed vertically on a horizontal metal rod 20.5 cm long, 1.2 cm in diameter, and 8.5 cm above the workbench. The rat's hind limbs were then gently placed on the test box and the timing was started.

[0563] When the rat's forelimbs moved or climbed down the metal pole, the timing was stopped, and the duration of the rat's forelimbs maintaining the posture on the metal pole was recorded, which was recorded as the freezing latency. If the rat's forepaws did not put down, the observation was terminated at 600 seconds, and the freezing latency of the animal was recorded as 600 seconds. When the climbing pole test was completed on the same day, 5 animals from each group were perfused, fixed, and the whole brain was taken for preservation.

[0564] Freeze latency: the time that the rat's two forelimbs remain motionless on the metal rod. The longest freeze latency is 600 seconds.

[0565] Freeze rate % = (freeze latency / 600) * 100%

[0566] All quantitative data were expressed as mean ± standard error (mean ± SEM) and analyzed using Prism 8.0 statistical software.

[0567] 3. Results and Conclusions

[0568] Table 21 Effects of the compounds of the present application on the freezing latency of rats

[0569]

[0570] Table 22 Effects of the compounds of the present application on the rigidity rate of rats

[0571]

[0572]

[0573] The test results showed that in rats with haloperidol-induced spasticity, after a single oral administration of three doses of 3, 10 and 30 mg / kg of compound 22 acetate, the experimental rats were able to significantly shorten the spasticity latency (reduce the spasticity rate), indicating that compound 22 acetate can counteract or prevent the spasticity symptoms induced by haloperidol, and there is a dose-effect relationship. In addition, the control drug istradefylline showed roughly the same efficacy in the test. The haloperidol-induced rat Parkinson's disease spasticity model test supports the application of compound 22 acetate in the treatment of Parkinson's disease.

[0574] 7.3 Pharmacodynamic evaluation of compound 40 benzenesulfonate in the haloperidol-induced catalepsy model in Wistar rats

[0575] 1. Experimental Design and Methods

[0576] Five groups were set up in the experiment, including the modeling groups Vehicle, Compound 40 benzenesulfonate (3 mg / kg, 10 mg / kg, 30 mg / kg), and istradefylline 10 mg / kg, n=8, 8, 9, 8, 8. Vehicle, Compound 40 benzenesulfonate (3 mg / kg, 10 mg / kg, 30 mg / kg), and istradefylline 10 mg / kg were administered by gavage, and the administration volume was 10 mL / kg. After 30 minutes, intraperitoneal injection of Haloperidol 1 mg / kg was started, and the administration volume was 5 mL / kg. The number of standing times was tested by rearing 1 hour after modeling. After the rearing test, the rats rested for 30 minutes before the bar test.

[0577] 2. Experimental Procedure

[0578] 1) Drug preparation

[0579] Solvent selection: 40% HP-β-CD (2-hydroxypropyl)-β-cyclodextrin)

[0580] Vehicle solvent control (Ctrl): 40% HP-β-CD

[0581] 1.40% HP-β-CD: weigh 120.2 g and add about 250 mL ultrapure water. Stir magnetically at 900 rpm and completely dissolve at room temperature. Add ultrapure water to 300 mL and store in a 500 mL brown bottle at room temperature away from light.

[0582] Compound 40 benzenesulfonate: prepare immediately before use, prepare 3 mg / mL, weigh 0.1777 g, add 42.5 mL 40% HP-β-CD, vortex, ultrasonicate at room temperature for 10 min, blow evenly, then use a 20 mL syringe (replace with a 2.5 mL needle), blow evenly, prepare 1 mg / mL, dilute 3 times, prepare 0.3 mg / mL, dilute 10 times.

[0583] Istradefylline: Prepare immediately before use, prepare 1 mg / mL, weigh 0.0312 g, add 31.2 mL 40% HP-β-CD, and vortex.

[0584] Haloperidol induction: prepare immediately before use, prepare 0.2 mg / mL, weigh 0.0148 g, add 100 μL acetic acid, add 2 mL ultrapure water, add 390 μL 14 M NaOH, and pour to about 35 mL, measure pH (pen pH meter), pH = 5.86, add ultrapure water to 74 mL.

[0585] 2) Bar Test

[0586] Wistar rats were gavaged with different concentrations of compound 40 benzenesulfonate and istradefylline, with a dosing volume of 10 mL / kg. After 30 minutes, Haloperidol (1 mg / kg) was intraperitoneally administered, with a dosing volume of 5 mL / kg. One hour after Haloperidol administration, the Rearing test was performed on the number of standing times. After the Rearing test, the rats rested for 30 minutes before the Bar Test.

[0587] Bar test time: Test the rigidity of rats with the Bar test, which uses a wooden bar with a diameter of 8 mm, fixed between two iron racks, 10 cm above the tabletop. Then grab the rat's tail with your hands, so that its front paws are placed on the bar and its hind paws or buttocks are on the ground. Generally, rats need to adapt several times when tested for the first time. The test is used to test the time it takes for the forelimbs to drop the bar or the time it takes for the hind limbs to leave the tabletop. One rat is tested 3 times.

[0588] 3)Rearing

[0589] Number of standing: The number of standing of rats was tested by rearing, that is, the rats were placed in a 27*52cm cage, adapted for 5 minutes, and the number of standing of rats' spontaneous activities within 10 minutes was recorded. When the rat's hind legs were upright and both forelimbs were raised over the shoulders and touched the ground, it was considered as standing once, and one forelimb lifting was not counted. Usually, rats would raise their forelimbs to touch the cage wall. If the rat raised its forelimbs over the shoulders for several consecutive times without touching the ground, it was counted only once.

[0590] The Bar test time is used as a measurement indicator, including the average time and the maximum time. A long time indicates severe rigidity, and a short time indicates mild rigidity. After drug treatment, the shorter the Bar test time, the better the drug is in relieving rigidity.

[0591] Rearing counted the number of standing times. The more standing times, the more frequent the rat's spontaneous exploration activities and the lower the degree of rigidity.

[0592] 4) Statistical analysis

[0593] Prism GraphPad 7.0 was used to perform independent sample T test for statistical analysis of the data among the groups, and P < 0.05 was considered to be a significant difference.

[0594] 5. Results and Conclusions

[0595] Table 23 Effects of the compounds of the present application on the freezing time of rats

[0596]

[0597] Note: Compared with the Vehicle group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0598] The results in Table 23 show that, compared with the Vehicle group, the mean and maximum freezing time of rats in the 10 mg / kg and 30 mg / kg treatment groups of compound 40 benzenesulfonate and the 10 mg / kg istradefylline treatment groups had significantly shortened mean and maximum freezing time, and significantly increased the number of standing times, all with significant differences (the mean freezing time P values ​​after treatment were <0.0001, <0.0001, <0.0001, respectively; the maximum freezing time P values ​​were <0.0001, <0.0001, <0.0001, respectively; the number of standing times P values ​​were <0.0001, <0.0001, <0.0001, respectively). However, compared with the Vehicle group, the mean and maximum freezing time of rats in the 3 mg / kg treatment group of compound 40 benzenesulfonate had no significant statistical difference, but the number of standing times had significant differences (mean freezing time after treatment, P=0.2741; maximum freezing time after treatment, P=0.0521; number of standing times after treatment, P=0.0351). The haloperidol-induced Parkinson's disease rigidity model experiment in rats supports the application of compound 40 benzenesulfonate in the treatment of Parkinson's disease.

[0599] Example 8 Pharmacodynamic evaluation of the compounds of the present application in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse Parkinson's disease model

[0600] 1. Experimental Principle

[0601] By injecting the selective poison MPTP in the substantia nigra, lipid peroxidation is induced by interfering with mitochondria, resulting in disordered membrane structure and affecting cell function, which ultimately leads to selective destruction of substantia nigra DA neurons, resulting in massive death of substantia nigra DA neurons, massive loss of striatal tyrosine hydroxylase-positive fibers, and significant decreases in the levels of striatal DA and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. There is also hyperplasia of microglia and astrocytes in the substantia nigra and striatum, and damage to the locus coeruleus and hypothalamus, which are basically the same as the changes in patients with Parkinson's disease. Evaluate the effect of the test substance on the improvement of the symptoms of Parkinson's disease rigidity caused by MPTP-induced mice.

[0602] Group Number of animals Drug administration dose Dosage Dosing frequency Group 1 6 Vehicle N / A po once Group 2 7 Compound 22 10mpk po once Group 3 7 Compound 22 30mpk po once

[0603] 3. Experimental Procedure

[0604] C57BL / 6 male mice, 8 weeks old, were selected and 8-week-old C57 mice were injected intraperitoneally (ip) daily for 5 days with vehicle (normal saline) or MPTP (30 mg / kg body weight) dissolved in saline to induce Parkinson's disease.

[0605] Two doses (10 mpk and 30 mpk) were given by intragastrically administration 0.5 hours after the last MPTP injection.

[0606] One and a half hours after the last MPTP injection, a 10-min open field test (total distance traveled and number of rearings) was performed.

[0607] 4. Experimental results and conclusions

[0608] Group Movement distance (cm) Number of standing Model group (n=6) 139.5 0.83 Compound 22 (10 mg / kg) (n=6) 647.0 18.3 Compound 22 (30 mg / kg) (n=6) 1107.3 31.0

[0609] The results showed that in the MPTP-induced mouse Parkinson's model, compound 22 significantly alleviated the Parkinson's motor symptoms of mice at doses of 10 mg / kg and 30 mg / kg, and significantly improved the movement distance and the number of standing times, showing a dose-effect relationship and demonstrating good efficacy. The MPTP-induced mouse Parkinson's model experiment supports the application of the test compound 22 in the treatment of Parkinson's disease.

[0610] Example 9 Evaluation of the efficacy of the compounds of the present application in the 6-OHDA-induced rat rotation test and their effect on overcoming L-DOPA resistance

[0611] 1. Experimental Principle

[0612] After long-term use of levodopa, patients with Parkinson's disease are prone to drug resistance (wearing-off phenomenon), that is, the duration of its efficacy tends to gradually shorten (shortening of the on-time period), the efficacy will become weaker and weaker, and the patient's symptoms will become increasingly uncontrolled. By combining with the test compound, the efficacy of levodopa can be restored for patients with Parkinson's disease in the off-time period who have already experienced wearing-off phenomenon. The Parkinson's rat model was established by using the method of 6-hydroxydopamine (6-OHDA) to damage the substantia nigra and striatum by targeted injection. Then the animals were given levodopa (L-DOPA) / benserazide (BID) for a long time (planned 3-4 weeks). After the animals showed resistance to levodopa, that is, when the rotation time caused by levodopa was significantly reduced (after 3-4 weeks), the animals were given the test compound and levodopa / benserazide at the same time, and the rotation time of the animals was measured to investigate the recovery of the rat's rotation symptoms by the test compound.

[0613] 2. Experimental Design

[0614] Phase I: The same batch of model rats (a total of 15) were given different doses (0, 1, 3, 10, 30 and 100 mg / kg) of compound 22 acetate for rotation test. After counting with a rotation counter for 60 minutes, L-DOPA (5 mg / kg) / Benserazide (1.25 mg / kg) was intraperitoneally injected, and then the rotation counter was used again for rotation counting for 60 minutes. There was at least one day between the rotation tests of each dose. A total of 6 tests were conducted in the first phase.

[0615] Animal grouping and drug treatment in the first phase:

[0616]

[0617] Phase II: The experimental animals in groups 2, 3, 4, 5 and 6 were intraperitoneally injected with high doses of L-DOPA (25 mg / kg) / Benserazide (6.25 mg / kg) twice a day to induce drug tolerance. Five rotation tests were performed on days 0 (day 0 was the day before the high dose of L-DOPA / Benserazide was started), 7, 14, 21 and 22. On the day of the test, all experimental animals were intraperitoneally injected with L-DOPA (20 mg / kg) / Benserazide (5 mg / kg) once to induce rotation. On day 22, 60 minutes before the intraperitoneal injection of L-DOPA (20 mg / kg) / Benserazide (5 mg / kg), each experimental group was first orally administered with a vehicle, a test drug or a positive drug. In order to evaluate whether compound 22 acetate can prevent animals from developing resistance to L-DOPA / Benserazide, in addition to L-DOPA (25 mg / kg) / Benserazide (6.25 mg / kg) intraperitoneally twice a day, the test animals in groups 7 and 8 were orally administered with the test drug compound 22 acetate and the control drug Istradefylline twice a day, respectively, and four rotation tests were performed on days 0, 7, 14, and 21, respectively. On the test day, the test drug and the control drug were only orally administered once; on this day, all test animals were also intraperitoneally injected with L-DOPA (20 mg / kg) / Benserazide (5 mg / kg) once to induce rotation. In the second stage, the rotation counting time using the rotation counter was 180 minutes.

[0618]

[0619]

[0620] 3. Experimental reagents and animals:

[0621] Phase I: Wistar rats, 6-8 weeks, male, 178-213 g, experimental animal provider: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0006, quality certificate number: 1100112011004974;

[0622] Reagents and consumables Article number / Batch number supplier Methylcellulose 079K0054V Sigma-Aldrich glacial acetic acid / Tianjin Guangfu Technology Development Co., Ltd. Sodium acetate trihydrate WXBC8551V Sigma-Aldrich 6-Hydroxydopamine MKCH0942 Sigma-Aldrich L-Ascorbic Acid SLBS0713V Sigma-Aldrich Apomorphine Hydrochloride / China Inspection and Quarantine Institute Levodopa SLCB0627 Sigma-Aldrich Benserazide hydrochloride BCBZ1809 Sigma-Aldrich

[0623] 4. Experimental steps and methods

[0624] Parkinson's rat model:

[0625] L-DOPA injection amount: 4 μl of 6-OHDA solution (2.5 μg / μl) was injected into the left medial forebrain bundle (MFB) of each rat.

[0626] L-DOPA injection coordinates: With bregma as the standard reference point and referring to the rat brain stereotaxic atlas, the left medial forebrain bundle was injected: anterior (A), -2.5 mm; lateral (L), +2.0 mm; ventral (V), -8.5 mm.

[0627] L-DOPA injection speed: injection speed 1μl / min, leave the needle in situ for 5 minutes after injection, then slowly withdraw the needle at a speed of 1mm / min and suture the wound.

[0628] The groups and medication were determined according to the experimental design.

[0629] The animals were tested only on the number of rotations in the first phase and only on the rotation reaction time in the second phase.

[0630] In the second stage, after the experimental animals were intraperitoneally injected with L-DOPA / Benserazide, the total number of rotations was recorded every 5 minutes, and the number of rotations per 5 minutes was calculated accordingly. The 5 minutes with the most rotations were found, and the number of rotations within the 5 minutes was defined as the peak number of rotations. The first 5 minutes when the number of rotations increased to 20% of the peak number of rotations was the starting point of the rotation reaction time, and the first 5 minutes when the number of rotations decreased to 20% of the peak number of rotations was the ending point of the rotation reaction time. The time from the starting point to the ending point was calculated as the rotation reaction time.

[0631] The test results were expressed as "mean ± standard deviation". SPSS16.0 software package was used for statistical analysis of the data, and one-way ANOVA, paired sample T test and independent sample T test were used for comparison. P < 0.05 was considered statistically significant.

[0632] 6. Results and Conclusions

[0633] The results of the first phase are shown in Tables 25 and 26.

[0634] Table 25 Total rotations after administration of acetate salt of compound 22 alone

[0635]

[0636] Note: *P<0.05 compared with the vehicle group (Compound 22 acetate, 0 mg / kg).

[0637] Table 26 Total rotation number of compound 22 acetate combined with L-DOPA (mean ± standard deviation)

[0638]

[0639] Note: One hour after oral administration of acetate salt of compound 22, L-DOPA (5 mg / kg) / Benserazide (1.25 mg / kg) was intraperitoneally injected. Compared with the vehicle group (acetate salt of compound 22, 0 mg / kg), *P<0.05.

[0640] The first phase of the study was designed to evaluate the efficacy of different doses (0, 1, 3, 10, 30 and 100 mg / kg) of the test drug compound 22 acetate on 6-OHDA-induced contralateral rotation in rats.

[0641] The results showed that compared with the administration of the vehicle (Compound 22 acetate, 0 mg / kg), the number of rotation circles of the animals increased at high doses after administration of different doses of the test drug Compound 22 acetate alone, among which the test drug could significantly increase the number of rotation circles on the contralateral side at a dose of 30 mg / kg.

[0642] Although compound 22 acetate did not cause high rotations in animals at all doses when used alone, and the efficacy of L-DOPA / Benserazide alone at low doses was not obvious, the efficacy of compound 22 acetate combined with L-DOPA was significant when L-DOPA / Benserazide was kept at the same low dose. The test drug compound 22 acetate could increase the number of contralateral rotations caused by L-DOPA / Benserazide at all five doses in a dose-dependent manner. At doses of 30 mg / kg and 100 mg / kg, compound 22 acetate could significantly increase the number of contralateral rotations of animals.

[0643] The results of the second stage are shown in Table 27.

[0644] Table 27 Rotational reaction time of animals in each test group (mean ± standard deviation, min)

[0645]

[0646]

[0647] The results showed that after giving 6-OHDA Parkinson's model rats high doses of L-DOPA (25 mg / kg, BID) / Benserazide (6.25 mg / kg, BID), the rats developed resistance to L-DOPA on the 7th day, as evidenced by a significantly shortened L-DOPA-induced rotation reaction time, which lasted until the 22nd day when the experiment ended.

[0648] On the 22nd day, after a single administration of different doses of compound 22 acetate (7.5, 15 and 30 mg / kg) to 6-OHDA Parkinson's model rats that have developed drug resistance, the test drug can restore or increase the animals' rotation reaction time to varying degrees compared with the 0th and 21st days. The test drug can significantly increase the rotation reaction time at doses of 15 and 30 mg / kg (compared with the 21st day), and there is a dose-dependent relationship. This shows that after animals develop drug resistance to L-DOPA / Benserazide, compound 22 acetate can effectively restore or improve the original efficacy of L-DOPA in drug-resistant animals.

[0649] The test results also showed that the acetate of compound 22 may also effectively prevent or slow down the drug resistance of 6-OHDA Parkinson's model rats to L-DOPA / Benserazide.

[0650] In addition, compared with the model control group, there was no significant change in the body weight of the rats in each test group during the test, indicating that the test drug compound 22 acetate had no significant effect on the body weight of the test animals.

[0651] The model test shows that when Parkinson's disease patients develop drug resistance due to long-term use of levodopa, levodopa may restore its therapeutic efficacy for these patients with Parkinson's disease in the off-phase by combining it with the acetate of compound 22. This supports the application of the treatment of Parkinson's disease in the claims.

[0652] Example 10 Pharmacodynamic evaluation of the compounds of the present application in a subcutaneous homologous transplantation C57BL / 6 female mouse model of murine colon cancer MC38 cell line

[0653] 1. Test Principle

[0654] The MC38 mouse colon cancer cell model is a commonly used model for colon cancer drug efficacy validation. It is often used to study the occurrence and metastasis of colorectal cancer and has become a common way to validate tumor drug efficacy. The anti-tumor effect of the test drug in the subcutaneous homologous transplantation of the murine colon cancer MC38 cell line in female C57BL / 6 mice was evaluated.

[0655] 2. Experimental Design

[0656]

[0657] 3. Experimental Materials and Reagents

[0658] Experimental animals: C57BL / 6 mice, female, 7-9 weeks (age of mice at the time of tumor cell inoculation), weighing 16.4-20.1 g, purchased from Shanghai Lingchang Biotechnology Co., Ltd. (LC), production license number: SCXK (Shanghai) 2013-0018, animal certificate number: 2013001837385. Breeding environment: SPF grade.

[0659] Anti-PD-1 antibody: Batch number: 695318A1B, packaging specification: 18 mg, provided by BioXcell, colorless solution, stored at 4°C.

[0660] Solvent preparation: Weigh 16.8 g of hydroxypropyl-beta-cyclodextrin (Zibo Qianhui Biotechnology Co., Ltd., batch number: 160101), add 42 ml of sterile water, and set aside after complete dissolution.

[0661] 4. Experimental Methods

[0662] MC38 cells were cultured in DMEM medium containing 10% fetal bovine serum. MC38 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0663] Female mice were inoculated subcutaneously on the right side with 1 × 10 6 MC38 cells. The day of inoculation was defined as day 0. On the second day (Day 1) of MC38 cell inoculation, the cells were randomly divided into groups and administered according to their body weight. 3 mg / mL and 10 mg / mL solutions of compound 22 were prepared for use.

[0664] Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 )(where a represents the major diameter and b represents the minor diameter);

[0665] Tumor proliferation rate, T / C%, is the percentage of tumor volume or tumor weight between the treatment group and the control group at a certain time point.

[0666] The calculation formula is as follows:

[0667] T / C% = T TV / C TV ×100%(T TV : The average TV of the treatment group at a specific time point; C TV : the average TV of the vehicle control group at a specific time point);

[0668] Or T / C% = T TW / C TW ×100%(T TW : Average tumor weight at the end of the experiment in the treatment group; C TW: Average tumor weight at the end of the experiment in the vehicle control group).

[0669] Tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (TV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

[0670] All experimental results are expressed as mean tumor volume ± SEM (mean standard error). Statistical analysis between different groups selected the best drug treatment point (usually after the last administration). Independent sample T test method was used to compare whether there was a significant difference in tumor volume between the treatment group and the control group. All data were analyzed using SPSS18.0. p < 0.05 was considered to be significantly different.

[0671] 5. Experimental results and discussion

[0672] Table 28 Efficacy of the compounds of the present application on colon cancer mouse model

[0673]

[0674] The test results showed that in the MC38 mouse colon cancer cell model, the test compound 22 showed good tumor inhibition activity on the 22nd day at a dose of 30 mg / kg and 100 mg / kg, with tumor inhibition rates (TGI) of 34% and 48%, respectively. The test compound 22, combined with the Anti-PD-1 antibody, also showed excellent anti-tumor activity in vivo, with a tumor inhibition rate (TGI) of 85%, and no animal deaths or significant changes in body weight were observed. The above experiments support the use of compound 22 as a single drug or in combination with Anti-PD-1 antibodies in the corresponding tumor indications.

[0675] Example 11 Pharmacodynamic evaluation of the compounds of the present application in the B16F10 mouse melanoma model

[0676] 1. Test Principle

[0677] The B16F10 mouse melanoma model is a classic melanoma drug efficacy evaluation model, which is created by inoculating the same type of B16F10 tumor cells into the subcutaneous tissue of female C57BL / 6 mice. The test substance is administered to evaluate the inhibitory effect on tumor growth in the model.

[0678] 2. Experimental Design

[0679]

[0680] 3. Experimental Materials and Reagents

[0681] Experimental animals: Species: Rodent; Strain: C57BL / 6mice; Grade: SPF; Age: 6-8 weeks; Gender:

[0682] Female; Weight: 18-22 g; Laboratory animal provider: Beijing Weitonglihua Biotechnology Co., Ltd.; Production license number: SCXK (Beijing) 2016-0006; Quality certificate number: 1100111911039475;

[0683] Anti PD-1 antibody: Provided by: Pharmaron (Beijing) Pharmaceutical Technology Co., Ltd.; Batch: 717919M1:

[0684] Solvent: 40% hydroxypropyl-β-cyclodextrin (SIGMA, batch: Z08D9Y76902; weigh 120 g HP-beta-CD and dissolve it in distilled water, and finally use a beaker to make the volume to 300 ml to prepare a 40% hydroxypropyl-β-cyclodextrin solution).

[0685] 4. Experimental Methods

[0686] The cells were incubated at 37°C with 5% CO in DMEM containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 2 mM glutamine. 2 B16F10 tumor cells were cultured in an incubator at 4°C and subcultured every 3 to 4 days after the cells were fully grown. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[0687] B16F10 tumor cells resuspended in serum-free DMEM culture medium were cultured at a concentration of 1×10 5 / 100μl was inoculated subcutaneously on the right rib of the experimental animals. In the second group, when the tumor grew to 75mm 3 Twelve animals with relatively uniform tumor volumes were selected and administrated according to the experimental design.

[0688] 31.99 mg (purity 93.79%) of compound 22 was weighed and added to 6 ml of 40% HP-beta-CD solution. The mixture was vortexed and ultrasonically mixed to prepare a drug concentration of 5 mg / ml for later use.

[0689] Use a pipette to draw up 0.418 ml of 7.18 mg / ml Anti PD-1 and add it to 1.082 ml of pH 7.0 PBS solution. Gently invert and mix to prepare a 2 mg / ml concentration solution for later use.

[0690] The first day of tumor inoculation was designated as D0. Groups 1, 3, and 4 were given medication from the second day of tumor inoculation. Group 2 was given medication when the tumor volume reached 75 mm 3 Start dosing.

[0691] Tumor volume: The tumor volume was measured three times a week using a vernier caliper to measure the long and short diameters of the tumor. The volume calculation formula was: volume = 0.5 × long diameter × short diameter 2 .

[0692] Tumor growth inhibition rate (TGI, %):

[0693] Tumor growth inhibition rate (TGI, %) = (1-T / C) × 100.

[0694] TGI = 100-T / C ​​× 100,

[0695] T / C (%) = (mean tumor volume of the treatment group on the same day - mean tumor volume of the treatment group on initial D0) / (mean tumor volume of the control group on the same day - mean tumor volume of the control group on initial D0) × 100.

[0696] Graphpad Prism 8.0 was used for survival data analysis.

[0697] 5. Experimental results and discussion

[0698] Table 29 Tumor inhibitory efficacy of the compounds of the present application on melanoma mouse model

[0699]

[0700] a Mean ± standard error; b vs. solvent control group;

[0701] The experimental results showed that in the B16F10 mouse melanoma model, the treatment results on the 18th day after tumor inoculation showed that the compound 22 combined with Anti PD-1 treatment group (average tumor volume 1176mm 3 ) had a significant anti-tumor effect (TGI = 48.1%, P = 0.004, vs. solvent control group); the compound 22 alone treatment group showed a greater anti-tumor trend (the average tumor volume was 1764mm 3 , TGI=22.1%, P=0.173, vs. solvent control group); Anti PD-1 alone treatment group (average tumor volume was 2071mm 3 , TGI = 8.6%, P = 0.586, vs. solvent control group) had no anti-tumor effect, which is related to the fact that the model itself is an Anti PD-1 resistance model. No animal death or significant changes in body weight were observed in the experiment. The above experiments support the use of compound 22 as a single drug or in combination with Anti-PD-1 antibodies in corresponding tumor indications.

[0702] Example 12 Pharmacodynamic evaluation of the compounds of the present application in the A20 murine lymphoma (B lymphocyte) homologous transplant model

[0703] 1. Experimental Principle

[0704] The A20 murine lymphoma (B lymphocyte) allograft model is a classic B lymphocyte cancer drug efficacy evaluation model. It is created by inoculating A20 tumor cells subcutaneously in the right rib of female BALB / c mice. The test substance is administered to evaluate the inhibitory effect on tumor growth in the model.

[0705] 2. Experimental Design

[0706]

[0707] 3. Experimental Materials and Reagents

[0708] Experimental animals: Species: Rodent; Strain: BALB / c mice; Grade: SPF; Age: 6-8 weeks; Gender:

[0709] Female; Weight: 18-21 g; Laboratory animal provider: Beijing Weitonglihua Biotechnology Co., Ltd.; Production license number: SCXK (Beijing) 2016-0006; Quality certificate number: 1100111911036383;

[0710] Solvent: 40% hydroxypropyl-β-cyclodextrin (SIGMA, batch: Z08D9Y76902; weigh 120 g HP-beta-CD and dissolve it in distilled water, and finally use a beaker to make the volume to 300 ml to prepare a 40% hydroxypropyl-β-cyclodextrin solution).

[0711] 4. Experimental Methods

[0712] The cells were incubated at 37°C with 5% CO in RPMI 1640 medium containing 10% inactivated fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 0.05 mM 2-mercaptoethanol. 2 A20 tumor cells were cultured in an incubator at 4°C and subcultured every 3 to 4 days after the cells were fully grown. Tumor cells in the logarithmic growth phase were used for inoculation of tumors in vivo.

[0713] A20 tumor cells resuspended in serum-free RPMI 1640 culture medium were cultured at a concentration of 3×10 5 / 100μl was inoculated subcutaneously on the right flank of the experimental animals, and a total of 30 animals were inoculated. On the first day after tumor cell inoculation, 24 animals with relatively uniform weight were selected according to their weight and divided into groups for drug administration, with 12 animals in each group. The specific drug administration scheme was carried out according to the experimental design.

[0714] 15.99 mg (purity 93.79%) of compound 22 was weighed and added to 3 ml of 40% HP-beta-CD solution. The mixture was vortexed and ultrasonically mixed to prepare a 5 mg / mL compound 22 solution for use.

[0715] Tumor volume: The tumor volume was measured three times a week using a vernier caliper to measure the long and short diameters of the tumor. The volume calculation formula was: volume = 0.5 × long diameter × short diameter 2 .

[0716] Tumor growth inhibition rate (TGI, %):

[0717] Tumor growth inhibition rate (TGI, %) = (1-T / C) × 100,

[0718] TGI = 100 - T / C × 100;

[0719] T / C (%) = (mean tumor volume of the treatment group on the same day - mean tumor volume of the treatment group on initial D0) / (mean tumor volume of the control group on the same day - mean tumor volume of the control group on initial D0) × 100.

[0720] SPSS T-test was used to analyze the tumor volume among the groups.

[0721] 5. Experimental results and conclusions

[0722] Table 30 Tumor inhibitory efficacy of the compounds of the present application in the lymphoma mouse model

[0723]

[0724] The test results showed that the compound 22 (50 mg / kg) treatment group showed a clear anti-tumor trend and showed significant anti-tumor effects on the 15th and 17th days after tumor inoculation (P < 0.05), with TGIs of 29.4% and 32.6%, respectively. In addition, during the administration period, the general state of the experimental animals in each group, such as activity and eating, was good, and no obvious clinical abnormalities were found. The overall weight of the animals increased before and after treatment. The above experiments support the use of compound 22 as a single drug in the corresponding tumor indications.

[0725] Example 13 Pharmacodynamic evaluation of the compounds of the present application in a homograft model of CT26 murine colon cancer model

[0726] 1. Experimental Principle

[0727] The CT26 murine colon cancer syngeneic model is a classic colon cancer drug efficacy evaluation model, which is created by subcutaneously inoculating CT26 in BALB / c mice. The test substance is administered to evaluate the inhibitory effect on tumor growth in the model.

[0728] 2. Experimental Design

[0729]

[0730]

[0731] 3. Experimental Materials and Reagents

[0732] Experimental animals: BALB / c mice, female, 7-8 weeks (age of mice at the time of tumor cell inoculation), weight 17.4-23.5 g, 160 mice (100 mice plus 60 surplus mice). Purchased from Shanghai Lingchang Biotechnology Co., Ltd. (LC), production license number: SCXK (Shanghai) 2013-0018, animal certificate number: 2013001837385. Breeding environment: SPF grade. ;

[0733] Solvent: 40% hydroxypropyl-β-cyclodextrin (Zibo Qianhui Biotechnology Co., Ltd.; batch number: 160101; weigh 16.8 g hydroxypropyl-β-cyclodextrin, add 42 ml sterile water, and dissolve completely for later use.).

[0734] 4. Experimental Methods

[0735] CT26 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. CT26 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0736] Female mice were inoculated subcutaneously on the right side with 5 × 10 5 CT26 cells. The day of inoculation was defined as day 0. MC38 cells were inoculated on the second day (Day 1) and randomly divided into groups according to body weight for drug administration.

[0737] Weigh 64.35 mg of compound 22 (60 mg free base), add 6.0 ml of solvent, vortex and sonicate to obtain 6.0 ml of suspension, and prepare a 10 mg / mL solution of compound 22 for use.

[0738] Tumor proliferation rate, T / C%, is the percentage of tumor volume or tumor weight between the treatment group and the control group at a certain time point.

[0739] The calculation formula is as follows:

[0740] T / C% = T TV / C TV ×100%(T TV : The average TV of the treatment group at a specific time point; C TV : the average TV of the vehicle control group at a specific time point);

[0741] Or T / C% = T TW / CTW ×100%(T TW : Average tumor weight at the end of the experiment in the treatment group; C TW : Average tumor weight at the end of the experiment in the vehicle control group).

[0742] Tumor inhibition rate, TGI (%), was calculated as follows: TGI% = (1-T / C) × 100%. (T and C are the relative tumor volume (TV) or tumor weight (TW) of the treatment group and the control group at a specific time point, respectively).

[0743] All data were analyzed using SPSS 18.0.

[0744] 5. Experimental results and conclusions

[0745] Table 31 Tumor inhibitory efficacy of the compounds of the present application in the colon cancer mouse model

[0746]

[0747] The test results showed that the compound 22 (50 mg / kg) treatment group showed anti-tumor effects on the 26th day, with a TGI of 19%. In addition, during the administration period, the general state of the experimental animals in each group, such as activity and eating, was good, and no obvious clinical abnormalities were found. The above experiments support the application of compound 22 as a single drug in the corresponding tumor indications.

[0748] Example 14 Pharmacokinetics of the compounds of the present application in ICR mice

[0749] 14.1 Pharmacokinetics of Compound 27, Compound 40, Compound 22 and Compound 41 in Different Administration Routes

[0750] 1. Experimental Information and Design

[0751]

[0752] 2. Computational software

[0753] use 6.4 as PK parameter calculation software.

[0754] 3. Results and Conclusions

[0755] Table 32 Pharmacokinetic results of the compounds of the present application in ICR mice

[0756]

[0757]

[0758] The experimental results show that the compounds in the list have excellent pharmacokinetic performance in ICR mice. Compound 27, compound 40, compound 22, and compound 41 show higher oral in vivo exposure and higher oral bioavailability. Compound 27, compound 22, compound 40 and compound 41 show higher oral in vivo exposure, among which compound 27, compound 22 and compound 40 have no obvious drug accumulation after continuous oral administration at a dose of 30 mg / kg.

[0759] 14.2 Pharmacokinetics of Compound 40 and Its Benzenesulfonate in ICR Mice

[0760] 1. Experimental information

[0761]

[0762] 2. Experimental Design

[0763]

[0764]

[0765] 3. Experimental results and conclusions

[0766] Table 33 Pharmacokinetic results of the compounds of the present application in ICR mice

[0767]

[0768] The data in Table 33 show that the tested compounds have excellent pharmacokinetic parameters in ICR mice. The in vivo exposure of compound 40 and its besylate salt are both high.

[0769] Example 15 Pharmacokinetics of the compounds of the present application in SD rats

[0770] 15.1 Pharmacokinetics of Compound 27, Compound 16, Compound 14 and Compound 22 in SD rats

[0771] 1. Experimental Information and Design

[0772]

[0773] 2. Computational software

[0774] use 6.4 as PK parameter calculation software.

[0775] 3. Results and Conclusions

[0776] Table 34 Pharmacokinetic results of the compounds of the present application in SD rats

[0777]

[0778] The experimental results showed that the compounds in the list had excellent pharmacokinetic performance in SD rats. 1 / 2 About 0.5 to 1 hour, T of oral administration 1 / 2 It exhibits higher oral exposure and higher oral bioavailability in about 3-5 hours.

[0779] 15.2 Pharmacokinetics of the Test Substance in SD Rats

[0780] 1. Experimental Design

[0781] Dosage:

[0782]

[0783] Dosage: The compounds in this group were all administered in a mixed manner, with 6 compounds in one group administered in a mixed PO manner.

[0784]

[0785]

[0786] Detection model and detection method

[0787]

[0788]

[0789] 2. Test materials and equipment

[0790] Reagent name source batch number Formic acid Aladdin F1627037 Acetonitrile Merck KGaA Co. JA050830 PEG400 Mecoxlin Chemical Reagent Co., Ltd. C10083707 Verapamil Aladdin K1629079

[0791] 2) Main instruments

[0792]

[0793] 3. Analytical methods

[0794] 1) Liquid phase conditions

[0795] Chromatographic column: ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm)

[0796] Flow rate: Compound 40, Compound 27, Compound 40 and Compound 47: 0.4 mL / min, column temperature: 40°C, injection volume: 5 μL; Compound 44 and Compound 48: 0.3 mL / min, column temperature: 35°C, injection volume: 1 μL.

[0797] Compound 40, Compound 27, Compound 40 and Compound 47: Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile (0.075% formic acid);

[0798] Compound 44 and Compound 48: Mobile phase A: water (0.1% formic acid), mobile phase B: acetonitrile (0.1% formic acid).

[0799] Gradient elution, elution program see the table below.

[0800]

[0801]

[0802] 2) Mass spectrometry conditions

[0803] Electrospray ion source (Turbo spray), positive ion detection mode, multiple reaction monitoring scan (MRM) mode was selected for mass spectrometry analysis. The mass spectrometry ion source parameters and compound detection parameters are shown in the table below.

[0804]

[0805] 4. Test methods

[0806] Blood was collected according to the blood sampling points in the experimental protocol. 50 μL of plasma sample was taken, 3 volumes of acetonitrile (containing 5 ng / mL of verapamidine) were added, vortexed for 30 seconds, centrifuged at 15000 rpm and 4°C for 15 minutes, and 10 μL of supernatant was taken for LC-MS / MS analysis.

[0807] Compound formulations were performed according to the vehicle in the experimental protocol.

[0808] 3 SD rats / compound, male, weighing 200-220 g, fasted overnight before administration but allowed to drink water.

[0809] Oral gavage: Blood was collected from the orbitals before gavage, 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h and 24h after administration (Compound 40, Compound 27, Compound 40 and Compound 47), or 0.25h, 0.5h, 1h, 4h, 6h and 8h after administration (Compound 44 and Compound 48) into sodium heparin-treated test tubes, and the supernatant plasma was collected after centrifugation for LC-MS / MS analysis.

[0810] The plasma samples were processed according to the method in the "Sample Processing" section and analyzed by LC-MS / MS, along with the standard curve and quality control samples. The time points exceeding the upper limit of quantification were diluted 10 or 100 times for analysis.

[0811] All the measured data were collected and processed by Analyst 1.6.3 software, and the data were calculated and processed by Microsoft Excel. The pharmacokinetic parameters were calculated by DAS 3.2.8 software using the statistical moment method. The main parameters included were kinetic parameters T max ,t 1 / 2 , C max , AUC (0-t) wait.

[0812] 5. Test results and conclusions

[0813] Table 35 Pharmacokinetic results of the compounds of the present application in SD rats

[0814]

[0815] Table 36 Pharmacokinetic results of the compounds of the present application in SD rats

[0816]

[0817] The experimental results of Table 35 and Table 36 show that the compounds in the list have excellent pharmacokinetic performance in SD rats, with AUC last The higher the value, the higher the oral exposure and the higher the oral bioavailability. The excellent pharmacokinetic properties of the compounds in the list can support further clinical human experiments.

[0818] 15.3 Pharmacokinetics of Compound 22 Acetate in SD Rats after Single and Repeated Dosing

[0819] 1. Experimental Design

[0820]

[0821]

[0822] Dosing information is as follows:

[0823]

[0824] a: The solvent was 10% DMAC / 15% Solutol HS 15 / 75% PBS (pH=7.4).

[0825] b: The solvent is acetic acid-sodium acetate (pH=4.0) buffer containing 0.5% MC.

[0826] 2. Experimental Materials

[0827] Animal Information:

[0828]

[0829] name source batch number DMAC SIGMA BCBT8964 Solutol HS15 BASF SE 69889088QO PBS GIBCO 1948177 glacial acetic acid H O Heywell SZBF2670V Sodium acetate trihydrate SIGMA BC13W4910 Methylcellulose SIGMA SLCB1319 Deionized water Kanglong Chemical Laboratory 08142019

[0830] 3. Experimental Methods

[0831] 1) Preparation

[0832] Preparation of intravenous injection: Accurately weigh 7.13 mg of compound 22 acetate into a container, add 1.5 mL of DMAC, and vortex to completely dissolve it; add 2.25 mL of Solutol HS 15 and vortex, then add 11.25 mL of PBS to the final volume (15 mL), vortex for 1 minute, and obtain a clear solution with a final concentration of 0.4 mg / mL (pH = 3.96).

[0833] Preparation of oral group (Groups 2-5): In the second group, 69.91 mg of the acetate salt of compound 22 was accurately weighed into a container, and 20 mL of acetic acid-sodium acetate (pH=4.0) buffer containing 0.5% MC was added, stirred for 66 minutes, and ultrasonicated for 30 minutes to a final volume (28 mL) to obtain a clear solution with a final concentration of 2.1 mg / mL (PH=3.99). In the third group, 209.7 mg of the acetate salt of compound 22 was accurately weighed into a container, and 20 mL of acetic acid-sodium acetate (pH=4.0) buffer containing 0.5% MC was added, stirred for 66 minutes, and ultrasonicated for 30 minutes to a final volume (28 mL) to obtain a clear solution with a final concentration of 6.3 mg / mL (pH=4.15). The fourth group accurately weighed 629.2 mg of compound 22 acetate into a container, added 20 mL of acetic acid-sodium acetate (pH = 4.0) buffer containing 0.5% MC, stirred for 66 minutes, and ultrasonicated for 30 minutes to a final volume (28 mL) to obtain a clear solution with a final concentration of 18.9 mg / mL (pH = 4.35). The fifth group accurately weighed 1348.3 mg of compound 22 acetate into a container, added 150 mL of acetic acid-sodium acetate (pH = 4.0) buffer containing 0.5% MC, stirred for 66 minutes, and ultrasonicated for 30 minutes to a final volume (180 mL) to obtain a clear solution with a final concentration of 6.3 mg / mL (pH = 4.15). The dosing preparations were freshly prepared under yellow light before daily administration. Among them, the preparations of Group 5 were prepared before administration on the first day, and the daily dosage was divided and stored at 5 ± 3 ° C. After the preparations were taken out on the day of administration, they were placed at room temperature for at least 30 minutes before administration.

[0834] 2) Drug administration

[0835] Drug administration was carried out according to the experimental design.

[0836] 3) Sample processing

[0837] After blood collection, invert the blood collection tube containing anticoagulant several times to mix thoroughly and place it on wet ice before centrifugation. Within 60 minutes after blood collection, centrifuge at 2000g for 10 minutes at 2 to 8°C to separate red blood cells and obtain plasma samples. Transfer plasma samples to cryovials and store at -75±15°C until analysis.

[0838] Blood samples were collected from SD rats at room temperature before and within 24 hours after administration. All collected samples were placed on wet ice before centrifugation and stored at -75±15℃ until analysis.

[0839] Sample processing method:

[0840]

[0841] NA: Not applicable

[0842] ①. According to the table above, take the required volume and transfer it to a 1.1mL plastic tube.

[0843] ②. Mix the sample using a vortexer, shake for 10 minutes, and centrifuge at 4000 rpm and 4°C for 10 minutes.

[0844] ③. After centrifugation, transfer 20 μL of supernatant to a 1.1 mL plastic tube containing 140 μL of diluent and vortex mix. Transfer 60 μL of mixed sample to a 1.1 mL plastic tube containing 100 μL of diluent and vortex mix.

[0845] ④. Transfer 160 μL of sample to a 96-well plate.

[0846] ⑤. Inject 5.0 μL of sample into LC-MS / MS.

[0847] 4) Analysis methods and conditions

[0848]

[0849]

[0850] Mass spectrometry parameters

[0851]

[0852] Liquid chromatography-mass spectrometry was used to measure the concentration of acetate of compound 22 in SD rat plasma using Verapamil as the internal standard compound. The mass spectrometer was Applied Biosystems / AB Sciex Triple Quad 4500, and the mass-to-charge ratios of the parent ion and daughter ion of acetate of compound 22 and Verapamil were monitored to be 393.2→146.0 and 455.2→165.0, respectively, in the positive ion mode of the electrospray ion source.

[0853] The pharmacokinetic parameters of acetate salt of compound 22 were calculated by Phoenix software. TM (Version 8.1) was calculated using a non-compartmental model. The pharmacokinetic data were calculated using the linear logarithmic trapezoidal method with a weight of 1 / (Y*Y). Samples with concentrations below the lower limit of quantification were not included in the calculation of pharmacokinetic parameters. Microsoft Excel 2010 was used to calculate the average values ​​of various parameters, which were determined by the pharmacokinetic parameters of each animal. The peak time (T max ) is represented by the median.

[0854] 4. Experimental results and conclusions

[0855] Table 37 Main pharmacokinetic parameters in SD rats after administration of compound 22 acetate (mean ± standard deviation)

[0856]

[0857]

[0858] NA: Not applicable.

[0859] a:T max The mean is the median.

[0860] *: Standard deviation calculation is not performed when the number of individuals is less than 3.

[0861] The results are shown in Table 36. After intravenous injection of 2.0 mg / kg of compound 22 acetate, the drug exposure AUC of compound 22 acetate in SD rats was 0-t The half-life is 5363hr*ng / mL, T 1 / 2 The clearance rate (CL) is 6.02 mL / min / kg, which is much lower than the hepatic blood flow (55.2 mL / min / kg). It is a low-clearance drug. ss ) was 0.457 L / kg, which was close to the total body fluid volume (0.668 L / kg), indicating that the drug was widely distributed in SD rats.

[0862] After oral administration of 3 dose levels of compound 22 acetate (21 mg / kg, 63 mg / kg and 189 mg / kg) to SD rats, the drug exposure (AUC 0-t ) were 42765hr*ng / mL, 111910hr*ng / mL and 241332hr*ng / mL respectively, and the maximum blood concentration (C max) were 18367ng / mL, 27367ng / mL and 51850ng / mL respectively. When the dose increased from 21mg / kg to 189mg / kg, AUC 0-t and C max The increase ratio was lower than the dose ratio.

[0863] After oral administration of 21, 63, and 189 mg / kg of the acetate salt of Compound 22 to SD rats, the average bioavailability of the acetate salt of Compound 22 was 76.5%, 65.5%, and 47.9%, respectively.

[0864] After daily administration of 63 mg / kg of compound 22 acetate for 7 consecutive days, the AUC on the 1st and 7th days 0-t The values ​​were 110818 hr*ng / mL and 127358 hr*ng / mL, respectively. The exposure ratio on the 7th day was less than 2 times that on the 1st day, indicating that after repeated administration of 63 mg / kg of compound 22 acetate for 7 consecutive days, there was no obvious accumulation of compound 22 acetate in SD rats.

[0865] The experimental results showed that the acetate of compound 22 has excellent pharmacokinetic properties in rats: low clearance, wide distribution, in vivo exposure and maximum blood concentration increase with increasing dose, high oral bioavailability, and no obvious accumulation in the body after 7 days of administration.

[0866] 15.4 Pharmacokinetics of Compounds 22, 27 and 40 in SD rats after 7-day oral administration

[0867] 1. Experimental Design

[0868] Twelve male SD rats (about 7-9 weeks old on the day of administration, 283.0-298.1 g) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and randomly divided into 3 groups, 4 rats in each group, and orally administered once a day for 7 consecutive days, wherein the first group was administered with 30 mg / kg of compound 27, the second group was administered with 30 mg / kg of compound 22, and the third group was administered with 30 mg / kg of compound 40. All animals had free access to water and food during the administration process.

[0869]

[0870] 2. Experimental Materials

[0871] Species / Strain / Grade: Sprague-Dawley (SD) rats [Crl: CD (SD)] (SPF / VAF) source: Sibeifu (Beijing) Biotechnology Co., Ltd. Experimental Animal Production License: SCXK(Beijing)2016-0002 Animal quality certificate number: 11401500037777 Age at first dose: About 7 to 9 weeks Body weight at first dose: Male 283.0~298.1g

[0872] name source batch number PEG400 Sigma BCBV8368 PBS Gibco 1951153

[0873] name: Compound 27 Compound 22 Compound 40 purity: 98.43％ 99.00％ 97.76％ Expiration Date: Untested Untested Untested Storage conditions: Room temperature, away from light Room temperature, away from light Room temperature, away from light Properties: White powder White powder White powder Correction Factor: 1.02 1.01 1.02

[0874] Correction factor = FW / MW*1 / purity

[0875] 3. Experimental Methods

[0876] 1) Preparation steps:

[0877] Weigh 0.3366g of compound 27, 0.3333g of compound 22 and 0.3366g of compound 40 respectively, dissolve in 44mL of PEG400, stir and ultrasonically dissolve, add 50mL of PBS, stir and ultrasonically dissolve until completely dissolved, add PBS to a final volume of 110mL, and obtain a target preparation solution with a final concentration of 3.0mg / mL. The prepared preparation solution was divided into 7 portions, each 15mL, and stored in a refrigerator at 4°C. Before administration, the preparation was placed at room temperature until the preparation temperature returned to room temperature and stirred at room temperature for at least 10 minutes, and continued stirring during administration. The entire preparation preparation process was operated under yellow light, and the prepared preparation was wrapped in tin foil to avoid light when transferred.

[0878] 2) Pharmacokinetic sample collection:

[0879]

[0880] 3) Sample analysis

[0881] Liquid chromatography-mass spectrometry was used to measure the concentrations of compound 27, compound 22 and compound 40 in SD rat plasma using Verapamil as an internal standard compound. The mass spectrometer was scanned in the positive ion mode of the electrospray ion source, and the parent ion and daughter ion mass-to-charge ratios of compound 27, compound 40, compound 22 and Tolbutamide were 395.2→178.2, 404.2→158.9, 393.4→146.2 and 455.2→165.0, respectively.

[0882] The pharmacokinetic parameters of compounds 27, 40 and 22 were calculated by Phoenix software. TM WinNonlin 6.1 version was used for calculation using a non-compartmental model. The pharmacokinetic data were calculated using the linear logarithmic trapezoidal method with a weight of 1 / Y*Y. Samples with concentrations below the lower limit of quantification were not included in the calculation of pharmacokinetic parameters. Microsoft Excel 2010 version was used to calculate the average values ​​of various parameters, which were determined by the pharmacokinetic parameters of each animal. The peak time (T max ) is represented by the median.

[0883] 4. Analytical methods

[0884] 1) LC-MS bioanalysis of compound 27

[0885] Standard curve and quality control sample information:

[0886]

[0887] Liquid phase conditions:

[0888]

[0889] Mass spectrometry conditions:

[0890]

[0891]

[0892] 2) LC-MS bioanalysis of compound 40

[0893] Standard curve and quality control sample information:

[0894]

[0895] Liquid phase conditions:

[0896]

[0897]

[0898] Mass spectrometry conditions

[0899]

[0900] 3) LC-MS bioanalysis of compound 22

[0901] Standard curve and quality control sample information:

[0902]

[0903] Liquid phase conditions:

[0904]

[0905]

[0906] Mass spectrometry conditions:

[0907]

[0908] 5. Experimental results and conclusions

[0909] Table 38 Main PK parameters (average values) of compound 27, compound 22, and compound 40 after oral administration for 7 consecutive days

[0910]

[0911]

[0912] a:T max The value is the median.

[0913] "-":not applicable.

[0914] SD male rats were orally administered 30 mg / kg of Compound 27, Compound 22, and Compound 40 once a day for 7 consecutive days, and their plasma drug concentrations were measured on the 1st and 7th days after administration.

[0915] After SD male rats were orally administered 30 mg / kg of compound 27, compound 22, and compound 40 once a day for 7 consecutive days, the drug exposure AUC on the first day 0-t The maximum blood concentration is 29550hr*ng / mL, 6630hr*ng / mL and 5154hr*ng / mL respectively; the maximum blood concentration is C max The drug exposure AUC of compound 27, compound 22 and compound 40 in SD rat plasma on the 7th day of continuous administration was 7103 ng / mL, 1022 ng / mL and 503 ng / mL respectively. 0-t The maximum blood concentration was 22194 hr*ng / mL, 6953 hr*ng / mL and 6584 hr*ng / mL respectively; max The results showed that after oral administration of 30 mg / kg of compound 27, compound 22, and compound 40 once a day for 7 consecutive days, compound 27, compound 22, and compound 40 had no obvious accumulation in SD rats (Table 38).

[0916] The data showed that the test compound had excellent pharmacokinetic parameters in SD rats, high in vivo exposure, and no obvious accumulation after continuous administration. The excellent rat pharmacokinetic properties of the test compound can support further clinical human experiments.

[0917] Example 16 Pharmacokinetic evaluation of the compounds of the present application in female BALB / c mice

[0918] Group Number of animals Drug group Dosage (mg / kg) Dosage Planned dosing cycle Actual dosing cycle 1 12 Compound 22 100 po QDx1 times QDx1 times 2 12 Compound 22 100 po QDx1 times QDx1 times

[0919] 2. Experimental Materials

[0920] 1) Experimental animals

[0921] BALB / c mice, female, 7-9 weeks (age of mice at the beginning of the experiment), weight 19.2-22.1 g, 51 mice (34 mice plus 10 surplus mice). Purchased from Shanghai Lingchang Biotechnology Co., Ltd. (LC), animal certificate number: 2013001835211. Breeding environment: SPF grade.

[0922] 2) Test sample

[0923] Test sample: Compound 22: Batch number: F, packaging specification: 38.0 mg / vial + 5045.1 mg / vial, purity 98.96%, moisture 5.38%, provided by Zhejiang Chunhe Pharmaceutical Technology Co., Ltd., powder, sealed and stored at RT.

[0924] 3. Experimental Methods

[0925] 1) Randomization

[0926] Before dosing, all animals were weighed and the weight of the mice was calculated using StudyDirector. TM Randomization was performed using the Studylog System (version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA).

[0927] 2) Preparation of test sample and solvent

[0928]

[0929] 4) Experimental observation and data collection

[0930] The experimental protocols for animal experiments in this experiment were reviewed and approved by the CrownBio IACUC Committee. During the experiment, animal experimental operations were in accordance with AAALAC requirements. After grouping, routine monitoring included the effects of treatment on the normal behavior of the animals, including the activity of the experimental animals, food and water intake, eyes, fur and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data. StudyDirector was used in the experiment TM The software (version 3.1.399.19, supplier Studylog System, Inc.) collects animal weight data. The raw data is directly imported into the software after being measured by the balance, and any changes in the data will be recorded in this software. The entire process of drug administration and body weight weighing is carried out in a biological safety cabinet or ultra-clean workbench.

[0931] 5) Experimental endpoint

[0932] After a single dose, plasma was collected from each group at 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h, with 3 mice at each time point, and the experiment was terminated after 24h. The remaining 10 mice were used to collect blank plasma.

[0933] 5. Experimental results and discussion

[0934] Table 39 Pharmacokinetic data of compound 22 in different solvents

[0935]

[0936] After a single dose of G1 and G2 mice, plasma was collected at 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h and 24h in each group to detect the PK response of compound 22 in mice. The results showed that the concentration of compound 22 in the plasma of mice in group G2 at different time points was significantly higher than that in the plasma of mice in group G1. 40% (2-hydroxypropyl)-β-cyclodextrin has a good effect as a solvent for compound 22. At the same time, under the dosage of 100mg / kg, there is a higher drug exposure in mice, and the pharmacokinetic performance is excellent.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease, wherein the disease is lymphoma, wherein the compound is: 2-(furan-2-yl)-N5-(4-(oxetan-3-ylamino)phenethyl)-[1,2,4]triazolo[1,5-a][1,3,5]triazine-5,7-diamine.

2. The use according to claim 1, wherein the pharmaceutically acceptable salt of the compound comprises acetate.

3. The use according to claim 1, wherein the medicament is prepared for oral administration. The use according to claim 1 , wherein the medicament further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 4, wherein the pharmaceutically acceptable carrier comprises cyclodextrin.

Images