Use of pyrazolopyrimidines

Abstract

The present application relates to the use of pyrazolopyrimidine compounds of general formula (I). The compounds are effective in inhibiting the activity of CD73 and have good application prospects as CD73 small molecule inhibitors in the treatment and / or prevention of CD73-related diseases, disorders and conditions, particularly cancer and immune disorders.

Description

Technical Field

[0001] This invention relates to CD73 inhibitors, and more particularly to the use of a class of pyrazolopyrimidine compounds in the preparation of CD73 inhibitors, especially in the preparation of medicaments for the treatment and / or prevention of CD73-related diseases, disorders and conditions, particularly cancer and immune disorders. Background Technology

[0002] Tumor immune escape refers to the process by which tumor cells evade the surveillance and elimination of the immune system through their own or non-self means. The realization of tumor immune escape is related to changes in the tumor cell's own cellular epitopes and alterations in the tumor immune microenvironment. Tumor-related changes refer to antigen blockade, hypoxic metabolism, or epithelial-mesenchymal transition caused by gene mutations, making them more difficult for the body's immune system to recognize. The tumor's influence on the microenvironment stems from the interaction between tumor cells and surrounding normal tissues or immune factors, creating an immunosuppressive environment. In this context, regulatory T cells (Tregs) significantly inhibit the killing effect of effector T cells on tumor cells. Due to the limitations of current tumor immunotherapy drugs, the mechanism of immune escape is increasingly attracting the attention of researchers. Based on the above information, there is a need to develop tumor immunotherapy drugs with novel mechanisms and to develop combination therapies that simultaneously inhibit multiple immunosuppressive pathways to improve clinical response and better meet patient needs.

[0003] CD73 is an extracellular nucleotidase. Highly expressed CD73 in tumors can hydrolyze adenosine monophosphate (AMP) to produce adenosine (ADO), significantly altering the tumor microenvironment. CD73 also acts as an adhesion signaling molecule on the cell surface (MINOR M, ALCEDO KP, BATTAGLIA RA, et al. Cell type-and tissue-specific functions of ecto-5'-nucleotidase (CD73)[J]. Am J Physiol Cell Physiol, 2019, 317(6):C1079-C1092.). Extracellular adenosine production is one of the most important immunosuppressive regulatory pathways in the tumor microenvironment, primarily generated through a cascade hydrolysis of CD39 (belonging to NTPdases) and CD73. As the main enzyme producing extracellular adenosine, CD73 becomes an important regulator in various tissue mechanisms of cellular homeostasis, stress, injury, and inflammation. ATP is released from stressed or dying cells, representing a "dangerous" inflammatory signal. Release ceases when this molecule is rapidly converted into the anti-inflammatory signaling molecule adenosine, thus preventing severe inflammatory responses that could harm the body. In the hypoxic tumor microenvironment, high levels of extracellular ATP accumulate. CD39 (exonucleoside triphosphate diphosphate hydrolase 1, NTPDase 1, or EC 3.6.1.5) and CD73 can continuously hydrolyze it into ADP, AMP, and adenosine (ADO), altering the microenvironment and upregulating adenosine concentration. Adenosine receptor A2AR (belonging to the P1 receptor) is widely distributed in human tissues. After binding to adenosine, the receptor enhances Treg cell proliferation, increases its immunosuppressive capacity, inhibits T cell proliferation and NK cell activation and function, and promotes macrophage M2 differentiation (LAPPAS CM, RIEGER JM, LINDEN J. A2A Adenosine Receptor Induction Inhibits IFN-γ Production in Murine CD4). +T Cells[J].The Journal of Immunology,2005,174(2):1073-1080.). A2BR signal transduction can promote the proliferation of myeloid-derived suppressor cells (MDSCs), and CD73 on MDSCs can also suppress T cells and NK cells. During tumor development, the hypoxic and inflammatory environment in the tumor microenvironment is maintained, and tumor cells continuously enhance immunosuppression by increasing the expression of related genes (such as NT5E, the gene encoding CD73). The significant upregulation of CD73 on the surface of tumor tissue cells is one of the important reasons for tumor immune escape (NEO SY, YANG Y, RECORD J, et al. CD73 immune checkpoint defines regulatory NK cells within the tumor microenvironment[J].J Clin Invest,2020,130(3):1185-1198.). Therefore, inhibiting CD39 and CD73 will effectively block adenosine-mediated immunosuppression.

[0004] CD73, encoded by the NT5E gene, is widely expressed in normal human tissues such as lymphocytes and endothelial cells. The Cancer Genome Atlas (TCGA) shows that NT5E is highly upregulated in most solid cancer cells compared to normal cells, especially on the surface of Treg cells (YANG H, YAO F, DAVIS PF, et al. CD73, Tumor Plasticity and Immune Evasion in Solid Cancers[J]. Cancers (Basel), 2021, 13(2).). NT5E expression is most abundant in thyroid cancer, sarcoma, and acute myeloid leukemia, and is also high in breast cancer and bladder tumors. Evidence suggests that high CD73 expression is positively correlated with breast cancer cell density, and its enhanced activity may lead to adenosine accumulation, thereby enhancing purine signaling pathway activation (Zhou Tingting, Zhou Ping, Yin Lianhua. Effects of CD73 on the invasiveness and motility of human breast cancer cells[J]. Chinese Journal of Pathophysiology, 2006, 22(2): 360-364.). This evidence suggests that CD73 should be prioritized as a potential target in these solid tumor cancers.

[0005] Furthermore, increasing evidence suggests that CD73 is associated with tumor cell metastasis. Stagg et al. found that CD73 is a driving factor for tumor metastasis in a mouse model of breast cancer (STAGG J, DIVISEKERA U, MCLAUGHLINN, et al. Anti-CD73 antibody therapy inhibits breast tumor growth and metastasis[J]. Proc Natl Acad Sci USA, 2010, 107(4):1547-1552.). This process is still mainly mediated by adenosine as a signaling molecule, exerting an immunosuppressive effect on lymphocytes while also enhancing the migration ability of tumor cells. Experimental settings have confirmed that targeting CD73 can also effectively prevent gastric cancer metastasis (XU Z, GU C, YAO X, et al. CD73 promotes tumor metastasis by modulating RICS / RhoA signaling and EMT in gastric cancer[J]. Cell Death Dis, 2020, 11(3):202.).

[0006] Tu and colleagues demonstrated through experiments that, compared to anti-PD-L1 or anti-CD73 therapy alone, combination therapy with anti-PD-L1 and anti-CD73 can promote the T cell immune response to EGFR-mutant non-small cell lung cancer (NSCLC) and significantly shrink tumors (TU E, MCGLINCHEY K, LAZDUN Y, et al. Abstract 911: Anti-PD-L1 and anti-CD73 combination therapy promotes T cell response to EGFR mutant NSCLC[J]. Cancer Research, 2020, 80(16_Supplement):911-911.). Compared with isotype controls, combination therapy increased MART1 (Melanoma antigen recognized by T-cells 1)-specific CD8+ in tumors. + The number of T cells also affects the CD8 count in the spleen. + T cells CD62L + / CD45RO + / CCR7 + The incidence of this phenotype has increased. Their findings could provide a theoretical basis for CD73 combination therapy.

[0007] Therefore, as a highly promising cancer therapeutic target in recent years, CD73 inhibitors have the potential to be used in combination with other tumor immunotherapy drugs or chemotherapy drugs to form a synergistic treatment strategy. Although several monoclonal antibody CD73 drugs are already in clinical trials, CD73 small molecule inhibitors still have important research value due to their unique physicochemical properties, metabolic distribution characteristics, and non-immunogenicity. Summary of the Invention

[0008] This invention provides the use of pyrazolopyrimidine compounds of general formula (I), or their prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, pharmaceutically acceptable salts, polymorphs, solvates, or isotopically labeled compounds, in the preparation of CD73 inhibitors, particularly in the preparation of medicaments for the treatment and / or prevention of CD73-related diseases, disorders, and conditions, especially cancer and immune dysregulations:

[0009]

[0010] in:

[0011] A is selected from hydrogen, halogen, C1-C6 alkyl, -C(=O)NR 5 R 6 -C(=S)NR 5 R 6 -SO2NR 5 R 6 -S(=O)NR 5 R 6 -CH2NR 5 R 6 、or -C(=O)OR 5 , where R 5 and R 6 Independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, 3-10 membered heterocyclic groups, C6-C 12 aryl or 5-10 heteroaryl; the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, 3-10 membered heterocyclic groups, C6-C 12 The aryl or 5-10 heteroaryl group is optionally substituted by one or more substituents selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, and C1-C4 alkyl.

[0012] B is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, C6-C12 aryl, 5-15 membered heteroaryl, 3-10 membered heterocyclic; the C1-C6 alkyl, C2-C6 alkenyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, C6-C 12 The aryl, 5-15-membered heteroaryl, and 3-10-membered heterocyclic groups are optionally substituted by one or more substituents selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, and C1-C4 alkyl.

[0013] R 1 R 2 Independently selected from hydrogen, halogen, C 1- C6 alkyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups;

[0014] R 3 and R 4 Each is independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, C6-C 12 aryl, 5-10 membered heteroaryl, 3-10 membered heterocyclic, wherein the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups, C6-C 12 The aryl, 5-10-membered heteroaryl, and 3-10-membered heterocyclic groups are optionally substituted by one or more substituents selected from the W1 group; or

[0015] R 3 and R 4 The N atom attached thereto forms a monocyclic, spirocyclic, or bridged ring containing 1-4 heteroatoms selected from N, O, and S, wherein the monocyclic, spirocyclic, or bridged ring is optionally substituted by one or more substituents selected from the W1 group; the W1 group substituents include D, halogen, oxo (=O), C1-C6 alkyl, hydroxyl, C1-C6 hydroxyalkyl, -NR 7 R 8 , cyano, nitro, carboxyl;

[0016] R 7 R 8 Each is independently selected from hydrogen, C1-C6 alkyl, C3-C 10 Saturated or unsaturated cyclic hydrocarbon groups; the C1-C6 alkyl, C3-C 10 The saturated or unsaturated cyclic hydrocarbon group is optionally substituted by one or more substituents selected from the W2 group, which includes D, halogen, hydroxyl, -NH2, and C1-C3 alkyl groups.

[0017] In some embodiments, A is selected from hydrogen, halogen, C1-C4 alkyl, -C(=O)NR 5 R 6 .

[0018] In some embodiments, A is selected from hydrogen, In some implementations, R 5 and R 6 Independently selected from hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups, 3-7 membered heterocyclic groups, C6-C 12 aryl or 5-10 membered heteroaryl; wherein the C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C7 saturated or unsaturated cyclic hydrocarbon group, 3-7 membered heterocyclic group, C6-C 12 The aryl or 5-10 heteroaryl group is optionally substituted with one or more substituents selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, and C1-C4 alkyl, and particularly optionally substituted with one or more substituents selected from D, halogen, hydroxyl, -NH2, and C1-C4 alkyl.

[0019] In some implementations, R 5 and R 6 It is independently selected from hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, and C3-C6 saturated or unsaturated cyclic hydrocarbon groups.

[0020] In some embodiments, B is selected from hydrogen, halogen, C1-C6 alkyl, C2-C6 alkenyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups, C6-C 12 aryl, 5-15 membered heteroaryl, 3-7 membered heterocyclic group; wherein the C1-C6 alkyl, C2-C6 alkenyl, C3-C7 saturated or unsaturated cyclic hydrocarbon group, C6-C 12 The aryl, 5-15-membered heteroaryl, and 3-7-membered heterocyclic groups are optionally substituted with one or more substituents selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, and C1-C4 alkyl, and particularly optionally substituted with one or more substituents selected from D, halogen, hydroxyl, -NH2, Boc, and C1-C4 alkyl.

[0021] In some implementations, B is selected from: Where i is selected from 0, 1, 2, 3, 4; R 12 Each is independently selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, C1-C4 alkyl, and particularly from D, halogen, hydroxyl, -NH2, Boc, and C1-C4 alkyl.

[0022] In some implementations, B is selected from

[0023] In some implementations, R 1 R 2 It is independently selected from hydrogen, halogen, C1-C4 alkyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups, especially independently selected from hydrogen and halogen.

[0024] In some implementations, R 3 and R 4 Each is independently selected from hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups, C6-C 12 aryl, 5-10 membered heteroaryl, 3-7 membered heterocyclic group, wherein the C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C7 saturated or unsaturated cyclic hydrocarbon group, C6-C 12 The aryl group, 5-10-membered heteroaryl group, and 3-7-membered heterocyclic group are optionally substituted by one or more substituents selected from the W1 group; or

[0025] R 3 and R 4 The N atom connected thereto forms a monocyclic, spirocyclic, or bridged ring containing 1-4 heteroatoms selected from N, O, and S, wherein the monocyclic, spirocyclic, or bridged ring is optionally substituted by one or more substituents selected from the W1 group;

[0026] The W1 substituent includes D, halogen, oxo (=O), C1-C4 alkyl, hydroxyl, C1-C4 hydroxyalkyl, and -NR. 7 R 8 , cyano, nitro, carboxyl;

[0027] R 7 R 8 Each is independently selected from hydrogen, C1-C4 alkyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups, wherein the C1-C4 alkyl, C3-C7 saturated or unsaturated cyclic hydrocarbon groups are optionally substituted by one or more substituents selected from the W2 group, wherein the W2 group substituents include D, halogen, hydroxyl, -NH2, and C1-C3 alkyl.

[0028] In some implementations, R 3 and R 4 The N atoms bonded to it together form ring structures selected from the following:

[0029]

[0030] In particular, R 3 and R 4The N atoms bonded to it together form ring structures selected from the following:

[0031]

[0032] Where n is selected from 1, 2, 3, 4; m is selected from 0, 1, 2, 3, 4, 5, 6;

[0033] X is selected from N, O, and S atoms;

[0034] R 9 Each is independently selected from D, halogen, hydroxyl, cyano, oxo (=O), C1-C4 hydroxyalkyl, -NR 10 R 11 ;

[0035] R 10 R 11 Independently selected from hydrogen, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, and C3-C6 saturated or unsaturated cyclic hydrocarbon groups; said C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, and C3-C6 saturated or unsaturated cyclic hydrocarbon groups are optionally substituted with substituents selected from D, halogen, hydroxyl, -NH2, and cyano. In some embodiments, R 3 and R 4 The N atoms bonded to it together form ring structures selected from the following:

[0036]

[0037] In some embodiments, the compound represented by formula (I) is selected from the following structures:

[0038]

[0039] Among them, A and R 1 R 2 As defined above,

[0040] R 9 Selected from hydroxyl, oxo (=O), C1-C4 hydroxyalkyl, -NR 10 R 11 ;

[0041] R 10 R 11 Independently selected from hydrogen and C1-C4 alkyl groups; the C1-C4 alkyl groups are optionally substituted with substituents selected from D, halogens, hydroxyl groups, and -NH2 groups;

[0042] R 9’ Selected from H, hydroxyl, and C1-C4 hydroxyalkyl;

[0043] R 12Selected from halogens, hydroxyl groups, -NH2, C1-C4 alkylamino groups, and C1-C4 alkyl groups;

[0044] B1 is selected from C3-C7 saturated or unsaturated cyclic hydrocarbon groups, 3-7 membered heterocyclic groups, and C2-C6 alkenyl groups; the C3-C7 saturated or unsaturated cyclic hydrocarbon groups, 3-7 membered heterocyclic groups, and C2-C6 alkenyl groups are optionally substituted by one or more substituents selected from D, halogens, hydroxyl groups, -NH2, C1-C4 alkylamino groups, C1-C4 alkylcarbonyloxy groups, and C1-C4 alkyl groups, particularly optionally substituted by one or more substituents selected from D, halogens, hydroxyl groups, -NH2, Boc, and C1-C4 alkyl groups; specifically, B1 is selected from:

[0045] In some embodiments, the compound represented by formula (I) is selected from the following structures:

[0046]

[0047]

[0048]

[0049]

[0050] The compounds of the present invention can be used alone, in combination with other compounds of the present invention, or in combination with one or more, preferably one or two other therapeutic agents, simultaneously or sequentially.

[0051] In addition to the pyrazolopyrimidine compound of general formula (I) of the present invention, or its prodrug, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, pharmaceutically acceptable salt, polymorph, solvate, or isotopically labeled compound, the CD73 inhibitor or the drug may also contain a pharmaceutically acceptable carrier and optionally, one or more other therapeutic agents.

[0052] The present invention also provides the use of a pharmaceutical composition in the preparation of a CD73 inhibitor, particularly in the preparation of a medicament for the treatment and / or prevention of CD73-related diseases, disorders and conditions, particularly cancer and immune disorders, said pharmaceutical composition comprising a therapeutically effective dose of one or more selected from compounds of general formula (I), their prodrugs, tautomers, mesosomes, racemates, enantiomers, diastereomers or mixtures thereof, pharmaceutically acceptable salts, polymorphs, solvates or isotopically labeled compounds, and at least one pharmaceutically acceptable carrier.

[0053] In some embodiments, the composition may also contain one or more other therapeutic agents.

[0054] The present invention also provides a method for inhibiting CD73 enzyme activity or for treating and / or preventing CD73-related diseases, disorders, and conditions, particularly cancer and immune dysregulation, said method comprising administering to an individual in need a therapeutically effective amount of one or more, or a combination thereof, selected from compounds of general formula (I), their prodrugs, tautomers, meso compounds, racemates, enantiomers, diastereomers, or mixtures thereof, pharmaceutically acceptable salts thereof, polymorphs, solvates, or isotopically labeled compounds. In some embodiments, the method further includes administering to the individual an effective dose of one of a variety of other therapeutic agents.

[0055] In the embodiments, the other therapeutic agents include, but are not limited to, immune checkpoint PD-1 / PD-L1 monoclonal antibodies, immune checkpoint small molecule inhibitors, antitumor drugs that act on the chemical structure of DNA, antitumor drugs that affect nucleic acid synthesis, antitumor drugs that affect nucleic acid transcription, antitumor drugs that act on microtubule synthesis, aromatase inhibitors, and cell signaling pathway inhibitors.

[0056] Specifically, the other therapeutic agents include cisplatin, methotrexate (MTX), 5-fluorouracil (5FU), doxorubicin, epirubicin, aclarubicin, sclerosomycin, paclitaxel, vinorelbine, aminoglutethimide, lantron, letrozole, reninide, imatinib, gefitinib, erlotinib, and lapatinib.

[0057] In some implementations, the CD73-related diseases, disorders, and conditions include cancer and immune dysregulation.

[0058] Preferably, the cancer includes bladder cancer, breast cancer, bile duct cancer, colorectal cancer, colon cancer, stomach cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, kidney cancer, glioblastoma, sarcoma, leukemia, lymphoma, or melanoma.

[0059] Beneficial effects

[0060] The pyrazolopyrimidine compounds of the present invention can effectively inhibit the activity of CD73 and can be used as small molecule inhibitors of CD73. Detailed Implementation

[0061] The invention will be aided in by referring to the following examples, which are used to illustrate the invention and should not be construed as limiting the scope of the invention in any way.

[0062] In the context of this invention (especially in the context of the claims), singular terms used should be understood to include plural meanings, unless otherwise specifically indicated herein or clearly contradicted by the context.

[0063] As used herein, "halogen" or "halogenated" refers to fluorine, chlorine, bromine, and iodine. Fluorine and chlorine are preferred halogens as substituents.

[0064] As used herein, "alkyl" refers to a fully saturated straight-chain or branched monovalent hydrocarbon group. Alkyl groups preferably contain 1-20 carbon atoms, more preferably 1-16 carbon atoms, 1-10 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, 1-4 carbon atoms, or 1-3 carbon atoms. The number preceding the alkyl group indicates the number of carbon atoms. For example, "C1-C6 alkyl" indicates an alkyl group having 1-6 carbon atoms, "C1-C4 alkyl" indicates an alkyl group having 1-4 carbon atoms, "C1-C3 alkyl" indicates an alkyl group having 1-3 carbon atoms, and so on. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc. This definition applies whether the term "alkyl" appears alone or as part of other groups such as haloalkyl, alkoxy, etc.

[0065] As used herein, "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one double bond. Alkenyl groups preferably contain 2-20 carbon atoms, more preferably 2-10, 2-8, 2-6, or 2-4 carbon atoms. The number preceding the alkenyl group indicates the number of carbon atoms. "C2-C6 alkenyl" refers to an alkenyl group with 2-6 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, and hexenyl.

[0066] As used herein, "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group containing at least one triple bond. The alkynyl group preferably contains 2-20 carbon atoms, more preferably 2-10 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. The number preceding the alkynyl group indicates the number of carbon atoms. "C2-C6 alkynyl" refers to an alkynyl group with 2-6 carbon atoms, representative examples including but not limited to ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, pentyynyl, isopentenynyl, and hexynyl.

[0067] As used herein, "cyclic hydrocarbon group" refers to a saturated or partially saturated non-aromatic carbon ring, including mono-, di-, or tri-rings, preferably having 3-12 ring carbon atoms, more preferably 3-10 ring carbon atoms, for example 3-8, 3-7, 3-6, 4-10, or 4-8 ring carbon atoms. "C3-C 10 "Saturated or unsaturated cyclic hydrocarbon groups" are intended to include C3, C4, C5, C6, C7, C8, C9 ... 10 Cyclic hydrocarbon groups; "C3-C7 cyclic hydrocarbon groups" are intended to include C3, C4, C5, C6, and C7 cyclic hydrocarbon groups; and so on. Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl. Exemplary dicyclic hydrocarbon groups include borneol, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, and bicyclo[2.2.2]octyl. Exemplary tricyclic hydrocarbon groups include adamantyl, etc.

[0068] As used herein, "aryl" refers to an aryl group consisting of one or more fused rings having 6-20, preferably 6-14, more preferably 6-12, and most preferably 6-10 rings. An aryl group having 6-10 ring carbon atoms is C6-C... 10 Aryl groups include: monocyclic aryl groups (e.g., phenyl); or fused bicyclic systems, wherein one ring is an aromatic ring and the other ring is an aromatic ring (e.g., in naphthalene, biphenyl) or a non-aromatic ring (e.g., in dihydroindene, tetrahydronaphthalene). Non-limiting examples of aryl groups include phenyl, biphenyl, naphthyl, tetrahydronaphthyl, indene, dihydroindene, or anthracene.

[0069] As used herein, "heteroaryl" refers to a 5-15 member, preferably 5-13 member, containing 1-4, preferably 1-3, cyclic heteroatoms selected from N, O, or S, including monocyclic, bicyclic, or fused polycyclic rings, with the remaining ring atoms being carbon atoms. Examples of heteroaryl groups include, but are not limited to: pyrrole, furanyl, thiophene, pyrazolyl, imidazolyl, triazolyl, thiazolyl, isothiazolyl, oxazolyl, pyridinyl, pyranyl, pyrazinyl, pyridazinyl, pyrimidinyl, oxazinyl, quinolinyl, isoquinolinyl, borazolinyl, quinazolinyl, quinoxalinyl, benzoxazinyl, 2H-chromene, benzopyranyl, benzothiophene, indole, inazolyl, benzopyrazole, benzimidazolyl, imidazopyridyl, and so on. Benzooxazolyl, benzothiazolyl, 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl, 1H-benzo[d][1,2,3]triazolyl, [1,2,4]triazolo[1,5-a]pyridyl, [1,2,4]triazolo[4,3-a]pyridine, pyrazolo[1,5-a]pyridine, dibenzofuran, dibenzo[b,d]furan, 9H-carbazole, dibenzo[b,d]thiophene, etc.

[0070] As used herein, "heterocyclic group" refers to a group obtained by replacing one or more carbon atoms in a cyclic hydrocarbon group as defined in this application with a heteroatom selected from N, O, or S, such as -O-, -N=, -NR-, -S-, -S(=O)-, and -S(=O)2-, where R is hydrogen, C is carbon, and D is carbon. 1-4Alkyl or nitrogen-protecting groups (e.g., benzyloxycarbonyl, p-methoxybenzylcarbonyl, tert-butoxycarbonyl, acetyl, benzoyl, benzyl, p-methoxy-benzyl, p-methoxy-phenyl, 3,4-dimethoxybenzyl, etc.). Preferably, the heterocyclic group is a monocyclic, bicyclic, or tricyclic saturated or partially unsaturated non-aromatic ring having 3-20 ring atoms, for example 3-12 ring atoms, for example 3-8 ring atoms, for example 3-7 ring atoms. More preferably, the heterocyclic group preferably contains 1, 2, or 3 heteroatoms selected from N, O, or S and is a 4- to 12-membered heterocyclic group, preferably a 4- to 8-membered heterocyclic group, more preferably a 3- to 7-membered, 4- to 6-membered, or 5- to 6-membered heterocyclic group, wherein the heteroatoms are substituted or unsubstituted, for example, substituted by C1-C4 alkyl groups. Examples of heterocyclic groups include, but are not limited to: ethylene oxide, aziridinyl, and aziridine. Alkyl, oxacyclobutyl, azircyclopentyl (pyrrolidinyl), tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydrothiophenyl 1,1-dioxide, tetrahydropyridyl, pyrazolyl, imidazoyl, oxazolyl, thiazoyl, isothiazolyl, pyrrolidinyl-2-one, imidazolide, piperidinyl (hexahydropyridine), N-methylpiperidinyl, tetrahydropyranyl, oxazinyl, 1,3-oxazinyl, hexahydropyrimidinyl, piperazine, piperidinylone, 1,4-dioxa-8-aza-spiro[4.5]decane-8-yl, morpholinyl, thiomorpholinyl, thiomorpholino-S-monoxide (sulfanomorpholino), thiomorpholino-S,S-dioxide (sulfonomorpholino), octahydropyrrolo[3,2-b]pyrrolidinyl, etc.

[0071] As used in this article, "Boc" stands for tert-butyloxycarbonyl.

[0072] As used herein, the term "oxo" refers to an oxygen atom being bonded to another atom via a double bond, and can be represented as "=O". The terms "-C(=O)" represent a carbonyl group, "-S(=O)" represent a sulfoxide group, and "-S(=O)2" represent a sulfone group.

[0073] As used in this article, "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

[0074] As used herein, the terms "optional," "optional," or "optionally" mean that the events described below may or may not occur, and the description includes both cases where the events occur and cases where they do not occur. For example, "optionally substituted alkyl" includes both "unsubstituted alkyl" and "substituted alkyl" as defined herein. "Optionally halogenated" includes both cases where the alkyl is substituted with a halogen and cases where it is not substituted with a halogen, such as substitution with 0-3 halogens. It will be understood by those skilled in the art that, for any group containing one or more substituents, the group does not include any substitution pattern that is spatially impractical, chemically incorrect, synthetically infeasible, and / or inherently unstable.

[0075] As used herein, the terms “substituted,” “substituted,” or “replaced by” mean that one or more hydrogen atoms on a given atom or group are replaced by one or more substituents selected from the given group of substituents, provided that the substitution does not exceed the normal valence of the given atom. When the substituent is oxo (i.e., =O), the two hydrogen atoms on a single atom are replaced by oxygen. Oxo-substituents are not present on aromatic moieties. When a ring system (e.g., a carbocyclic or heterocyclic ring) is substituted by a carbonyl group or double bond, it is intended that the carbonyl group or double bond is part of the ring (i.e., within the ring). Such combinations are permitted only if the combination of substituents and / or variables results in a chemically correct and stable compound. A chemically correct and stable compound means that the compound is stable enough to be isolated from the reaction mixture and its chemical structure can be determined, and it can subsequently be formulated into an agent with at least practical utility. For example, where no substituent is explicitly listed, the terms “substituted,” “substituted by,” or “replaced by” as used herein mean that one or more hydrogen atoms on a given atom or group are independently replaced by one or more, for example, 1, 2, 3, or 4 substituents. When an atom or group is replaced by multiple substituents, the substituents may be the same or different.

[0076] Unless otherwise specified, the term "compound of the present invention" or "compound of the present invention" refers to one or more compounds of formula (I) or its subforms as defined herein, such as formulas (I-1), (I-2), etc., or pharmaceutically acceptable salts thereof, and all isomers such as stereoisomers (including diastereomers, enantiomers, and racemates), geometric isomers, conformational isomers (including rotational isomers and rotation-blocked isomers), tautomers, internal addition products of isomers, prodrugs, and isotopically labeled compounds (including deuterium-substituted compounds) and inherently formed moieties (e.g., polymorphs, solvates, and / or hydrates). Salts, particularly pharmaceutically acceptable salts, are also included where salt-forming moieties are present. The presence of tautomers or internal addition products of isomers can be identified by those skilled in the art using tools such as NMR. Compounds of formula (I) of the present invention are capable of readily forming internal addition products of tautomers and isomers as described herein.

[0077] Those skilled in the art will recognize that the compounds of the present invention may contain a chiral center, thereby allowing for different isomeric forms. As used herein, "isomer" refers to different compounds having the same molecular formula but differing in the arrangement and configuration of their atoms.

[0078] As used herein, an enantiomer is a pair of stereoisomers that are non-overlapping mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. Where appropriate, the term is used to refer to racemic mixtures. When indicating the stereochemistry of the compounds of the present invention, a single stereoisomer with known relative and absolute configurations (e.g., (1S,2S)) having two chiral centers is designated using the conventional RS system; a single stereoisomer with a known relative configuration but an unknown absolute configuration is indicated by an asterisk (e.g., (1R*,2R*)); and a racemic mixture with two letters (e.g., (1RS,2RS) is a racemic mixture of (1R,2R) and (1S,2S); (1RS,2SR) is a racemic mixture of (1R,2S) and (1S,2R)). A "diastereomer" is a stereoisomer having at least two asymmetric atoms but not being mirror images of each other. Absolute stereochemistry is indicated according to the Cahn-Lngold-Prelog RS system. When the compound is a pure enantiomer, the stereochemistry at each chiral carbon can be described by R or S. The resolved compounds with unknown absolute configurations can be designated as (+) or (-) based on the direction (dextrorotatory or levorotatory) of their rotational plane-polarized light at the sodium D line wavelength. Alternatively, the resolved compounds can be defined by the respective retention times of the corresponding enantiomers / diastereomers via chiral HPLC.

[0079] Some of the compounds described herein contain one or more asymmetric centers or axes, thus producing enantiomers, diastereomers, and other stereoisomers that can be defined by absolute stereochemistry as (R)- or (S)-.

[0080] Geometric isomerism can occur when a compound contains a double bond or other features that give the molecule a certain degree of structural rigidity. If the compound contains a double bond, the substituent can be in the E or Z conformation. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent can have a cis or trans configuration.

[0081] Conformational isomers are isomers that differ by rotation of one or more valence bonds. Rotational isomers are conformational isomers that differ by rotation of only a single valence bond.

[0082] "Restricted rotation isomers" refer to structural isomers that exhibit axial or planar chirality due to rotational restriction within the molecule.

[0083] Unless otherwise stated, the compounds of this invention are intended to include all such possible isomers, including racemic mixtures, optically active forms, and intermediate mixtures. Optically active (R)- and (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.

[0084] The compounds of the present invention can be isolated into optically active or racemic forms. The optically active form can be prepared by resolution of the racemic form or by synthesis from optically active starting materials. All methods used to prepare the compounds of the present invention and the intermediates prepared therein are considered part of the present invention. When preparing enantiomers or diastereomers, they can be separated by conventional methods such as chromatography or stepwise crystallization.

[0085] Depending on the method conditions, the end products of the present invention are obtained in free (neutral) or salt form. Both the free and salt forms of these end products are within the scope of the present invention. If desired, one form of the compound can be converted to another form. Free bases or acids can be converted to salts; salts can be converted to free compounds or other salts; mixtures of isomers of the present invention can be separated into individual isomers.

[0086] As used herein, "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compounds of the present invention, and that is not biologically or otherwise undesirable. Non-limiting examples of such salts include non-toxic, inorganic or organic addition salts of bases or acids of the compounds of the present invention. In many cases, the compounds of the present invention are capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or similar groups. Pharmaceutically acceptable acid addition salts can be formed using inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc., especially, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. The pharmaceutically acceptable salts of this invention can be synthesized from the parent compound (basic or acidic moiety) using conventional chemical methods. Generally, the salts described herein can be prepared by reacting the free acidic form of the compound with a stoichiometric amount of an appropriate base (e.g., hydroxides, carbonates, bicarbonates, etc. of Na, Ca, Mg, or K) or by reacting the free basic form of the compound with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent or a mixture of both. Generally, when feasible, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Other suitable salts can be found in Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, Easton, Pa., (1985), which is incorporated herein by reference.

[0087] The arbitrary formulas given herein are also intended to represent the unlabeled form and the isotopically labeled form of the compound. Except that one or more atoms are replaced by atoms having a selected atomic mass or mass number, the isotopically labeled compound has the structure described by the formulas given herein. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, for example, respectively. 2 H (i.e., D) 3 H (i.e., T) 11 C13 C 14 C 15 N、 18 F 31 P, 32 P, 35 S, 36 Cl、 125 I. This invention includes compounds labeled with different isotopes as defined herein, for example, compounds containing radioactive isotopes such as... 3 H, 13 C and 14 Those with C. These isotope-labeled compounds can be used for metabolic studies (using...). 14 C) Reaction kinetic studies (e.g., using...) 2 H or 3 H) Detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or those applicable to the patient's radiation therapy. In particular, 18 F-labeled compounds are particularly desirable for PET or SPECT studies. The isotopically labeled compounds of the present invention can typically be prepared by performing the methods described in the procedures or examples and preparation examples below, using readily available isotopically labeled reagents instead of unlabeled reagents.

[0088] Moreover, it is affected by heavier isotopes, especially deuterium (i.e., 2 Substitution with H or D can also yield certain therapeutic benefits due to greater metabolic stability, such as prolonged in vivo half-life, reduced dose requirements, or improved therapeutic index. It is understood that deuterium in the context can be considered a substituent in the compounds of this invention. The concentration of such heavier isotopes, particularly deuterium, can be defined by isotope enrichment factors.

[0089] As used herein, the "therapeutic effective dose" of the compounds of this invention refers to the amount of the compounds of this invention that can elicit a biological or medical response in an individual or improve symptoms, slow or delay disease progression, or prevent disease. The "therapeutic effective dose" can be determined by the participating physician or veterinary practitioner and will vary depending on factors such as the compound, the disease state being treated, the severity of the disease being treated, the individual's age and related health conditions, the route and form of administration, and the judgment of the attending physician or veterinary practitioner.

[0090] As used herein, "individual" refers to an animal. Preferably, the animal is a mammal. "Individual" also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In a preferred embodiment, the individual is a human being.

[0091] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0092] As used in this article, “inhibition” refers to the reduction or suppression of a specific patient, symptom, condition, or disease, or a significant reduction in biological activity or baseline activity of a process.

[0093] The following embodiments illustrate the present invention as described above; however, they do not limit the scope of the invention in any way. The beneficial effects of combinations of the present invention can also be determined using other test models known to those skilled in the art.

[0094] Compound synthesis:

[0095] In this invention, room temperature refers to ambient temperature, which is 10℃-35℃. Overnight refers to 8-15 hours. Reflux temperature refers to the solvent reflux temperature under normal pressure.

[0096] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) are given in units of 10⁻⁶ (ppm). NMR determinations were performed using a Bruker AVANCE-400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), and deuterated methanol (CD₃OD) as solvents, and tetramethylsilane (TMS) as the internal standard. MS determinations were performed using a Finnigan LCQ / Deca (ESI) mass spectrometer. High-performance liquid chromatography (HPLC) analysis was performed using a Gilson Nebula Series HPLC system.

[0097] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF 254 or Qingdao GF 254 silica gel plates. The silica gel plates used in TLC are 0.15mm-0.2mm in diameter, while those used for separating and purifying products are 0.4mm-0.5mm in diameter. Silica gel column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0098] The known starting materials of this invention can be synthesized using methods known in the art, or can be purchased from companies such as Shanghai Haohong Biomedical Technology Co., Ltd. and Bid Pharmaceuticals. Unless otherwise specified in the examples, all reactions can be carried out under an argon or nitrogen atmosphere.

[0099] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system used for column chromatography to purify the compounds, and the developing solvent system for TLC included dichloromethane / methanol and petroleum ether / ethyl acetate systems. The volume ratio of the solvent was adjusted according to the polarity of the compounds. Small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0100] Example 1

[0101]

[0102] first step

[0103] Ethyl benzoyl ester (1 g, 5.2 mmol) and ethyl 5-amino-pyrazole-3-carboxylate (673 mg, 4.34 mmol) were reacted in acetic acid solvent at 80 °C for 8 h. After removing acetic acid under reduced pressure, the reaction solution was separated by column chromatography (DCM:MeOH = 20:1) to give ethyl 7-hydroxy-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylate as a white solid in yield of 61.07%.

[0104] 1 H NMR (400MHz, DMSO-d6) δ7.96–7.79(m,2H),7.73–7.49(m,3H),6.56(s,1H),6.18(s,1H),4.34(q,J=7.1Hz,2H),1.33(t,J=7.1Hz,3H).

[0105] Step 2

[0106] Ethyl 7-hydroxy-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylate (750 mg, 2.65 mmol) was dissolved in phosphorus oxychloride (15 ml) in an ice bath. After stirring for five minutes, the ice bath was removed, and the reaction was carried out at 80 °C for 3 h until TLC showed complete consumption of the starting material. Phosphorus oxychloride was removed under reduced pressure, and the reaction solution was poured into ice water. Saturated sodium bicarbonate aqueous solution was added to adjust the pH to about 7. The mixture was extracted with ethyl acetate (EA), dried over anhydrous sodium sulfate, and separated by column chromatography (PE:EA = 5:1, v:v) to give ethyl 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylate as a white solid with a yield of 52.58%.

[0107] 1 H NMR (400MHz, DMSO-d6) δ8.33–8.24(m,3H),7.59(p,J=3.9Hz,3H),7.35(s,1H),4.42(q,J=7.1Hz,2H),1.37(t,J=7.2Hz,3H).

[0108] Step 3

[0109] Ethyl 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylate (420 mg, 1.39 mmol) was dissolved in dioxane (10 mL), and 3-hydroxypyrrolidine (146 mg, 1.67 mmol) and diisopropylethylamine (DIPEA) (0.5 mL) were added. The reaction was carried out at room temperature for 8 h. TLC showed that the starting material was completely consumed. Column chromatography (DCM:MeOH = 30:1, v:v) yielded compound 1: 7-(3-hydroxypyrrolidine-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylate, a white solid with a yield of 67.28%.

[0110] 1 H NMR(400MHz,Chloroform-d)δ7.99–7.93(m,2H),7.54–7.40(m,3H),6.94(s,1H),6.18(s,1H),4.67(s,1H),4.43(q,J=7.1Hz,2H),4.28(d,J=2.8Hz ,2H),4.09(q,J=9.4,8.9Hz,1H),3.99(dq,J=10.8,6.3,5.0Hz,1H),2.16( tt,J=9.2,5.1Hz,2H),1.43(t,J=7.1Hz,3H).MS(ESI):m / z=353.18[M+H]+

[0111] Step 4

[0112] Ethyl 7-(3-hydroxypyrrolidone-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid (330 mg, 1.39 mmol) was dissolved in a methanol / water solution (10 mL, v:v = 1:1). Lithium hydroxide (10%) solution was then added dropwise, and the mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, followed by extraction with EA. The aqueous phase was adjusted to pH 2 with 2 M hydrochloric acid, and then extracted three more times with EA. The organic phases were combined, dried over anhydrous sodium sulfate, and separated by column chromatography (DCM:MeOH = 15:1, v:v) to obtain 7-(3-hydroxypyrrolidone-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid as a white solid in 72.43% yield.

[0113] 1 H NMR(400MHz, Methanol-d4)δ8.09–7.92(m,2H),7.49(dd,J=8.8,3.9Hz,3H),6.77(s,1H),6.29(s,1H),4.62–4 .55(m,1H),4.31(d,J=2.9Hz,2H),4.14(dd,J=9.0,5.0Hz,2H),2.21–2.12(m,2H).MS(ESI):m / z=325.20[M+H]+

[0114] Step 5

[0115] 7-(3-hydroxypyrrolidone-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxylic acid (80 mg, 0.25 mmol), DIPEA (64 mg, 0.49 mmol), and cyclopropylamine (16 mg, 0.28 mmol) were dissolved in DMF, and HATU (283 mg, 0.74 mmol) was added. The reaction was carried out at room temperature for 12 h. The reaction solution was extracted with EA, washed with a saturated sodium chloride system, and the organic phase was washed successively with 2 M hydrochloric acid, water, and 5% sodium bicarbonate. After drying the organic phase with anhydrous sodium sulfate, column chromatography (DCM:MeOH = 20:1) was used to separate compound 1: N-cyclopropyl-7-(3-hydroxypyrrolidone-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxamide. The product was a white solid with a yield of 44.62%.

[0116] 1 H NMR (400MHz, DMSO-d6) δ8.29(d,J=4.1Hz,1H),8.15(dd,J=7.3,2.5Hz,2H),7.50(dd,J=5.0,2.2Hz,3H),6.69(s,1H),6.50( s,1H),5.12(d,J=3.4Hz,1H),4.46(s,1H),4.19–4.09(m,2H),2.95(s,2H),2.84(dt,J=7.2,3.8Hz,2H),2.08–1.97(m,4H).

[0117] MS(ESI): m / z = 364.26[M+H]+

[0118] Example 2

[0119]

[0120] Using the synthetic route of Example 1, cyclopropylamine, the starting material in step 5, was replaced with cyclohexylamine to obtain compound 2: N-cyclohexyl-7-(3-hydroxypyrrolidine-1-yl)-5-phenylpyrazolo[1,5-a]pyrimidine-2-carboxamide. The product was a white solid with a yield of 45.55%.

[0121] 1 H NMR (400MHz, DMSO-d6) δ8.21–8.12(m,2H),7.94(d,J=8.4Hz,1H),7.50(dd,J= 5.1,2.1Hz,3H),6.70(s,1H),6.51(s,1H),5.14(d,J=3.4Hz,1H),4.47(s,1H), 4.24–4.00(m,3H),3.80(d,J=6.9Hz,1H),2.04(d,J=26.0Hz,2H),1.82(d,J=1 1.5Hz,2H),1.74(d,J=12.6Hz,2H),1.62(d,J=12.9Hz,1H),1.47–1.20(m,8H).

[0122] MS(ESI): m / z = 406.20[M+H]+

[0123] Example 3

[0124]

[0125] first step

[0126] Ethyl benzoyl acetate (500 mg, 2.6 mmol) and 3-aminopyrazole (180.12 mg, 2.17 mmol) were reacted in acetic acid solvent at 80 °C for 8 h to give 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol. After removing acetic acid under reduced pressure, the product was washed with water and dichloromethane to give the crude product 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol, which did not require further purification and was a white solid with a yield of 65.52%.

[0127] 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),7.90(d,J=1.9Hz,1H),7.89–7.78(m,2H),7.59(dd,J=5.2,1.9Hz,3H),6.22(d,J=2.0Hz,1H),6.06(s,1H).

[0128] Step 2

[0129] 330 mg (1.56 mmol) of 5-phenylpyrazolo[1,5-a]pyrimidine-7-ol was carefully added in portions to phosphorus oxychloride (15 ml) in an ice bath. After stirring for a period of time, the ice bath was removed, and the mixture was heated to 80 °C for 3 h until TLC showed that the starting material was completely consumed. The phosphorus oxychloride was removed under reduced pressure, and the reaction solution was poured into ice water. A saturated sodium bicarbonate aqueous solution was added to adjust the pH to about 7. The mixture was extracted with EA, dried over anhydrous sodium sulfate, and separated by column chromatography (PE:EA = 5:1, v:v) to give 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine as a white solid with a yield of 55.74%.

[0130] 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=2.3Hz,1H),8.27–8.21(m,2H),8.05(s,1H),7.60–7.49(m,3H),6.91(d,J=2.2Hz,1H).

[0131] Step 3

[0132] 7-Chloro-5-phenylpyrazolo[1,5-a]pyrimidine (200 mg, 0.87 mmol) was dissolved in dioxane, and 3-hydroxypyrrolidine (151.74 mg, 1.74 mmol) and DIPEA (337.65 mg, 2.61 mmol) were added. The mixture was reacted at room temperature for 8 h to give compound 3:1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 65.54%.

[0133] 1 H NMR (400MHz, Methanol-d4) δ8.01–7.93(m,3H),7.53–7.45(m,3H),6.38(d,J=2.3Hz,1H),6.25(s,1H ),4.56(tt,J=4.1,2.5Hz,1H),4.22(d,J=4.0Hz,2H),4.11(dd,J=9.7,5.9Hz,2H),2.22–2.10(m,2H).

[0134] Example 4

[0135]

[0136] Using the synthetic route of Example 1, the third step starting material pyrrolanol was replaced with (S)-3-pyrrolanol to obtain compound 4: (S)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolan-3-ol. The product was a white solid with a yield of 66.54%.

[0137] 1 H NMR(400MHz,Chloroform-d)δ8.01–7.94(m,2H),7.92(d,J=2.3Hz,1H),7.51–7.40(m,3H),6.48(d,J=2.3Hz,1H),6.06(s,1H),4.63( p,J=3.2Hz,1H),4.20(d,J=3.1Hz,2H),4.05(dt,J=10.3,8.6Hz,1H),3.97(dt,J=10.6,5.5Hz,1H),2.13(ddt,J=8.9,5.8,2.5Hz,2H).

[0138] Example 5

[0139]

[0140] Using the synthetic route of Example 1, the third step starting material pyrrolanol was replaced with (R)-3-pyrrolanol to obtain compound 5: (R)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolan-3-ol. The product was a white solid with a yield of 53.25%.

[0141] 1 H NMR(400MHz,Chloroform-d)δ8.02–7.96(m,2H),7.94(d,J=2.2Hz,1H),7.51–7.41(m,3H),6.50(d,J=2.3Hz,1H),6.13(s,1H ),4.67(p,J=3.1Hz,1H),4.24(d,J=2.9Hz,2H),4.11(td,J=9.9,8.0Hz,1H),4.02(dt,J=10.7,5.0Hz,1H),2.18–2.13(m,2H).

[0142] Example 6

[0143]

[0144] Using the synthetic route of Example 1, the third step starting material pyrrolanol was replaced with (3S,5S)-5-(hydroxymethyl)pyrrolan-3-ol to obtain compound 6: (3S,5S)-5-(hydroxymethyl)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolan-3-ol. The product was a white solid with a yield of 39.22%.

[0145] 1H NMR(400MHz,Chloroform-d)δ7.69–7.51(m,3H),7.30(d,J=6.9Hz,3H),6.21(d,J=2.2Hz,1H),5.74(s,1H),4.81(tt,J=7 .4,3.5Hz,1H),4.72–4.47(m,2H),4.39(q,J=4.3,3.6Hz,1H),3.61(td,J=8.9,4.4Hz,2H),2.11(dt,J=12.6,5.4Hz,1H).

[0146] Example 7

[0147]

[0148] Using the synthetic route of Example 1, the third step starting material pyrrolanol was replaced with (3R,4R)-pyrrolidine-3,4-diol to obtain compound 7: (3R,4R)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3,4-diol. The product was a white solid with a yield of 37.50%.

[0149] 1 H NMR (400MHz, DMSO-d6) δ8.18–8.11(m,2H),8.03(d,J=2.3Hz,1H),7.54–7.44(m,3H),6 .43–6.38(m,2H),5.31(d,J=3.0Hz,2H),4.09(t,J=3.3Hz,2H),4.03(q,J=7.1Hz,2H).

[0150] Example 8

[0151]

[0152] Compound 3 (50 mg, 0.18 mmol) was dissolved in THF in an ice bath. A THF solution of Dysmart oxidant (DMP) (381 mg, 0.90 mmol) was added dropwise. After stirring for five minutes, the ice bath was removed, and the mixture was allowed to rise to room temperature and react for 4 hours until TLC showed complete consumption of the starting material. The mixture was washed with saturated sodium bicarbonate solution, followed by washing with saturated sodium thiosulfate solution. The mixture was extracted with EA, and the organic phase was dried over anhydrous sodium sulfate. Column chromatography (DCM:MeOH = 50:1) yielded compound 8: 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-one, a white solid with a yield of 41.11%.

[0153] Example 9

[0154]

[0155] Following the synthetic route of Example 1, the starting material in step 3, pyrrolanol, was replaced with (S)-pyrrolidine-3-ylcarbamate tert-butyl ester. After step 3, the boc protecting group was removed using dioxane hydrochloride to yield compound 9: (S)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-amine, a white solid, with a two-step yield of 54.33%.

[0156] 1 H NMR (400MHz, DMSO-d6) δ8.47(s,3H),8.20(d,J=2.2Hz,1H),8.12(dd,J=6.7,3.0Hz,2H),7.59(h,J=5.0,4.4Hz,3H),6.56(d,J= 3.0Hz,2H),4.51(s,2H),4.16(d,J=36.8Hz,2H),2.37(dt,J=11.0,4.2Hz,1H),2.28–2.17(m,1H),1.99(dt,J=13.3,6.9Hz,1H).

[0157] Example 10

[0158]

[0159] Using the synthetic route of Example 9, the third-step starting material (S)-pyrrolidine-3-ylcarbamate tert-butyl ester was replaced with (R)-pyrrolidine-3-ylcarbamate tert-butyl ester to obtain compound 10: (R)-1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-amine. The product was a white solid, and the two-step yield was 49.33%.

[0160] 1 H NMR (400MHz, DMSO-d6) δ8.51(s,3H),8.23–8.13(m,2H),8.06(d,J=2.3Hz,1H),7.56–7.46(m,3H),6.45(d,J=1.8Hz,2H),4.4 1(d,J=4.5Hz,2H),4.08(q,J=8.6,8.0Hz,1H),3.97(s,2H),2.34(dt,J=14.3,6.9Hz,1H),2.21(dq,J=12.0,6.9,5.6Hz,1H).

[0161] Example 11

[0162]

[0163] Using the synthetic route of Example 3, the starting material pyrrolanol in the third step was replaced with (S)-2-aminoprop-1-ol to obtain compound 11: (S)-2-((5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)amino)prop-1-ol. The product was a white solid with a yield of 34.24%.

[0164] 1H NMR(400MHz,Chloroform-d)δ8.00(dd,J=5.6,2.6Hz,3H),7.47(q,J=6.9Hz,3H),6.55(q,J=2.3Hz,1H),6.42(d,J =5.3Hz,1H),4.09–3.94(m,1H),3.89(dt,J=10.6,4.7Hz,1H),3.77(dt,J=11.1,5.5Hz,1H),1.43(t,J=6.1Hz,3H).

[0165] Example 12

[0166]

[0167] Using the synthetic route of Example 3, the starting material pyrrolanol in the third step was replaced with azircyclobutane-3-ol to obtain compound 12: 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)azircyclobutane-3-ol. The product was a white solid with a yield of 36.65%.

[0168] 1 H NMR(500MHz,DMSO-d6)δ8.16–8.11(m,2H),8.03(d,J=2.3Hz,1H),7.53–7.45(m,3H),6.41(d,J=2.3Hz,1H),6 .32(s,1H),5.87(d,J=6.3Hz,1H),4.75(s,2H),4.67–4.62(m,1H),4.26(s,2H).MS(ESI):m / z=267.20[M+H]+

[0169] Example 13

[0170]

[0171] Following the synthetic route of Example 3, the third-step starting material, pyrrolidine alcohol, was replaced with azircyclobutane-3-ylmethanol to obtain compound 13: (1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)azircyclobutane-3-yl)methanol. The product was a white solid with a yield of 49.16%.

[0172] 1H NMR(400MHz,Chloroform-d)δ8.02–7.96(m,2H),7.94(d,J=2.3Hz,1H),7.52–7.42(m,3H),6.47(d,J=2.3Hz,1H),5. 96(s,1H),4.63(t,J=8.7Hz,2H),4.43–4.30(m,2H),3.03(ddq,J=14.3,8.3,6.0Hz,1H).MS(ESI):m / z=281.26[M+H]+

[0173] Example 14

[0174]

[0175] Following the synthetic route of Example 3, the third-step starting material, pyrrolidine alcohol, was replaced with piperidine-4-ol to obtain compound 14: 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperidine-4-ol. The product was a white solid with a yield of 29.16%.

[0176] 1 H NMR(400MHz,Chloroform-d)δ8.08(d,J=2.3Hz,1H),8.05–7.99(m,2H),7.53–7.43(m,3H),6.63(d,J=2.3Hz,1H),6.56(s,1H),4.16(dt,J=1 2.4,3.4Hz,2H),4.06(td,J=8.5,8.0,4.5Hz,1H),3.49(ddd,J=12.3,8.8,3.3Hz,2H),2.20–2.10(m,2H),1.86(dtd,J=12.5,8.5,3.7Hz,2H).

[0177] Example 15

[0178]

[0179] Following the synthetic route of Example 3, the third-step starting material, pyrrolidine alcohol, was replaced with piperidine-3-ol to obtain compound 15: 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)piperidine-3-ol. The product was a white solid with a yield of 39.16%.

[0180] 1H NMR (400MHz, DMSO-d6) δ8.23–8.09(m,3H),7.59–7.46(m,3H),6.82(s,1H),6.57(d,J=2.3Hz,1H),5.01(d,J=4.3Hz,1H),4.35(dd,J=12.3,3.8Hz,1H) ,4.13(d,J=12.8Hz,1H),3.75(tt,J=8.7,4.3Hz,1H),3.19(dd,J=12.2,8.5 Hz,1H),2.02–1.85(m,2H),1.73–1.58(m,1H),1.49(q,J=10.5,8.8Hz,1H).

[0181] Example 16

[0182]

[0183] Following the synthetic route of Example 3, the third-step starting material, pyrrolidone, was replaced with morpholine to obtain compound 16: 4-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)morpholine. The product was a white solid with a yield of 32.77%.

[0184] 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J=2.3Hz,1H),8.00(dd,J=7.5,2.0Hz,2H),7.50–7.42 (m,3H),6.63(d,J=2.3Hz,1H),6.50(s,1H),4.04–3.93(m,4H),3.74(dd,J=5.8,3.4Hz,4H).

[0185] Example 17

[0186]

[0187] first step

[0188] Diethyl malonate (3.86 g, 24.07 mmol) and 3-aminopyrazole (2.00 g, 24.07 mmol) were dissolved in ethanol (20 ml), and freshly prepared sodium ethoxide (16.38 g, 240.69 mmol) was added. The mixture was refluxed for 6 h, and the pH was adjusted to 3. After washing with water and DCM, the solid was dried and directly proceeded to the next step of the reaction without further purification. The product was white and the yield was 60.48%.

[0189] 1H NMR (400MHz, DMSO-d6) δ12.26(s,1H),10.53(s,1H),7.60(d,J=2.3Hz,1H),6.48(d,J=2.3Hz,1H).MS(ESI):m / z=152.02[M+H]+

[0190] Step 2

[0191] 2.2 g (14.56 mmol) of pyrazolo[1,5-a]pyrimidine-5,7-diol was dissolved in 25 ml of phosphorus oxychloride in an ice bath. After stirring for five minutes, the ice bath was removed, and the reaction was carried out at 80 °C for 3 h until TLC showed complete consumption of the starting material. Phosphorus oxychloride was removed under reduced pressure, and the reaction solution was poured into ice water. Saturated sodium bicarbonate aqueous solution was added to adjust the pH to about 7. Extraction was performed with EA, and column chromatography was used for separation (PE:EA = 5:1, v:v) to obtain 5,7-dichloropyrazolo[1,5-a]pyrimidine. The product was a white solid with a yield of 40.19%.

[0192] 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=2.3Hz,1H),7.67(s,1H),6.89(d,J=2.3Hz,1H).

[0193] Step 3

[0194] 5,7-Dichloropyrazolo[1,5-a]pyrimidine (1.10 g, 5.85 mmol) was dissolved in dioxane (15 mL), and 3-hydroxypyrrolidine (612 mg, 7.02 mmol) and DIPEA were added. The mixture was reacted at room temperature for 3 h. TLC showed that the starting material was completely consumed. The mixture was extracted with EA, dried over anhydrous sodium sulfate, and separated by column chromatography (DCM:MeOH = 30:1, v:v) to give 1-(5-chloropyrazolo[1,5-a]pyrimidine-7-yl)pyrrolidine-3-ol, which was a white solid with a yield of 57.29%.

[0195] 1 H NMR (400MHz, DMSO-d6) δ8.04(d,J=2.2Hz,1H),6.33(d,J=2.2Hz,1H),5.95(s,1H),5.12(d ,J=3.5Hz,1H),4.44–4.34(m,1H),4.00(s,3H),1.98(dddd,J=19.1,14.5,7.7,3.6Hz,2H).

[0196] Step 4

[0197] 1-(5-chloropyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol (40 mg, 0.17 mmol), 2,2-aminophenylboronic acid pinacol ester (47 mg, 0.21 mmol), and K2CO3 (71 mg, 0.51 mmol) were dissolved in a solvent (5 mL, dioxane:water = 3:1). After degassing for 5 minutes, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride catalyst (10 mg, 0.01 mmol) was added. Under N2 protection, the reaction was carried out at 80 °C for 8 h. After the starting material was completely consumed, the mixture was extracted using an EA / H2O system and separated by column chromatography (DCM:MeOH = 30:1) to give compound 17:1-(5-(2-aminophenyl)pyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol, which was a white solid with a yield of 19.39%.

[0198] 1 H NMR(400MHz,Chloroform-d)δ7.92(d,J=2.3Hz,1H),7.52(dd,J=7.7,1.6Hz,1H),7.21–7.14(m,1H),6.75(t,J=8.6Hz,2H),6.37(d,J=2.3Hz,1H),6.06 (s,1H),4.65(dq,J=5.8,2.8Hz,1H),4.26–4.16(m,2H),4.09(td,J=9.9,7. 4Hz, 1H), 4.00 (ddd, J=10.7, 7.5, 3.8Hz, 1H), 2.15 (dt, J=10.3, 3.9Hz, 2H).

[0199] Example 18

[0200]

[0201] Following the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with 3-fluorophenylboronic acid to obtain compound 18: 1-(5-(3-fluorophenyl)pyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 41.60%.

[0202] 1H NMR(400MHz,Chloroform-d)δ7.97(d,J=2.3Hz,1H),7.79(dt,J=7.8,1.3Hz,1H),7.73( dt,J=10.2,2.2Hz,1H),7.45(td,J=8.0,5.8Hz,1H),7.20–7.10(m,1H),6.52(d,J=2.3Hz ,1H),6.11(s,1H),4.70(p,J=3.2Hz,1H),4.28(d,J=3.3Hz,2H),4.20–4.10(m,1H),4.05 (dt,J=10.7,5.5Hz,1H),2.19(ddt,J=8.7,5.7,2.4Hz,2H).MS(ESI):m / z=299.24[M+H]+

[0203] Example 19

[0204]

[0205] Using the synthetic route of Example 17, the fourth step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with 3-aminophenylboronic acid pinacol ester to obtain compound 19: 1-(5-(3-aminophenyl)pyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 40.41%.

[0206] 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=2.3Hz,1H),7.78(d,J=8.6Hz,2H),6.64(d,J=8.6Hz,2H),6.26(d, J=2.3Hz,1H),6.17(s,1H),5.49–5.33(m,3H),4.41(s,2H),2.01(dq,J=9.1,4.4Hz,1H),1.94(s,2H).

[0207] Example 20

[0208]

[0209] Using the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with 4-aminophenylboronic acid pinacol ester to obtain compound 20: 1-(5-(4-aminophenyl)pyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 27.16%.

[0210] 1H NMR(500MHz,Chloroform-d)δ7.91(d,J=2.3Hz,1H),7.88–7.83(m,2H),6.78–6.72(m,2H),6.42(d,J=2.3Hz ,1H),6.10(s,1H),4.66(s,1H),4.29–4.16(m,2H),4.14–4.09(m,1H),4.03–3.96(m,1H),2.19–2.11(m,2H).

[0211] Example 21

[0212]

[0213] Following the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with (1-(tert-butoxycarbonyl)-1,2,3,6-tetrahydropyridin-4-yl)boronic acid to obtain compound 21: 4-(7-(3-hydroxypyrrolidine-1-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-3,6-dihydropyridin-1(2H)-carboxylic acid tert-butyl ester. The product was a white solid with a yield of 43.34%.

[0214] 1 H NMR(400MHz,Chloroform-d)δ7.89(t,J=2.0Hz,1H),6.53(s,1H),6.38(t,J=1.9Hz,1H),5.75(s,1H),4.63(d,J=4.2Hz,1H),4.26–4.09(m,4H ),4.03(q,J=9.1Hz,1H),3.92(dt,J=10.4,5.5Hz,1H),3.62(t,J=5.7Hz,2H),2.63(s,2H),2.13(p,J=4.1Hz,2H).MS(ESI):m / z=386.14[M+H]+

[0215] Example 22

[0216]

[0217] Following the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with dibenzo[b,d]furan-4-ylboronic acid to obtain compound 22: 1-(5-(dibenzo[b,d]furan-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 54.13%.

[0218] 1H NMR(600MHz,Chloroform-d)δ8.27(dd,J=7.8,1.3Hz,1H),8.00(dd,J=7.6,1.3Hz,1 H),7.99–7.95(m,2H),7.62(d,J=8.2Hz,1H),7.48(ddd,J=10.2,8.7,7.4Hz,2H),7.3 8(t,J=7.5Hz,1H),6.90(s,1H),6.55(d,J=2.2Hz,1H),4.72(d,J=4.0Hz,1H),4.38– 4.28(m,2H),4.22(q,J=9.0Hz,1H),4.16(dt,J=11.1,5.8Hz,1H),1.55–1.47(m,2H).

[0219] MS(ESI): m / z = 325.25[M+H]+

[0220] Example 23

[0221]

[0222] Following the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with (2-methylprop-1-en-1-yl)boronic acid to obtain compound 23: 1-(5-(2-methylprop-1-en-1-yl)pyrazol[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 53.90%.

[0223] 1 H NMR(400MHz,Chloroform-d)δ7.84(d,J=2.3Hz,1H),6.31(d,J=2.3Hz,1H),6.18–6.10(m,1H),5.50(s,1H),4.58(dd,J=5.1,2.5Hz,1 H),4.19–4.05(m,2H),3.98(td,J=10.0,7.4Hz,1H),3.84(ddd,J=10.6,7.5,3.6Hz,1H),2.14–2.07(m,2H),2.06(s,3H),1.92(s,3H).

[0224] Example 24

[0225]

[0226] Using the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with cyclopropylboronic acid to obtain compound 24: 1-(5-cyclopropylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 60.61%.

[0227] 1 H NMR(400MHz,Chloroform-d)δ7.84(d,J=2.3Hz,1H),6.25(d,J=2.3Hz,1H),5.54(s,1H),4.62(p,J=3.2Hz,1H),4.14(d,J=2.9Hz,2H),4.07–3.95(m,1H) ,3.89(dt,J=10.6,5.6Hz,1H),2.12(ddd,J=8.8,6.0,3.5Hz,2H),1.93–1.8 9(m,1H),1.05(td,J=6.1,5.4,2.7Hz,2H),0.97(dq,J=8.1,2.6,1.8Hz,3H).

[0228] Example 25

[0229]

[0230] first step

[0231] Ethyl 3-cyclobutyl-3-oxopropionate (250 mg, 1.47 mmol) and 3-aminopyrazole (102 mg, 1.22 mmol) were dissolved in acetic acid and reacted at 80 °C for 8 h to obtain 5-cyclobutylpyrazolo[1,5-a]pyrimidin-7-ol. After removing acetic acid under reduced pressure, the product was washed with water and dichloromethane to obtain the crude product. No further purification was required. The product was a white solid with a yield of 51.81%.

[0232] MS(ESI): m / z = 190.27[M+H]+

[0233] Step 2

[0234] 5-Cyclobutylpyrazolo[1,5-a]pyrimidine-7-ol (120 mg, 0.63 mmol) was carefully added in portions to phosphorus oxychloride (10 ml) at 0°C. After stirring for a period of time, the ice bath was removed, and the mixture was heated to 80°C for 3 h until TLC showed complete consumption of the starting material. Phosphorus oxychloride was removed under reduced pressure, and the reaction solution was poured into ice water. Saturated sodium bicarbonate aqueous solution was added to adjust the pH to about 7. Extraction was performed with EA, and the organic phase was dried over anhydrous sodium sulfate. The mixture was then separated by column chromatography (PE:EA = 5:1, v:v) to give 7-chloro-5-cyclobutylpyrazolo[1,5-a]pyrimidine as a white solid with a yield of 49.36%.

[0235] 1 H NMR(400MHz,Chloroform-d)δ8.16(d,J=2.3Hz,1H),6.84(s,1H),6.71(d,J=2.3Hz ,1H),3.77–3.61(m,1H),2.46–2.36(m,4H),2.19–2.04(m,1H),2.02–1.88(m,1H).

[0236] Step 3

[0237] 7-Chloro-5-cyclobutylpyrazolo[1,5-a]pyrimidine (65 mg, 0.3 mmol) was dissolved in dioxane (3 ml), and 3-hydroxypyrrolidine (33 mg, 0.38 mmol) and DIPEA (121 mg, 0.94 mmol) were added. The mixture was reacted at room temperature for 8 h to give compound 25:1-(5-cyclobutylpyrazolo[1,5-a]pyrimidine-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 55.65%.

[0238] 1 H NMR(500MHz,Chloroform-d)δ7.82(d,J=2.2Hz,1H),6.30(d,J=2.3Hz,1H),5. 47(s,1H),4.59(dp,J=4.3,2.0Hz,1H),4.23–4.07(m,2H),3.98(td,J=10.0,7. 2Hz,1H),3.85(ddd,J=10.6,7.9,3.2Hz,1H),3.58–3.48(m,1H),2.29(tdd,J= 9.8,5.4,4.2Hz,4H),2.14–2.05(m,2H),2.04–1.96(m,1H),1.91–1.79(m,1H).

[0239] Example 26

[0240]

[0241] Following the synthetic route of Example 17, the fourth-step starting material, 2-aminophenylboronic acid pinacol ester, was replaced with cyclopent-1-en-1-ylboronic acid to obtain compound 26: 1-(5-(cyclopent-1-en-1-yl)pyrazol[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 54.44%.

[0242] 1H NMR(400MHz,Chloroform-d)δ7.85(d,J=2.3Hz,1H),6.59(p,J=2.3Hz,1H),6.38(d,J=2.2 Hz,1H),5.73(s,1H),4.61(dq,J=4.1,2.1Hz,1H),4.24–4.09(m,2H),4.00(td,J=10.0,7. 5Hz, 1H), 3.87 (ddd, J=10.6, 7.4, 3.7Hz, 1H), 2.77 (tq, J=7.1, 2.3Hz, 2H), 2.57 (ddt, J=10 .2,7.6,2.6Hz,2H),2.15–2.10(m,2H),2.04(q,J=7.6Hz,3H).MS(ESI):m / z=271.43[M+H]+

[0243] Example 27

[0244]

[0245] Compound 26 (50 mg, 0.18 mmol) was dissolved in methanol (15 ml), and palladium on carbon (8 mg, 15%) was added. The reaction was carried out at room temperature under normal pressure for 3 hours. After the reaction, palladium on carbon was removed by filtration (used palladium on carbon is flammable and needs to be disposed of separately). The solvent was dried under reduced pressure, and the product was separated by column chromatography (DCM:MeOH = 30:1, v:v) to give a transparent oily product, yielding compound 27: 1-(5-cyclopentylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol, with a yield of 66%.

[0246] 1 H NMR(500MHz,Chloroform-d)δ7.79(d,J=2.2Hz,1H),6.27(d,J=2.2Hz,1H),5.55(s,1H),4.56(dq,J=4.3,2.2Hz,1H),4.15–4.05(m,2H),3. 98(td,J=9.9,7.4Hz,1H),3.85(ddd,J=10.6,7.5,3.7Hz,1H),3.06–2.94(m,1H),2.11–1.97(m,4H),1.84–1.69(m,4H),1.69–1.58(m,2H).

[0247] Example 28

[0248]

[0249] Following the synthetic route of Example 25, the first step of ethyl 3-cyclobutyl-3-oxopropionate was replaced with ethyl 3-cyclohexyl-3-oxopropionate to obtain compound 28: 1-(5-cyclohexylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidin-3-ol 1-(5-cyclohexylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 59.86%.

[0250] 1 H NMR (400MHz, Chloroform-d) δ7.86 (dd, J=2.4, 1.0Hz, 1H), 6.33 (dd, J=2.3, 1.0Hz, 1H), 5.62 (d, J=1. 2Hz,1H),4.63(p,J=3.2Hz,1H),4.17(t,J=2.6Hz,2H),4.09–3.97(m,1H),3.92(dt,J=10.7,5.2Hz,1 H),2.57(tt,J=12.0,3.4Hz,1H),2.12(dt,J=8.4,3.6Hz,2H),1.94(d,J=12.7Hz,2H),1.84(dt,J=12 .8,3.2Hz,2H),1.79–1.66(m,2H),1.52(qd,J=12.3,3.0Hz,2H),1.39(ddt,J=15.6,12.4,6.2Hz,2H).

[0251] Example 29

[0252]

[0253] first step

[0254] Ethyl benzoyl acetate (500 mg, 2.6 mmol) and 3-aminopyrazole (180.12 mg, 2.17 mmol) were dissolved in acetic acid and reacted at 80 °C for 8 h to give 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol. After removing acetic acid under reduced pressure, the product was washed with water and dichloromethane to give the crude product 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol, which did not require further purification and was a white solid with a yield of 65.52%.

[0255] 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),7.90(d,J=1.9Hz,1H),7.89–7.78(m,2H),7.59(dd,J=5.2,1.9Hz,3H),6.22(d,J=2.0Hz,1H),6.06(s,1H).

[0256] Step 2

[0257] 330 mg (1.56 mmol) of 5-phenylpyrazolo[1,5-a]pyrimidine-7-ol was carefully added in portions to phosphorus oxychloride (15 ml) in an ice bath. After stirring for a period of time, the ice bath was removed, and the mixture was heated to 80 °C for 3 h until TLC showed complete consumption of the starting material. Phosphorus oxychloride was removed under reduced pressure, and the reaction mixture was poured into ice water. Saturated sodium bicarbonate solution was added to adjust the pH to about 7. Extraction was performed with EA, and the mixture was dried over anhydrous sodium sulfate. Column chromatography (PE:EA = 5:1, v:v) was used to separate the product into 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine, which was a white solid with a yield of 55.74%.

[0258] 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=2.3Hz,1H),8.27–8.21(m,2H),8.05(s,1H),7.60–7.49(m,3H),6.91(d,J=2.2Hz,1H).

[0259] Step 3

[0260] The intermediate 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine (150 mg, 0.65 mmol) was dissolved in chloroform, and NCS (105 mg, 0.78 mmol) was added in portions. The mixture was heated to 60 °C for 6 h, and the organic phase was extracted with EA. After drying, the product 3,7-dichloro-5-phenylpyrazolo[1,5-a]pyrimidine was obtained by column chromatography (DCM:MeOH = 50:1). The product was a white solid with a yield of 46.38%.

[0261] Step 4

[0262] 3,7-Dichloro-5-phenylpyrazolo[1,5-a]pyrimidine (274 mg, 0.87 mmol) was dissolved in dioxane, and 3-hydroxypyrrolidine (151.74 mg, 1.74 mmol) and DIPEA (337.65 mg, 2.61 mmol) were added. The mixture was reacted at room temperature for 8 h to give compound 29: 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was a white solid with a yield of 44.31%.

[0263] 1H NMR(500MHz,Chloroform-d)δ8.07–7.99(m,2H),7.88(s,1H),7.51–7.43(m,3H),6.12(s,1H),4. 65(p,J=3.2Hz,1H),4.19(d,J=3.5Hz,2H),4.10–3.97(m,2H),2.14(ddd,J=8.7,6.4,3.5Hz,2H).

[0264] Example 30

[0265]

[0266] first step

[0267] Ethyl benzoyl acetate (500 mg, 2.6 mmol) and 3-aminopyrazole (180.12 mg, 2.17 mmol) were dissolved in acetic acid and reacted at 80 °C for 8 h to obtain 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol. After removing acetic acid under reduced pressure, the product was washed with water and dichloromethane to obtain the crude product 5-phenylpyrazolo[1,5-a]pyrimidin-7-ol, which did not require further purification and was a white solid with a yield of 65.52%.

[0268] 1 H NMR (400MHz, DMSO-d6) δ12.54(s,1H),7.90(d,J=1.9Hz,1H),7.89–7.78(m,2H),7.59(dd,J=5.2,1.9Hz,3H),6.22(d,J=2.0Hz,1H),6.06(s,1H).

[0269] Step 2

[0270] 330 mg (1.56 mmol) of 5-phenylpyrazolo[1,5-a]pyrimidine-7-ol was carefully added in portions to phosphorus oxychloride (15 ml) in an ice bath. After stirring for a period of time, the ice bath was removed, and the mixture was heated to 80 °C for 3 h until TLC showed complete consumption of the starting material. Phosphorus oxychloride was removed under reduced pressure, and the reaction mixture was poured into ice water. Saturated sodium bicarbonate solution was added to adjust the pH to about 7. Extraction was performed with EA, and the mixture was dried over anhydrous sodium sulfate. Column chromatography (PE:EA = 5:1, v:v) was used to separate the product into 7-chloro-5-phenylpyrazolo[1,5-a]pyrimidine, which was a white solid with a yield of 55.74%.

[0271] 1H NMR (400MHz, DMSO-d6) δ8.35(d,J=2.3Hz,1H),8.27–8.21(m,2H),8.05(s,1H),7.60–7.49(m,3H),6.91(d,J=2.2Hz,1H).

[0272] Step 3

[0273] 7-Chloro-5-phenylpyrazolo[1,5-a]pyrimidine (200 mg, 0.87 mmol) was dissolved in dioxane, and 3-hydroxypyrrolidine (151.74 mg, 1.74 mmol) and DIPEA (337.65 mg, 2.61 mmol) were added. The mixture was reacted at room temperature for 8 h to give 1-(5-phenylpyrazolo[1,5-a]pyrimidine-7-yl)pyrrolidine-3-ol. The product was white and the yield was 65.54%.

[0274] 1 H NMR (400MHz, Methanol-d4) δ8.01–7.93(m,3H),7.53–7.45(m,3H),6.38(d,J=2.3Hz,1H),6.25(s,1H ),4.56(tt,J=4.1,2.5Hz,1H),4.22(d,J=4.0Hz,2H),4.11(dd,J=9.7,5.9Hz,2H),2.22–2.10(m,2H).

[0275] Step 4

[0276] 1-(5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol (80 mg, 0.29 mmol) was dissolved in chloroform (5 ml), and NCS (46 mg, 0.34 mmol) was added in portions. The mixture was heated to 60 °C for 6 h, and the organic phase was extracted with EA. After drying, the mixture was separated by column chromatography (DCM:MeOH = 50:1) to give compound 30:1-(6-chloro-5-phenylpyrazolo[1,5-a]pyrimidin-7-yl)pyrrolidine-3-ol. The product was white, and the yield was 55.66%.

[0277] 1H NMR(400MHz,Chloroform-d)δ8.03(d,J=2.3Hz,1H),7.73–7.67(m,2H),7.47(qd,J=4.3,1.5Hz,3H),6.64(d,J=2.4Hz,1H),4.60(dt,J=4.9,3. 1Hz,1H),4.33–4.28(m,1H),4.15(dt,J=10.2,8.2Hz,1H),3.99–3.92(m,1H),3.88(dd,J=12.0,3.6Hz,1H),2.20(ddt,J=7.2,5.9,2.9Hz,2H).

[0278] MS(ESI): m / z = 315.20[M+H]+

[0279] Experimental Example: Bioactivity Testing

[0280] CD73 enzyme activity assay

[0281] 1. Test method: CD73 activity was determined using the Promega CTG-KIT kit;

[0282] 2. Detection Procedure: Add 4 μL of enzyme and 1 μL of different concentrations of the test compound to a 384 reaction plate and pre-incubate for 0.5 hours. Add 4 μL of substrate (ATP and AMP) and incubate at room temperature for 40 minutes. Detect enzyme activity using a CTG-KIT kit. Simultaneously, set up a solvent control group and a blank control group by replacing the test compound with DMSO. The final reaction volume was 15 μL. The specific reaction system consisted of 2% DMSO, 50 ng / ml CD73, 100 μM ATP, and 300 μM AMP. Data processing: Plot the logarithm of concentration against the percentage of activity.

[0283] 3. Data Processing: The fitted curve was calculated using nonlinear regression, and the IC was obtained using the formula log(inhibitor) vs. response-variable slope in GraphPad Prism 5 software. 50 Values. The results are shown in Table 1.

[0284] Table 1. Results of the compounds' inhibition of CD73 enzyme activity.

[0285] compound <![CDATA[IC 50 ]]> compound <![CDATA[IC 50 ]]> 1 + 16 + 2 + 17 + 3 ++ 18 ++ 4 ++ 19 + 5 ++ 20 ++ 6 + 21 ++ 7 ++ 22 + 8 + 23 ++ 9 + 24 +++ 10 + 25 ++ 11 + 26 ++ 12 ++ 27 +++ 13 ++ 28 ++ 14 + 29 + 15 + 30 +

[0286] Note: "+" indicates that the compound inhibits CD73 at IC50. 50 Value > 10 μ M; "++" indicates IC 50 Values ​​between 1 and 10 μ Between M; "++" indicates IC50 Value < 1 μ M.

[0287] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of pyrazolopyrimidine compounds of general formula (I), or their racemates, enantiomers, or pharmaceutically acceptable salts thereof, in the preparation of CD73 inhibitors: in: A is hydrogen; B is selected from , , , , , , Where i is selected from 0, 1, 2, 3, 4; R 12 Each is independently selected from D, halogen, hydroxyl, -NH2, C1-C4 alkylamino, C1-C4 alkoxycarbonyl, and C1-C4 alkyl; R 1 R 2 Independently selected from hydrogen and halogens; R 3 and R 4 The N atoms bonded to it together form ring structures selected from the following: ； Where n is selected from 1, 2, 3, 4; m is selected from 0, 1, 2, 3, 4, 5, 6; X is selected from N, O, and S atoms; R 9 Each is independently selected from D, halogen, hydroxyl, cyano, oxo (=O), C1-C4 hydroxyalkyl, -NR 10 R 11 ; R 10 R 11 It is independently selected from hydrogen and C1-C4 alkyl groups.

2. The use according to claim 1, characterized in that, R 12 Each is independently selected from D, halogen, hydroxyl, -NH2, Boc, C1-C4 alkyl; and / or R 3 and R 4 The N atoms bonded to it together form ring structures selected from the following: 、 、 、 ； Where m is selected from 0, 1, 2, 3, 4, 5, 6.

3. Use of pyrazolopyrimidine compounds of general formula (I), or their racemates, enantiomers, or pharmaceutically acceptable salts thereof, in the preparation of CD73 inhibitors: in: A is hydrogen; B is selected from , , , , , , , , , , , ; R 1 R 2 Independently selected from hydrogen and halogens; R 3 and R 4 The N atoms bonded to it together form ring structures selected from the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 。 4. Use of pyrazolopyrimidine compounds selected from the following structures, or their racemic mixtures, enantiomers, or pharmaceutically acceptable salts, in the preparation of CD73 inhibitors: in, A, R 1 R 2 As defined in claim 1, R 9 Selected from hydroxyl, oxo (=O), C1-C4 hydroxyalkyl, -NR 10 R 11 ; R 10 R 11 Independently selected from hydrogen and C1-C4 alkyl groups; R 9’ Selected from H, hydroxyl, and C1-C4 hydroxyalkyl; R 12 Selected from halogens, hydroxyl groups, -NH2, C1-C4 alkylamino groups, and C1-C4 alkyl groups; B1 is selected from: , , , , , .

5. Use of pyrazolopyrimidine compounds selected from the following structures or pharmaceutically acceptable salts thereof in the preparation of CD73 inhibitors: 。 6. The use according to any one of claims 1-5, characterized in that, The CD73 inhibitor comprises a pharmaceutically acceptable carrier and, optionally, one or more other therapeutic agents.

7. The use according to any one of claims 1-5, characterized in that, The CD73 inhibitor is a drug for the treatment and / or prevention of CD73-related diseases, disorders, and conditions.

8. The use according to claim 7, characterized in that, The CD73-related diseases, disorders, and conditions are selected from cancer and immune disorders.

9. The use according to claim 8, characterized in that, The cancers mentioned are selected from bladder cancer, breast cancer, bile duct cancer, colorectal cancer, stomach cancer, lung cancer, liver cancer, pancreatic cancer, prostate cancer, kidney cancer, glioblastoma, sarcoma, leukemia, lymphoma, and melanoma.

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