Pyrimidine cyclic compounds, their preparation methods and applications

Abstract

This invention discloses a pyrimidine cyclocyclic compound, its preparation method, and its applications. This invention provides a compound as shown in Formula I, its pharmaceutically acceptable salt, its tautomer, or a solvate thereof. The compound of Formula I of this invention can act as an adenosine A2A receptor antagonist or a histone deacetylase HDAC inhibitor. Further, the compound of this invention can simultaneously possess adenosine A2A receptor antagonistic activity and histone deacetylase HDAC inhibitory activity. The compound of Formula I of this invention can be used to treat and / or prevent diseases associated with adenosine A2A receptor and / or histone deacetylase HDAC, such as tumors and central nervous system diseases.

Description

Technical Field

[0001] This invention relates to a pyrimidine cyclic compound, its preparation method, and its application. Background Technology

[0002] Adenosine is an important endogenous signaling molecule involved in the regulation of various important physiological functions (Ohta, Gorelik et al., Proceedings of the National Academy of Sciences, 2006, 103(35), 13132-13137). As a purine nucleoside, adenosine acts on adenosine receptors on the cell membrane for downstream signal transduction. Adenosine receptors belong to G protein-coupled receptors (GPCRs) and are divided into four subtypes: A1, A2, A3, A4, A5, A6, A7, A8, A9, A1, A2, A1, A2, A3, A4, A5, A6, A1, A2, A3, A4, A5, A6, A7, A8, A9, A1, A2, A2, A3, A2, A3, A4, A5, A6, A1, A2, A2, A3, A4, A5, A6, A1, A2, A2, A3, A4, A5, A6, A7, A8, A9, A1, A2, A2, A2, A3, A2, A3, A4, A5, A6, A1, A2, A2, A3, A4, A5, A6, A2, A3, A4, A5, A6, A7, A8, A9, A1, A2, A2, A2, A3, A2, A2, A3, A4, A5, A6 ...3, A4, A5, A6, A2, A3, A4, A5, A6, A7, A8, A9, A1, A2, A2, A2, A3, A4, A5 2A A 2B And A3 (Antonioli, Fornai et al., Pharmacology and Therapeutics, 2008, 120(3), 233-532), where A 2A The receptor couples to the Gs protein, activating downstream adenylate cyclase, which increases cAMP concentration (Markus Klinger, Cellular Signalling, 2002, 14, 99–108). Adenosine A 2A Receptors are densely distributed in the central nervous system and are closely related to the pathogenesis of various degenerative central nervous system diseases such as Parkinson's disease, Alzheimer's disease, and Huntington's disease (Gomes et al., Biochimica et Biophysica Acta, 2011, 1808, 1380-1399). In Parkinson's disease, A... 2A The receptor is highly expressed in the substantia nigra striatum and can co-localize with the dopamine D2 receptor and form a heterodimer. 2A Activation of the receptor inhibits dopamine D2 receptor signaling (Shook, Jackson, ACS Chemical Neuroscience, 2011, 2, 555-567). Correspondingly, A 2A Receptor antagonists can relieve this inhibition, enhance downstream signaling of the D2 receptor, and thus enhance dopaminergic activity, making them potential treatments for Parkinson's disease. 2A Receptor antagonists, as a treatment for Parkinson's disease, can also reduce the motor dysfunction side effects induced by levodopa (L-DOPA). Multiple A 2ASmall molecule antagonists of the receptor have entered clinical trials as drugs for the treatment of Parkinson's disease (Aren van Waarde et al., Medicinal Research Reviews, 2018, 38, 5-56). Among them, itradefylline (KW-6002) was approved for marketing in Japan in 2013 as an adjunct therapy for Parkinson's disease, and subsequently received FDA approval in August 2019 for the treatment of Parkinson's disease. Compounds such as preladenant (MK-3814) and tozadenant (SYN-115) have also entered clinical trials. Although most A... 2A Clinical trials of receptor antagonists as monotherapy for Parkinson's disease have shown poor results, but A 2A The potential of receptor antagonists in treating related degenerative central nervous system diseases has been preliminarily demonstrated.

[0003]

[0004] In recent years, A 2A Receptors have attracted much attention as potential targets for tumor immunotherapy. Although current tumor immunotherapies have achieved very good results in the treatment of specific cancer types, and many drugs such as PD-1 and PD-L1 antibodies Keytruda, Opdivo, and Tecentriq, and CTLA4 antibody Yervoy have been approved for marketing in the United States (Hoos, Nature Reviews Drug Discovery 2016, 15, 235-247), the efficacy of these drugs is still low due to the presence of various immunosuppressive mechanisms in the tumor microenvironment. The development of new tumor immunotherapies remains urgent. In the tumor microenvironment, adenosine is an important immunosuppressive factor involved in immunosuppression (Antonioli, Blandizzi et al., Nature Reviews Cancer, 2013, 13(12), 842-857). The hypoxic microenvironment of tumors restricts energy utilization and induces the accumulation of extracellular ATP. ATP can be hydrolyzed into adenosine by nucleotidases CD39 and CD73, thus significantly increasing the concentration of adenosine around the tumor, forming an "adenosine protective ring" that helps cancer resist the attack of the body's immune system (Linden, American Journal of Physiology Cell Physiology, 2006, 291(3), 405-406). Adenosine and adenosine A1, A... 2A A 2B Binding to the A3 receptor can activate the receptor and thus exert different regulatory functions. Among them, A2A As immune checkpoint proteins on the surface of T cells, receptors mainly exert their immunosuppressive function by inhibiting the action of effector T cells (Jacobson, Gao, Nature Reviews Drug Discovery, 2006, 5(3), 247-264). As early as 2001, Ohta and Sitkovsky et al. discovered that blocking A receptors... 2A Receptors can reverse immunosuppression (Sitkovsky, Ohta, Nature, 2001, 414, 916-919). Adenosine activates A receptors on T cells. 2A Upon receptor activation, intracellular cAMP levels rise, thereby suppressing T cell immune function. The addition of A... 2A Receptor antagonists, blocking A 2A The receptor reduces cAMP concentration, which can reverse this immunosuppression (Antonioli, Blandizzi et al., Nature Review Cancer, 2013, 13(12), 842-857). Many studies have shown that A 2A A2A receptors are a promising target for developing tumor immunotherapy. Several known A2A receptor antagonists, such as vipadenant, CPI-444, PBF-509, and AZD4635, have entered clinical trials as agents for tumor immunotherapy. However, A... 2A Receptor antagonists do not have a direct killing effect on tumor cells, and are currently not used clinically. 2A Receptor antagonists must be used in combination with other anticancer drugs to treat tumors (Vecchio, White et al., Pharmacology and Therapeutics, 2019, 198, 20-33). Further breakthroughs are needed to enhance their anticancer effects.

[0005] Histone deacetylases (HDACs) are another drug target closely related to both tumors and degenerative central nervous system diseases. HDACs and histone acetyltransferases (HATs) are two key enzymes regulating epigenetics, capable of reversibly acetylating histone lysine residues, and they play opposite roles (Kazantsev and Thompson, Nature Reviews Drug Discovery, 2008, 7, 854-868). HATs catalyze the acetylation of lysine residues at the N-terminus of histones, placing chromatin in a relatively loose and open state to facilitate access to DNA by transcription factors and promote gene expression; they are transcriptional coactivators. HDACs, on the other hand, catalyze the removal of acetyl groups from lysine residues of histones, placing chromatin in a compact conformation and thus blocking DNA transcription and gene expression; they are transcriptional corepressors (Ellmeier and Seiser, Nature Reviews Immunology, 2018, 18(10), 617-634). Currently, 18 subtypes of human HDACs have been identified, which can be divided into four subfamilies: Class I-IV. Class I includes HDAC 1, 2, 3, and 8; Class II is further divided into Class IIa (HDAC 4, 5, 7, and 9) and Class IIb (HDAC 6 and 10); Class IV has only one member, HDAC 11. All three subfamilies are Zn... 2+ HDACs that depend on NAD are also known as classic HDACs. Class III, also known as sirtuins, includes SIRT 1-7 and depends on NAD. + Exercising catalytic activity (Wilfried Ellmeier and Christian Seiser, Nature Reviews Immunology, 2018, 18(10), 617-634).

[0006] Currently, HDAC inhibitors are widely used in anti-tumor therapy. Overexpression of HDAC can inhibit the expression of a series of tumor suppressor genes, thereby promoting the growth of tumor cells. For example, abnormal HDAC function can lead to the deacetylation of p53 protein, blocking its binding to DNA and thus blocking the transcription of apoptosis genes; it can also lead to a decrease in the expression of the cell cycle repressor p21, resulting in cell cycle arrest; in addition, HDAC is also related to angiogenesis in tumor tissues and the regulation of immune cell function (Falkenberg and Johnstone, Nature Reviews Drug Discovery, 2014, 13, 673-691). Given the significant potential of HDAC inhibitors in inhibiting tumor proliferation, their research and application as anti-tumor drugs have received widespread attention. Currently, four HDAC inhibitors (vorinostat / SAHA, romidepsin / FK228, belinostat / PDX-101, and panobinostat / LBH-589) have been approved for marketing by the US FDA: SAHA, developed by Merck, is used to treat refractory cutaneous T-cell lymphoma (CTCL); romidepsin, developed by Celgene, is used to treat CTCL and peripheral T-cell lymphoma (PTCL); Panobinostat, developed by Novartis, is used orally in combination with bortezomib and dexamethasone to treat multiple myeloma; and Belinostat, developed by Spectrum, is used to treat PTCL. Chidamide, a subtype-selective HDAC inhibitor developed and marketed by Shenzhen Chipscreen Biosciences, was approved by the CFDA in December 2014 for patients with relapsed or refractory peripheral T-cell lymphoma who have previously received at least one prior systemic chemotherapy. Other HDAC inhibitors, such as abexinostat / PCI024781, givinostat / ITF2375, and mocetinostat / MGCD-0103, are in different stages of clinical trials.

[0007]

[0008] Besides their applications in anti-tumor therapy, genetic evidence shows that HDACs and HATs are associated with maintaining central nervous system homeostasis, and these are related to neurological disorders such as Rubinstein-Taybi syndrome and Rett syndrome (Kazantsev and Thompson, Nature Reviews Drug Discovery, 2008, 7(10), 854-868). Therefore, the application of HDAC inhibitors in neurological diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease has also received increasing attention (Falkenberg and Johnstone, Nature Reviews Drug Discovery, 2014, 13, 673-691). For example, HDAC6 can regulate the phosphorylation level of tau protein, thereby affecting the development of tau protein-driven neurological diseases (Selenica et al., Alzheimer's Research & Therapy, 2014, 6, 12). HDAC6 can also regulate the degradation of misfolded proteins by modulating protein aggregation and HSP90 function. The accumulation of misfolded proteins is a pathological feature of many neurological diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Similarly, HDAC2 has been shown in various models to regulate brain function and the development and deterioration of the nervous system; overexpression of HDAC2 can negatively regulate synaptic plasticity and number, as well as dendritic spine density, leading to the decline of learning and cognitive functions (Guan et al., Nature, 2009, 459, 55-60). Existing literature has confirmed that HDAC inhibitors can have therapeutic effects on neurological diseases. For example, panobinostat can reverse the symptoms of Huntington's disease in animal models by inhibiting HDAC function (Siebzehnrübl et al., Proceedings of the National Academy of Sciences, 2018, 115(37), E8765-8774), and SAHA can significantly improve cognition in animal models (Guan et al., Nature, 2009, 459, 55-60). However, HDAC inhibitors are difficult to achieve sufficiently potent anti-tumor therapeutic effects when used alone, and their indications are also relatively narrow. Currently, the clinical application of HDAC inhibitors is mostly carried out in combination with other anti-tumor drugs (Botsand Johnstone, Clinical Cancer Research, 2009, 15(12), 3970-7 2009).

[0009] For diseases with complex pathogenesis, such as tumors and neurodegenerative diseases, designing and developing single drug molecules with multiple pharmacological activities is a superior and more effective strategy. Given A 2A Receptors and HDACs have similar effects on tumors and various central nervous system diseases, and their synergistic use is likely to produce a more effective therapeutic effect in the treatment of related diseases.

[0010] Chinese patent applications CN201911152880.3, CN201911153069.7, and CN202010168088.3 report A-type structures with different parent nuclei. 2A Compounds with dual receptor antagonistic and HDAC inhibitory effects. Yan et al. published compounds with bicyclic or tricyclic parent nuclei containing A... 2A Compounds with dual receptor antagonistic and HDAC inhibitory effects exhibit good in vivo antitumor activity (J Med Chem 2021, 64, 16573-16597). However, the types and structures of the aforementioned compounds are limited. Obtaining a wider variety of compounds with A 2A Compounds with both receptor antagonistic and HDAC inhibitory effects are a problem that urgently needs to be solved by engineers in the field. Summary of the Invention

[0011] The problem this invention aims to solve is the limitation of existing technologies that rely on dual-target small molecule structures based on A2A receptor antagonists and HDAC inhibitors, and it provides a pyrimidine cyclic compound, its preparation method, and its applications. The pyrimidine cyclic compound of this invention can be used as adenosine A... 2A Receptor antagonists or histone deacetylase HDAC inhibitors, while also containing adenosine A 2A Receptor antagonistic activity and histone deacetylase HDAC inhibitory activity can be used to treat and / or prevent adenosine A receptor antagonism. 2A Diseases associated with receptors and / or histone deacetylases (HDACs), such as tumors and neurodegenerative diseases.

[0012] This invention provides a compound as shown in Formula I, a pharmaceutically acceptable salt thereof, a tautomer thereof, or a solvate thereof:

[0013]

[0014] Where A and B are independently CH or N;

[0015] R 1 and R 2 Independently H or C1-C6 alkyl;

[0016] R 3 It is a 5-10 member heteroaryl group or is surrounded by one or more R groups.3-1 The substituted 5-10-membered heteroaryl group; wherein the heteroaryl group has one, two or three heteroatoms, and the heteroatoms are selected from one, two or three of N, O and S;

[0017] L is -C1-C 10 Alkylene-*, -C1-C 10 Alkylene-C6-C 10 Aromatic-*, -C1-C 10 Alkylene-C6-C 10 arylene-C2-C4-ene-*, -C1-C 10 Alkylene-O-C6-C 10 Aromatic-*, -C1-C 10 Alkylene-C6-C 10 Aromatic-O-C1-C 10 Alkylene-* or -C1-C 10 alkylene-5-10-membered heteroaryl-O-C1-C 10 Alkylene-*; In the 5-10 alkylene-*, the number of heteroatoms is 1, 2 or 3, and the heteroatoms are selected from one, two or three of N, O and S; wherein the end with "*" is connected to ZBG;

[0018] ZBG is

[0019] R 3-1 It is independently a cyano or C1-C6 alkyl group;

[0020] L 1 L 2 Independently for -C1-C 10 Alkylene or -O-C1-C 10 Alkylene;

[0021] R 6 Independently hydrogen or halogen;

[0022] m can be 1, 2, 3 or 4.

[0023] In some embodiments, certain groups in the compounds of Formula I or their pharmaceutically acceptable salts described above have the following definitions, and the definitions of groups not mentioned are as described in any of the other embodiments (hereinafter referred to as "in one embodiment"):

[0024] R 3 In this context, the heteroaryl group is preferably a 5-6 membered heteroaryl group; the heteroatom in the heteroaryl group is preferably O, and the number of heteroatoms is preferably 1 or 2. The heteroaryl group is preferably furanyl, and more preferably...

[0025] In one particular scheme, R 1 R 2 and R 3-1 In this context, the C1-C6 alkyl groups are independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.

[0026] In one scheme, in L, the C1-C 10 The alkylene group is independently a C1-C7 alkylene group, and is more preferably methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene, and more preferably methylene, n-ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, or n-heptylene.

[0027] In one scheme, in L, the C6-C 10 The arylene group is independently phenylene, and is further preferably...

[0028] In one embodiment, in L, the C2-C4 alkenyl group is independently vinylidene, more preferably...

[0029]

[0030] In one embodiment, in L, the 5-10-membered heteroaryl group is a 5-6-membered heteroaryl group; the heteroatom in the 5-10-membered and 5-6-membered heteroaryl groups is preferably N, and the number of heteroatoms is preferably 1 or 2. The 5-6-membered heteroaryl group is preferably pyridyl, and more preferably...

[0031] In one particular scheme, R 6 In this context, the halogen is fluorine, chlorine, bromine, or iodine.

[0032] In one scheme, in ZBG, the aforementioned for

[0033] In one particular scheme, L is

[0034]

[0035] In one particular scheme, R 1 and R 2 For H.

[0036] In one particular scheme, R 3 for

[0037] In one embodiment, the compound represented by Formula I is a substituted pyrimidine compound represented by Formula I-1, a substituted pyrimidine compound represented by Formula I-2, a substituted pyrimidine compound represented by Formula I-3, or a substituted pyrimidine compound represented by Formula I-4.

[0038]

[0039] In one embodiment, the compound represented by Formula I is any of the following compounds:

[0040]

[0041]

[0042]

[0043]

[0044] The present invention also provides a pharmaceutical composition comprising a compound as shown in Formula I as described above, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

[0045] The present invention also provides the use of a compound of Formula I as described above or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the preparation of adenosine A2A receptor antagonists and / or histone deacetylase HDAC inhibitors.

[0046] In the described applications, the adenosine A2A receptor antagonist and / or histone deacetylase HDAC inhibitor can be used in mammalian organisms; they can also be used in vitro, primarily for experimental purposes, such as providing comparisons as standard or control samples, or preparing kits according to conventional methods in the art to provide rapid detection of the antagonistic effect of adenosine A2A receptor and / or the inhibitory effect of histone deacetylase HDAC.

[0047] The present invention also provides the use of a compound of Formula I as described above or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the preparation of a medicament for treating and / or preventing diseases.

[0048] The dosage of the compound as shown in Formula I or a pharmaceutically acceptable salt thereof, or the dosage of the pharmaceutical composition, may be a therapeutically effective amount.

[0049] The disease mentioned can be cancer or a central nervous system disease.

[0050] In some embodiments, when the ZBG in the compound shown in Formula I is When the histone deacetylase HDAC is histone deacetylase HDAC1 or histone deacetylase HDAC6; when the ZBG in the compound shown in Formula I is In this case, the histone deacetylase HDAC is histone deacetylase HDAC1.

[0051] The cancers mentioned can be head and neck cancers (such as thyroid cancer, nasopharyngeal cancer, meningeal cancer, or intracranial metastases), respiratory cancers (such as small cell lung cancer or non-small cell lung cancer), digestive system cancers (such as liver cancer, stomach cancer, esophageal cancer, rectal cancer, colon cancer, or pancreatic cancer), urinary system cancers (such as kidney cancer, bladder cancer, prostate cancer, or testicular cancer), bone cancer, gynecological cancers (such as breast cancer, cervical cancer, or ovarian cancer), hematological cancers (such as leukemia, lymphoma, or myeloma), or other types of cancer (such as melanoma, glioma, or skin cancer).

[0052] The central nervous system diseases mentioned can be Parkinson's disease, Alzheimer's disease, or Huntington's disease.

[0053] The compounds of Formula I as described above, or their pharmaceutically acceptable salts, or the pharmaceutical compositions described herein, may also be used for any disease process characterized by abnormal cell proliferation, such as benign prostatic hyperplasia, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, inflammatory bowel disease, transplant rejection, endotoxic shock, and fungal infection.

[0054] The present invention also provides the use of a compound of Formula I as described above, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of articles for regulating the activity of adenosine A2A receptor and / or histone deacetylase HDAC.

[0055] In some embodiments, the histone deacetylase HDAC is histone deacetylase HDAC1.

[0056] The selection of pharmaceutical excipients varies depending on the route of administration and characteristics of action, and can generally include fillers, diluents, binders, wetting agents, disintegrants, lubricants, emulsifiers, suspending agents, etc., which are common in the field.

[0057] The pharmaceutical composition can be administered orally, by injection (intravenous, intramuscular, subcutaneous, and intracoronary), sublingually, buccally, rectally, urethra, vaginally, nasally, by inhalation, or topically, with oral administration being the preferred route.

[0058] The present invention also provides a method for preparing a compound as shown in Formula I, comprising the following steps: in an organic solvent, a compound as shown in Formula II is reacted as shown below to obtain the compound as shown in Formula I.

[0059]

[0060] Among them, A, B, R 1 R 2 R 3 L and ZBG are as defined above; R a It is H or C1-C6 alkyl.

[0061] The reaction conditions in the preparation method of the compound shown in Formula I are conventional in the art.

[0062] The present invention also provides a compound as shown in Formula II.

[0063]

[0064] Among them, A, B, R 1 R 2 R 3 L and R a The definition is as described above.

[0065] In one aspect of the present invention, the compound represented by Formula II is the following compound:

[0066]

[0067]

[0068]

[0069] In this invention, unless otherwise stated, the following terms appearing in the specification and claims have the following meanings:

[0070] In this invention, the term "substitution" or "substituent" means that one or more hydrogen atoms are replaced by a specified group. When the substitution position is not specified, substitution can be at any position, but it is only permitted if a stable or chemically viable chemical is formed.

[0071] In this invention, the terms "optional" or "optionally" refer to events or conditions described subsequently that may but are not required to occur, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur. For example, the term "optionally substituted" means that something may or may not be substituted, and unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis.

[0072] When any variable (e.g., R) 3-1 When a group appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is surrounded by 1-2 R... 3-1 If the substituent is replaced, the group may optionally be replaced by up to two Rs, and each R has an independent option. Furthermore, combinations of substituents and / or their variants are permitted only if such combinations produce a stable compound.

[0073] In this invention, the term "alkyl" refers to a saturated, straight-chain or branched monovalent hydrocarbon group having a specified number of carbon atoms, such as C1-C2. 10 Alkyl refers to a straight-chain or branched alkyl group having 1 to 10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl).

[0074] In this invention, the term "alkylene" refers to a saturated straight-chain or branched divalent hydrocarbon group having a specified number of carbon atoms. Thus, C1 alkylene (i.e., methylene) refers to -CH2-, C2 alkylene (i.e., ethylene) refers to -CH2-CH2-, and C3 alkylene refers to -CH2-CH2-CH2-.

[0075] In this invention, the term "alkenyl" refers to a straight-chain divalent hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond, wherein the carbon-carbon double bond can be located at any position within the alkenyl group. Thus, C2 alkenyl (i.e., vinylene) refers to -CH=CH-, C3 alkenyl refers to -CH2-CH=CH- and -CH2=CH-CH2-, and C4 alkenyl refers to -CH2-CH=CH-CH2-, -CH2=CH-CH2-CH2-, and -CH2-CH-CH2=CH2-.

[0076] In this invention, the term "aryl" refers to any stable monocyclic or polycyclic (e.g., bicyclic or tricyclic) carbocyclic ring containing up to 6-10 atoms in each ring, wherein at least one ring is an aromatic ring. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindenyl, biphenyl, phenanthryl, anthracene, or acenaphthyl. It is understood that in cases where the aryl substituent is a bicyclic substituent and one of the rings is a non-aromatic ring, the linkage occurs through the aromatic ring.

[0077] In this invention, the term "arylene" refers to a divalent aryl group. 1,4-Phenylene... Or 1,3-phenylene

[0078] In this invention, the term "heteroaryl" refers to a stable monocyclic or polycyclic (e.g., bicyclic or tricyclic) carbon ring containing up to 6-10 atoms in each ring, wherein at least one ring is an aromatic ring and contains at least one heteroatom selected from O, N, and S. Heteroaryl groups can be linked to other parts of a molecule via heteroatoms or carbon atoms. Examples of heteroaryl groups include, but are not limited to, acridinel, carbazolyl, cenolinyl, quinoxalinyl, pyrazolyl, indoleyl, benzotriazolyl, furanyl, thiophenel, benzothiophenel, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indoleyl, pyrazinyl, pyridinyl, pyrimidinel, and pyrroloyl (e.g., pyrrolo-1-yl). pyrrole-2-yl ), tetrahydroquinolinyl. It can be understood that in the case where the heteroaryl substituent is a bicyclic substituent and one of the rings is a non-aromatic ring, the linkage occurs through the aromatic ring.

[0079] In this invention, the term "heteroaryl" refers to a divalent heteroaryl group. The 6-membered heteroaryl in 1,4-(6-membered heteroaryl) is monocyclic, where 1 and 4 do not refer to the original numbering of the ring atoms in the 6-membered heteroaryl, but rather to the para position of the two linking sites. Examples of 1,4-(6-membered heteroaryl) include, but are not limited to, those... The same applies to 1,3-(5-membered heteroaryl), and examples of 1,3-(5-membered heteroaryl) include, but are not limited to, those mentioned above.

[0080] When the linking groups listed in this invention do not specify their linking direction, the linking direction is the same as the reading order from left to right. Examples are given below. The linker group L1 is -CD-, and -CD- connects ring A and ring B in the same direction as the reading order from left to right to form... and does not constitute When L is listed hour, The structure formed is Instead

[0081]

[0082] The combination of linking groups, substituents, and / or their variants is permitted only if such a combination produces a stable compound.

[0083] In this invention, unless otherwise stated, the term "halogen" refers to F, Cl, Br, and I.

[0084] In this invention, the term "pharmaceutically acceptable salt" refers to a salt formed from a suitable nontoxic organic acid, inorganic acid, organic base, or inorganic base with a compound of Formula I, which retains the biological activity of the compound of Formula I. The organic acid may be one or more of the conventional salt-forming organic acids in the art, preferably methanesulfonic acid, p-toluenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, malic acid, lactic acid, formic acid, acetic acid, propionic acid, trifluoroacetic acid, oxalic acid, succinic acid, benzoic acid, hydroxyethylsulfonic acid, naphthalenesulfonic acid, and salicylic acid. The inorganic acid may be one or more of the conventional salt-forming inorganic acids in the art, preferably hydrochloric acid, sulfuric acid, and phosphoric acid. The organic base may be one or more of the conventional salt-forming organic bases in the art, preferably pyridines, imidazoles, pyrazines, indoles, purines, tertiary amines, and anilines. The tertiary amine organic base is preferably triethylamine and / or N,N-diisopropylethylamine. The aniline-based organic base is preferably N,N-dimethylaniline. The pyridine-based organic base is preferably one or more of pyridine, methylpyridine, 4-dimethylaminopyridine, and 2-methyl-5-ethylpyridine. The inorganic base can be any salt-forming inorganic base conventional in the art, preferably one or more of alkali metal hydrides, alkali metal hydroxides, alkali metal alkoxides, potassium carbonate, sodium carbonate, lithium carbonate, cesium carbonate, potassium bicarbonate, and sodium bicarbonate. The alkali metal hydrides are preferably sodium hydride and / or potassium hydride. The alkali metal hydroxides are preferably one or more of sodium hydroxide, potassium hydroxide, and lithium hydroxide. The alkali metal alkoxides are preferably one or more of sodium methoxide, sodium ethoxide, potassium tert-butoxide, and sodium tert-butoxide. In some embodiments of the invention, the pharmaceutically acceptable salt is a hydrochloride salt.

[0085] The term "solvent" refers to a substance formed by a compound of Formula I with a suitable solvent. The solvent is preferably water or an organic solvent.

[0086] The compounds and their structures of the present invention also represent all isomers (e.g., enantiomers, diastereomers, geometric isomers, and conformational isomers) that can be defined according to the absolute stereochemical definition for an amino acid as (R)- / (S)-, (D)- / (L)-, or (R,R)- / (R,S)- / (S,S)-. The present invention includes all such possible isomers, as well as their racemic, enantiomer-enriched, and optionally pure forms. Optical (+) and (-), (R)- and (S)-, and (R,R)- / (R,S)- / (S,S)- or (D)- and (L)- isomers can be prepared using chiral synthesis, chiral resolution, or can be resolved using conventional techniques such as, but not limited to, high-performance liquid chromatography (HPLC) using chiral columns. When the compounds described herein contain an alkenyl double bond or other geometrically asymmetric center, unless otherwise stated, the compounds include both E and Z geometric isomers. Similarly, all tautomer forms are also included.

[0087] In this invention, the term "tautomer" refers to the movement of a proton from one atom of a molecule to another position within the same molecule. This invention includes tautomers of any of the compounds described.

[0088] For pharmaceuticals or pharmacologically active agents, the term "therapeutic effective amount" refers to a sufficient quantity of a drug or agent that is non-toxic but achieves the desired effect. For the oral dosage form of this invention, the "therapeutic effective amount" of one active substance in the composition refers to the quantity required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the recipient's age and general condition, as well as the specific active substance. A suitable effective amount in any given case can be determined by a person skilled in the art through routine testing.

[0089] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0090] The reagents and raw materials used in this invention are all commercially available.

[0091] The significant advantages of this invention are: it provides a thiazocyclic compound, its preparation method, intermediates, and applications. The thiazocyclic compound of this invention can act as an adenosine A2A receptor antagonist or a histone deacetylase HDAC inhibitor. Furthermore, the thiazocyclic compound of this invention can simultaneously possess adenosine A2A receptor antagonistic activity and histone deacetylase HDAC inhibitory activity. The thiazocyclic compound of this invention can be used to treat and / or prevent diseases related to adenosine A2A receptor and / or histone deacetylase HDAC, such as tumors and central nervous system diseases. Attached Figure Description

[0092] Figure 1 The relative tumor growth rate of I-14 administered by gavage.

[0093] Figure 2 The relative tumor growth rate after intraperitoneal administration of compound I-14. Detailed Implementation

[0094] The invention will be further illustrated in the following embodiments. These embodiments are for illustrative purposes only and do not limit the scope of protection of the invention in any way. All parameters and other descriptions in the embodiments, unless otherwise stated, are in units of mass (grams).

[0095] Example 1: Preparation of 5-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxypentanamide (compound I-1)

[0096] Step 1: Preparation of methyl 5-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)valerate (intermediate Int-1)

[0097]

[0098] The starting material, compound A1, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 5-bromopentanoate (218 mg, 1.12 mmol), K₂CO₃ (206 mg, 1.49 mmol), and compound C-1 (150 mg, 0.745 mmol) were dissolved in DMF (15 mL) and stirred overnight at 100 °C. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellow oily intermediate, Int-1 (84 mg, yield 34%). 1 H NMR(600MHz,DMSO-d6)δ8.23(s,1H),8.05(dd,J=1.7,0.8Hz,1H),7.42(dd,J=3.5,0.8Hz,1H),6.87(brs,2H),6.78(dd,J=3 .5,1.7Hz,1H),4.19(t,J=6.9Hz,2H),3.55(s,3H),2.33(t,J=7.4Hz,2H),1.84–1.78(m,2H),1.52–1.45(m,2H).HRMS(ESI)C 15 H 18 N5O3 + [M+H] +Calculated value: 316.1404, measured value: 316.1406.

[0099] Step 2: Preparation of 5-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxypentanamide (compound I-1)

[0100]

[0101] An anhydrous methanol solution of potassium hydroxide (1.29 g, 23 mmol) in 3.22 mL was added dropwise to an anhydrous methanol solution of hydroxylamine hydrochloride (1.07 g, 15.4 mmol) in 5.52 mL at 0 °C. The mixture was slowly heated to room temperature and stirred for 0.5 hours. The solid was removed by filtration to obtain a methanol solution of hydroxylamine. Methyl 5-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)valerate (intermediate Int-1) (0.076 g, 0.241 mmol) was dissolved in the above methanol solution of hydroxylamine and stirred at room temperature for 1 hour. The reaction mixture was neutralized to pH 7.4 with a 1,4-dioxane solution of hydrogen chloride (4 M). The solvent was evaporated under reduced pressure. Water was added to the solid residue, and the mixture was stirred at room temperature for 1 hour. The residue was filtered to obtain a yellow solid (I-1) (0.030 g, yield 38%). 1 H NMR (800MHz, DMSO-d6) δ10.33(s,1H),8.66(s,1H),8.23(s,1H),8.05(d,J=1.7Hz,1H),7.42(d,J=3.4Hz,1H),6.88(brs,2H) ,6.78(dd,J=3.5,1.7Hz,1H),4.18(t,J=7.1Hz,2H),1.98(t,J=7.4Hz,2H),1.78–1.73(m,2H),1.49–1.44(m,2H).HRMS(ESI)C 14 H 17 N6O3 + [M+H] + Calculated value: 317.1357, measured value: 317.1360.

[0102] Example 2: Preparation of 6-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxyhexanoamide (compound I-2)

[0103]

[0104] In Example 1, methyl 5-bromopentanoate was replaced with methyl 6-bromoheptanoate in step 1. The other raw materials, reagents and preparation methods were the same as in Example 1, yielding a white solid compound (I-2) (0.116 g, yield 84%). 1 H NMR (600MHz, DMSO-d6) δ10.31(s,1H),8.65(d,J=1.7Hz,1H),8.22(s,1H),8.05(dd,J=1.8,0.8Hz,1H),7.42(dd,J=3.6,0.8Hz,1H),6.87(brs,2H),6 .78(dd,J=3.5,1.7Hz,1H),4.17(t,J=7.1Hz,2H),1.92(t,J=7.4Hz,2H),1 .79(p,J=7.4Hz,2H),1.51(p,J=7.5Hz,2H),1.26–1.19(m,2H).HRMS(ESI)C 15 H 19 N6O3 + [M+H] + Calculated value: 331.1513, measured value: 331.1515.

[0105] Example 3: Preparation of 7-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxyheptamide (compound I-3)

[0106]

[0107] In Example 1, methyl 5-bromopentanoate was replaced with methyl 7-bromoheptanoate in step 1. The other raw materials, reagents and preparation methods were the same as in Example 1, yielding a white solid compound (I-3) (0.190 g, yield 88%). 1 H NMR (800MHz, DMSO-d6) δ10.31(s,1H),8.64(s,1H),8.22(s,1H),8.05(d,J=1.6Hz,1H),7.42(dd,J=3.5,0.7Hz,1H),6.86(brs,2H),6.78(dd, J=3.5,1.7Hz,1H),4.17(t,J=7.1Hz,2H),1.91(t,J=7.4Hz,2H),1.78(p,J=7.2Hz,2H),1.45(p,J=7.4Hz,2H),1.28–1.21(m,4H).HRMS(ESI)C 16 H 21 N6O3 + [M+H] + Calculated value: 345.1670, measured value: 345.1678.

[0108] Example 4: Preparation of 5-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxypentanamide (compound I-4)

[0109] Step 1: Preparation of methyl 5-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)valerate (intermediate Int-2)

[0110]

[0111] Starting material compound A2 was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Compound C-1 in step 1 of Example 1 was replaced with compound C-2, and the remaining raw materials, reagents, and preparation methods were the same as in step 1 of Example 1, yielding a yellow oily intermediate Int-2 (42 mg, yield 18%). 1 H NMR(600MHz,DMSO-d6)δ8.23(s,1H),7.35(d,J=3.4Hz,1H),6.82(brs,2H),6.41(dd,J=3.3,1.2Hz,1H),4.18(t, J=6.9Hz,2H),3.55(s,3H),2.45(s,3H),2.33(t,J=7.4Hz,2H),1.84–1.76(m,2H),1.52–1.45(m,2H).HRMS(ESI)C 16 H 20 N5O3 + [M+H] + Calculated value: 330.1561, measured value: 330.1562.

[0112] Step 2: Preparation of 5-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxypentanamide (compound I-4)

[0113]

[0114] The reagents and preparation method were the same as in step 2 of Example 1, yielding a white solid compound (I-4) (0.048 g, yield 67%). 1H NMR (800MHz, DMSO-d6) δ10.33(s,1H),8.66(s,1H),8.23(s,1H),7.35(d,J=3.4Hz,1H),6.82(brs,2H),6.41(dd,J=3.4, 1.2Hz,1H),4.17(t,J=7.1Hz,2H),2.45(s,3H),1.97(t,J=7.4Hz,2H),1.78–1.73(m,2H),1.49–1.43(m,2H).HRMS(ESI)

[0115] C 15 H 19 N6O3 + [M+H] + Calculated value: 331.1513, measured value: 331.1515.

[0116] Example 5: Preparation of 6-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxyhexanoamide (compound I-5)

[0117]

[0118] In Example 1, methyl 5-bromopentanoate was replaced with methyl 6-bromoheptanoate in step 1. The other raw materials, reagents and preparation methods were the same as in Example 4, yielding a white solid compound (I-5) (0.186 g, yield 81%). 1 H NMR (600MHz, DMSO-d6) δ10.31(s,1H),8.65(d,J=1.8Hz,1H),8.22(s,1H),7.34(d,J=3.4Hz,1H),6.82(brs,2H),6.41(dd,J=3.4,1.1Hz,1 H),4.16(t,J=7.1Hz,2H),2.45(s,3H),1.92(t,J=7.4Hz,2H),1.78(p,J=7.3Hz,2H),1.51(p,J=7.5Hz,2H),1.27–1.18(m,2H).HRMS(ESI)C 16 H 21 N6O3 + [M+H] + Calculated value: 345.1670, measured value: 345.1679.

[0119] Example 6: Preparation of 7-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-N-hydroxyheptamide (compound I-6)

[0120]

[0121] In Example 4, step 1, methyl 5-bromopentanoate was replaced with methyl 7-bromoheptanoate. The other required raw materials, reagents and preparation methods were the same as in Example 4, yielding a white solid compound (I-6) (0.105 g, yield 85%). 1 H NMR (800MHz, DMSO-d6) δ10.32(s,1H),8.65(s,1H),8.22(s,1H),7.34(d,J=3.3Hz,1H),6.81(brs,2H),6.40(dd,J=3.4,1.1Hz,1H),4 .16(t,J=7.1Hz,2H),2.45(s,3H),1.91(t,J=7.4Hz,2H),1.77(p,J=7.2Hz,2H),1.45(p,J=7.4Hz,2H),1.28–1.21(m,4H).HRMS(ESI)C 17 H 23 N6O3 + [M+H] + Calculated value: 359.1826, measured value: 359.1822.

[0122] Example 7: Preparation of 7-(2-amino-6-(furan-2-yl)-9H-purine-9-yl)-N-hydroxyheptanamide (compound I-7)

[0123] Step 1: Preparation of methyl 7-(2-amino-6-(furan-2-yl)-9H-purine-9-yl)heptaate (intermediate Int-3)

[0124]

[0125] The starting material, compound A3, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 7-bromoheptanoate (133 mg, 0.596 mmol), K₂CO₃ (137 mg, 0.994 mmol), and compound C-3 (100 mg, 0.497 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellow oily intermediate, Int-3 (35 mg, yield 21%). 1H NMR (800MHz, DMSO-d6) δ8.12(s,1H),7.95–7.94(m,1H),7.72(d,J=3.3Hz,1H),6.74(dd,J=3.4,1.7Hz,1H),6.54(brs,2H),4.04(t,J=7.2H z,2H),3.56(s,3H),2.27(t,J=7.4Hz,2H),1.77(p,J=7.4Hz,2H),1.49(p,J=7.4Hz,2H),1.31–1.26(m,2H),1.27–1.22(m,2H).HRMS(ESI)C 17 H 22 N5O3 + [M+H] + Calculated value: 344.1717, measured value: 344.1715.

[0126] Step 2: Preparation of 7-(2-amino-6-(furan-2-yl)-9H-purine-9-yl)-N-hydroxyheptanamide (compound I-7)

[0127]

[0128] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-3. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a white solid compound (I-7) (0.039 g, yield 57%). 1 H NMR (800MHz, DMSO-d6) δ10.32(s,1H),8.65(s,1H),8.12(s,1H),7.96–7.94(m,1H),7.72(d,J=3.4Hz,1H),6.74(dd,J=3.4,1.7Hz,1H),6 .55(brs,2H),4.04(t,J=7.2Hz,2H),1.92(t,J=7.4Hz,2H),1.77(p,J=7.3Hz,2H),1.47(p,J=7.3Hz,2H),1.29–1.22(m,4H).HRMS(ESI)C 16 H 21 N6O3 + [M+H] + Calculated value: 345.1670, measured value: 345.1669.

[0129] According to the methods listed in Examples 1-7, the compounds listed in Examples 8-10 can be prepared by changing the corresponding raw materials using the same methods, as detailed in Table 1.

[0130] Table 1

[0131]

[0132]

[0133] Example 11: Preparation of 4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-11)

[0134] Step 1: Preparation of methyl 4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzoate (intermediate Int-4)

[0135]

[0136] In Example 1, step 1, methyl 5-bromopentanoate was replaced with methyl 4-(bromomethyl)benzoate, and the reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 1, step 1, to obtain a yellow solid intermediate (Int-4) (0.152 g, yield 26%). 1 H NMR(800MHz,Chloroform-d)δ8.28(s,1H),7.98–7.95(m,2H),7.72(dd,J=1.7,0.8Hz,1H),7.41(d,J= 3.6Hz,1H),7.34–7.31(m,2H),6.62(dd,J=3.5,1.7Hz,1H),5.51(s,2H),5.36(brs,2H),3.88(s,3H). HRMS(ESI)C 18 H 16 N5O3 + [M+H] + Calculated value: 350.1248, measured value: 350.1245.

[0137] Step 2: Preparation of 4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzoic acid (intermediate Int-5)

[0138]

[0139] The ester intermediate Int-4 (0.096 g, 0.274 mmol) was dissolved in a mixed solution of tetrahydrofuran (12 mL) and water (3 mL), and lithium hydroxide (0.036 g, 0.151 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was neutralized to pH 7.4 with a 1,4-dioxane solution of hydrogen chloride (4 M), and the solvent was evaporated under reduced pressure to obtain crude carboxylic acid intermediate Int-5, which was used directly in the next reaction. HRMS(ESI)C 17 H 14 N5O3 + ([M+H)) + Calculated value: 336.1091, measured value: 336.2810.

[0140] Step 3: Preparation of 4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-11)

[0141]

[0142] The mixture of all intermediates Int-5 obtained in step 2 and lithium chloride was dissolved in anhydrous DMF (15 mL), HATU (0.209 g, 0.548 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. Then o-phenylenediamine (0.119 g, 1.10 mmol) and DIPEA (0.106 g, 0.823 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction solution was diluted with water, extracted three times with ethyl acetate, the organic phases were combined, the solvent was removed by vacuum distillation, and the remaining solid was purified by silica gel column chromatography to give compound I-11 (0.063 g, yield 54%). 1 H NMR(800MHz, DMSO-d6)δ9.60(s,1H),8.31(s,1H),8.07–8.06(m,1H),7.92(d,J=7.9Hz,2H),7.46(dd,J=3.5,0.8Hz,1H),7.30(d,J=8.1Hz,2H),7 .17–7.13(m,1H),6.98–6.94(m,3H),6.80(dd,J=3.5,1.8Hz,1H),6.76(d d, J=8.0, 1.4Hz, 1H), 6.58 (t, J=7.5Hz, 1H), 5.51 (s, 2H), 4.89 (brs, 2H). HRMS(ESI)

[0143] C 23 H 20 N7O2 + ([M+H)) + Calculated value: 426.1673, measured value: 426.1670.

[0144] Example 12: Preparation of 4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-hydroxybenzamide (compound I-12)

[0145]

[0146] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-4. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellowish-white solid compound (I-12) (0.053 g, yield 97%). 1 H NMR (800MHz, DMSO-d6) δ11.16(s,1H),8.95(brs,1H),8.30(s,1H),8.06(d,J=1.6Hz,1H),7.68(d,J=8.2Hz, 2H), 7.46 (d, J = 3.5Hz, 1H), 7.23 (d, J = 8.1Hz, 2H), 6.95 (brs, 2H), 6.79 (dd, J = 3.5, 1.8Hz, 1H), 5.47 (s, 2H). HRMS(ESI)C 17 H 15 N6O3 + [M+H] + Calculated value: 351.1200, measured value: 351.1187.

[0147] Example 13: Preparation of 4-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-13)

[0148] Step 1: Preparation of methyl 4-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)benzoate (intermediate Int-6)

[0149]

[0150] In Example 4, step 1, methyl 5-bromopentanoate was replaced with methyl 4-(bromomethyl)benzoate, and the reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 4, step 1, to obtain a white solid intermediate (Int-6) (0.050 g, yield 14%). 1H NMR (800MHz, DMSO-d6) δ8.32(s,1H),7.90(d,J=8.2Hz,2H),7.38(d,J=3.4Hz,1H),7.27(d,J=8 .1Hz,2H),6.91(brs,2H),6.42(dd,J=3.4,1.2Hz,1H),5.51(s,2H),3.82(s,3H),2.46(s,3H). HRMS(ESI)C 19 H 18 N5O3 + [M+H] + Calculated value: 364.1404, measured value: 364.1387.

[0151] Steps 2-3: Preparation of 4-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-13)

[0152]

[0153] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-6. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellowish-white solid compound (I-13) (0.073 g, yield 55%), obtained according to step 3 of Example 11. 1 H NMR (800MHz, DMSO-d6) δ9.60 (s, 1H), 8.32 (s, 1H), 7.92 (d, J = 7.9Hz, 2H), 7.38 (d,J=3.4Hz,1H),7.28(d,J=8.0Hz,2H),7.15(d,J=8.4Hz,1H),6.96(td,J=7. 6,1.5Hz,1H),6.90(brs,2H),6.76(dd,J=8.0,1.4Hz,1H),6.58(td,J=7.6,1. 4Hz, 1H), 6.43 (dd, J = 3.4, 1.1Hz, 1H), 5.50 (s, 2H), 4.89 (s, 2H), 2.46 (s, 3H). HRMS(ESI)C 24 H 22 N7O2 + [M+H] + Calculated value: 440.1829, measured value: 440.1811.

[0154] Example 14: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-14)

[0155] Step 1: Preparation of methyl 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)benzoate (intermediate Int-7)

[0156]

[0157] In Example 1, step 1, methyl 5-bromopentanoate was replaced with methyl 4-(2-bromoethyl)benzoate. The reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 1, step 1, to obtain a yellow solid intermediate (Int-7) (0.096 g, yield 35%). 1 H NMR(800MHz,Chloroform-d)δ8.23(s,1H),7.92–7.90(m,2H),7.72(dd,J=1.7,0.8Hz,1H),7.42(s,1H),7.26–7.23(m,2H),6.62(dd,J=

[0158] 3.5, 1.7Hz, 1H), 5.31 (brs, 2H), 4.53 (t, J = 7.6Hz, 2H), 3.87 (s, 3H), 3.27 (d, J = 7.6Hz, 2H). HRMS(ESI)C 19 H 18 N5O3 + [M+H] + Calculated value: 364.1404, measured value: 364.1413.

[0159] Steps 2-3: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-14)

[0160]

[0161] In step 2 of Example 11, intermediate Int-4 was replaced with intermediate Int-7. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 11. The product obtained was a brownish-yellow solid compound (I-14) (0.039 g, yield 70%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.57(s,1H),8.24(s,1H),8.05(d,J=1.7Hz,1H),7.86(d,J= 7.7Hz,2H),7.42(d,J=3.5Hz,1H),7.30(d,J=7.8Hz,2H),7.14(d,J=7.8Hz,1H),6.9 5(t,J=7.5Hz,1H),6.88(s,2H),6.78(dd,J=3.5,1.7Hz,1H),6.77(d,J=8.8Hz,1H), 6.58(t,J=7.5Hz,1H), 4.87(brs,2H), 4.48(t,J=7.2Hz,2H), 3.26(t,J=7.2Hz,2H). HRMS(ESI)C 24 H 22 N7O2 + [M+H] + Calculated value: 440.1829, measured value: 440.1826.

[0162] Example 15: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-hydroxybenzamide (compound I-15)

[0163]

[0164] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-7. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellow solid compound (I-15) (0.070 g, yield 80%) was further purified by preparative HPLC. 1 H NMR (800MHz, DMSO-d6) δ11.11(s,1H),8.95(brs,1H),8.24(s,1H),8.05(d,J=1.7Hz,1H),7.63(d,J=8.0Hz,2H),7.43(d,J= 3.5Hz, 1H), 7.23 (d, J = 7.8Hz, 2H), 6.88 (brs, 2H), 6.78 (dd, J = 3.5, 1.7Hz, 1H), 4.45 (t, J = 7.3Hz, 2H), 3.21 (t, J = 7.3Hz, 2H). HRMS(ESI)C 18 H 17 N6O3 + [M+H] + Calculated value: 365.1357, measured value: 365.1355.

[0165] Example 16: Preparation of 4-(2-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-16)

[0166] Step 1: Preparation of methyl 4-(2-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)benzoate (intermediate Int-8)

[0167]

[0168] In Example 4, step 1, methyl 5-bromopentanoate was replaced with methyl 4-(2-bromoethyl)benzoate, and the reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 4, step 1, to obtain a yellowish-white solid intermediate (Int-8) (0.083 g, yield 32%). 1 H NMR (800MHz, DMSO-d6) δ8.22(s,1H),7.83–7.80(m,2H),7.33(d,J=3.4Hz,1H),7.30–7.27(m,2H),6.80(br s, 2H), 6.41 (dd, J = 3.4, 1.1Hz, 1H), 4.45 (t, J = 7.2Hz, 2H), 3.81 (s, 3H), 3.23 (t, J = 7.2Hz, 2H), 2.45 (s, 3H). HRMS(ESI)C 20 H 20 N5O3 + [M+H] + Calculated value: 378.1561, measured value: 378.1557.

[0169] Steps 2-3: Preparation of 4-(2-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-16)

[0170]

[0171] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-8. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-16) (0.056 g, yield 55%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.59(s,1H),8.24(s,1H),7.86(d,J=7.9Hz,2H),7.34(d,J= 3.4Hz,1H),7.29(d,J=8.0Hz,2H),7.15(d,J=7.8Hz,1H),6.96(td,J=7.6,1.6Hz,1H) ,6.82(brs,2H),6.78(dd,J=8.0,1.5Hz,1H),6.60(t,J=7.5Hz,1H),6.41(dd,J=3.3 ,1.1Hz,1H),4.98(s,2H),4.47(t,J=7.2Hz,2H),3.25(t,J=7.2Hz,2H),2.45(s,3H). HRMS(ESI)C 25 H 24 N7O2 + [M+H] + Calculated value: 454.1986, measured value: 454.1980.

[0172] Example 17: Preparation of 4-(2-(6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-N-hydroxybenzamide (compound I-17)

[0173]

[0174] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-8. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellow solid compound (I-17) (0.041 g, yield 56%) was further purified by preparative HPLC. 1 H NMR (800MHz, DMSO-d6) δ11.11(s,1H),8.95(s,1H),8.24(s,1H),7.62(d,J=8.2Hz,2H),7.35(d,J=3.4Hz,1H),7.23(d ,J=8.3Hz,2H),6.83(brs,2H),6.42(dd,J=3.3,1.2Hz,1H),4.44(t,J=7.2Hz,2H),3.20(t,J=7.2Hz,2H),2.45(s,3H). HRMS(ESI)C 19 H 19 N6O3 + [M+H] + Calculated value: 379.1513, measured value: 379.1501.

[0175] Example 18: Preparation of 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-N-(2-aminophenyl)benzamide (compound I-18)

[0176] Steps 1-4: Preparation of methyl 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)benzoate (intermediate Int-9)

[0177]

[0178] Compound D-1 was synthesized according to the literature method (Yan et al., J Med Chem., 2021, 64, 16573-16597). Compound D-1 (363 mg, 1.18 mmol), K₂CO₃ (260 mg, 1.57 mmol), and compound C-1 (189 mg, 0.79 mmol) were dissolved in DMF (20 mL) and stirred overnight at 100 °C. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid Int-9 (0.105 g, yield 35%). 1 H NMR(800MHz, DMSO-d6)δ8.23(s,1H),8.05(dd,J=1.7,0.8Hz,1H),7.87–7.85(m,2H),7.42(dd,J=3.5,0.8Hz,1H),7.37–7.34( m,2H),6.88(s,2H),6.78(dd,J=3.5,1.7Hz,1H),4.21(t,J=6.9Hz,2H),3.82(s,3H),2.66(t,J=7.6Hz,2H),2.18–2.12(m,2H).

[0179] Steps 5-6: Preparation of 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propyl)-N-(2-aminophenyl)benzamide (compound I-18)

[0180]

[0181] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-9. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellowish-white solid compound (I-18) (0.104 g, yield 69%), obtained according to step 3 of Example 11. 1H NMR(800MHz,DMSO-d6)δ9.60(s,1H),8.27(s,1H),8.06(dd,J=1.8,0.8Hz,1H),7.91(d,J=7.8 Hz,2H),7.43(dd,J=3.5,0.7Hz,1H),7.36(d,J=8.1Hz,2H),7.16(d,J=7.8Hz,1H),6.96(td,J =7.6,1.6Hz,1H),6.89(brs,2H),6.79–6.78(m,1H),6.77(d,J=1.5Hz,1H),6.59(td,J=7.5,1 .4Hz,1H),4.88(brs,2H),4.23(t,J=7.0Hz,2H),2.67(t,J=7.6Hz,2H),2.16(p,J=7.3Hz,2H). HRMS(ESI)C 25 H 24 N7O2 + [M+H] + Calculated value: 454.1986, measured value: 454.1988.

[0182] Example 19: Preparation of 4-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-19)

[0183] Step 1: Preparation of methyl 4-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)benzoate (intermediate Int-10)

[0184]

[0185] The starting material, compound C-4, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 4-(bromomethyl)benzoate (434 mg, 1.90 mmol), K₂CO₃ (349 mg, 2.53 mmol), and compound C-4 (253 mg, 1.26 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellowish-white solid intermediate, Int-10 (207 mg, yield 47%). 1H NMR(600MHz,DMSO-d6)δ7.97(dd,J=1.8,0.8Hz,1H),7.93–7.89(m,2H),7.29–7.26(m,3H),7.22(d,J=3 .6Hz,1H),6.77(d,J=3.6Hz,1H),6.72(dd,J=3.5,1.7Hz,1H),6.28(brs,2H),5.38(s,2H),3.82(s,3H). HRMS(ESI)C 19 H 17 N4O3 +

[0186] [M+H] + Calculated value: 349.1295, measured value: 349.1278.

[0187] Step 2: Preparation of 4-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-19)

[0188]

[0189] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-10. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-19) (0.050 g, yield 41%), obtained according to step 3 of Example 11. 1 H NMR (800MHz, DMSO-d6) δ9.61(s,1H),7.96(d,J=1.8Hz,1H),7.92(d,J=7.9Hz,2H),7.30( d,J=8.0Hz,2H),7.28(dd,J=3.5,0.8Hz,1H),7.24(d,J=3.6Hz,1H),7.15(d,J=7.8Hz,1H) ,6.96(td,J=7.6,1.6Hz,1H),6.78(d,J=3.6Hz,1H),6.77(dd,J=8.1,1.6Hz,1H),6.72(d d, J=3.4, 1.7Hz, 1H), 6.58 (td, J=7.5, 1.4Hz, 1H), 6.27 (s, 2H), 5.37 (s, 2H), 4.87 (s, 2H). HRMS(ESI)C 24 H 21 N6O2 + [M+H] + Calculated value: 425.1721, measured value: 425.1737.

[0190] Example 20: Preparation of 4-((2-amino-6-(furan-2-yl)-9H-purin-9-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-20)

[0191] Step 1: Preparation of methyl 4-((2-amino-6-(furan-2-yl)-9H-purin-9-yl)methyl)benzoate (intermediate Int-11)

[0192]

[0193] In Example 7, step 1, methyl 7-bromoheptanoate was replaced with methyl 4-(bromomethyl)benzoate, and the reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 7, step 1, to obtain a pale yellow solid intermediate (Int-11) (0.289 g, yield 88%). 1 H NMR(800MHz, DMSO-d6)δ8.24(s,1H),7.96(dd,J=1.7,0.8Hz,1H),7.94–7.92(m,2H),7.74(dd,J=3.4, 0.9Hz,1H),7.37–7.34(m,2H),6.75(dd,J=3.4,1.8Hz,1H),6.59(brs,2H),5.42(s,2H),3.83(s,3H). HRMS(ESI)

[0194] C 18 H 16 N5O3 + [M+H] + Calculated value: 350.1248, measured value: 350.1253.

[0195] Step 2: Preparation of 4-((2-amino-6-(furan-2-yl)-9H-purin-9-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-20)

[0196]

[0197] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-11. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a pale yellow solid compound (I-20) (0.080 g, yield 63%) according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.61 (s, 1H), 8.26 (s, 1H), 7.96 (dd, J = 1.8, 0.8Hz, 1H), 7 .94(d,J=8.0Hz,2H),7.74(dd,J=3.4,0.8Hz,1H),7.38(d,J=8.0Hz,2H),7.15(d ,J=7.8Hz,1H),6.96(ddd,J=8.4,7.3,1.6Hz,1H),6.77(d,J=1.4Hz,1H),6.76–6 .75(m,1H),6.60(s,2H),6.58(dd,J=7.5,1.4Hz,1H),5.41(s,2H),4.88(s,2H). HRMS(ESI)C 23 H 20 N7O2 + [M+H] + Calculated value: 426.1673, measured value: 426.1654.

[0198] Example 21: Preparation of 4-(2-(2-amino-6-(furan-2-yl)-9H-purin-9-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-21)

[0199] Step 1: Preparation of methyl 4-(2-(2-amino-6-(furan-2-yl)-9H-purin-9-yl)ethyl)benzoate (intermediate Int-12)

[0200]

[0201] In Example 7, step 1, methyl 7-bromoheptanoate was replaced with methyl 4-(2-bromoethyl)benzoate. The other raw materials, reagents and preparation methods were the same as in Example 7, step 1, to obtain a pale yellow solid intermediate (Int-12) (0.187 g, yield 34%). 1 H NMR (800MHz, DMSO-d6) δ7.94(dd,J=1.7,0.8Hz,1H),7.90(s,1H),7.88–7.85(m,2H),7.68(dd,J=3.4,0.8Hz,1H),7.3 4–7.31(m,2H),6.73(dd,J=3.4,1.7Hz,1H),6.58(brs,2H),4.36(t,J=7.2Hz,2H),3.82(s,3H),3.23(t,J=7.2Hz,2H). HRMS(ESI)C 19 H 18 N5O3 + [M+H] +Calculated value: 364.1404, measured value: 364.1402.

[0202] Step 2: Preparation of 4-(2-(2-amino-6-(furan-2-yl)-9H-purin-9-yl)ethyl)-N-(2-aminophenyl)benzamide (compound I-21)

[0203]

[0204] In step 2 of Example 11, intermediate Int-4 was replaced with intermediate Int-12. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 11. The product obtained was a pale yellow solid compound (I-21) (0.060 g, yield 49%), obtained according to step 3 of Example 11. 1 H NMR(800MHz,DMSO-d6)δ9.60(s,1H),7.95(dd,J=1.7,0.8Hz,1H),7.93(s,1H),7.90(d,J=7 .9Hz,2H),7.69(dd,J=3.4,0.8Hz,1H),7.32(d,J=8.0Hz,2H),7.14(d,J=7.8Hz,1H),6.96( ddd,J=8.6,7.3,1.6Hz,1H),6.77(dd,J=8.0,1.5Hz,1H),6.73(dd,J=3.4,1.7Hz,1H),6.59 (brs, 2H), 6.58 (d, J = 8.1Hz, 1H), 4.87 (s, 2H), 4.38 (t, J = 7.2Hz, 2H), 3.24 (t, J = 7.2Hz, 2H). HRMS(ESI)C 24 H 22 N7O2 + [M+H] + Calculated value: 440.1829, measured value: 440.1814.

[0205] Example 22: Preparation of 4-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-22)

[0206] Step 1: Preparation of methyl 4-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)benzoate (intermediate Int-13)

[0207]

[0208] The starting material, compound C-5, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 4-(bromomethyl)benzoate (153 mg, 0.669 mmol), K₂CO₃ (123 mg, 0.892 mmol), and compound C-5 (96 mg, 0.446 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid intermediate, Int-13 (85 mg, 52% yield). 1 H NMR (600MHz, DMSO-d6) δ8.20 (s, 1H), 7.95–7.91 (m, 2H), 7.68 (d, J = 3.3Hz, 1H), 7.35 (d, J = 8. 4Hz,2H),6.57(brs,2H),6.38(dd,J=3.3,1.1Hz,1H),5.40(s,2H),3.83(s,3H),2.40(s,3H). HRMS(ESI)C 19 H 18 N5O3 + [M+H] + Calculated value: 364.1404, measured value: 364.1400.

[0209] Step 2: Preparation of 4-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)-N-(2-aminophenyl)benzamide (compound I-22)

[0210]

[0211] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-13. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-22) (0.039 g, yield 40%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.62 (s, 1H), 8.22 (s, 1H), 7.94 (d, J = 7.9Hz, 2H), 7. 68(d,J=3.3Hz,1H),7.37(d,J=8.0Hz,2H),7.15(d,J=7.8Hz,1H),6.96(td,J =7.6,1.6Hz,1H),6.77(dd,J=8.0,1.4Hz,1H),6.59(d,J=8.1Hz,1H),6.57(b rs, 2H), 6.39 (dd, J = 3.4, 0.9Hz, 1H), 5.39 (s, 2H), 4.93 (s, 2H), 2.41 (s, 3H). HRMS(ESI)C 24 H 22 N7O2 + [M+H] + Calculated value: 440.1829, measured value: 440.1822.

[0212] Example 23: Preparation of 4-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)-N-hydroxybenzamide (compound I-23)

[0213]

[0214] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-13. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-23) (0.077 g, yield 87%). 1 H NMR (800MHz, DMSO-d6) δ11.18(s,1H),9.02(s,1H),8.20(s,1H),7.72–7.69(m,2H),7.67(d,J=3.3H z,1H),7.30(d,J=8.2Hz,2H),6.57(brs,2H),6.38(dd,J=3.4,1.2Hz,1H),5.35(s,2H),2.40(s,3H). HRMS(ESI)C 18 H 17 N6O3 + [M+H] + Calculated value: 365.1357, measured value: 365.1334.

[0215] Example 24: Preparation of 4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)-N-(2-aminophenyl))benzamide (compound I-24)

[0216] Step 1: Preparation of methyl 4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)benzoate (intermediate Int-14)

[0217]

[0218] The starting material, compound C-6, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 4-(bromomethyl)benzoate (293 mg, 1.25 mmol), Cs₂CO₃ (651 mg, 2.00 mmol), and compound C-6 (165 mg, 0.816 mmol) were dissolved in CH₃CN and DMF (1:1 v / v, 10 mL each) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellow solid intermediate, Int-14 (23 mg, 8% yield). 1 HNMR(800MHz,DMSO)δ8.16–8.12(m,1H),7.98–7.95(m,2H),7.75(d,J=3.5Hz,1 H), 7.49 (d, J = 8.5Hz, 2H), 7.15 (s, 2H), 6.85 (dd, 1H), 6.00 (s, 2H), 3.84 (s, 3H). HRMS(ESI)C 17 H 15 N6O3 + [M+H] + Calculated value: 351.1200, measured value: 351.1201.

[0219] Step 2: Preparation of 4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)-N-(2-aminophenyl))benzamide (compound I-24)

[0220]

[0221] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-14. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-24) (0.015 g, yield 12%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.65(s,1H),8.13(dd,J=1.7,0.8Hz,1H),7.98(d,J=7.9Hz,2H),7.74(dd,J=3.5,0.8Hz,1H),7.51(d,J=8.1Hz,2H) ,7.15(dd,1H),7.05(s,2H),6.96(m,1H),6.85(dd,J=3.5,1.7Hz,1H),6.76(dd,J=8.0,1.5Hz,1H),6.58(m,1H),5.98(s,2H),4.88(s,2H). HRMS(ESI)C 22 H 19 N8O2 + [M+H] + Calculated value: 427.1625, measured value: 427.1624.

[0222] Example 25: Preparation of 4-((5-amino-7-(5-methylfuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)-N-hydroxybenzamide (compound I-25)

[0223] Step 1: Preparation of 4-((5-amino-7-(5-methylfuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)benzoate (intermediate Int-15)

[0224]

[0225] The starting material compound C-7 was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 4-(bromomethyl)benzoate (427 mg, 1.87 mmol), Cs₂CO₃ (967 mg, 2.97 mmol), and 7-(5-methylfuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine (262 mg, 1.21 mmol) were dissolved in CH₃CN and DMF (1:1 v / v, 10 mL each) and stirred overnight at room temperature. The reaction mixture was diluted with water, extracted three times with ethyl acetate, and the combined organic phases were dried once with anhydrous Na₂SO₄. The solvent was removed under reduced pressure, and the remaining solid was purified by silica gel column chromatography to give a yellow solid intermediate Int-14 (21 mg, 7% yield). 1HNMR(800MHz,DMSO-d6)δ7.94(m,2H),7.88(d,J=3.3Hz,1H),7.37(m,2H),7.34– 7.19(s,2H),6.51(dd,J=3.4,1.1Hz,1H),5.75(s,2H),3.83(s,3H),2.45(s,3H). HRMS(ESI)C 18 H 17 N6O3 + [M+H] + Calculated value: 365.1357, measured value: 365.1403.

[0226] Step 2: Preparation of 4-((5-amino-7-(5-methylfuran-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)-N-hydroxybenzamide (compound I-25)

[0227]

[0228] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-15. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-25) (0.018 g, yield 69%). 1 H NMR(800MHz,DMSO-d6)δ11.17 9.03(s,1H),9.03(s,1H),7.87(d,J=3.4Hz,1H),7.73–7.69(m,2H),7.40(s,2 H),7.32–7.29(m,2H),6.51(dd,J=3.4,1.1Hz,1H),5.70(s,2H),2.45(s,3H). HRMS(ESI)C 17 H 16 N7O3 + [M+H] + Calculated value: 366.1309, measured value: 366.1329.

[0229] Example 26: Preparation of (E)-3-(4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-26)

[0230] Step 1: Preparation of methyl(E)-3-(4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)acrylate (intermediate Int-16)

[0231]

[0232] In Example 1, step 1, methyl 5-bromopentanoate was replaced with methyl (E)-3-(4-(bromomethyl)phenyl)acrylate, and the reaction conditions were changed from 100°C to room temperature. The other raw materials, reagents and preparation methods were the same as in Example 1, step 1, to obtain a yellow solid intermediate (Int-7) (0.081 g, yield 29%). 1 H NMR (800MHz, DMSO-d6) δ8.29(s,1H),8.06(dd,J=1.7,0.8Hz,1H),7.67(d,J=7.7Hz,2H),7.62(d,J=16.0Hz,1H),7.45(dd,J=3.5, 0.8Hz,1H),7.21(d,J=7.2Hz,2H),6.95(brs,2H),6.79(dd,J=3.5,1.7Hz,1H),6.60(d,J=16.0Hz,1H),5.45(s,2H),3.71(s,3H). HRMS(ESI)C 20 H 18 N5O3 +

[0233] [M+H] + Calculated value: 376.1404, measured value: 376.1405.

[0234] Step 2: Preparation of (E)-3-(4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-26)

[0235]

[0236] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-16. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-26) (0.047 g, yield 55%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.38(s,1H),8.30(s,1H),8.07(dd,J=1.7,0.8Hz,1H),7.57(d,J=8.0Hz ,2H),7.51(d,J=15.7Hz,1H),7.46(dd,J=3.5,0.8Hz,1H),7.32(dd,J=8.0,1.6Hz,1H),7.24(d,J =8.0Hz,2H),6.96(brs,2H),6.91(dd,J=7.6,1.5Hz,1H),6.85(d,J=15.7Hz,1H),6.80(dd,J=3.5 ,1.7Hz,1H),6.74(dd,J=7.9,1.5Hz,1H),6.57(td,J=7.5,1.4Hz,1H),5.46(s,2H),4.93(s,2H). HRMS(ESI)C 25 H 22 N7O2 + [M+H] + Calculated value: 452.1829, measured value: 452.1851.

[0237] Example 27: Preparation of (E)-3-(4-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (compound I-27)

[0238]

[0239] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-16. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellow solid compound (I-27) (0.026 g, yield 38%) was further purified by preparative HPLC. 1 H NMR (800MHz, DMSO-d6) δ10.74(s,1H),9.02(s,1H),8.29(s,1H),8.06(dd,J=1.7,0.8Hz,1H),7.51(d,J=8.0Hz,2H),7.45(dd,J=3.5,0.8 Hz,1H),7.41(d,J=15.8Hz,1H),7.21(d,J=8.0Hz,2H),6.95(brs,2H),6.79(dd,J=3.5,1.7Hz,1H),6.41(d,J=15.8Hz,1H),5.44(s,2H). HRMS(ESI)C 19 H 17 N6O3 + [M+H]+ Calculated value: 377.1357, measured value: 377.1359.

[0240] Example 28: Preparation of (E)-3-(4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-28)

[0241] Step 1: Preparation of methyl(E)-3-(4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl))phenyl)acrylate (intermediate Int-17)

[0242]

[0243] In Example 24, step 1, methyl 4-(bromomethyl)benzoate was replaced with methyl (E)-3-(4-(bromomethyl)phenyl)acrylate. The other required raw materials, reagents and preparation methods were the same as in step 1 of Example 24, yielding a yellow solid intermediate (Int-7) (0.083 g, yield 11%). 1 H NMR (800MHz, DMSO) δ8.12(d,J=0.9Hz,1H),7.91(d,J=3.3Hz,1H),7.71(d,J=8.3Hz,2H),7.63(d,J=16.0Hz,1H),7 .36(s,2H),7.31(d,J=8.3Hz,2H),6.86(dd,J=3.5,1.7Hz,1H),6.62(d,J=16.0Hz,1H),5.70(s,2H),3.71(s,3H). HRMS(ESI)C 19 H 17 N6O3 + [M+H] + Calculated value: 377.1357, measured value: 377.1361.

[0244] Step 2: Preparation of (E)-3-(4-((5-amino-7-(furan-2-yl)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-28)

[0245]

[0246] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-17. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a brownish-yellow solid compound (I-28) (0.006 g, yield 4%), obtained according to step 3 of Example 11. 1 H NMR(800MHz,DMSO-d6)δ9.40(s,1H),8.13(dd,J=1.8,0.8Hz,1H),7.91(dd,J=3.5,0.8Hz, 1H),7.60(d,J=8.2Hz,2H),7.53-1.51(d,J=15.7Hz,1H),7.38(s,2H),7.33(d,J=8.3Hz,2 H),7.32(d,J=1.5Hz,1H),6.91(m,1H),6.88-6.86(d,J=15.8,1H),6.88–6.86(d,J=15.1H z, 1H), 6.74 (dd, J = 8.0, 1.5Hz, 1H), 6.57 (td, J = 7.5, 1.4Hz, 1H), 5.71 (s, 2H), 4.94 (s, 2H). HRMS(ESI)C 24 H 21 N8O2 + [M+H] + Calculated value: 453.1782, measured value: 453.1721.

[0247] Example 29: Preparation of (E)-3-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-29)

[0248] Step 1: Preparation of methyl(E)-3-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)acrylate (intermediate Int-18)

[0249]

[0250] 3-(bromomethyl)benzaldehyde (0.285 g, 1.43 mmol), K₂CO₃ (275 mg, 1.99 mmol), and 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-amine (160 mg, 0.795 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid intermediate Int-18a (0.105 g, 41% yield).1 H NMR(800MHz, DMSO-d6)δ9.96(s,1H),8.31(s,1H),8.07(dd,J=1.7,0.8Hz,1H),7.83(dt,J=7.5,1.5Hz,1H),7.68(q,J=1.3,0.9Hz,1H),7 .58(t,J=7.6Hz,1H),7.54(dt,J=7.9,1.5Hz,1H),7.45(dd,J=3.5,0.8Hz,1H),6.96(brs,2H),6.79(dd,J=3.5,1.7Hz,1H),5.53(s,2H). HRMS(ESI)C 17 H 14 N5O2 + [M+H] + Calculated value: 320.1142, Measured value: 320.1144. Methyl 2-(dimethoxyphosphoryl)acetate (111 mg, 0.608 mmol) and C4H9OK (55 mg, 0.498 mmol) were dissolved in DMF (15 mL), stirred at 0 °C for 15 minutes, and then a DMF solution of Int-18a was added dropwise. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid intermediate Int-18 (0.092 g, yield 75%). 1 H NMR(800MHz, DMSO-d6)δ8.29(s,1H),8.06(dd,J=1.7,0.8Hz,1H),7.65–7.63(m,1H),7.62(d,J=16.1Hz,1H),7.59–7.58(m,1H),7.45(dd,J=3.5,0.8 Hz,1H),7.37(t,J=7.7Hz,1H),7.18(d,J=7.8Hz,1H),6.95(brs,2H),6.79 (dd,J=3.5,1.7Hz,1H),6.59(d,J=16.1Hz,1H),5.45(s,2H),3.71(s,3H). HRMS(ESI)C 20 H 17 N5O3 + [M+H] + Calculated value: 376.1404, measured value: 376.1397.

[0251] Step 2: Preparation of (E)-3-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-29)

[0252]

[0253] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-18. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-29) (0.028 g, yield 26%), obtained according to step 3 of Example 11. 1 H NMR (800MHz, DMSO-d6) δ9.38(s,1H),8.30(s,1H),8.06(d,J=1.7Hz,1H),7.52(d,J=8.9Hz,1H),7.51 (d,J=15.9Hz,2H),7.45(dd,J=3.5,0.8Hz,1H),7.40(t,J=7.7Hz,1H),7.32(dd,J=7.8,1.6Hz,1H),7. 21(d,J=7.7Hz,1H),6.95(brs,2H),6.91(td,J=7.8,1.8Hz,1H),6.85(d,J=15.8Hz,1H),6.79(dd,J=3 .5, 1.8Hz, 1H), 6.74 (dd, J=8.0, 1.5Hz, 1H), 6.57 (td, J=7.5, 1.4Hz, 1H), 5.47 (s, 2H), 4.93 (brs, 2H). HRMS(ESI)C 25 H 22 N7O2 + [M+H] + Calculated value: 452.1829, measured value: 452.1830.

[0254] Example 30: Preparation of (E)-3-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (compound I-30)

[0255]

[0256] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-18. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The compound (I-30) was further purified by preparative HPLC to obtain a white solid compound (I-30) (0.060 g, yield 84%). 1H NMR (800MHz, DMSO-d6) δ10.76(s,1H),9.04(s,1H),8.29(s,1H),8.06(d,J=1.7Hz,1H),7.46(d,J=7.9Hz,1H),7.45(d,J=3.5Hz,1H),7.43(s,1H),7. 40(d,J=15.8Hz,1H),7.36(t,J=7.7Hz,1H),7.17(d,J=7.7Hz,1H),6.94(b rs, 2H), 6.79 (dd, J = 3.6, 1.7Hz, 1H), 6.41 (d, J = 15.8Hz, 1H), 5.44 (s, 2H). HRMS(ESI)C 19 H 17 N6O3 + [M+H] + Calculated value: 377.1357, measured value: 377.1354.

[0257] Example 31: Preparation of (E)-3-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-31)

[0258] Step 1: Preparation of (E)-3-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)methyl acrylate (intermediate Int-19)

[0259]

[0260] In step 1 of Example 29, 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine was replaced with 4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine. The other raw materials, reagents and preparation methods were the same as in step 1 of Example 29, yielding a pale yellow solid compound (I-31) (0.313 g, yield 57%). 1 H NMR (600MHz, DMSO-d6) δ8.30(s,1H),7.64–7.60(m,2H),7.57(t,J=1.8Hz,1H),7.39–7.34(m,2H),7.17(dt,J=7.8,1. 3Hz, 1H), 6.90 (brs, 2H), 6.58 (d, J = 16.0Hz, 1H), 6.42 (dd, J = 3.4, 1.1Hz, 1H), 5.44 (s, 2H), 3.71 (s, 3H), 2.45 (s, 3H). HRMS(ESI)C 21H 20 N5O3 + [M+H] + Calculated value: 390.1561, measured value: 390.1563.

[0261] Step 2: Preparation of (E)-3-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)-N-(2-aminophenyl)acrylamide (compound I-31)

[0262]

[0263] In Example 11, step 2, intermediate Int-4 was replaced with intermediate Int-19. The other required raw materials, reagents and preparation methods were the same as in Example 11, step 2. The product obtained was a yellow solid compound (I-31) (0.064 g, yield 49%), obtained according to Example 11, step 3. 1 H NMR(600MHz,DMSO-d6)δ9.38(s,1H),8.31(s,1H),7.54–7.47(m,3H),7.42–7 .36(m,2H),7.32(dd,J=7.9,1.5Hz,1H),7.20(d,J=7.6Hz,1H),6.93–6.88(m, 3H), 6.84 (d, J = 15.7Hz, 1H), 6.74 (dd, J = 8.0, 1.5Hz, 1H), 6.57 (td, J = 7.5, 1. 4Hz, 1H), 6.43 (dd, J = 3.3, 1.2Hz, 1H), 5.46 (s, 2H), 4.93 (s, 2H), 2.46 (s, 3H). HRMS(ESI)C 26 H 24 N7O2 + [M+H] + Calculated value: 466.1986, measured value: 466.1982.

[0264] Example 32: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethoxy)-N-(2-aminophenyl)benzamide (compound I-32)

[0265] Step 1: Preparation of methyl 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethoxy)benzoate (intermediate Int-20)

[0266]

[0267] Compound D-2 was synthesized according to the literature method (Yan et al., J Med Chem., 2021, 64, 16573-16597). Compound C-1 (0.081 g, 0.40 mmol), compound D-2 (0.125 g, 0.48 mmol), and K₂CO₃ (111 mg, 0.80 mmol) were dissolved in DMF (15 mL) and stirred overnight at 100 °C. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid intermediate Int-20 (0.099 g, yield 65%). 1 H NMR(800MHz,Chloroform-d)δ8.27(s,1H),7.96–7.92(m,2H),7.74(dd,J=1.7,0.8Hz,1H),7.51–7.41(m,1H),6.89– 6.87(m,2H),6.64(dd,J=3.5,1.7Hz,1H),5.28(brs,2H),4.70(t,J=5.9Hz,2H),4.49(t,J=5.9Hz,2H),3.86(s,3H). HRMS(ESI)C 19 H 18 N5O4 + [M+H] + Calculated value: 380.1353, measured value: 380.1351.

[0268] Step 2: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethoxy)-N-(2-aminophenyl)benzamide (compound I-32)

[0269]

[0270] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-20. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-32) (0.025 g, yield 42%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.53(s,1H),8.28(s,1H),8.05(d,J=1.7Hz,1H),7.93(d,J=8.5 Hz,2H),7.44(d,J=3.5Hz,1H),7.13(d,J=7.8Hz,1H),7.00(d,J=8.8Hz,2H),6.96(dd,J =7.7,1.6Hz,1H),6.94(brs,2H),6.78(dd,J=3.5,1.7Hz,1H),6.77(dd,J=8.1,1.5Hz,1 H), 6.59 (t, J = 7.5Hz, 1H), 4.85 (brs, 2H), 4.61 (t, J = 5.5Hz, 2H), 4.53 (t, J = 5.5Hz, 2H). HRMS(ESI)C 24 H 22 N7O3 + [M+H] + Calculated value: 456.1779, measured value: 456.1773.

[0271] Example 33: Preparation of 4-(2-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethoxy)-N-hydroxybenzamide (compound I-33)

[0272]

[0273] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-20. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The compound (I-33) was further purified by preparative HPLC to obtain a yellowish-white solid compound (I-33) (0.066 g, yield 49%). 1 H NMR (800MHz, DMSO-d6) δ11.03(s,1H),8.86(brs,1H),8.26(s,1H),8.04(d,J=1.7Hz,1H),7.68(d,J=8.9Hz,2H),7.43(d,J= 3.5Hz, 1H), 6.94 (d, J = 8.8Hz, 2H), 6.91 (brs, 2H), 6.78 (dd, J = 3.5, 1.7Hz, 1H), 4.58 (t, J = 5.5Hz, 2H), 4.49 (t, J = 5.5Hz, 2H). HRMS(ESI)C 18 H 17 N6O4 + [M+H] + Calculated value: 381.1306, measured value: 381.1317.

[0274] Example 34: Preparation of 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)-N-(2-aminophenyl)benzamide (compound I-34)

[0275] Step 1: Preparation of methyl 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)benzoate (intermediate Int-21)

[0276]

[0277] Replace 1,2-dibromoethane in step 1 of Example 32 with 1,3-dibromopropane. The other required raw materials, reagents and preparation methods are the same as in step 1 of Example 32, to obtain a white solid intermediate Int-21 (0.060 g, yield 28%). 1 H NMR(800MHz, DMSO-d6)δ8.24(s,1H),8.05(dd,J=1.7,0.8Hz,1H),7.89–7.87(m,2H),7.42(dd,J=3.5,0.8Hz,1H),7.01–6.98(m,2 H), 6.86 (brs, 2H), 6.78 (dd, J = 3.5, 1.7Hz, 1H), 4.38 (t, J = 6.9Hz, 2H), 4.09 (t, J = 6.1Hz, 2H), 3.80 (s, 3H), 2.29 (p, J = 6.5Hz, 2H).

[0278] HRMS(ESI)C 20 H 20 N5O4 + [M+H] + Calculated value: 394.1510, measured value: 394.1515.

[0279] Step 2: Preparation of 4-(3-(6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)propoxy)-N-(2-aminophenyl)benzamide (compound I-34)

[0280]

[0281] Replace intermediate Int-4 in step 2 of Example 11 with intermediate Int-21. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 11. The product obtained is a yellow solid compound (I-34) (0.032 g, yield 32%), obtained according to step 3 of Example 11. 1H NMR (800MHz, DMSO-d6) δ9.53 (s, 1H), 8.25 (s, 1H), 8.05 (d, J = 1.6Hz, 1H), 7.94 (d, J=8.3Hz,2H),7.43(d,J=3.5Hz,1H),7.14(d,J=7.5Hz,1H),7.00(d,J=8.9Hz,2H), 6.96(td,J=7.6,1.5Hz,1H),6.88(s,2H),6.80–6.77(m,2H),6.60(t,J=7.5Hz,1H) ,4.93(s,2H),4.40(t,J=6.9Hz,2H),4.10(t,J=6.1Hz,2H),2.30(p,J=6.5Hz,2H). HRMS(ESI)C 25 H 24 N7O3 + [M+H] + Calculated value: 470.1935, measured value: 470.1982.

[0282] According to the methods provided in Examples 11-34, the compounds listed in Examples 35-44 can be prepared by changing the corresponding raw materials using the same methods, as detailed in Table 2.

[0283] Table 2

[0284]

[0285]

[0286]

[0287]

[0288] Example 45: Preparation of 5-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxypentanamide (compound I-45)

[0289] Step 1: Preparation of methyl 5-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)valerate (intermediate Int-22)

[0290]

[0291] m-Cresol (1 g, 9.25 mmol), K₂CO₃ (2.58 g, 18.49 mmol), and methyl 5-bromopentanoate (2.18 g, 11.10 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a colorless liquid intermediate Int-22a (1.559 g, 76% yield). HRMS(ESI)C 13 H 19 O3 + [M+H] + Calculated value: 223.1441, Measured value: 223.1466. Int-22a (1.559 g, 7.01 mmol), 1-bromopyrrolidone-2,5-dione (1.5 g, 8.42 mmol), and benzoic acid peroxyanhydride (85 mg, 0.350 mmol) were dissolved in CCl4 (30 mL) and refluxed overnight. The reaction solution was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na2SO4, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a pale yellow oily intermediate, Int-22b (1.461 g, yield 69%). HRMS(ESI)C 13 H 18 BrO3 + [M+H] + Calculated value: 301.0434, Measured value: 301.0389. Int-22b (1.461 g, 4.85 mmol), K₂CO₃ (466 mg, 3.37 mmol), and 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine (339 mg, 1.68 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellow oily intermediate, Int-22 (0.140 g, yield 20%). 1H NMR (800MHz, DMSO-d6) δ8.28 (s, 1H), 8.06 (dd, J=1.8, 0.8Hz, 1H), 7.44 (dd, J=3.5, 0.8Hz,1H),7.21(t,J=7.9Hz,1H),6.93(brs,2H),6.83–6.80(m,1H),6.79(dd,J=3. 5,1.7Hz,1H),6.74(t,J=2.0Hz,1H),6.73–6.70(m,1H),5.38(s,2H),3.90(t,J=6. 1Hz, 2H), 3.57 (s, 3H), 2.35 (t, J = 7.2Hz, 2H), 1.70–1.66 (m, 2H), 1.66–1.62 (m, 2H). HRMS(ESI)C 22 H 24 N5O4 + [M+H] + Calculated value: 422.1823, measured value: 422.1829.

[0292] Step 2: Preparation of 5-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxypentanamide (compound I-45)

[0293]

[0294] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-22. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellowish-white solid compound (I-45) (0.075 g, yield 59%). 1 H NMR (800MHz, DMSO-d6) δ10.35(s,1H),8.67(d,J=1.7Hz,1H),8.28(s,1H),8.06(dd,J=1.7 ,0.8Hz,1H),7.44(dd,J=3.5,0.8Hz,1H),7.21(t,J=7.9Hz,1H),6.94(brs,2H),6.83–6.8 1(m,1H),6.79(dd,J=3.5,1.7Hz,1H),6.76(t,J=2.0Hz,1H),6.71(d,J=7.7Hz,1H),5.38( s,2H),3.90(t,J=6.2Hz,2H),1.99(t,J=7.2Hz,2H),1.68–1.63(m,2H),1.63–1.58(m,2H). HRMS(ESI)C 21 H 23 N6O4 +[M+H] + Calculated value: 423.1775, measured value: 423.1787.

[0295] Example 46: Preparation of 6-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyhexanoamide (compound I-46)

[0296] Step 1: Preparation of 6-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyhexanoamide (intermediate Int-23)

[0297]

[0298] In Example 45, methyl 5-bromopentanoate was replaced with methyl 6-bromohexanoate in step 1. The other required raw materials, reagents and preparation methods were the same as in step 1 of Example 45, and a yellow oily intermediate Int-23 (0.173 g, yield 40%) was obtained. 1 H NMR (800MHz, DMSO-d6) δ8.28(s,1H),8.06(dd,J=1.8,0.7Hz,1H),7.44(dd,J=3.5,0.8 Hz,1H),7.20(t,J=7.9Hz,1H),6.94(brs,2H),6.81(ddd,J=8.2,2.6,0.9Hz,1H),6.79 (dd,J=3.5,1.7Hz,1H),6.74–6.70(m,2H),5.38(s,2H),3.89(t,J=6.4Hz,2H),3.56(s ,3H),2.30(t,J=7.4Hz,2H),1.69–1.64(m,2H),1.58–1.52(m,2H),1.40–1.35(m,2H). HRMS(ESI)C 23 H 26 N5O4 + [M+H] + Calculated value: 436.1979, measured value: 436.1980.

[0299] Step 2: Preparation of 6-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyhexanoamide (compound I-46)

[0300]

[0301] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-23. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-46) (0.130 g, yield 81%). 1 H NMR (800MHz, DMSO-d6) δ10.33(s,1H),8.65(s,1H),8.28(s,1H),8.06(d,J=1.7Hz,1H),7.44(d d,J=3.5,0.8Hz,1H),7.20(t,J=7.9Hz,1H),6.94(brs,2H),6.81(dd,J=8.2,2.6Hz,1H),6.79(d d,J=3.5,1.7Hz,1H),6.74(t,J=2.1Hz,1H),6.71(d,J=7.6Hz,1H),5.38(s,2H),3.88(t,J=6.4 Hz,2H),1.95(t,J=7.4Hz,2H),1.66(p,J=6.6Hz,2H),1.52(p,J=7.5Hz,2H),1.37–1.32(m,2H). HRMS(ESI)C 22 H 25 N6O4 + [M+H] + Calculated value: 437.1932, measured value: 437.1930.

[0302] Example 47: Preparation of 7-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-47)

[0303] Step 1: Preparation of methyl 7-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)heptanoate (intermediate Int-24)

[0304]

[0305] In Example 45, step 1, methyl 5-bromopentanoate was replaced with methyl 7-bromoheptanoate. The remaining raw materials, reagents, and preparation methods were the same as in Step 1 of Example 45, yielding a yellow oily intermediate, Int-24 (0.067 g, 50% yield). ¹H NMR (800 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.06 (dd, J = 1.8, 0.8 Hz, 1H), 7.44 (dd, J = 3.5, 0.8 Hz, 1H), 7.20 (t, J = 7.9 Hz, 1H), 6.94 (brs, 2H), 6.81 (ddd, J = 8.2, 2.6, 0.9 Hz, 1H), 6.79 (dd, J = 3... .5,1.7Hz,1H),6.73–6.70(m,2H),5.38(s,2H),3.88(t,J=6.5Hz,2H),3.57(s,3H),2.28(t ,J=7.4Hz,2H),1.67–1.62(m,2H),1.54–1.49(m,2H),1.38–1.33(m,2H),1.30–1.26(m,2H). HRMS(ESI)C 24 H 28 N5O3 + [M+H] + Calculated value: 450.2136, Measured value: 450.2136.

[0306] Step 2: Preparation of 7-(3-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-47)

[0307]

[0308] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-24. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellowish-white solid compound (I-47) (0.076 g, yield 52%). 1H NMR(800MHz,DMSO-d6)δ10.32(s,1H),8.64(brs,1H),8.28(s,1H),8.06(d,J=1.7Hz,1H),7.46–7.43 (m,1H),7.20(t,J=7.9Hz,1H),6.94(brs,2H),6.81(dd,J=8.2,2.5Hz,1H),6.79(dd,J=3.5,1.7Hz,1 H),6.74(t,J=2.0Hz,1H),6.71(d,J=7.6Hz,1H),5.38(s,2H),3.88(t,J=6.5Hz,2H),1.93(t,J=7.4H z,2H),1.64(p,J=6.7Hz,2H),1.48(p,J=7.5Hz,2H),1.35(tt,J=9.4,6.6Hz,2H),1.29–1.22(m,2H). HRMS(ESI)C 23 H 27 N6O4 + [M+H] + Calculated value: 451.2088, measured value: 451.2082.

[0309] Example 48: Preparation of 5-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxypentanamide (compound I-48)

[0310] Step 1: Preparation of methyl 5-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)valerate (intermediate Int-25)

[0311]

[0312] In step 1 of Example 45, 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine was replaced with 4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine. The other raw materials, reagents and preparation methods were the same as in step 1 of Example 45, and a yellow oily intermediate Int-25 (0.070 g, yield 24%) was obtained. 1H NMR(800MHz,DMSO-d6)δ8.29(s,1H),7.37(d,J=3.3Hz,1H),7.20(t,J=7.9Hz,1 H),6.88(brs,2H),6.81(ddd,J=8.2,2.6,0.9Hz,1H),6.72(t,J=2.0Hz,1H),6. 70(dt,J=7.7,1.3Hz,1H),6.42(dd,J=3.4,1.1Hz,1H),5.37(s,2H),3.90(t,J= 6.1Hz,2H),3.57(s,3H),2.46(s,3H),2.35(t,J=7.2Hz,2H),1.70–1.62(m,4H). HRMS(ESI)C 23 H 26 N5O4 + [M+H] + Calculated value: 436.1979, measured value: 436.1973.

[0313] Step 2: Preparation of 5-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxypentanamide (compound I-48)

[0314]

[0315] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-25. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-48) (0.027 g, yield 39%). 1 H NMR (600MHz, DMSO-d6) δ10.35(s,1H),8.68(d,J=1.9Hz,1H),8.29(s,1H),7.37( d,J=3.4Hz,1H),7.20(t,J=7.9Hz,1H),6.88(brs,2H),6.82(dd,J=8.2,2.5Hz,1H ),6.74(t,J=2.0Hz,1H),6.70(d,J=7.6Hz,1H),6.42(d,J=3.1Hz,1H),5.37(s,2 H), 3.90 (t, J = 6.1Hz, 2H), 2.46 (s, 3H), 1.99 (t, J = 7.1Hz, 2H), 1.71–1.58 (m, 4H). HRMS(ESI)C 22 H 25 N6O4 + [M+H] + Calculated value: 436.1979, measured value: 436.1973.

[0316] Example 49: Preparation of 7-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-49)

[0317] Step 1: Preparation of methyl 7-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)heptanoate (intermediate Int-26)

[0318]

[0319] In step 1 of Example 47, 4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine was replaced with 4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-6-amine. The other raw materials, reagents and preparation methods were the same as in step 1 of Example 47, and a yellow oily intermediate Int-26 (0.043 g, yield 30%) was obtained. 1 H NMR(800MHz,DMSO-d6)δ8.28(s,1H),7.36(d,J=3.4Hz,1H),7.19(d,J=8.0Hz,1H), 6.89(brs,2H),6.81–6.79(m,1H),6.72–6.70(m,2H),6.41(dd,J=3.4,1.1Hz,1H),5 .38(s,2H),3.87(t,J=6.5Hz,2H),3.56(s,3H),2.45(s,3H),2.27(t,J=7.4Hz,2H), 1.63(p,J=6.6Hz,2H), 1.50(p,J=7.4Hz,2H), 1.38–1.32(m,2H), 1.30–1.23(m,2H). HRMS(ESI)C 25 H 30 N5O4 + [M+H] + Calculated value: 464.2292, measured value: 464.2282.

[0320] Step 2: Preparation of 7-(3-((6-amino-4-(5-methylfuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-49)

[0321]

[0322] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-26. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The white solid compound (I-49) (0.036 g, yield 55%) was further purified by preparative HPLC. 1 H NMR (800MHz, DMSO-d6) δ10.31(s,1H),8.64(d,J=1.8Hz,1H),8.29(s,1H),7.37(d,J=3.4Hz,1H ),7.20(t,J=7.9Hz,1H),6.88(brs,2H),6.82–6.80(m,1H),6.72(s,1H),6.70(d,J=7.7Hz,1H) ,6.42(dd,J=3.4,1.1Hz,1H),5.37(s,2H),3.88(t,J=6.5Hz,2H),2.46(s,3H),1.93(t,J=7.4H z,2H),1.64(p,J=6.6Hz,2H),1.48(p,J=7.5Hz,2H),1.35(p,J=7.1Hz,2H),1.29–1.24(m,2H). HRMS(ESI)C 24 H 29 N6O4 + [M+H] + Calculated value: 465.2245, measured value: 465.2242.

[0323] Example 50: Preparation of 7-(3-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-50)

[0324] Step 1: Preparation of 7-(3-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)heptanoate (intermediate Int-27)

[0325]

[0326] The starting material, compound C-8, was synthesized according to the literature (Gillespie, Cliffe et al., Bioorg Med ChemLett., 2008, 18, 2924-2929). Methyl 7-(3-(bromomethyl)phenoxy)heptanate (218 mg, 1.12 mmol), K₂CO₃ (206 mg, 1.49 mmol), and compound C-8 (150 mg, 0.745 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a white solid intermediate, Int-27 (28 mg, 34% yield). 1 H NMR (800MHz, DMSO-d6) δ8.53(t,J=1.7Hz,1H),8.49(dt,J=8.0,1.4Hz,1H),8.42(s,1H),8.06(dt, J=7.7,1.4Hz,1H),7.80(t,J=7.8Hz,1H),7.21(t,J=7.9Hz,1H),7.10(brs,2H),6.83–6.80(m,1H), 6.76(t,J=2.1Hz,1H),6.74(d,J=7.9Hz,1H),5.42(s,2H),3.89(t,J=6.4Hz,2H),3.56(s,3H),2.2 7(t,J=7.4Hz,2H),1.67–1.62(m,2H),1.51(p,J=7.5Hz,2H),1.38–1.33(m,2H),1.31–1.26(m,2H). HRMS(ESI)C 27 H 29 N6O3 + [M+H] + Calculated value: 485.2296, measured value: 485.2291.

[0327] Step 2: Preparation of 7-(3-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-50)

[0328]

[0329] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-27. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellowish-white solid compound (I-50) (0.007 g, yield 31%) was further purified by preparative HPLC. 1H NMR (800MHz, DMSO-d6) δ10.33(s,1H),9.20(s,1H),8.50(s,1H),8.46(dt,J=7.9,1.4Hz,1H),8.40(t,J=1 .8Hz,1H),7.91(dt,J=7.8,1.4Hz,1H),7.82(t,J=7.8Hz,1H),7.22(t,J=7.9Hz,1H),7.10(brs,2H),6.83 (dd,J=8.3,2.5Hz,1H),6.77(t,J=2.1Hz,1H),6.74(d,J=7.6Hz,1H),5.43(s,2H),3.90(t,J=6.5Hz,2H), 1.93(t,J=7.4Hz,2H), 1.65(p,J=6.7Hz,2H), 1.48(p,J=7.5Hz,2H), 1.39–1.33(m,2H), 1.29–1.24(m,2H). HRMS(ESI)C 26 H 28 N7O3 + [M+H] + Calculated value: 485.2296, measured value: 485.2291.

[0330] Example 51: Preparation of 7-((6-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptamide (compound I-51)

[0331] Step 1: Preparation of methyl 7-((6-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)heptanoate (intermediate Int-28)

[0332]

[0333] 6-Methylpyridin-2-ol (0.96 g, 8.80 mmol), K₂CO₃ (2.43 g, 17.59 mmol), methyl 7-bromoheptanoate (2.36 g, 10.56 mmol), and sodium iodide (200 mg, 0.15 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a colorless liquid intermediate Int-28a (1.317 g, 60% yield). 1H NMR(800MHz,Chloroform-d)δ7.44(dd,J=8.2,7.2Hz,1H),6.69(d,J=7.2Hz,1H),6.50(d,J=8.2Hz,1H),4.24(t,J=6.6Hz,2H),3 .66(s,3H),2.43(s,3H),2.32(t,J=7.5Hz,2H),1.81–1.74(m,2H),1.66(p,J=7.6Hz,2H),1.51–1.44(m,2H),1.42–1.37(m,2H). HRMS(ESI)C 14 H 22 O3 + [M+H] + Calculated value: 252.1594, Measured value: 252.1645. Int-28a (1.317 g, 5.24 mmol), 1-bromopyrrolidone-2,5-dione (1.165 g, 6.29 mmol), and benzoic acid peroxyanhydride (66 mg, 0.262 mmol) were dissolved in CCl4 (30 mL) and refluxed overnight. The reaction solution was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na2SO4, the solvent was removed under reduced pressure, and the remaining solid was purified by silica gel column chromatography to give a colorless oily intermediate, Int-28b (0.715 g, yield 33%). HRMS(ESI)C 14 H 21 BrO3 + [M+H] + Calculated value: 330.0699, Measured value: 330.0700. Int-28b (0.475 g, 1.44 mmol), K₂CO₃ (351 mg, 2.54 mmol), and 3-(6-amino-1H-pyrazolo[3,4-d]pyrimidin-4-yl)benzyl nitrile (200 mg, 0.846 mmol) were dissolved in DMF (15 mL) and stirred overnight at room temperature. The reaction mixture was diluted with water and extracted three times with ethyl acetate. The combined organic phases were dried once with anhydrous Na₂SO₄, and the solvent was removed under reduced pressure. The remaining solid was purified by silica gel column chromatography to give a yellowish-white solid intermediate, Int-28 (0.045 g, yield 11%). 1H NMR(800MHz,DMSO-d6)δ8.55(t,J=1.8Hz,1H),8.51(dt,J=7.9,1.4Hz,1H),8.46(s,1H),8 .07(dt,J=7.7,1.4Hz,1H),7.80(t,J=7.8Hz,1H),7.60(dd,J=8.2,7.4Hz,1H),7.06(brs,2 H),6.64(d,J=8.2Hz,1H),6.56(d,J=7.3Hz,1H),5.47(brs,2H),4.02(t,J=6.8Hz,2H),3. 57(s,3H),2.21(t,J=7.4Hz,2H),1.52–1.48(m,2H),1.45–1.39(m,2H),1.21–1.16(m,4H). HRMS(ESI)C 26 H 28 N7O3 + [M+H] + Calculated value: 486.2248, measured value: 486.2247.

[0334] Step 2: Preparation of 7-((6-((6-amino-4-(3-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptamide (compound I-51)

[0335]

[0336] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-28. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellowish-white solid compound (I-51) (0.003 g, yield 10%) was further purified by preparative HPLC. 1H NMR(800MHz,DMSO-d6)δ10.35(brs,1H),9.20(brs,1H),8.54(s,1H),8.47(dt,J=7.8,1.4Hz,1 H),8.42(t,J=1.8Hz,1H),7.92(dt,J=7.9,1.4Hz,1H),7.83(t,J=7.8Hz,1H),7.61(dd,J=8.2, 7.4Hz,1H),7.09(brs,2H),6.66(d,J=8.2Hz,1H),6.51(d,J=7.4Hz,1H),5.47(s,2H),4.06(t, J=6.7Hz,2H),1.91(t,J=7.4Hz,2H),1.58–1.53(m,2H),1.46–1.40(m,2H),1.27–1.19(m,4H). HRMS(ESI)C 25 H 27 N8O3 + [M+H] + Calculated value: 487.2201, measured value: 487.2199.

[0337] Example 52: Preparation of 5-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxypentanamide (compound I-52)

[0338] Step 1: Preparation of methyl 5-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)valerate (intermediate Int-29)

[0339]

[0340] In Example 45, step 1, methyl 5-bromopentanoate was replaced with methyl 5-((6-(bromomethyl)pyridin-2-yl)oxy)pentanoate. The other raw materials, reagents and preparation methods were the same as in Example 45, step 1, to obtain a white solid intermediate Int-29 (0.108 g, yield 30%). 1H NMR (800MHz, DMSO-d6) δ8.31 (s, 1H), 8.06 (dd, J = 1.8, 0.8Hz, 1H), 7.59 (t, J = 7. 8Hz,1H),7.45(dd,J=3.5,0.8Hz,1H),6.91(brs,2H),6.79(dd,J=3.5,1.7Hz,1 H),6.65(d,J=8.2Hz,1H),6.48(d,J=7.4Hz,1H),5.43(s,2H),4.09(t,J=6.4Hz ,2H),3.56(s,3H),2.28(t,J=7.3Hz,2H),1.60–1.56(m,2H),1.55–1.51(m,2H). HRMS(ESI)C 21 H 23 N6O4 + [M+H] + Calculated value: 423.1775, measured value: 423.1779.

[0341] Step 2: Preparation of 5-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxypentanamide (compound I-52)

[0342]

[0343] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-29. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The yellowish-white solid compound (I-52) (0.088 g, yield 88%) was further purified by preparative HPLC. 1 H NMR(600MHz,DMSO-d6)δ10.35(s,1H),8.68(d,J=1.7Hz,1H),8.32(s,1H),8.07( d,J=1.8Hz,1H),7.59(dd,J=8.3,7.4Hz,1H),7.45(dd,J=3.6,0.7Hz,1H),6.92( brs,2H),6.80(dd,J=3.5,1.7Hz,1H),6.66(d,J=8.2Hz,1H),6.43(d,J=7.4Hz,1 H), 5.42 (s, 2H), 4.11 (t, J = 6.3Hz, 2H), 1.96 (t, J = 7.1Hz, 2H), 1.62–1.51 (m, 4H). HRMS(ESI)C 20 H 22 N7O4 + [M+H] +Calculated value: 424.1728, measured value: 424.1723.

[0344] Example 53: Preparation of 7-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptamide (compound I-53)

[0345] Step 1: Preparation of methyl 7-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)heptarate (intermediate Int-30)

[0346]

[0347] In Example 45, step 1, methyl 5-bromopentanoate was replaced with methyl 7-((6-(bromomethyl)pyridin-2-yl)oxy)heptanoate. The other required raw materials, reagents and preparation methods were the same as in step 1 of Example 45, yielding a yellow solid intermediate Int-30 (0.058 g, yield 26%). 1 H NMR(800MHz,DMSO-d6)δ8.30(s,1H),8.06(dd,J=1.7,0.8Hz,1H),7.60(dd,J=8.2,7.4Hz, 1H),7.44(dd,J=3.5,0.8Hz,1H),6.90(brs,2H),6.79(dd,J=3.5,1.7Hz,1H),6.64(dd,J=8 .2,0.8Hz,1H),6.52(dd,J=7.3,0.8Hz,1H),5.42(s,2H),4.04(t,J=6.8Hz,2H),3.57(s,3 H), 2.24 (t, J = 7.4Hz, 2H), 1.51 (p, J = 7.3Hz, 2H), 1.45 (p, J = 7.3Hz, 2H), 1.23–1.17 (m, 4H). HRMS(ESI)C 23 H 27 N6O4 + [M+H] + Calculated value: 451.2088, measured value: 451.2086.

[0348] Step 2: Preparation of 7-((6-((6-amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptanamide (compound I-53)

[0349]

[0350] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-30. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellowish-white solid compound (I-53) (0.026 g, yield 52%). 1 H NMR(800MHz,DMSO-d6)δ10.32(s,1H),8.65(s,1H),8.31(s,1H),8.08–8.05(m,1H),7. 59(t,J=8.7Hz,1H),7.45(dd,J=3.5,0.8Hz,1H),6.90(brs,2H),6.79(dd,J=3.5,1.8Hz ,1H),6.64(d,J=8.2Hz,1H),6.49(d,J=7.4Hz,1H),5.43(s,2H),4.04(t,J=6.8Hz,2H), 1.91(t,J=7.4Hz,2H), 1.53(p,J=6.8Hz,2H), 1.43(p,J=7.4Hz,2H), 1.26–1.17(m,4H). HRMS(ESI)C 22 H 26 N7O4 + [M+H] + Calculated value: 452.2041, measured value: 452.2038.

[0351] Example 54: Preparation of 7-(3-((2-amino-6-(furan-2-yl)-9H-purine-9-yl)methyl)phenoxy)-N-hydroxyheptanamide (compound I-54)

[0352] Step 1: Preparation of methyl 7-(3-((2-amino-6-(furan-2-yl)-9H-purin-9-yl)methyl)phenoxy)heptanoate (intermediate Int-31)

[0353]

[0354] In Example 7, step 1, methyl 7-bromoheptanate was replaced with methyl 7-(3-(bromomethyl)phenoxy)heptanate. The other raw materials, reagents and preparation methods were the same as in Example 7, step 1, to obtain a white solid intermediate Int-31 (0.073 g, yield 28%). 1H NMR(800MHz,DMSO-d6)δ8.21(s,1H),7.96(dd,J=1.7,0.8Hz,1H),7.73(dd,J=3.4,0.8Hz,1H) ,7.26–7.22(m,1H),6.84(tdd,J=3.8,2.5,0.9Hz,2H),6.81(dt,J=7.8,1.1Hz,1H),6.75(dd, J=3.4,1.7Hz,1H),6.60(brs,2H),5.27(s,2H),3.91(t,J=6.5Hz,2H),3.57(s,3H),2.29(t,J =7.4Hz,2H),1.70–1.63(m,2H),1.53(p,J=7.5Hz,2H),1.40–1.34(m,2H),1.32–1.27(m,2H). HRMS(ESI)C 24 H 28 N5O3 + [M+H] + Calculated value: 450.2136, measured value: 450.2198.

[0355] Step 2: Preparation of 7-(3-((2-amino-6-(furan-2-yl)-9H-purine-9-yl)methyl)phenoxy)-N-hydroxyheptanamide (compound I-54)

[0356]

[0357] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-31. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a white solid compound (I-54) (0.050 g, yield 88%). 1 H NMR (800MHz, DMSO-d6) δ10.32(d,J=1.8Hz,1H),8.65(d,J=1.8Hz,1H),8.21(s,1H),7.96(d,J= 1.6Hz,1H),7.72(d,J=3.3Hz,1H),7.27–7.20(m,1H),6.84(d,J=6.5Hz,2H),6.80(d,J=7.6Hz,1 H),6.75(dd,J=3.4,1.7Hz,1H),6.59(brs,2H),5.26(s,2H),3.91(t,J=6.5Hz,2H),1.93(t,J= 7.4Hz, 2H), 1.66 (p, J = 6.7Hz, 2H), 1.49 (p, J = 7.5Hz, 2H), 1.40–1.33 (m, 2H), 1.30–1.24 (m, 2H). HRMS(ESI)C23 H 27 N6O4 + [M+H] + Calculated value: 451.2088, measured value: 451.2082.

[0358] Example 55: Preparation of 7-((6-((2-amino-6-(furan-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptanamide (compound I-55)

[0359] Step 1: Preparation of methyl 7-((6-((2-amino-6-(furan-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)heptarate (intermediate Int-32)

[0360]

[0361] In Example 54, step 1, methyl 7-(3-(bromomethyl)phenoxy)heptanate was replaced with methyl 7-((6-(bromomethyl)pyridin-2-yl)oxy)heptanate. The other raw materials, reagents and preparation methods were the same as in step 1 of Example 54, yielding a yellow oily intermediate Int-32 (0.129 g, yield 37%). 1 H NMR(800MHz,DMSO-d6)δ8.19(s,1H),7.96–7.92(m,1H),7.74(dd,J=3.4,0.9Hz,1H) ,7.64(dd,J=8.2,7.3Hz,1H),6.74(dd,J=3.4,1.7Hz,1H),6.73–6.71(m,1H),6.66( d,J=8.2Hz,1H),6.52(brs,2H),5.33(s,2H),4.04(t,J=6.8Hz,2H),3.57(s,3H),2. 23(t,J=7.4Hz,2H),1.49(p,J=6.9Hz,2H),1.44(p,J=7.4Hz,2H),1.21–1.17(m,4H). HRMS(ESI)C 23 H 27 N6O4 + [M+H] + Calculated value: 451.2088, measured value: 451.2088.

[0362] Step 2: Preparation of 7-((6-((2-amino-6-(furan-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptanamide (compound I-55)

[0363]

[0364] In step 2 of Example 1, intermediate Int-1 was replaced with intermediate Int-32. The other required raw materials, reagents and preparation methods were the same as in step 2 of Example 1. The compound (I-55) was further purified by preparative HPLC to obtain a white solid compound (I-55) (0.051 g, yield 41%). 1 H NMR (800MHz, DMSO-d6) δ10.34–10.30(m,1H),8.66(d,J=1.8Hz,1H),8.20(s,1H),7.95(t,J=1. 2Hz,1H),7.74(d,J=3.6Hz,1H),7.64(d,J=7.9Hz,1H),6.75(dd,J=3.4,1.7Hz,1H),6.70(d,J=7 .4Hz,1H),6.67(d,J=8.2Hz,1H),6.52(brs,2H),5.33(s,2H),4.04(t,J=6.8Hz,2H),1.90(t,J= 7.4Hz,2H),1.51(p,J=6.9Hz,2H),1.42(p,J=7.4Hz,2H),1.25–1.20(m,2H),1.20–1.16(m,2H). HRMS(ESI)C 22 H 26 N7O4 + [M+H] + Calculated value: 452.2041, measured value: 452.2037.

[0365] Example 56: Preparation of 7-(3-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)phenoxy)-N-hydroxyheptanamide (compound I-56)

[0366] Step 1: Preparation of methyl 7-(3-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)phenoxy)heptarate (intermediate Int-33)

[0367]

[0368] In Example 22, step 1, methyl 7-(4-(bromomethyl)benzoate was replaced with methyl 7-(3-(bromomethyl)phenoxy)heptanoate. The other raw materials, reagents and preparation methods were the same as in Example 22, step 1, to obtain a yellowish-white solid intermediate Int-33 (0.147 g, yield 46%). 1H NMR(800MHz,DMSO-d6)δ8.17(s,1H),7.67(d,J=3.3Hz,1H),7.24–7.21(m,1H),6.8 4–6.82(m,2H),6.80(dt,J=7.8,1.2Hz,1H),6.57(brs,2H),6.37(dd,J=3.3,1.0Hz, 1H),5.25(s,2H),3.90(t,J=6.5Hz,2H),3.57(s,3H),2.40(s,3H),2.28(t,J=7.4H z,2H),1.68–1.62(m,2H),1.54–1.49(m,2H),1.39–1.33(m,2H),1.31–1.26(m,2H). HRMS(ESI)C 25 H 30 N5O4 + [M+H] + Calculated value: 464.2292, measured value: 464.2290.

[0369] Step 2: Preparation of 7-(3-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)phenoxy)-N-hydroxyheptanamide (compound I-56)

[0370]

[0371] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-33. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a white solid compound (I-56) (0.129 g, yield 96%). 1 H NMR (800MHz, DMSO-d6) δ10.32(s,1H),8.65(s,1H),8.17(s,1H),7.66(d,J=3.3Hz,1H) ,7.25–7.22(m,1H),6.85–6.82(m,2H),6.79(d,J=7.6Hz,1H),6.56(brs,2H),6.38(dd, J=3.3,1.2Hz,1H),5.25(s,2H),3.90(t,J=6.5Hz,2H),2.40(s,3H),1.93(t,J=7.4Hz,2 H),1.65(p,J=6.7Hz,2H),1.49(p,J=7.5Hz,2H),1.39–1.34(m,2H),1.30–1.24(m,2H). HRMS(ESI)C 24 H 29 N6O4 + [M+H] +Calculated value: 465.2245, measured value: 465.2249.

[0372] Example 57: Preparation of 7-((6-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)N-hydroxyheptanamide (compound I-57)

[0373] Step 1: Preparation of methyl 7-((6-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)heptanate (intermediate Int-34)

[0374]

[0375] In Example 56, step 1, methyl 7-(3-(bromomethyl)phenoxy)heptanate was replaced with methyl 7-((6-(bromomethyl)pyridin-2-yl)oxy)heptanate. The other raw materials, reagents and preparation methods were the same as in step 1 of Example 56, yielding a yellowish-white solid intermediate Int-34 (0.127 g, yield 42%). 1 H NMR(800MHz,DMSO-d6)δ8.16(s,1H),7.68(d,J=3.3Hz,1H),7.64(t,J=7.7Hz,1H ),6.72(d,J=7.3Hz,1H),6.66(d,J=8.2Hz,1H),6.51(brs,2H),6.37(dd,J=3.3, 1.2Hz,1H),5.32(s,2H),4.04(t,J=6.8Hz,2H),3.57(s,3H),2.40(s,3H),2.22( t,J=7.5Hz,2H),1.49(p,J=6.9Hz,2H),1.44(p,J=7.4Hz,2H),1.23–1.15(m,4H). HRMS(ESI)C 24 H 29 N6O4 + [M+H] + Calculated value: 465.2245, measured value: 465.2267.

[0376] Step 2: Preparation of 7-((6-((2-amino-6-(5-methylfuran-2-yl)-9H-purine-9-yl)methyl)pyridin-2-yl)oxy)-N-hydroxyheptanamide (compound I-57)

[0377]

[0378] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-34. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-57) (0.105 g, yield 80%). 1 H NMR (800MHz, DMSO-d6) δ10.32(s,1H),8.66(s,1H),8.17(s,1H),7.68(d,J=3.2Hz,1 H),7.64(t,J=7.8Hz,1H),6.69(d,J=7.3Hz,1H),6.67(d,J=8.2Hz,1H),6.51(brs,2 H),6.38(dd,J=3.3,1.2Hz,1H),5.32(s,2H),4.05(t,J=6.8Hz,2H),2.40(s,3H),1. 91(t,J=7.4Hz,2H),1.52(p,J=6.9Hz,2H),1.43(p,J=7.4Hz,2H),1.25–1.17(m,4H). HRMS(ESI)C 23 H 28 N7O4 + [M+H] + Calculated value: 466.2197, measured value: 466.2183.

[0379] Example 58: Preparation of 7-(3-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)phenoxy)-N-hydroxyheptamide (compound I-58)

[0380] Step 1: Preparation of methyl 7-(3-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)phenoxy)heptanoate (intermediate Int-35)

[0381]

[0382] In Example 19, step 1, methyl 4-(bromomethyl)benzoate was replaced with methyl 7-(3-(bromomethyl)phenoxy)heptanoate. The other raw materials, reagents and preparation methods were the same as in Example 19, step 1, to obtain a yellow solid intermediate Int-35 (0.061 g, yield 18%). 1H NMR(800MHz, DMSO-d6)δ7.96(d,J=1.8Hz,1H),7.26(d,J=3.4Hz,1H),7.22–7.19(m, 2H),6.80(dd,J=8.3,2.3Hz,1H),6.75–6.72(m,3H),6.72(dd,J=3.4,1.7Hz,1H),6. 27(brs,2H),5.23(s,2H),3.88(t,J=6.5Hz,2H),3.56(s,3H),2.28(t,J=7.4Hz,2H) ,1.64(p,J=6.6Hz,2H),1.51(p,J=7.4Hz,2H),1.38–1.33(m,2H),1.30–1.26(m,2H). HRMS(ESI)C 25 H 29 N4O4 + [M+H] + Calculated value: 449.2183, measured value: 449.2185.

[0383] Step 2: Preparation of 7-(3-((2-amino-4-(furan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)phenoxy)-N-hydroxyheptanamide (compound I-58)

[0384]

[0385] Replace intermediate Int-1 in step 2 of Example 1 with intermediate Int-35. The other required raw materials, reagents and preparation methods are the same as in step 2 of Example 1, to obtain a yellow solid compound (I-58) (0.034 g, yield 53%). 1 H NMR (800MHz, DMSO-d6) δ10.32 (s, 1H), 8.65 (s, 1H), 7.96 (d, J = 1.7Hz, 1H), 7.2 6(d,J=3.4Hz,1H),7.22–7.19(m,2H),6.81(dd,J=8.1,2.5Hz,1H),6.76–6.70( m,4H),6.27(brs,2H),5.23(s,2H),3.88(t,J=6.5Hz,2H),1.93(t,J=7.4Hz,2H ),1.67–1.62(m,2H),1.51–1.46(m,2H),1.38–1.33(m,2H),1.29–1.23(m,2H). HRMS(ESI)C 24 H 28 N5O4 + [M+H] +Calculated value: 450.2136, measured value: 450.2134.

[0386] According to the methods provided in Examples 45-58, the compounds listed in Examples 59-66 can be prepared by changing the corresponding raw materials using the same methods, as detailed in Table 3.

[0387] Table 3

[0388]

[0389]

[0390]

[0391] Biological Assay Example 1: Determination of the inhibitory activity of the compound against histone deacetylase HDAC. The specific procedure is as follows:

[0392] (1) Prepare the experimental buffer solution (50mM Tris pH, 0.01% Tween-20, 50mM NaCl);

[0393] (2) Prepare a DMSO solution of the compound to be tested at a concentration of 10 mM, then dilute it to 1 mM with DMSO, and then perform a 3-fold serial dilution for 10 concentration points.

[0394] (3) Different concentrations of the test compound were transferred to a 384-well plate (Perkin Elmer, Cat. No. 6007279) using an Echo, 250 nL per well (final DMSO content was 1%).

[0395] (4) Prepare histone deacetylase solutions using the buffer solution from step (1). The final concentration of HDAC1 (BPS bioscience, Cat. No. 50051) is 4 nM, and the final concentration of HDAC6 (BPS bioscience, Cat. No. 50056) is 5 nM.

[0396] (5) Prepare a mixed solution of substrate (LGK(Ac)-AMC, Trypsin) using the buffer solution in step (1). For the determination of HDAC1 activity: the concentration of LGK(Ac)-AMC (Jier Biochemical) is 8 μM and the concentration of Trypsin is 0.05 μM. For the determination of HDAC6 activity: the final concentration of LGK(Ac)-AMC is 11 μM and the final concentration of Trypsin is 0.01 μM.

[0397] (6) Add 15 μL of the enzyme solution prepared in step (4) to each well of the 384-well plate for testing. For the low control group, add 15 μL of the buffer solution prepared in step (1). Centrifuge at 1000 rpm for 1 minute and then incubate at room temperature for 15 minutes.

[0398] (7) Add 10 μL of the enzyme solution prepared in step (5) to each well of the 384-well plate, centrifuge at 1000 rpm for 1 minute, and then incubate at room temperature for 60 minutes.

[0399] (8) Read the values ​​using Synergy MX (maximum excitation light: 355nm, maximum emission light: 460nm);

[0400] (9) Process the data using GraphPad Prism5 and calculate IC. 50 The values ​​are shown in Table 4.

[0401] Table 4 shows the inhibitory activity of the compounds against HDAC1.

[0402]

[0403] Example 2 of biological testing: Determination of the binding activity of the compound of the present invention to the A2A receptor. The binding activity of the compound to the human A2A receptor was determined using a radioisotope ligand competitive binding assay. The specific procedure is as follows:

[0404] (1) Prepare DMSO solutions of the test compound at the corresponding 10 mM concentration. Then dilute to 10 μM with buffer, and then serially dilute 3-fold with buffer to obtain 10 concentration points;

[0405] (2) Use Echo to transfer different concentrations of the test compound to a 384-well plate, 50 nL per well;

[0406] (3) Prepare a suspension of human A2A receptor cell membrane (RBHA2AM400UA; Perkin Elmer) and wheat germ lectin-coated yttrium silicate SPA beads (RPNQ0023; Perkin Elmer): (0.0334 mg / mL A2a cell membrane, 3.33 mg / mL SPA beads, 0.02 mg / mL ADA, experimental buffer containing 1×DPBS, 10 mM MgCl2, 1% DMSO), and incubate at room temperature for 20 minutes;

[0407] (4) Add 20 μL to each well 3 H SCH58261 (ART2128; ARC) solution (15 nM SCH 58261, experimental buffer containing 1×DPBS, 10 mM MgCl2, 1% DMSO), centrifuged at 1000 rpm for 1 minute;

[0408] (5) Add 30 μL of A2A cell membrane / SPA bead suspension to each well, centrifuge at 1000 rpm for 1 minute, seal the plate and incubate at room temperature with continuous shaking for 60 minutes.

[0409] (6) Read the CPM value using Microbeta 2 (Perkin Elmer);

[0410] (7) Process the data using GraphPad Prism 5 and calculate IC. 50 Value, to obtain K i The results are shown in Table 5.

[0411] Table 5 shows the competitive binding strength of the compounds in the examples to the radioisotope ligands of the A2A receptor.

[0412]

[0413] Biological Testing Example 3: Test of the inhibitory activity of the compound of the present invention on tumor cell proliferation. The inhibitory activity of the compound on tumor cell proliferation was measured using MC-38 and CT-26 cell lines.

[0414] (1) Cell plating

[0415] a. Prepare the complete culture medium and mix thoroughly.

[0416] b. Select cell lines in good growth condition.

[0417] c. Remove the cell culture flask from the incubator and check the cell name, culture medium type, and cell passage number marked on the flask.

[0418] d. Use a pipette to transfer the cell suspension into a centrifuge tube and centrifuge at 800-1000 rpm for 3-5 minutes.

[0419] e. Discard the cell supernatant from the centrifuge tube, add an appropriate volume of culture medium to the centrifuge tube, and gently pipette to resuspend the cells evenly.

[0420] f. Use a Vi-Cell XR cell counter to count the cells and adjust the cell suspension to the appropriate concentration.

[0421] g. Add the cell suspension to 36 μL / well of a 384-well plate containing a bottom-permeable cell culture medium. Label the cell names, seeding density, date, and other details, and incubate the plate overnight in a CO2 incubator.

[0422] (2) Cell experiments:

[0423] a. Prepare the test compound to 200× using DMSO, and then dilute the compound 3 times with DMSO to obtain 10 concentration gradients of the compound.

[0424] b. After seeding cells for 24 hours, add 1 μL of the compound to 19 μL of culture medium to prepare a 10× intermediate plate. Then add 4 μL of the corresponding 10× compound to each well and incubate at 37°C for 72 hours.

[0425] c. Observe cell morphology under an inverted microscope.

[0426] d. Place the cell culture plate at room temperature for equilibration for 30 minutes, add 25 μL of CTG to each well, and then mix on a vibratory plate for 10 minutes to induce cell lysis.

[0427] e. Place the 384-well plate at room temperature for 10 minutes to allow the luminescence signal to stabilize. Then, attach the white substrate to the bottom of the culture plate and read the plate using a Flexstation 3 (with the following settings: luminescence, integration time 500ms).

[0428] f. Record the experimental results obtained from the analysis. The results are shown in Table 6.

[0429] Table 6. Experimental results of compounds inhibiting tumor cell proliferation.

[0430]

[0431]

[0432] Biological Test Example 4: Pharmacokinetic Properties of the Compounds of the Present Invention as a Single Administered by Gavage and Intravenous Injection to C57 Male Mice

[0433] (1) Experimental Objective

[0434] After a single dose of the compound of the present invention was administered to male C57 mice, blood samples were collected at different time points. The concentration of the compound in mouse plasma was determined by LC-MS / MS and relevant pharmacokinetic parameters were calculated to investigate the pharmacokinetic behavior of the compound in mice.

[0435] (2) Experimental Design

[0436] Three male C57 mice were provided by Suzhou Zhaoyan Experimental Animal Co., Ltd., and the experiments were conducted according to the dosing regimens in Table 7 below.

[0437] Table 7. Dosing regimens for the compounds

[0438]

[0439] (3) Sample collection

[0440] 0.030 mL of blood was collected from each animal via the orbital cavity, anticoagulated with EDTA-K2, at the following time points: 0, 5, 15, 30 min, 1, 2, 4, 6, 8, and 24 h after administration of the test substance. Blood samples were placed on ice after collection and centrifuged within 30 minutes to separate the plasma (centrifugation conditions: 5000 rpm, 10 minutes, 4°C). The plasma was stored at –80°C before analysis.

[0441] (4) Data processing

[0442] The data acquisition and control system software was Analyst 1.5.1 (Applied Biosystem). Peak integration of the chromatograms was automatic; the ratio of the sample peak area to the internal standard peak area was used as an indicator, and regression was performed with the sample concentration. The regression method was linear regression with a weighting coefficient of 1 / X². Pharmacokinetic parameters were analyzed using a non-compartmental model with WinNonlin Professional v6.3 (Pharsight, USA). Cmax represents the measured maximum plasma concentration, the area under the plasma concentration-time curve (AUC(0→t)) was calculated using the trapezoidal rule, and Tmax represents the time to peak plasma concentration after administration. Experimental data are expressed as mean ± standard deviation (Mean ± ICR, n≥3) or mean (Mean, n=2).

[0443] (5) Experimental Results

[0444] The pharmacokinetic results of the compounds of this invention are shown in the table below. It can be seen that the compounds exhibit favorable pharmacokinetic properties in C57 male mice. See Table 8 for details.

[0445] Table 8. Oral pharmacokinetic parameters of compound I-14

[0446]

[0447] Note: "--" indicates that it is not applicable.

[0448] Biological Test Example 4: Inhibitory Activity of Compound I-14 on the Growth of Subcutaneous Colon Cancer X-rays in Mice (MC38)

[0449] (1) Laboratory animals

[0450] C57BL / 6 mice, 6-8 weeks old, female, were purchased from Beijing Huafukang Biotechnology Co., Ltd. The use and welfare of these laboratory animals were conducted in accordance with the regulations of the International Committee for Assessment and Accreditation of Laboratory Animals (AAALAC). The health status and mortality of the animals were monitored daily. Routine examinations included observing the effects of test substances and drugs on the animals' daily behavior, such as activity levels, weight changes, and physical appearance.

[0451] (2) Experimental steps

[0452] Mouse MC38 colon cancer cells were divided into groups of 5.0 × 10⁻⁶. 5 Cells / mouse were subcutaneously injected into the right axilla of mice. Mice were monitored daily, and measurements were taken with calipers when tumors became visible. The width (W) and length (L) of each tumor were measured using calipers, and the equation V = (L × W²) / 2 was used. The average tumor volume was determined when the tumor grew to approximately 100 mm. 3 Mice were then randomly divided into groups. One group received intraperitoneal injection of compound I-14 (30 mg / kg, bid; 60 mg / kg, bid), while the other group received I-14 via gavage (90 mg / kg, bid; 120 mg / kg, qd). The control group received an equal volume of the solvent via intraperitoneal injection or gavage daily. Tumor volume and mouse weight were recorded every two days. Relative tumor volume (RTV) was calculated as Vt / V0, where Vt represents the tumor volume on the day of measurement, and V0 represents the initial tumor volume before administration. The evaluation index for tumor inhibition was the relative tumor growth rate: T / C (%) = (mean tumor growth volume in the treatment group / mean tumor growth volume in the control group) × 100%.

[0453] (3) Experimental Results

[0454] Compound I-14 of this invention exhibits an inhibitory effect on the growth of subcutaneous xenografts of colon cancer in mice (MC38). For example... Figure 1 As shown, the relative tumor growth rates (T / C%) of mice administered I-14 by gavage (90 mg / kg, bid; 120 mg / kg, qd) were 56% and 74%, respectively (P < 0.05), and there was no significant difference in body weight gain among the groups of mice. Figure 1 The asterisk (*) indicates P < 0.05. For example... Figure 2 As shown, the relative tumor growth rates (T / C%) of intraperitoneal administration of compound I-14 (30 mg / kg, bid; 60 mg / kg, bid) were 32% and 15%, respectively (P < 0.01). Figure 2 ** is represented as P<0.01.

Claims

1. A compound as shown in Formula I or a pharmaceutically acceptable salt thereof: in, A and B are independently CH or N; R 1 and R 2 Independently H or C1-C6 alkyl; R 3 It is a 5-10 member heteroaryl group or is surrounded by one or more R groups. 3-1 The substituted 5-10-membered heteroaryl group; wherein the heteroaryl group has one, two or three heteroatoms, and the heteroatoms are selected from one, two or three of N, O and S; L is -C1-C 10 Alkylene-*, -C1-C 10 Alkylene-C6-C 10 Aromatic-*, -C1-C 10 Alkylene-C6-C 10 arylene-C2-C4-ene-*, -C1-C 10 Alkylene-O-C6-C 10 Aromatic-*, -C1-C 10 Alkylene-C6-C 10 Aromatic-O-C1-C 10 Alkylene -* or -C1-C 10 alkylene-5-10-membered heteroaryl-O-C1-C 10 Alkylene-*; In the 5-10 membered heteroaryl group, the number of heteroatoms is 1, 2 or 3, and the heteroatoms are selected from one, two or three of N, O and S; wherein, the "*" end is connected to ZBG; ZBG is R 3-1 It is independently a cyano or C1-C6 alkyl group; R 6 Independently hydrogen or halogen; m can be 1, 2, 3 or 4.

2. The compound of formula I as claimed in claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1)R 3 In this context, the heteroaryl group is independently a 5-6 member heteroaryl group; (2)R 1 R 2 and R 3-1 In this context, the C1-C6 alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; (3) In L, the C1-C 10 The alkylene group is independently a C1-C7 alkylene group; (4) In L, the C6-C 10 The arylene group is independently a phenylene group; (5) In L, the C2-C4 alkenyl group is independently an vinylidene; (6) In L, the 5-10-membered heteroaryl group is a 5-6-membered heteroaryl group; (7)R 6 In this context, the halogen is fluorine, chlorine, bromine, or iodine.

3. The compound of formula I as claimed in claim 2, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1)R 3 In the above, the heteroatom in the heteroaryl group is independently O, and the number of heteroatoms is independently 1 or 2; (2) In L, the C1-C 10 The alkylene group is independently methylene, ethylene, propylene, butylene, pentylene, hexylene, or heptylene; (3) In L, the C6-C 10 Alpha-aryl is independently In (4)L, the C2-C4 subene group is independently... (5) In L, the heteroatom in the 5-6 methyl aryl group is N, and the number of heteroatoms is 1 or 2.

4. The compound of formula I as claimed in claim 3, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1)R 3 In this context, the heteroaryl group is independently a furanyl group; (2) In L, the C1-C 10 The alkylene group is independently methylene, n-ethylene, n-propylene, n-butylene, n-pentylene, n-hexylene, or n-heptylene; (3) In L, the 5-6 member heteroaryl group is pyridinyl.

5. The compound of formula I as claimed in claim 4, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or two of the following conditions: (1)R 3 In this context, the heteroaryl group is independently... (2) In L, the 5-6 member heteroaryl group is 6. The compound of formula I as claimed in any one of claims 2-5, or a pharmaceutically acceptable salt thereof, characterized in that, It meets one or more of the following conditions: (1) L is (2)R 1 and R 2 For H; (3)R 3 for (4) The compound shown in Formula I is a substituted pyrimidine compound shown in Formula I-1, a substituted pyrimidine compound shown in Formula I-2, a substituted pyrimidine compound shown in Formula I-3, or a substituted pyrimidine compound shown in Formula I-4. (5) In ZBG, the following for 7. The compound of formula I as claimed in claim 6, or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by Formula I is any of the following compounds:

8. A pharmaceutical composition comprising a compound of Formula I as described in any one of claims 1-7 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutical excipient.

9. The use of a compound of Formula I as described in any one of claims 1-7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 8, in the preparation of a histone deacetylase HDAC inhibitor.

10. The use of a compound of Formula I as described in any one of claims 1-7, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 8, in the preparation of a medicament for treating and / or preventing a disease, wherein the disease is cancer.

11. The application as described in claim 10, characterized in that, The cancers mentioned include head and neck cancers, respiratory system cancers, digestive system cancers, urinary system cancers, bone cancers, gynecological cancers, hematological cancers, gliomas, or skin cancers.

12. The application as described in claim 10, characterized in that, The cancer in question is melanoma.

13. A method for preparing a compound as shown in Formula I, comprising the following steps: in an organic solvent, reacting a compound as shown in Formula II as described below to obtain the compound as shown in Formula I. in, A, B, R 1 R 2 R 3 L and ZBG are as described in any one of claims 1 to 7; R a It is H or C1-C6 alkyl.

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