Substituted alkynyl heterocyclic compounds

Abstract

The present application relates to substituted alkynyl heterocyclic compounds having SHP2 inhibitory activity represented by Formula (I), methods of making the same, pharmaceutical compositions thereof, and to the use of such compounds and pharmaceutical compositions thereof for the treatment of diseases that benefit from inhibition of SHP2 enzyme.

Description

Technical Field

[0001] This invention generally relates to novel substituted alkyne heterocyclic compounds having SHP2 inhibitory activity, methods for their preparation, pharmaceutical compositions thereof, and uses of such compounds and pharmaceutical compositions thereof for treating diseases that benefit from SHP2 enzyme inhibition, such as cancer. Background Technology

[0002] Cancer is a serious disease that severely threatens human health and life. In recent years, its incidence and mortality rates have been rising rapidly, surpassing cardiovascular disease to become the leading cause of death worldwide. Tumor proliferation, apoptosis, and metastasis are closely related to abnormalities in certain stages of a series of intracellular and extracellular signaling pathways. Among these pathways, protein phosphorylation and dephosphorylation are crucial, and this reversible process is jointly regulated by kinases and phosphatases. Phosphorylation of protein tyrosine kinase (PTK) and dephosphorylation of protein tyrosine phosphatase (PTP) are such a pair of reversible processes, maintaining a dynamic balance to sustain normal cellular physiological functions. Conversely, abnormal phosphorylation can lead to cancer, inflammation, diabetes, and other diseases.

[0003] SHP2 protein is a non-receptor protein tyrosine phosphatase encoded by the ptpn11 gene. It is widely expressed in various tissues and participates in important physiological and pathological processes such as embryonic development, metabolism, immune response, and tumorigenesis.

[0004] The SHP2 protein consists of two tandem SH2 domains (N-SH2 and C-SH2) at the N-terminus, a PTP catalytic domain, and a regulatory C-terminal tail. The SH2 domain acts as a conformational switch, mediating interactions between the SHP2 protein and phosphotyrosine-containing activators (such as insulin receptor substrate 1-IRS1 and GRB2-associated binding protein 1-GAB1), as well as intramolecular interactions between the SH2 domain and the PTP catalytic domain. In the unstimulated state, the SHP2 domain binds to the PTP domain, blocking the catalytic active site and maintaining SHP2 phosphatase activity in an autoinhibited state. When the SH2 domain binds to an activator, the inhibitory intramolecular interaction is released, and the SHP2 phosphatase enters an open conformation, allowing the SHP2 substrate to localize to the catalytic active site and perform its phosphatase function. This activity-switching characteristic of SHP2 means that various mutations in SHP2 can disrupt its autoinhibitory state, leading to overactivation of SHP2 protein phosphatase activity and potentially inducing carcinogenesis. Both experimental and clinical data have confirmed that SHP2 plays a promoting role in most cancers. As the first discovered tyrosine phosphatase to promote cancer development, it has received great attention in the field of cancer, and its phosphatase activity plays an important role in intracellular signal regulation.

[0005] SHP2 participates in regulating cell signaling pathways activated by cytokines, growth factors, and hormones, including the RAS / ERK, JAK / STAT, PI3K / AKT, and NF-κB signaling pathways, thereby regulating physiological functions such as cell proliferation, differentiation, cell cycle maintenance, and migration. Simultaneously, SHP2 mediates compensatory activation pathways following the inhibition of kinases such as MEK, thus promoting tumor drug resistance. As a downstream molecule of the PD-1 receptor, SHP2 also participates in the transduction of inhibitory signals in T cells. Previous studies have shown that SHP2 is a downstream molecule of PD-1 signaling, inhibiting not only T cell activation but also promoting T cell dysfunction. Therefore, targeting SHP2 can restore or enhance T cell-mediated anti-tumor immune function. Furthermore, SHP2 can inhibit IFN-γ-mediated immune responses by inactivating signal transduction and transcription activator STAT1.

[0006] In recent years, SHP2 activating mutations and high expression have been successively discovered in leukemia, solid tumors, melanoma, malignant glioma, lung cancer, breast cancer, and Norman Syndrome, and are closely related to the occurrence, development, and prognosis of tumors. Currently, SHP2 has been studied as a target molecule for clinical oncology. The mechanism of action of traditional SHP2 inhibitors (such as II-B08 and PHPS1) is to bind to the PTP catalytic domain of SHP2, preventing tyrosine phosphorylated substrates from entering the catalytic site, thereby inhibiting the phosphatase activity of SHP2. However, due to the highly conserved, polar, and charged environment of the PTP catalytic domains of various phosphatases, traditional SHP2 inhibitors have significant deficiencies in terms of specificity and bioavailability, limiting their clinical application. Therefore, developing SHP2 inhibitors with high specificity, high safety, and strong cell membrane permeability is crucial to determining whether SHP2 can become a novel tumor intervention target, and SHP2 protein allosteric inhibitors have become the main research direction.

[0007] Currently, no SHP2 inhibitors have been approved for marketing. There are three compounds in the clinical research stage. Among them, JAB-3068 from Jacobio is progressing the fastest and has been approved for Phase II clinical trials. Novartis' TNO155 and RMC-4630 from Synnex are both in Phase I clinical trials. Summary of the Invention

[0008] This invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite, or prodrug thereof.

[0009]

[0010] in,

[0011] A is selected from the following di- or tri-heterocyclic rings;

[0012]

[0013] R1 and R2 are each independently selected from hydrogen, halogens, and C. 1-6 alkyl;

[0014] X is selected from hydrogen, C 1-6 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl and C 5-10 heteroaryl, the C 1-6 Alkyl and C 3-8 The cycloalkyl group may optionally be reacted with one or more halogens, -OH, -OC. 1-6 Alkyl, -NH2, or C 1-6 Alkyl substitution, the C 6-10 Aryl and C 5-10 Heteroaryl groups can fused with unsaturated alicyclic, heteroalicyclic, and spirocyclic rings, and can optionally be reacted with one or more halogens, -CF3, -OH, -CN, or -OC. 1-6 Alkyl groups, -NR3R4, -OC(O)NR3R4, -NH-(CO)-C 1-6 Alkyl, -S-CH2-CONH2, C 1-6 Alkyl, C 3-6 cycloalkyl, C 6-10 Aryl, or C 6-10 heteroaryl substitution;

[0015] R3 and R4 are each independently selected from hydrogen and C. 1-6 alkyl;

[0016] Y is selected from C 6-10 Aryl, C 5-10 heteroaryl and C 3-12 The aryl, heteroaryl, and heterocyclic groups may optionally be converted by one or more halogens, -OH, or -OC. 1-6 Alkyl, -NH2, C 1-6 Alkyl, or C 3-6 Cycloalkyl substitution, wherein the alkyl or cycloalkyl group may optionally be replaced by a halogen, -OH, or -OC. 1-6 Alkyl or -NH2 substituted;

[0017] Z is selected from single bonds, -S-, and -C≡C-.

[0018] According to some embodiments of the present invention, the compound of formula (I) of the present invention has the following formulas II to X:

[0019]

[0020] R1 and R2 are each independently selected from hydrogen, halogens, and C. 1-6alkyl;

[0021] X is selected from hydrogen, C 1-6 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl and C 5-10 heteroaryl, the C 1-6 Alkyl and C 3-8 The cycloalkyl group may optionally be reacted with one or more halogens, -OH, -OC. 1-6 Alkyl, -NH2, or C 1-6 Alkyl substitution, the C 6-10 Aryl and C 5-10 Heteroaryl groups can fused with unsaturated alicyclic, heteroalicyclic, and spirocyclic rings, and can optionally be reacted with one or more halogens, -CF3, -OH, -CN, or -OC. 1-6 Alkyl groups, -NR3R4, -OC(O)NR3R4, -NH-(CO)-C 1-6 Alkyl, -S-CH2-CONH2, C 1-6 Alkyl, C 3-6 cycloalkyl, C 6-10 Aryl, or C 6-10 heteroaryl substitution;

[0022] R3 and R4 are each independently selected from hydrogen and C. 1-6 alkyl;

[0023] Y is selected from C 6-10 Aryl, C 5-10 heteroaryl and C 3-12 The aryl, heteroaryl, and heterocyclic groups may optionally be converted by one or more halogens, -OH, or -OC. 1-6 Alkyl, -NH2, C 1-6 Alkyl, or C 3-6 Cycloalkyl substitution, wherein the alkyl or cycloalkyl group may optionally be replaced by a halogen, -OH, or -OC. 1-6 Alkyl or -NH2 substituted.

[0024] In some implementations, R1 and R2 are each independently selected from hydrogen and C. 1-6 alkyl;

[0025] In some embodiments, X is selected from hydrogen, C 1-6 Alkyl, C 3-8 cycloalkyl, C 6-10 Aryl and C 5-10 heteroaryl, the C 1-6 Alkyl and C 3-8 The cycloalkyl group may optionally be reacted with one or more halogens, -OH, -OC. 1-6 Alkyl, -NH2, or C 1-6 Alkyl substitution, the C6-10 Aryl and C 5-10 The heteroaryl group may optionally be reacted with one or more halogens, -CF3, -OH, -CN, -OC 1-6 Alkyl groups, -NR3R4, -OC(O)NR3R4, -NH-(CO)-C 1-6 Alkyl, -S-CH2-CONH2, C 1-6 Alkyl, C 3-6 cycloalkyl, C 6-10 Aryl, or C 5-10 heteroaryl substitution,

[0026] R3 and R4 are each independently selected from hydrogen and C. 1-6 alkyl;

[0027] In some implementations, Y is selected from C. 6-10 Aryl, C 5-10 heteroaryl and C 3-12 The heterocyclic group, wherein the aryl, heteroaryl, and heterocyclic groups may optionally be converted to one or more -OH, -NH2, or C groups. 1-6 Alkyl substitution;

[0028] In some implementations, Y is selected from C. 3-12 The heterocyclic group may optionally be converted to one or more -OH, -NH2, or C groups. 1-6 Alkyl substitution;

[0029] In some implementations, Y is selected from C. 3-12 Heterocyclic group, wherein the heterocyclic group may optionally be surrounded by one or more -NH2 or C groups. 1-6 Alkyl substitution;

[0030] According to some embodiments of the present invention, the present invention provides the following compounds:

[0031]

[0032]

[0033] On the other hand, the present invention provides a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof. In some embodiments, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable excipient.

[0034] On the other hand, the present invention provides a method for treating diseases related to SHP2, comprising administering to a mammal, preferably a human, a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer of the present invention or a pharmaceutical composition thereof.

[0035] On the other hand, the present invention provides the use of the compounds of the present invention or pharmaceutically acceptable salts, solvates, polymorphs, or tautomers thereof, or pharmaceutical compositions thereof, in the preparation of medicaments for treating diseases related to SHP2.

[0036] In some embodiments of the present invention, the SHP2-related diseases are leukemia, melanoma, malignant glioma, lung cancer, breast cancer, or Nursing syndrome.

[0037] Some chemical terms

[0038] The term "compound" as used in this invention includes all stereoisomers, geometric isomers, tautomers, and isotopes. The compounds of this invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of this invention containing asymmetric carbon atoms can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0039] The compounds of this invention also include tautomer forms. Tautomer forms arise from the exchange of a single bond with an adjacent double bond, accompanied by the migration of a proton.

[0040] This invention also includes atoms of all isotopes, whether in intermediates or final compounds. Isotopic atoms include those having the same number of atoms but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0041] In the above definition of compounds of general formula IV, the terms used herein have the following meanings:

[0042] The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

[0043] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting of carbon and hydrogen atoms, such as C... 1-20 Alkyl group, preferably C 1-6 Alkyl groups, such as methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylhexyl, etc. The alkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl, and hydroxyl groups.

[0044] Term "C" 1-6"Alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, connected to the rest of the molecule by single bonds, and having 1-6 carbon atoms. The alkyl group may be unsubstituted or substituted with one or more substituents selected from alkyl, alkoxy, amino, halogen, and hydroxyl groups. Non-limiting examples of unsubstituted alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-methylhexyl, etc.

[0045] The term "cycloalkyl" refers to a saturated hydrocarbon group consisting of a monocyclic carbon ring composed of carbon and hydrogen atoms, such as C. 3-20 Cycloalkyl, preferably C 3-6 Cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The cycloalkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl, and hydroxyl groups.

[0046] The term "aryl" refers to a fully carbon monocyclic or fused ring having a fully conjugated π-electron system, having 6-14 carbon atoms, preferably 6-12 carbon atoms, and most preferably 6 carbon atoms. The aryl group can be unsubstituted or substituted with one or more substituents, examples of which include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, sulfonyl, sulfinyl, phosphoryl, and heterocyclic groups. Non-limiting examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthraceneyl.

[0047] The term "heteroaryl" refers to a monocyclic or fused ring with 5-12 ring atoms, having 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms, of which 1, 2, 3, or 4 are selected from N, O, and S, and the remaining ring atoms are C, and possessing a fully conjugated π-electron system. Heteroaryl groups can be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, cyano, nitro, carbonyl, and heterocyclic groups. Non-limiting examples of unsubstituted heteroaryl groups include, but are not limited to, pyrrole, furanyl, thiophene, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, and triazineyl.

[0048] The term "heterocyclic ring" refers to a monocyclic or fused ring having 3 to 12 ring atoms, of which 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ring atoms are selected from N, O, and S(O). nThe ring consists of heteroatoms (where n is 0, 1, or 2), with the remaining ring atoms being carbon. Such a ring can be saturated or unsaturated (e.g., having one or more double bonds), but does not possess a fully conjugated π-electron system. Examples of ternary saturated heterocyclic rings include, but are not limited to, those shown below. Examples of 4-membered saturated heterocyclic rings include, but are not limited to, Examples of 5-membered saturated heterocyclic rings include, but are not limited to, those that are not part of the above. Examples of 6-membered saturated heterocyclic rings include, but are not limited to, those that are not part of the above. Examples of 7-membered saturated heterocyclic rings include, but are not limited to, Examples of 5-membered unsaturated heterocyclic rings include, but are not limited to, Examples of 6-membered unsaturated heterocyclic rings include, but are not limited to, those with 6-membered ...

[0049] The term "heterocyclic group" refers to the group remaining after removing one hydrogen atom from a "heterocyclic" molecule. Heterocyclic groups can be unsubstituted or have their hydrogen atoms optionally substituted by substituents, including but not limited to alkyl, alkoxy, =O, aryl, aralkyl, -COOH, -CN, amino, halogen, and hydroxyl groups.

[0050] The present invention also provides a method for preparing compounds of the above formula, comprising the following synthetic schemes:

[0051] Synthesis Scheme 1:

[0052]

[0053] Compounds 1-6 can be synthesized using Synthetic Scheme 1. 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine reacts with a suitable protecting agent to give intermediate 1-1. Intermediate 1-1 reacts with sodium hydroxide solution to give intermediate 1-2. Intermediate 1-2 reacts with iodomethane and a base to give intermediate 1-3. Intermediate 1-3 reacts with a compound containing NH or a borate ester to give intermediate 1-4. Deprotection of intermediate 1-4 gives intermediate 1-5. Intermediate 1-5 reacts with N-iodosuccinimide to give compound 1-6.

[0054] Synthesis Scheme 2:

[0055]

[0056] Compound 2-5 can be synthesized using synthetic scheme 2. 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine reacts with N-iodosuccinimide to give iodinated intermediate 2-1. Intermediate 2-1 reacts with ammonia to give intermediate 2-2. Intermediate 2-2 reacts with a suitable protecting agent to give intermediate 2-3. Intermediate 2-3 is cyclized with chloroacetaldehyde to give intermediate 2-4. Intermediate 2-4 reacts with a compound containing NH or a borate ester to give compound 2-5.

[0057] Synthesis Scheme 3:

[0058]

[0059] Compound 3-3 can be synthesized via synthetic scheme 3. 6-Chloro-1H-pyrazolo[3,4-b]pyrazine reacts with N-iodosuccinimide to give iodo intermediate 3-1. Intermediate 3-1 reacts with a suitable protecting agent to give intermediate 3-2. Intermediate 3-2 reacts with a compound containing NH or a borate ester to give compound 3-3.

[0060] Synthesis scheme 4:

[0061]

[0062] The final compound 4-1 can be synthesized using synthetic scheme 4. R1 represents terminal acetylene, boric acid or borate ester, or mercapto, and R2 represents iodine or bromine. The two starting materials react in the presence of a palladium catalyst and a base to give product 4-1. If the group on 4-1 has a protecting group, the protecting group is removed using an appropriate method.

[0063] The above synthesis schemes only illustrate some of the preparation methods of the compounds in this invention. Based on the known techniques in the art, those skilled in the art can also synthesize the compounds in this invention using similar methods.

[0064] The compounds or their salts of the present invention can be administered as active substances alone, preferably in the form of pharmaceutical compositions.

[0065] Another aspect of the present invention provides a pharmaceutical composition comprising a compound of general formula I, II, III, IV or V or a pharmaceutically acceptable salt, solvate, polymorph, or metabolite thereof as an active ingredient, and one or more pharmaceutically acceptable carriers.

[0066] "Pharmaceutical composition" refers to a formulation of one or more compounds of the present invention or salts thereof with a carrier commonly accepted in the art for delivering a bioactive compound to an organism (e.g., a human). The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of the present invention to an organism.

[0067] The term "pharmaceutically acceptable carrier" refers to carriers that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. "Pharmaceutically acceptable carrier" refers to an inert substance that is administered co-administered with the active ingredient and facilitates the administration of the active ingredient, including, but not limited to, any flow aids, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. Food and Drug Administration for use in humans or animals (e.g., livestock). Non-limiting examples of such carriers include calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0068] Administration of the compounds of the present invention, either in pure form or in the form of a suitable pharmaceutical composition, or of a pharmaceutically acceptable salt thereof, may be carried out by any acceptable mode of administration providing a pharmaceutical agent of similar use. The pharmaceutical compositions of the present invention may be prepared by combining the compounds of the present invention with a suitable pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions of the present invention may be formulated into solid, semi-solid, liquid, or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols, etc.

[0069] Typical routes of administration of the compounds of the present invention, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration. The preferred route of administration is oral.

[0070] The pharmaceutical compositions of the present invention can be manufactured using methods known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0071] In a preferred embodiment, the pharmaceutical composition is in an oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compounds of the present invention to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0072] Solid oral pharmaceutical compositions can be prepared using conventional mixing, filling, or tableting methods. For example, they can be obtained by mixing the active compound with a solid excipient, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain a tablet or sugar-coated core. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents. Examples include microcrystalline cellulose, glucose solution, gum arabic, gelatin solution, sucrose, and starch paste; talc, starch, magnesium stearate, calcium stearate, or stearic acid; lactose, sucrose, starch, mannitol, sorbitol, or dicalcium phosphate; silica; croscarmellose sodium, precrossyl cellulose, precrossyl starch, sodium starch glycolate, alginate, corn starch, potato starch, methylcellulose, agar, carboxymethyl cellulose, croscarmellose, etc. The sugar-coated core can be optionally coated using methods known in general pharmaceutical practice, particularly enteric coating.

[0073] The pharmaceutical composition may also be suitable for parenteral administration, such as in suitable unit dosage forms of sterile solutions, suspensions, or lyophilized products. Appropriate excipients, such as fillers, buffers, or surfactants, can be used.

[0074] Another aspect of this invention relates to the use of compounds of general formulas I to VI, or pharmaceutically acceptable salts, solvates, polymorphs, metabolites, etc., thereof, in medicaments for treating diseases that benefit from SHP2 inhibition. The diseases benefiting from SHP2 inhibition are selected from cancer.

[0075] The substituted alkynyl heterocyclic compounds provided by this invention have very good SHP2 inhibitory activity and are expected to become highly effective SHP2 inhibitor drugs. Detailed Implementation

[0076] The specific embodiments described below are intended to enable those skilled in the art to better understand and implement the present invention. They should not be considered as limiting the scope of the invention, but merely as exemplary illustrations and typical representatives. Those skilled in the art should understand that there are other synthetic routes for forming the compounds of the present invention; the examples provided below are non-limiting.

[0077] All operations involving easily oxidized or hydrolyzed raw materials are performed under nitrogen protection. Unless otherwise stated, the raw materials used in this invention are commercially available and used directly without further purification.

[0078] Column chromatography used silica gel (200-300 mesh) manufactured by Qingdao Chemical Co., Ltd. Thin-layer chromatography used pre-prepared plates (60PF silica gel) manufactured by E. Merck. 254Chiral compound separation and enantiomeric excess (ee) determination were performed using an Agilent LC 1200 series column (CHIRALPAK AD-H, 0.25 mm). (mm, 5 μm, 30℃). Nuclear magnetic resonance chromatography (NMR) was performed using a Varian VNMRS-400 NMR spectrometer; liquid chromatography-mass spectrometry (LC / MS) was performed using a FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18). (millimeter, 5 micrometer, 35℃), using ESI(+) ion mode.

[0079] Experimental Section

[0080] Intermediate 1: (3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine dihydrochloride

[0081]

[0082] (3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine dihydrochloride was synthesized according to the method of intermediate 14 in patent WO2017216706.

[0083] Intermediate 2: 6-Chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)- ketone

[0084]

[0085] Step 1: 4,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine

[0086] 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (2.7 g), 3,4-dihydro-2H-pyran (2.4 g), and p-toluenesulfonic acid (0.25 g) were added to tetrahydrofuran (25 mL), refluxed for 8 hours, cooled to room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 10:1) to give 4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (3.0 g). MS m / z [LC-MS]: 273.03 [M+1].

[0087] Step 2: 6-Chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

[0088] 3.0 g of 4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine and 4 mL of 20% sodium hydroxide solution were added to acetonitrile (40 mL), stirred overnight at room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 6:1) to give 2.4 g of 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-one. MS m / z [LC-MS]: 255.07 [M+1]. 1 HNMR (400MHz, DMSO-d6): δ = 13.300 (1H, brs), 8.113 (1H, s), 5.693 (1H, m), 3.908 (1H, m) , 3.645(1H,m), 2.286(1H,m), 1.967(1H,m), 1.820(1H,m), 1.708(1H,m), 1.523(2H,m).

[0089] Step 3: 6-Chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

[0090] 2.4 g of 6-chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one and 3.8 g of potassium carbonate were added to 40 mL of N,N-dimethylformamide, followed by dropwise addition of 1.0 mL of iodomethane. The mixture was stirred at room temperature for 4 hours. The reaction solution was poured into 200 mL of water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 8:1) to obtain 1.8 g of 6-chloro-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one. MS m / z [LC-MS]: 269.08 [M+1]. 1 HNMR (400MHz, DMSO-d6): δ = 8.150 (1H, s), 5.710 (1H, m), 3.911 (1H, m), 3.659 (1H, m), 3.567 (3H, s), 2.310 (1H, m), 1.974 (1H, m), 1.822 (1H, m), 1.708 (1H, m), 1.530 (2H, m).

[0091] Intermediate 3: 9-Iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine

[0092]

[0093] Step 1: 3-Iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine

[0094] 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (4.0 g) and N-iodosuccinimide (5.72 g) were added to acetonitrile (25 mL), heated to 100 °C, and microwaved for 25 minutes. After cooling to room temperature, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 8:1) to obtain 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (5.4 g). MS m / z [LC-MS]: 314.87 [M+1].

[0095] Step 2: 3-Iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidine-4-amine

[0096] 1.2 g of 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine and 2 mL of concentrated ammonia (25%-28%) were added to 20 mL of acetonitrile. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 3:1) to give 1.1 g of 3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidine-4-amine. MS m / z [LC-MS]: 295.92 [M+1].

[0097] Step 3: 3-Iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine

[0098] 1.1 g of 3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine and 1.3 g of potassium carbonate were added to 20 mL of N,N-dimethylformamide, followed by dropwise addition of 0.5 mL of 4-methoxybenzyl chloride. The mixture was stirred at room temperature for 2 hours. The reaction solution was poured into 150 mL of water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 4:1) to obtain 0.90 g of 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine. MS m / z [LC-MS]: 415.98 [M+1].

[0099] Step 4: 9-Iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine

[0100] 700 mg of 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine and 2 mL of chloroacetaldehyde were added to 20 mL of acetonitrile. The mixture was heated to 100 °C and the tube was sealed for 5 hours. The mixture was then poured into water, and the pH was adjusted to 9–10 with saturated sodium carbonate solution. The mixture was extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 8:1) to obtain 460 mg of 9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine. MS m / z [LC-MS]: 439.98 [M+1]. 1 HNMR (400MHz, DMSO-d6): δ=8.097 (1H, d, J=1.6Hz), 7.629 (1H, d, J=1.6Hz), 7.261 (2H, d, J=8.4Hz), 6.898 (2H, d, J=8.4Hz), 5.381 (2H, s), 3.72 (3H, s).

[0101] Intermediate 4: 6-Chloro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyrazine

[0102]

[0103] 6-Chloro-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[4,3-b]pyrazine was synthesized using the same intermediate as described in Example 29 on page 83 of patent WO2018057884.

[0104] Intermediate 5: 1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4- tert-butyl methylpiperidine-4-ylcarbamate

[0105]

[0106] Step 1: 4-Methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate tert-butyl ester

[0107] Intermediate 2 (538 mg), 4-methylpiperidin-4-ylcarbamate tert-butyl ester (514 mg), and diisopropylethylamine (1 mL) were added to tetrahydrofuran (20 mL), heated to 120 °C and stirred for 2 hours. The solvent was removed by rotary evaporation under reduced pressure. Separation was performed by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1) to obtain 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate tert-butyl ester (800 mg). MS m / z [LC-MS]: 447.27 [M+1].

[0108] Step 2: 4-Methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate tert-butyl ester

[0109] 445 mg of 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate tert-butyl ester was added to methanol (10 mL), followed by 2 mL of 1 M hydrochloric acid solution. The mixture was stirred overnight at room temperature. The pH was adjusted to 8–9 with saturated sodium bicarbonate aqueous solution. The organic solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate. The extract was dried, concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 4:1) to obtain 305 mg of 4-methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate. MS m / z [LC-MS]: 363.22 [M+1].

[0110] Step 3: 1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-ylcarbamate tert-butyl ester

[0111] 300 mg of 4-methyl-1-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-ylcarbamate tert-butyl ester and 177 mg of N-iodosuccinimide were added to acetonitrile (5 mL), heated to 100 °C and microwaved for 25 minutes. After cooling to room temperature, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether:ethyl acetate, 4:1) to obtain 320 mg of 1-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-ylcarbamate. MS m / z [LC-MS]: 489.11 [M+1]. 1H NMR (400MHz, DMSO-d6): δ = 13.545 (1H, s), 6.583 (1H, s), 3.317 (3H, s), 3.168 ( 2H, m), 2.994 (2H, m), 2.102 (1H, m), 1.529 (2H, m), 1.356 (9H, s), 1.230 (3H, s).

[0112] Intermediate 6: ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine- 6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)tert-butyl carbamate

[0113]

[0114] Step 1: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0115] Following the method in step 1 of intermediate 5, intermediate 1 was used to replace tert-butyl 4-methylpiperidin-4-ylcarbamate to yield 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one. MS m / z [LC-MS]: 403.25 [M+1].

[0116] Step 2: ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0117] 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (400 mg) and triethylamine (500 mg) were added to dichloromethane (10 mL). Di-tert-butyl dicarbonate (260 mg) was added dropwise under ice-water bath cooling. After the addition was complete, the mixture was brought to room temperature and stirred. After stirring for 4 hours, the mixture was washed successively with water and saturated brine. The organic phase was dried and concentrated, and then separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to obtain tert-butyl carbamate ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate (420 mg). MS m / z [LC-MS]: 503.3 [M+1].

[0118] Step 3: ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0119] Referring to step 2 of intermediate 5, 4-methyl-1-(5-methyl-4-oxo-1-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4,5]decane-4-yl)carbamate tert-butyl carbamate was used instead of 4-methyl-1-(5-methyl-4-oxo-1-( Tetrahydro-2H-pyran-2-yl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)piperidin-4-yl tert-butyl carbamate, yielding ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate. MS m / z [LC-MS]: 419.24 [M+1].

[0120] Step 4: ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0121] Referring to step 3 of intermediate 5, tert-butyl carbamate was substituted with ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate to obtain ((3S,4S)-8-(3-iodo-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate. MS m / z[LC-MS]: 545.14[M+1]. 1 HNMR (400MHz, DMSO-d6): δ = 13.562 (1H, s), 6.988 (1H, s), 4.134 (1H, m), 3.848 (1H, m), 3.638 (1H, d, J = 8.4Hz), 3.487 (1H, d, J = 8.4Hz), 3.328 (3H, s), 3.184 (1H, m), 3.092 (2H, m), 1.62-1.76 (3H, m), 1.50-1.60 (1H, m), 1.37 (9H, s), 0.994 (3H, d, J = 6.0Hz).

[0122] Intermediate 7: ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3- [e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)tert-butyl carbamate

[0123]

[0124] Step 1: (3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-amine

[0125] Following the method in step 1 of intermediate 5, intermediate 2 was replaced with intermediate 3, and tert-butyl 4-methylpiperidin-4-ylcarbamate was replaced with intermediate 1 to obtain (3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine. MS m / z [LC-MS]: 574.14 [M+1].

[0126] Step 2: ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0127] Referring to step 2 of intermediate 6, 6-((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine was substituted for 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl) -5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, yielding ((3S,4S)-8-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl) tert-butyl carbamate. MS m / z [LC-MS]: 674.2 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.707 (1H, d, J=1.2Hz), 7.437 (1H, d, J=1.2Hz), 7.251 (2H, d, J=8.4Hz), 6.850 (2H, d, J=8.4Hz), 5.391 (2H, s), 4 .163 (1H, m), 3.913 (1H, m), 3.673 (3H, s), 3.35-3.55 (6H, m), 1.72-1.90 (3H, m), 1.60-1.72 (1H, m), 1.352 (9H, s), 1.007 (3H, d, J = 6.4Hz).

[0128] Intermediate 8: ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazine- 6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)tert-butyl carbamate

[0129]

[0130] Step 1: (3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine

[0131] Following the method in step 1 of intermediate 5, intermediate 2 was replaced with intermediate 4, and tert-butyl 4-methylpiperidin-4-ylcarbamate was replaced with intermediate 1 to obtain (3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine. MS m / z [LC-MS]: 499.13 [M+1].

[0132] Step 2: ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl) tert-butyl carbamate

[0133] Referring to step 2 of intermediate 6, 6-((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine was substituted for 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl) -5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, yielding ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl) tert-butyl carbamate. MS m / z [LC-MS]: 599.19 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 8.410 (1H, s), 6.988 (1H, s), 5.71 (1H, m), 4.14 (1H, m), 3.84-3.92 (2H, m), 3.65 (2H, m), 3.472 (1H, d, J = 8.4 Hz), 3.18 (1H, m), 3.09 (2H, m), 2.31 (1H, m), 1.98 (1H, m), 1.62-1.83 (5H, m), 1.49-1.60 (3H, m), 1.38 (9H, s), 1.004 (3H, d, J = 6.0Hz).

[0134] Intermediate 9: ((3S,4S)-8-(3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]pyrazine- 6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)tert-butyl carbamate

[0135]

[0136] Step 1: 8-Bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine

[0137] 5-Bromo-6-chloro-2-(methylthio)pyrimidine-4-amine (1.0 g) and 40% aqueous chloroacetaldehyde (1.2 mL) were dissolved in dioxane (6 mL), refluxed under nitrogen protection for 14 hours, cooled to room temperature, and filtered to give 8-bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine (900 mg). MS m / z [LC-MS]: 277.92 [M+1].

[0138] Step 2: 8-Bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine

[0139] 8-Bromo-7-chloro-5-(methylthio)imidazo[1,2-c]pyrimidine (557 mg) was dissolved in dichloromethane (20 mL), and m-chloroperoxybenzoic acid (85%, 487 mg) was added. The mixture was stirred at room temperature for 6 hours. The reaction solution was washed successively with saturated sodium bicarbonate aqueous solution, water, and saturated brine. After drying with anhydrous sodium sulfate, the solution was filtered, and the filtrate was concentrated by rotary evaporation. The solution was purified by silica gel column chromatography (petroleum ether:ethyl acetate, 5:1) to give 8-bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine (420 mg). MS m / z [LC-MS]: 293.91 [M+1].

[0140] Step 3: (3S,4S)-8-(8-bromo-7-chloroimidozolo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-amine

[0141] Following the method in step 1 of intermediate 5, intermediate 2 was replaced with 8-bromo-7-chloro-5-(methylsulfinyl)imidazo[1,2-c]pyrimidine, and intermediate 1 was replaced with tert-butyl 4-methylpiperidin-4-ylcarbamate to obtain (3S,4S)-8-(8-bromo-7-chloroimidazo[1,2-c]pyrimidine-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine. MS m / z [LC-MS]: 400.06 [M+1].

[0142] Step 4: ((3S,4S)-8-(8-bromo-7-chloroimidozono[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0143] Referring to step 2 of intermediate 6, 6-((3S,4S)-8-(8-bromo-7-chloroimidozolo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine was substituted for 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl-1-(tetrahydro-2H-pyran-2-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one to obtain ((3S,4S)-8-(8-bromo-7-chloroimidozolo[1,2-c]pyrimidin-5-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate tert-butyl. MS m / z[LC-MS]: 500.11[M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.79 (1H, d, J=1.6Hz), 7.62 (1H, d, J=1.6Hz),

[0144] Intermediate 10: 2-Ethynyl-1,1-difluorocyclopropane

[0145]

[0146] 2-Ethynyl-1,1-difluorocyclopropane was synthesized according to the method of intermediate 3.1.35d in patent WO2015066413.

[0147] Intermediate 11: 1-Ethynyl-1-methylcyclopropane

[0148]

[0149] 1-Ethynyl-1-methylcyclopropane was synthesized according to the method in Example 4 of patent WO2011059784.

[0150] Intermediate 12: (2-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaneborane-2-yl)phenyl)ethyl (Alynyl)trimethylsilane

[0151]

[0152] Step 1: (2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane

[0153] 1-Bromo-2-chloro-3-iodobenzene (560 mg), ethynyltrimethylsilane (196 mg), cuprous iodide (34 mg), bis(triphenylphosphine)palladium dichloride (121 mg), and triethylamine (533 mg) were added to tetrahydrofuran (10 mL). The air in the reaction system was replaced three times with high-purity nitrogen, and the mixture was refluxed for 12 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure and separated by silica gel column chromatography (using n-hexane as the eluent) to obtain (2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane (424 mg). 1 HNMR (400MHz, CDCl3): δ=7.55 (1H, m), 7.43 (1H, m), 7.03 (1H, m), 0.25 (9H, m).

[0154] Step 2: (2-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)phenyl)ethynyl)trimethylsilane

[0155] (2-(3-bromo-2-chlorophenyl)ethynyl)trimethylsilane (232 mg), pinacol diboronate (244 mg), (1,1′-bis(diphenylphosphine)ferrocene)palladium dichloride (56 mg), and anhydrous potassium acetate (157 mg) were added to dioxane (2 mL). The air in the reaction system was replaced three times with high-purity nitrogen. The mixture was heated to 90 °C and reacted for 12 hours. The reaction mixture could be directly used for the next coupling reaction without further treatment. MS m / z [LC-MS]: 335.14 [M+1].

[0156] Intermediate 13: 5-(4,4,5,5-Tetramethyl-1,3,2-dioxacyclopentaborane-2-yl)-3H-spiro[benzofuran- 2,1′-cyclopropane]-3-one

[0157]

[0158] The raw material 5-bromo-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one was synthesized according to the literature Chemical and Pharmaceutical Bulletin; vol.32; nb.9; (1984); p.3532-3550.

[0159] 5-Bromo-3H-spiro[benzofuran-2,1′-cyclopropane]-3-one was reacted according to step 2 of intermediate 12 to obtain the corresponding borate ester. The reaction mixture could be directly used for the next coupling reaction without treatment. MS m / z [LC-MS]: 287.15 [M+1].

[0160] Intermediate 14: 2-(3-chloro-[1,1′-biphenyl]-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxanepentaneboron alkyl

[0161]

[0162] The raw material 4-bromo-3-chloro-1,1′-biphenyl was synthesized according to the literature Journal of the Chemical Society; (1964); pp. 3786-3790.

[0163] 4-Bromo-3-chloro-1,1′-biphenyl was reacted according to step 2 of intermediate 12 to obtain the corresponding borate ester. The reaction mixture could be directly used for the next coupling reaction without treatment. MS m / z [LC-MS]: 315.13 [M+1].

[0164] Example 1: 6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H- Pyrazolo[3,4-d]pyrimidin-4(5H)-one

[0165]

[0166] Step 1: 1-(3-(2-((2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)tert-butyl carbamate

[0167] Intermediate 5 (150 mg), 1-chloro-2-ethynylbenzene (50 mg), cuprous iodide (12 mg), triethylamine (100 mg), bis(triphenylphosphine)palladium dichloride (21 mg), and tetrahydrofuran (10 mL) were added to a sealed tube, purged with nitrogen, and heated to 80 °C with stirring overnight. After cooling to room temperature, the mixture was poured into water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separation was performed by silica gel column chromatography (petroleum ether:ethyl acetate, 6:1) to obtain tert-butyl 1-(3-((2-(2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)carbamate (75 mg). MS m / z [LC-MS]: 498.0 [M+1].

[0168] Step 2: 6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

[0169] 70 mg of 1-(3-(2-((2-chlorophenyl)ethynyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-4-methylpiperidin-4-yl)carbamate tert-butyl ester was added to a 4M solution of dioxane in hydrogen chloride (3 mL), stirred at room temperature for 1 hour, evaporated to dryness, and then extracted with 10% sodium carbonate aqueous solution (10 mL). The extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was separated by thin-layer silica gel chromatography (dichloromethane:methanol, 10:1) to obtain 35 mg of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2-(2-chlorophenyl)ethynyl)-5-methyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one. MS m / z[LC-MS]: 397.16[M+1]. 1 HNMR (400MHz, DMSO-d6): δ=7.70-8.40 (3H, brs), 7.66 (1H, dd, J=8.0Hz, 1.6Hz), 7.59 (1H, d, J=8.0Hz), 7.46 (1H, td, J=8.0H z, 1.6Hz), 7.41 (1H, t, J = 8.0Hz), 3.36-3.46 (5H, m), 3.08-3.16 (2H, m), 1.83-1.91 (2H, m), 1.72-1.80 (2H, m), 1.34 (3H, s).

[0170] Example 2: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2,4-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0171]

[0172] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 455.20 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.2-13.9 (1H, brs), 7.69 (1H, td, J=8.4Hz, 6.4Hz), 7.45 (1 H, td, J=9.6Hz, 2.8Hz), 7.19 (1H, td, J=8.4Hz, 2.8Hz), 6.71-6.84 (2H, brs), 4.13-4.1 7 (1H, m), 3.79 (1H, d, J = 8.8Hz), 3.59 (1H, d, J = 8.8Hz), 3.34-3.48 (6H, m), 2.85-2.96 ( 2H, m), 1.82-1.91 (2H, m), 1.66-1.73 (1H, m), 1.56-1.62 (1H, m), 1.17 (3H, d, J=6.4Hz).

[0173] Example 3: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-Chlorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0174]

[0175] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 453.18 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.66 (1H, dd, J=7.6Hz, 2.0Hz), 7.59 (1H, dd, J=8.0Hz, 1.6Hz), 7.4 6(1H, td, J=7.6Hz, 1.6Hz), 7.41(1H, td, J=7.6Hz, 1.6Hz), 6.71-6.84(3H, brs), 4.01-4.08( 1H, m), 3.64 (1H, d, J = 8.8Hz), 3.47 (1H, d, J = 8.8Hz), 3.39 (3H, s), 3.22-3.34 (2H, m), 2.90-3 .07(3H,m), 1.79-1.87(1H,m), 1.69-1.78(1H,m), 1.50-1.63(2H,m), 1.06(3H,d,J=6.0Hz).

[0176] Example 4: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((3-chlorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0177]

[0178] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 453.18 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.60 (1H, t, J=2.0Hz), 7.45-7.54 (3H, m), 4.01-4.06 (1H, m), 3.63 (1H, d, J=8.4Hz), 3.46 (1H, d, J=8.4Hz) , 3.39 (3H, s), 3.26-3.34 (2H, m), 2.89-3.07 (3H, m), 1.80-1.86 (1H, m), 1.69-1.77 (1H, m), 1.51-1.61 (2H, m), 1.06 (3H, d, J = 6.4Hz).

[0179] Example 5: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-Methoxyphenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0180]

[0181] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 449.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=12.80-13.80 (1H, brs), 7.46 (1H, dd, J=7.6Hz, 1.6Hz), 7.41 (1H, td, J=7.6Hz, 1.6Hz), 7.09 (1H, d, J=8.4Hz), 6.98 (1H, t, J=7.6Hz), 4.01-4.08 (1H, m), 3.84 ( 3H, s), 3.64 (1H, d, J = 8.8Hz), 3.47 (1H, d, J = 8.8Hz), 3.38 (3H, s), 3.26-3.34 (2H, m), 2.88-3 .05(3H,m), 1.79-1.88(1H,m), 1.69-1.76(1H,m), 1.50-1.62(2H,m), 1.07(3H,d,J=6.0Hz).

[0182] Example 6: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(phenylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0183]

[0184] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 419.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.54-7.57 (2H, m), 7.42-7.47 (3H, m), 4.03-4.09 (1H, m), 3.66 (1H, d, J=8.4Hz), 3.49 (1H, d, J=8.4Hz), 3 .39 (3H, s), 3.26-3.35 (2H, m), 2.90-3.04 (3H, m), 1.82-1.88 (1H, m), 1.72-1.78 (1H, m), 1.52-1.64 (2H, m), 1.08 (3H, d, J = 6.8Hz).

[0185] Example 7: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-Fluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0186]

[0187] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 437.21 [M+1]. 1 H NMR (400MH [z, DMSO-d6): δ = 7.62 (1H, td, J = 7.6Hz, 1.6Hz), 7.47-7.53 (1H, m), 7 .35 (1H, t, J = 8.8Hz), 7.28 (1H, t, J = 8.0Hz), 4.01-4.07 (1H, m), 3.63 (1H, d, J = 8. 4Hz), 3.46 (1H, d, J = 8.4Hz), 3.39 (3H, s), 3.25-3.34 (2H, m), 2.88-3.07 (3H, m), 1.80-1.87 (1H, m), 1.68-1.76 (1H, m), 1.50-1.62 (2H, m), 1.06 (3H, d, J=6.0Hz).

[0188] Example 8: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((3-aminophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0189]

[0190] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 434.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.3-13.8 (1H, brs), 7.05 (1H, t, J=8.0Hz), 6.73 (1 H, s), 6.65 (1H, d, J = 7.6Hz), 6.61 (1H, d, J = 7.6Hz), 5.30 (2H, s), 4.08-4.16 (1H , m), 3.75 (1H, d, J = 8.8Hz), 3.54 (1H, d, J = 8.8Hz), 3.39 (3H, s), 3.26-3.37 (3H, m), 2.88-2.98 (2H, m), 1.80-1.90 (2H, m), 1.56-1.70 (2H, m), 1.14 (3H, 5.6Hz).

[0191] Example 9: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(pyridin-3-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0192]

[0193] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 420.22 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=13.71 (1H, s), 8.74 (1H, s), 8.62 (1H, d, J=4.8Hz), 7.9 6-8.04 (3H, m), 7.49 (1H, dd, J = 8.0hz, 4.8Hz), 4.16-4.22 (1H, m), 3.83 (1H, d, J = 9 .2Hz), 3.64 (1H, d, J=9.2Hz), 3.40-3.50 (3H, m), 3.39 (3H, s), 2.84-2.94 (2H, m) , 1.82-1.92(2H,m), 1.70-1.76(1H,m), 1.58-1.64(1H,m), 1.20(3H,d,J=6.0Hz).

[0194] Example 10: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(pyridin-4-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0195]

[0196] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 420.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=8.65 (2H, d, J=5.6Hz), 7.51 (2H, d, J=5.6Hz), 4.10-4.16 (1H, m), 3.77 (1H, d, J=8.8Hz), 3.56 (1H, d, J=8.8Hz ), 3.40 (3H, s), 3.22-3.32 (3H, m), 2.87-2.99 (2H, m), 1.80-1.90 (2H, m), 1.65-1.70 (1H, m), 1.55-1.61 (1H, m), 1.15 (3H, d, J = 6.4Hz).

[0197] Example 11: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((3,5-dimethoxyphenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0198]

[0199] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 479.24 [M+1]. 1H NMR (400MHz, DMSO-d6): δ = 13.48-13.80 (1H, brs), 6.68 (2H, s), 6.58 (1H, s), 4.08-4.16 (1H, m), 3.76 (6H, s), 3.74 (1H, d, J = 8.8Hz), 3.56 (1H, d, J = 8.8Hz), 3.35-3.43 (5H, m), 3.16-3.22 (1H, m), 2.86-2.99 (2H, m), 1.78-1 .88(2H,m), 1.64-1.69(1H,m), 1.54-1.61(1H,m), 1.14(3H,d,J=6.4Hz).

[0200] Example 12: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- (3-hydroxy-3-methylbut-1-yn-1-yl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0201]

[0202] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 401.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 13.36-13.48 (1H, brs), 5.52 (1H, s), 4.08-4.14 (1H, m), 3.73 (1H, d, J = 8.8Hz), 3.55 (1H, d, J = 8.8Hz), 3.37 (3H , s), 3.11-3.21 (2H, m), 2.84-2.96 (3H, m), 1.77-1.86 (2H, m), 1.63-1.67 (1H, m), 1.54-1.58 (1H, m), 1.45 (6H, s), 1.13 (3H, d, J = 6.4Hz).

[0203] Example 13: 4-((6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)- 5-Methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-3-yl)ethynyl)benzonitrile

[0204]

[0205] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 444.22 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=7.91 (2H, d, J=8.0Hz), 7.73 (2H, d, J=8.0Hz), 4.06-4.13 (1H, m), 3.72 (1H, d, J=8.8Hz), 3.53 (1H, d, J=8.8Hz), 3.40 (3 H, s), 3.34-3.38 (2H, m), 3.09-3.13 (1H, m), 2.89-3.01 (2H, m), 1.77-1. 88 (2H, m), 1.62-1.66 (1H, m), 1.54-1.58 (1H, m), 1.12 (3H, d, J=6.8Hz).

[0206] Example 14: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((4-Chlorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0207]

[0208] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 453.18 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.57 (2H, d, J=8.0Hz), 7.51 (2H, d, J=8.0Hz), 4.08-4.14 (1H, m), 3.74 (1H, d, J=8.8Hz), 3.54 (1H, d, J=8.8Hz), 3.39 (3 H, s), 3.34-3.38 (2H, m), 3.15-3.18 (1H, m), 2.87-2.99 (2H, m), 1.79-1. 89 (2H, m), 1.63-1.67 (1H, m), 1.55-1.59 (1H, m), 1.14 (3H, d, J=6.0Hz).

[0209] Example 15: (3S,4S)-8-(3-((2-chlorophenyl)ethynyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3- Methyl-2-oxa-8-azaspiro[4.5]decane-4-amine

[0210]

[0211] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 423.17 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=8.30 (1H, s), 7.68 (1H, dd, J=7.2Hz, 2.0Hz), 7.43 (1H , d, J=8.0Hz), 7.23-7.32(2H,m), 4.17-4.22(1H,m), 3.96-4.06(2H,m), 3.82(1H , d, J=8.4Hz), 3.70 (1H, d, J=8.4Hz), 3.52-3.59 (1H, m), 3.42-3.49 (1H, m), 3.0 1(1H,d,J=4.0Hz), 1.88-2.03(2H,m), 1.67-1.79(2H,m), 1.24(3H,d,J=6.8Hz).

[0212] Example 16: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- (3-amino-3-methylbut-1-yn-1-yl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0213]

[0214] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 400.25 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.33-13.46 (1H, brs), 4.08-4.14 (1H, m), 3.73 (1H, d, J=8.8Hz), 3.55 (1H, d, J=8.8Hz), 3.37 (3H, s), 3 .11-3.21(2H,m), 2.85-2.96(3H,m), 1.76-1.86(2H,m), 1.63-1.67(1H,m), 1.54-1.59(1H,m), 1.43(6H,s), 1.14(3H,d,J=6.4Hz).

[0215] Example 17: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-((thiophen-2-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0216]

[0217] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 425.18 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=7.71 (1H, dd, J=5.2Hz, 0.8Hz), 7.45 (1H, dd, J=3.6Hz, 0.8 Hz), 7.14 (1H, dd, J = 5.2Hz, 3.6Hz), 4.07-4.13 (1H, m), 3.72 (1H, d, J = 8.8Hz), 3.53 (1 H, d, J = 8.8Hz), 3.39 (3H, s), 3.32-3.38 (2H, m), 3.11 (1H, d, J = 5.2Hz), 2.88-2.99 (2H , m), 1.77-1.88 (2H, m), 1.61-1.66 (1H, m), 1.53-1.58 (1H, m), 1.12 (3H, d, J=6.8Hz).

[0218] Example 18: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-Aminophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0219]

[0220] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 434.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.21 (1H, d, J=8.0Hz), 7.09 (1H, t, J=8.0Hz), 6.71 (1H, d, J=8.4H z), 6.51 (1H, t, J=7.2Hz), 6.01 (2H, s), 4.41-4.46 (1H, m), 4.12-4.20 (1H, m), 3.82 (1H, d, J =8.4Hz), 3.58 (1H, d, J = 8.4Hz), 3.45-3.49 (1H, m), 3.42 (3H, s), 3.13-3.15 (1H, m), 2.85-2 .95(2H,m), 1.84-1.98(2H,m), 1.66-1.72(2H,m), 1.58-1.63(2H,m), 1.19(3H,d,J=6.4Hz).

[0221] Example 19: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- (cyclopropylethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0222]

[0223] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 383.22 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=13.20-13.58 (1H, brs), 4.02-4.08 (1H, m), 3.90- 4.00 (1H, m), 3.65 (1H, d, J = 8.4Hz), 3.47 (1H, d, J = 8.4Hz), 3.35 (3H, s), 3.24 -3.30(1H,m), 3.11-3.15(1H,m), 2.86-2.99(3H,m), 1.70-1.85(2H,m), 1.5 0-1.62 (2H, m), 1.08 (3H, d, J=6.4Hz), 0.91-0.94 (2H, m), 0.75-0.83 (2H, m).

[0224] Example 20: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((3-amino-4-fluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0225]

[0226] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 452.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.07 (1H, d, J=8.0Hz), 6.94-6.99 (1H, m), 6.87-6.91 (1H, m), 4.26-4.32 (1H, m), 3.93 (1H, d, J=8.0Hz), 3.82 ( 1H, d, J = 8.4Hz), 3.47-3.58 (5H, m), 3.39-3.42 (1H, m), 2.96-3.08 (2H, m), 1.86-2.02 (3H, m), 1.71-1.75 (1H, m), 1.30 (3H, d, J = 7.2Hz).

[0227] Example 21: 2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4-5]decane-8-yl)-3- ((2-cyanophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0228]

[0229] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 444.22 [M+1]. 1H NMR (400MHz, DMSO-d6): δ = 7.75 (1H, d, J = 8.0Hz), 7.63-7.68 (1H, m), 7.51-7.58 (1H, m), 7.45 (1H, m), 4.14-4.20 (1H, m), 3.81 (1H, d, J=8.8Hz), 3.68 (1H, d, J=8.8Hz), 3.53 (3H, s), 3.30-3.43 (2H, m), 2.94-3.16 (3H, m), 1.52-1.86 (4H, m), 0.86 (3H, d, J=6.4Hz).

[0230] Example 22: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-((2-(trifluoromethyl)phenyl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0231]

[0232] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 487.21 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.82(1H,d,J=7.6Hz), 7.68(1H,d,J=8.4Hz), 7.52(1H,m), 7.44(1H,m), 4.13-4.19(1H,m), 3.79(1H,d , J=8.8Hz), 3.67 (1H, d, J=8.8Hz), 3.53 (3H, s), 3.31-3.41 (2H, m), 2.98-3.16 (3H, m), 1.46-1.90 (4H, m), 0.95 (3H, d, J=6.8Hz).

[0233] Example 23: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2,6-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0234]

[0235] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 455.2 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=7.51-7.59 (1H, m), 7.22-7.27 (2H, m), 4.04-4.1 1 (1H, m), 3.69 (1H, d, J = 8.4Hz), 3.51 (1H, d, J = 8.8Hz), 3.38 (3H, s), 3.32- 3.6(1H,m), 3.11-3.15(1H,m), 3.05-3.08(1H,m), 2.88-3.01(2H,m), 1.73 -1.86 (2H, m), 1.60-1.66 (1H, m), 1.52-1.58 (1H, m), 1.10 (3H, d, J = 6.4Hz).

[0236] Example 24: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(prop-1-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0237]

[0238] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 357.21 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.12-13.58 (1H, brs), 4.02-4.08 (1H, m), 3.65 (1H, d, J=8.4Hz), 3.47 (1H, d, J=8.4Hz), 3.35 (3H, s), 3 .24-3.30(1H,m), 3.11-3.15(1H,m), 2.86-2.99(3H,m), 2.05(3H,s), 1.70-1.85(2H,m), 1.50-1.62(2H,m), 1.07(3H,d,J=6.4Hz).

[0239] Example 25: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- (imidazo[1,2-b]pyridazin-3-ylethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0240]

[0241] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 460.22 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=13.66-1.84 (1H, brs), 8.67 (1H, dd, J=4.8Hz, 2.4Hz), 8.24 (1H, dd, J=8 .8Hz, 1.6Hz), 8.19 (1H, s), 7.37 (1H, dd, J=8.8Hz, 4.8Hz), 4.40-4.43 (1H, m), 4.16-4.20 (1H, m), 3 .83 (1H, d, J = 8.8Hz), 3.62 (1H, d, J = 8.8Hz), 3.43-3.50 (1H, m), 3.39 (3H, s), 3.34-3.36 (1H, m), 2 .85-2.94(2H,m), 1.85-1.94(2H,m), 1.70-1.73(1H,m), 1.58-1.62(1H,m), 1.19(3H,d,J=6.4Hz).

[0242] Example 27: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-ethyl alkynyl-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0243]

[0244] Step 1: ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-3-((trimethylsilyl)ethynyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0245] Following the method in step 1 of Example 1, intermediate 5 was replaced with intermediate 6, and 1-chloro-2-ethynylbenzene was replaced with trimethylsilylacetylene to obtain the target compound. MS m / z [LC-MS]: 515.28 [M+1].

[0246] Step 2: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-ethynyl-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0247] 40 mg of tert-butyl carbamate ((3S,4S)-3-methyl-8-(5-methyl-4-oxo-3-((trimethylsilyl)ethynyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-2-oxa-8-azaspiro[4.5]decane-4-yl)carbamate was dissolved in dichloromethane (1 mL), and 0.5 mL of 1 M tetrabutylammonium fluoride in tetrahydrofuran was added. The mixture was stirred at room temperature for 0.5 hours. Then, 3 mL of 4 M dioxane in hydrogen chloride was added, and the mixture was stirred at room temperature for 1 hour. The mixture was evaporated to dryness, and 10 mL of 10% sodium carbonate aqueous solution was added. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by thin-layer silica gel chromatography to obtain the target compound (15 mg). MS m / z [LC-MS]: 343.19 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 13.29-13.74 (1H, brs), 4.35 (1H, s), 4.05-4.12 (1H, m), 3.70 (1H, d, J = 8.8Hz), 3.52 (1H, d, J = 8.8Hz), 3.36 (3H, s) ), 3.22-3.35(2H,m), 3.08-3.12(1H,m), 2.86-2.98(2H,m), 1.74-1.88(2H,m), 1.60-1.64(1H,m), 1.52-1.56(1H,m), 1.11(3H,d,J=6.0Hz).

[0248] Example 28: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(3,3,3-trifluoroprop-1-yn-1-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0249]

[0250] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 452.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 4.06-4.12 (1H, m), 3.71 (1H, d, J = 8.8Hz), 3.52 (1H, d, J = 8.8Hz), 3.38 (3H, s), 3.24-3.36 (2H, m ), 3.10-3.14(1H,m), 2.89-3.01(2H,m), 1.76-1.86(2H,m), 1.61-1.65(1H,m), 1.53-1.57(1H,m), 1.11(3H,d,J=6.4Hz).

[0251] Example 30: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-aminopyridin-3-yl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0252]

[0253] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 435.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=7.97 (1H, d, J=5.2Hz), 7.60 (1H, dd, J=7.6Hz, 2.0Hz), 6.73 (2H, s), 6.55 (1H, dd, J=7.6Hz, 5.2Hz), 4.09-4.16 (1H, m), 3.77 (1H, d, J=8. 8Hz), 3.55 (1H, d, J=8.8Hz), 3.41 (3H, s), 3.22-3.39 (2H, m), 2.86-2.97 (3H, m), 1.82-1.92(2H,m), 1.65-1.68(1H,m), 1.56-1.61(1H,m), 1.15(3H,d,J=6.8Hz).

[0254] Example 31: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((1-hydroxycyclopropyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0255]

[0256] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 399.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.38-13.54(1H, brs), 6.35(1H, s), 4.05-4.11(1H, m), 3.68 (1H, d, J = 9.2Hz), 3.51 (1H, d, J = 8.4Hz), 3.35 (3H, s), 3.17-3.32 (2H, m ), 3.02-3.08(1H, m), 2.84-2.98(2H, m), 1.72-1.84(2H, m), 1.59-1.64(1H, m), 1.51-1.56 (1H, m), 1.09 (3H, d, J=6.0Hz), 0.97-0.99 (2H, m), 0.85-0.93 (2H, m).

[0257] Example 32: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-((1-methyl-1H-imidazol-4-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0258]

[0259] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 423.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 13.54 (1H, s), 7.69 (1H, s), 7.58 (1H, s), 4.15-4.20 (1H, m), 3.82 (1H, d, J = 8.8Hz), 3.66 (3H, s), 3.63 (1H, d, J=8.8Hz), 3.36-3.47 (5H, m), 2.82-2.93 (3H, m), 1.82-1.92 (2H, m), 1.70-1.74 (1H, m), 1.57-1.60 (1H, m), 1.19 (3H, d, J=6.8Hz).

[0260] Example 33: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-(pyridin-2-ylethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0261]

[0262] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 420.22 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=12.92-13.88 (1H, brs), 8.61 (1H, d, J=5.2Hz), 7.86 (1H, td , J=8.0Hz, 1.6Hz), 7.62 (1H, d, J=7.6Hz), 7.43 (1H, dd, J=7.6Hz, 5.2Hz), 4.04-4.10 ( 1H, m), 3.68 (1H, d, J = 8.8Hz), 3.51 (1H, d, J = 8.8Hz), 3.36-3.42 (5H, m), 2.89-3.06 (3 H, m), 1.72-1.84 (2H, m), 1.60-1.64 (1H, m), 1.52-1.56 (1H, m), 1.09 (3H, d, J=6.4Hz).

[0263] Example 34: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-((1-methyl-1H-pyrazol-4-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0264]

[0265] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 423.23 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=13.22-1.82 (1H, brs), 7.85 (1H, s), 7.70 (1H, s), 4.04-4.10 (1H, m), 3.85 (3H, s), 3.68 (1H, d, J=8.8Hz), 3.51 (1H, d, J = 8.8Hz), 3.32-3.42 (5H, m), 2.88-3.04 (3H, m), 1.72-1.86 (2H, m), 1.60-1.64 (1H, m), 1.52-1.56 (1H, m), 1.09 (3H, d, J = 6.4Hz).

[0266] Example 35: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-5-methyl 3-((1-methyl-1H-pyrazol-3-yl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0267]

[0268] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 423.23 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.22-1.82 (1H, brs), 7.78 (1H, d, J=2.4Hz), 6.52 (1H, d, J=2.4Hz), 4.04-4.10 (1H, m), 3.85 (3H, s), 3.68 (1H, d, J=8.8 Hz), 3.5) (1H, d, J = 8.8Hz), 3.32-3.42 (5H, m), 2.88-3.04 (3H, m), 1.72-1 .86(2H,m), 1.60-1.64(1H,m), 1.52-1.56(1H,m), 1.09(3H,d,J=6.4Hz).

[0269] Example 36: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- (2-Chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0270]

[0271] Step 1: ((3S,4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-yl) tert-butyl carbamate

[0272] Intermediate 6 (160 mg), reaction solution containing intermediate 12 (0.8 mL), tetra(triphenylphosphine)palladium (18 mg), and potassium phosphate (185 mg) were added to a mixed solvent of dioxane and water (10:1, 5 mL). The air in the reaction system was replaced three times with nitrogen, and the mixture was heated to 80 °C and stirred overnight. After cooling to room temperature, the mixture was poured into water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Separation was performed by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1) to obtain ((3S,4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-yl) tert-butyl carbamate (80 mg). MS m / z[LC-MS]: 625.27[M+1].

[0273] Step 2: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(2-chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0274] 80 mg of tert-butyl carbamate ((3S,4S)-8-(3-(2-chloro-3-((trimethylsilyl)ethynyl)phenyl)-5-methyl-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)-3-methyl-2-oxa-8-azaspiro[4,5]decane-4-yl)carbamate was added to a 4M solution of dioxane in hydrogen chloride (3 mL), stirred at room temperature for 1 hour, evaporated to dryness, and then a 10% sodium carbonate aqueous solution was added. (10 mL) was extracted with dichloromethane, the extract was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and separated by thin-layer silica gel chromatography (dichloromethane:methanol, 10:1) to give 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3-(2-chloro-3-ethynylphenyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (38 mg). MS m / z [LC-MS]: 453.18 [M+1]. 1H NMR (400MHz, CD3OD): δ = 7.70 (1H, d, J = 8.0Hz), 7.49 (1H, d, J = 7.6Hz), 7.34-7.40 (1H, m), 4.25-4.32 (1H, m), 3.94 (1H, d, J = 10.4Hz), 3.83 (1H, d, J = 10.4Hz), 3.71-3.73 (1H, m), 3.46-3.68 (6H, m), 2.94-3.09 (2H, m), 1.87- 2.05 (2H, m), 1.70-1.77 (1H, m), 1.56-1.62 (1H, m), 1.29 (3H, d, J=7.6Hz).

[0275] Example 37: 6-(4-amino-4-methylpiperidin-1-yl)-3-((2-fluoro-3,5-dimethoxyphenyl)acetylene 5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0276]

[0277] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 441.21 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.21-13.90 (1H, brs), 8.22 (2H, s), 6.81 (1H, dd, J=7.2Hz, 2.8Hz), 6.60 (1H, dd, J=4.4Hz, 2.8Hz), 3 .84(3H,s), 3.76(3H,s), 3.38-3.46(2H,m), 3.37(3H,s), 3.07-3.14(2H,m), 1.86-1.95(2H,m), 1.73-1.82(2H,m), 1.35(3H,s).

[0278] Example 38: 6-(4-amino-4-methylpiperidin-1-yl)-3-((2,5-difluorophenyl)ethynyl)-5-methyl- 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0279]

[0280] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 399.18 [M+1]. 1H NMR (400MHz, DMSO-d6): δ=13.63-13.90 (1H, brs), 8.13 (2H, s), 7.47-7.51 (1H, m), 7.34-7.44 (2H, m), 3.41-3.44(2H,m), 3.37(3H,s), 3.08-3.14(2H,m), 1.85-1.92(2H,m), 1.73-1.79(2H,m), 1.35(3H,s).

[0281] Example 40: 3-((1H-pyrazol-4-yl)ethynyl)-6-(4-amino-4-methylpiperidin-1-yl)-5-methyl- 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0282]

[0283] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 353.19 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.19-13.86 (1H, brs), 8.23 ​​(2H, s), 7.94 (2H, s), 3.37-3.44 ( 2H, m), 3.35 (3H, s), 3.05-3.13 (2H, m), 1.86-1.94 (2H, m), 1.72-7.8 (2H, m), 1.34 (3H, s).

[0284] Example 41: 6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-(pyrimidin-5-ylethynyl)-1,5-di Hydrogen-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0285]

[0286] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 365.18 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ = 13.61-13.94 (1H, brs), 9.22 (1H, s), 8.99 (2H, s), 7.78-8.22 (2H, brs), 3. 39-3.46 (2H, m), 3.38 (3H, s), 3.08-3.15 (2H, m), 1.83-1.91 (2H, m), 1.72-1.79 (2H, m), 1.34 (3H, s).

[0287] Example 42: 6-(4-amino-4-methylpiperidin-1-yl)-3-((3,5-bis(trifluoromethyl)phenyl)ethynyl)- 5-Methyl-1,5-dihydro-4Hpyrazolo[3,4-d]pyrimidin-4-one

[0288]

[0289] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 499.17 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=8.17-8.21(3H,m), 3.37(3H,s), 3.12-3.28(4H,m), 1.54-1.61(2H,m), 1.46-1.52(2H,m), 1.10(3H,s).

[0290] Example 43: 6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-((3-(trifluoromethyl)phenyl)acetylene) 1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0291]

[0292] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 431.18 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.58-13.86 (1H, brs), 8.08-8.18 (2H, brs), 7.85-7.87 (2H, m), 7.81 (1H, d, J=7.6Hz), 7.69 ( 1H, t, J = 7.6Hz), 3.40-3.46 (2H, m), 3.38 (3H, s), 3.08-3.14 (2H, m), 1.86-1.93 (2H, m), 1.73-1.80 (2H, m), 1.35 (3H, s).

[0293] Example 44: 6-(4-amino-4-methylpiperidin-1-yl)-5-methyl-3-(thiophen-3-ylethynyl)-1,5-di Hydrogen-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0294]

[0295] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 369.14 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=8.21 (2H, s), 7.95 (1H, d, J=1.6Hz), 7.69 (1H, dd, J=4.8Hz, 2.8Hz), 7.27 (1H, d, J=4 .8Hz), 3.65-3.73(2H, m), 3.39(3H, s), 3.01-3.06(2H, m), 1.89-1.97(2H, m), 1.76-1.84(2H, m), 1.38(3H, s).

[0296] Example 45: 6-(4-amino-4-methylpiperidin-1-yl)-3-((3-fluorophenyl)ethynyl)-5-methyl-1,5- Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0297]

[0298] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 381.19 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=12.74-13.90 (1H, brs), 7.70-8.90 (2H, brs), 7.46-7.51 (1H, m), 7.36-7.40 (2H, m), 7.28- 7.33 (1H, m), 3.38-3.46 (2H, m), 3.37 (3H, s), 3.07-3.14 (2H, m), 1.91-1.98 (2H, m), 1.75-1.80 (2H, m), 1.35 (3H, s).

[0299] Example 46: 6-(4-amino-4-methylpiperidin-1-yl)-3-((2,3-difluorophenyl)ethynyl)-5-methyl- 1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0300]

[0301] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 399.18 [M+1]. 1 H NMR (400MHz, CD3OD): δ=7.43-7.47(1H,m), 7.30-7.37(1H,m), 7.17-7.23(1H,m), 3.5 1-3.59 (5H, m), 3.21-3.29 (2H, m), 1.99-2.06 (2H, m), 1.88-1.94 (2H, m), 1.48 (3H, s).

[0302] Example 47: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2,3-difluorophenyl)ethynyl)-5-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0303]

[0304] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 455.20 [M+1]. 1H NMR (400MHz, CD3OD): δ=7.43-7.47(1H,m), 7.30-7.37(1H,m), 7.17-7.22(1H,m), 4.22-4.28(1H,m), 3.89(1H,d,J=8.8Hz), 3.76(1H,d,J =8.8Hz), 3.46-3.54(5H,m), 3.24-3.29(1H,m), 3.97-3.12(2H,m), 1.91-2.02(2H,m), 1.80-1.86(1H,m), 1.68-1.74(1H,m), 1.40(3H,s).

[0305] Example 48: 6-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-yl)-3- ((2-Fluorophenyl)ethynyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0306]

[0307] The target compound can be synthesized using suitable starting materials and intermediates, following the method described in Example 1. MS m / z [LC-MS]: 423.20 [M+1]. 1 H NMR (400MHz, DMSO-d6): δ=13.19-13.37 (1H, brs), 7.61 (1H, td, J=7.2Hz, 1.6Hz ), 7.46-7.52 (1H, m), 7.34 (1H, t, J = 8.4Hz), 7.27 (1H, td, J = 8.4Hz, 0.8Hz), 3.96 -4.19 (3H, m), 3.77 (1H, d, J = 8.8Hz), 3.54 (1H, d, J = 8.8Hz), 3.13-3.25 (m, 3H), 1.64-1.75 (2H, m), 1.53-1.62 (1H, m), 1.43-1.50 (1H, m), 1.14 (3H, d, J=6.0Hz).

[0308] Determination of the in vitro enzymatic activity inhibition of the compound on SHP2

[0309] In this patent, the enzymatic activity of SHP2 is detected using a rapid fluorescence method. DiFMUP is used as an alternative substrate, and a high-throughput screening platform has been optimized and established. The inhibitory activity of the compound against SHP2 is detected on this platform. The specific method is as follows: SHP2 at a final concentration of 1 nM is mixed with 2.5 μM of a diphosphorylated IRS1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) and pre-incubated at 23°C for 30 minutes. The compound is serially diluted 5-fold with 100% DMSO starting from 0.2 mM (total of 7 concentrations). 2 μL of each concentration is added to 48 μL of reaction buffer (60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Tween 20, 5 mM DTT) for dilution and mixing. Add 5 μL of the diluted compound to a black 384-well plate (OptiPlate-384, catalog number 6007270, purchased from PerkinElmer), then add 10 μL of pre-incubated SHP2 and IRS1 peptide mixture, centrifuge to mix, and incubate at 23°C for 30 min. Add 5 μL of the substrate substitute DiFMUP (final concentration 50 μM, catalog number D6567, purchased from Invitrogen) to the reaction and incubate at 23°C for 60 min. Then terminate the reaction by adding 5 μL of 160 μM bpV (Phen) solution (SC-22137, purchased from Santa). Immediately after the reaction is terminated, the fluorescence signal is detected using a microplate reader (PerkinElmer) at excitation and emission wavelengths of 340 nm and 450 nm, respectively. The IC50 of the compound is calculated using GraphPad Prism software. 50 value.

[0310] Determination of the inhibitory effect of the compound on the proliferation of SHP2 positive cells

[0311] Human non-small cell lung cancer cell line NCI-H358 cells were cultured in RPMI-1640 medium (catalog number C11875500BT, purchased from Biological Industries) with 10% fetal bovine serum (FBS, catalog number 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin antibiotics (P / S, catalog number 15070-063, purchased from Gibco) at 37°C and 5% CO2. The day before compound detection, NCI-H358 cells were seeded at a concentration of 2000 cells / 195 μL / well in 196-well plates (catalog number 3917, purchased from Corning). 24 hours later, the compound was serially diluted 3-fold starting at 10 mM with 100% DMSO (totaling 10 concentrations). 2 μL of each concentration was then added to 48 μL of serum- and antibiotic-free medium for further dilution. Five μL of each diluted compound was added to the prepared cell suspension. The compound and cells were incubated together in a cell culture incubator for 72 hours (3 days). After aspirating the culture medium, 25 μL of Cell-Titer Glo (G7570, purchased from Promega) reagent was added, and the cells were incubated again for 5-10 minutes. Fluorescence values ​​were then read on Envision, and the IC50 of the compound's inhibitory effect on cell proliferation was calculated using GraphPad Prism software. 50 value.

[0312] Kasumi-1 cells, a human acute myeloid leukemia cell line, were cultured in RPMI-1640 medium (C11875500BT, purchased from Biological Industries) with 20% fetal bovine serum (FBS, 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin antibiotics (P / S, 15070-063, purchased from Gibco) at 37°C and 5% CO2. The day before compound detection, Kasumi-1 cells were seeded at a concentration of 3000 cells / 195 μL / well in 196-well plates (Catalog No. 3599, purchased from Corning). Twenty-four hours later, the compound was serially diluted 3-fold with 100% DMSO, starting at 10 mM (totaling 10 concentrations). 2 μL of each concentration was then added to 48 μL of serum- and antibiotic-free medium for further dilution. Five μL of each diluted compound was added to the prepared cell suspension, and the compound and cells were incubated together in a cell culture incubator for 72 hours (3 days). Then, 35 μL of Cell-TiterBlue (G8082, purchased from Promega) reagent was added, and the cells were incubated again for 4 hours. Fluorescence values ​​were then read on a Flexstation III (excitation at 560 nm, detection at 590 nm). The IC50 of the compound's inhibitory effect on cell proliferation was calculated using GraphPad Prism software. 50 value.

[0313] Table 1. Inhibitory activity of the compounds in the examples against SHP2 enzyme and cell proliferation.

[0314]

[0315] The experimental data in Table 1 show that the compound provided by the present invention has excellent SHP2 kinase inhibitory activity and also has excellent inhibitory activity on the proliferation of SHP2 positive expression cells.

Claims

1. The following compounds or their pharmaceutically acceptable salts:

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