CN113004282A - Substituted alkynyl heterocyclic compounds - Google Patents

Substituted alkynyl heterocyclic compounds Download PDF

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Publication number
CN113004282A
CN113004282A CN201911314609.5A CN201911314609A CN113004282A CN 113004282 A CN113004282 A CN 113004282A CN 201911314609 A CN201911314609 A CN 201911314609A CN 113004282 A CN113004282 A CN 113004282A
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Prior art keywords
alkyl
methyl
pyrazolo
heteroaryl
pyrimidin
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Inventor
张晓军
闵汪洋
段小伟
陈曦
许新合
刘希杰
张凯
孙小亮
孙颖慧
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Shouyao Holdings Beijing Co Ltd
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Shouyao Holdings Beijing Co Ltd
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Priority to CN202311211012.4A priority Critical patent/CN117683033A/en
Priority to CN201911314609.5A priority patent/CN113004282A/en
Priority to PCT/CN2020/137662 priority patent/WO2021121397A1/en
Publication of CN113004282A publication Critical patent/CN113004282A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Abstract

The present application relates to substituted alkynyl heterocyclic compounds of formula I. The present invention relates to substituted alkynyl heterocyclic compounds having SHP2 inhibitory activity, processes for their preparation, pharmaceutical compositions thereof, and to the use of such compounds and pharmaceutical compositions thereof in the treatment of diseases where SHP2 enzyme inhibition is beneficial, for example in the treatment of cancer. During the preparation process, through iodination, amination, up-protection, coupling, deprotection,Ring-closing reaction, etc. to obtain the compound of formula I.

Description

Substituted alkynyl heterocyclic compounds
Technical Field
The present invention relates generally to novel substituted alkynyl heterocyclic compounds having SHP2 inhibitory activity, processes for their preparation, pharmaceutical compositions thereof, and to the use of such compounds and pharmaceutical compositions thereof in the treatment of diseases where SHP2 enzyme inhibition would be beneficial, such as in the treatment of cancer.
Background
Cancer is a serious disease seriously threatening human health and life, particularly, the morbidity and mortality of cancer in recent years are in a rapid rising trend, and the cancer becomes the first killer of human health beyond cardiovascular diseases. The proliferation, apoptosis, metastasis and the like of tumors are closely related to the abnormality of a certain link in a series of signal transduction pathways inside and outside cells. In these signaling pathways, protein phosphorylation and dephosphorylation are crucial, and this reversible process is co-regulated by kinases and phosphatases. Phosphorylation of Protein Tyrosine Kinases (PTKs) and dephosphorylation of Protein Tyrosine Phosphatases (PTPs) are a pair of reversible processes that maintain a dynamic equilibrium between them to maintain normal physiological functions of cells. Whereas abnormal phosphorylation can lead to the development of cancer, inflammation, diabetes and other diseases.
The SHP2 protein is an unresponsive protein tyrosine phosphatase encoded by ptpnll gene, is widely expressed in various tissues, and participates in important physiological and pathological processes such as embryonic development, metabolism, immune response, tumorigenesis and the like.
The SHP2 protein consists of two SH2 domains (N-SH2 and C-SH2) connected in series at the N terminal, a PTP catalytic domain and a C terminal tail with a regulating effect. The SH2 domain is a conformational switch that mediates the interaction of the SHP2 protein with phosphotyrosine-containing activators (e.g., insulin receptor substrate 1-IRS1 and GRB 2-related binding protein 1-GAB1) and the intramolecular interaction of the SH2 domain with the PTP catalytic domain. In the unstimulated state, the SHP2 domain binds to the PTP domain, blocking the catalytically active site, leaving SHP2 phosphatase activity in a self-inhibitory state. When the SH2 domain binds to the activator, inhibitory intramolecular interactions are released and the SHP2 phosphatase is in an open conformation, allowing the SHP2 substrate to localize to the catalytically active site and function as a phosphatase. The activity conversion property of SHP2 makes it possible for various mutations of SHP2 to destroy the self-inhibition state of SHP2, and thus to over-activate the phosphatase activity of SHP2 protein, which in turn can cause canceration. Both experimental and clinical data confirm that SHP2 plays a promoting role in most cancers, and as the first tyrosine phosphatase discovered to promote cancer progression, it has received considerable attention in the cancer field, and its phosphatase activity plays an important role in intracellular signaling.
SHP2 participates in regulating cell signal transduction pathways activated by cytokines, growth factors and hormones, including RAS/ERK, JAK/STAT, PI3K/AKT and NF-kB signal pathways, and further regulates physiological functions such as cell proliferation, differentiation, cell cycle maintenance and migration. Meanwhile, SHP2 mediates compensatory activation pathways after kinases such as MEK are inhibited, thereby promoting the occurrence of tumor drug resistance. As a downstream molecule of the PD-1 receptor, SHP2 is also involved in the transduction of T cell inhibitory signals. It has been shown that SHP2 is a downstream molecule of PD-1 signaling that not only inhibits T cell activation but also promotes T cell disability. Thus, targeting SHP2 could restore or enhance T cell-mediated anti-tumor immune function. In addition, SHP2 can inhibit IFN-gamma mediated immune response by inactivating signal transduction and activator of transcription STAT 1.
In recent years, SHP2 activating mutation and high expression are successively found in leukemia, solid tumor, melanoma, glioblastoma, lung cancer, breast cancer and knoop syndrome, and are closely related to the occurrence, development and prognosis of tumors. Currently, SHP2 has been studied as a target molecule for clinical tumors. The action mechanism of the traditional SHP2 inhibitor (such as II-B08 and PHPS1) is that the traditional SHP2 inhibitor is combined with PTP catalytic domain of SHP2, prevents tyrosine phosphorylation substrate from entering into the catalytic site, and thus inhibits the phosphatase activity of SHP 2. However, due to the highly conserved, polar and charged environment of the PTP catalytic domains of various phosphatases, the traditional inhibitor of SHP2 has a great defect in specificity and bioavailability, and the clinical application of the traditional inhibitor is limited. Therefore, the development of the SHP2 inhibitor with high specificity, high safety and strong cell membrane permeability is the key for determining whether SHP2 can become a novel tumor intervention target, and the SHP2 protein allosteric inhibitor becomes the main direction of current research.
There are three compounds currently in clinical research phase for which no SHP2 inhibitor is approved on the market, among which JAB-3068, gacanto, has progressed fastest, and phase II clinical research has been approved, and both TNO155, novice pharmaceutical and RMC-4630, are in phase I clinical research phase.
Disclosure of Invention
The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, tautomer, metabolite, or prodrug thereof,
Figure BSA0000197904800000021
wherein the content of the first and second substances,
a is selected from the following two-or three-heterocyclic ring;
Figure BSA0000197904800000022
R1and R2Each independently selected from hydrogen, halogen and C1-6An alkyl group;
x is selected from hydrogen and C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl groups may be fused to unsaturated alicyclic, heteroalicyclic, spirocyclic rings, and may optionally be substituted with one or more halogens, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C6-10Heteroaryl substitution;
R3and R4Each independently selected from hydrogen and C1-6An alkyl group;
y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more halogens, -OH, -O-C1-6Alkyl, -NH2、C1-6Alkyl, or C3-6Cycloalkyl optionally substituted by halogen, -OH, -O-C1-6Alkyl, or-NH2Substitution;
z is selected from the group consisting of a single bond, -S-and-C.ident.C-.
According to some embodiments of the invention, the compounds of formula (I) of the invention have the following formulae II to X:
Figure BSA0000197904800000031
wherein R is1And R2Each independently selected from hydrogen, halogen and C1-6An alkyl group;
x is selected from hydrogen and C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl groups may be fused to unsaturated alicyclic, heteroalicyclic, spirocyclic rings, and may optionally be substituted with one or more halogens, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C6-10Heteroaryl substitution;
R3and R4Each independently selected from hydrogen and C1-6An alkyl group;
y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more halogens, -OH, -O-C1-6Alkyl, -NH2、C1-6Alkyl, or C3-6Cycloalkyl optionally substituted by halogen, -OH, -O-C1-6Alkyl, or-NH2And (4) substitution.
In some embodiments, R1And R2Each independently selected from hydrogen and C1-6An alkyl group;
in some embodiments, X is selected from hydrogen, C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl may optionally be substituted by one or more halogen, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C5-10A heteroaryl group is substituted by a heteroaryl group,
R3and R4Each independently selected from hydrogen and C1-6An alkyl group;
in some embodiments, Y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more-OH, -NH2Or C1-6Alkyl substitution;
in some embodiments, Y is selected from C3-12A heteroalicyclic group, the heteroalicyclic group may beOptionally substituted by one or more-OH, -NH2Or C1-6Alkyl substitution;
in some embodiments, Y is selected from C3-12Heteroalicyclic optionally substituted with one or more-NH2Or C1-6Alkyl substitution;
according to some embodiments of the invention, the invention provides the following compounds:
Figure BSA0000197904800000041
Figure BSA0000197904800000051
Figure BSA0000197904800000061
in another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof. In some embodiments, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable excipient.
In another aspect, the present invention provides a method of treating a disease associated with SHP2, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, solvate, polymorph or tautomer thereof, or a pharmaceutical composition thereof.
In another aspect, the present invention provides the use of a compound of the present invention, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a disease associated with SHP 2.
In some embodiments of the invention, the disease associated with SHP2 is leukemia, melanoma, glioblastoma, lung cancer, breast cancer or knoop syndrome.
Certain chemical terms
The term "compound" as used herein includes all stereoisomers, geometric isomers, tautomers and isotopes.
The compounds of the invention may be asymmetric, e.g., having one or more stereoisomers. Unless otherwise indicated, all stereoisomers include, for example, enantiomers and diastereomers. The compounds of the 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 a racemic mixture or synthesized by using chiral starting materials or chiral reagents.
The compounds of the invention also include tautomeric forms. Tautomeric forms result from the exchange of one single bond with an adjacent double bond and the concomitant migration of one proton.
The invention also includes all isotopic atoms, whether in the intermediate or final compound. Isotopic atoms include those having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.
In the above definitions of the compounds of the formulae I to V, the terms used herein have the following meanings:
the term "halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group consisting of carbon and hydrogen atoms, such as C1-20Alkyl, preferably C1-6Alkyl 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, and the like. The alkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxy, aryl, heteroaryl, amino, halo, sulfonyl, sulfinyl, phosphoryl, and hydroxy.
The term "C1-6Alkyl "means a straight or branched chain saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms,which is connected to the rest of the molecule by a single bond, which has 1 to 6 carbon atoms. The alkyl group may be unsubstituted or substituted with one or more substituents selected from alkyl, alkoxy, amino, halo and hydroxy. Non-limiting examples of unsubstituted alkyl groups include, but are not limited to, groups such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-methylhexyl, and the like.
The term "cycloalkyl" refers to an all-carbon monocyclic saturated hydrocarbon group consisting of carbon and hydrogen atoms, such as C3-20Cycloalkyl, preferably C3-6Cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. The cycloalkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxy, aryl, heteroaryl, amino, halo, sulfonyl, sulfinyl, phosphoryl, and hydroxy.
The term "aryl" refers to an all-carbon monocyclic or fused ring having a fully conjugated pi-electron system, having 6 to 14 carbon atoms, preferably 6 to 12 carbon atoms, most preferably 6 carbon atoms. Aryl groups may be unsubstituted or substituted with one or more substituents, examples of which include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halo, hydroxy, sulfonyl, sulfinyl, phosphoryl, and heteroalicyclic. Non-limiting examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl.
The term "heteroaryl" refers to a monocyclic or fused ring of 5 to 12 ring atoms, having 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, containing 1, 2, 3 or 4 ring atoms selected from N, O, S, the remaining ring atoms being C, and having a fully conjugated pi-electron system. Heteroaryl groups may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halo, hydroxy, cyano, nitro, carbonyl, and heteroalicyclic. Non-limiting examples of unsubstituted heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolinyl, isoquinolinyl, tetrazolyl, triazinyl.
The term "heteroalicyclic" refers to a monocyclic or fused ring of 3 to 12 ring atoms having 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms wherein 1 or 2 ring atoms are selected from N, O, S (O)n(wherein n is 0, 1 or 2) and the remaining ring atoms are C. Such rings may be saturated or unsaturated (e.g. with one or more double bonds), but do not have a fully conjugated pi-electron system. Examples of 3-membered saturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000081
Examples of 4-membered saturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000082
Examples of 5-membered saturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000083
Examples of 6-membered saturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000084
Figure BSA0000197904800000085
Examples of 7-membered saturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000086
Examples of 5-membered unsaturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000087
Examples of 6-membered unsaturated heteroalicyclic include, but are not limited to
Figure BSA0000197904800000088
The term "heteroalicyclic" refers to a group that remains after 1 hydrogen atom has been removed from the "heteroalicyclic" molecule. Heteroalicyclic groups may be unsubstituted or have hydrogen atoms optionally substituted with substituents including, but not limited to, alkyl, alkoxy, ═ O, aryl, aralkyl, -COOH, -CN, amino, halogen, and hydroxy.
The present invention also provides a process for preparing a compound of the above formula comprising the following synthetic scheme:
synthesis scheme 1:
Figure BSA0000197904800000089
compounds of formulae 1-6 can be synthesized using synthesis scheme 1. 4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine is reacted with a suitable protecting agent to give an intermediate 1-1. And reacting the intermediate 1-1 with a sodium hydroxide solution to obtain an intermediate 1-2. The intermediate 1-2 reacts with methyl iodide and alkali to obtain an intermediate 1-3. The intermediate 1-3 reacts with a compound containing NH or borate to give an intermediate 1-4. Removing protecting groups from the intermediate 1-4 to obtain an intermediate 1-5. The intermediate 1-5 reacts with N-iodosuccinimide to obtain a compound 1-6.
Synthesis scheme 2:
Figure BSA0000197904800000091
compounds of formulae 2-5 can be synthesized using synthesis scheme 2. Reacting 4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine with N-iodosuccinimide to obtain an iodo intermediate 2-1. The intermediate 2-1 reacts with ammonia water to obtain an intermediate 2-2. The intermediate 2-2 is reacted with a suitable protecting agent to give an intermediate 2-3. The intermediate 2-3 is closed by chloral to obtain an intermediate 2-4. And reacting the intermediate 2-4 with a compound containing NH or boric acid ester to obtain a compound 2-5.
Synthesis scheme 3:
Figure BSA0000197904800000092
the compounds of formula 3-3 can be synthesized by synthesis scheme 3. Reacting 6-chloro-1H-pyrazolo [3, 4-b ] pyrazine with N-iodosuccinimide to obtain an iodo intermediate 3-1. The intermediate 3-1 is reacted with a suitable protecting agent to give the intermediate 3-2. And reacting the intermediate 3-2 with a compound containing NH or boric acid ester to obtain a compound 3-3.
Synthesis scheme 4:
Figure BSA0000197904800000093
the final compound 4-1 can be synthesized by synthetic scheme 4. R1Representing terminal acetylene, boric acid or boric acid ester, mercapto, R2Which represents iodine or bromine, and reacting the two raw materials in the presence of a palladium catalyst and a base to obtain a product 4-1. If the group on 4-1 carries a protecting group, the protecting group is removed by a corresponding method.
The above synthetic schemes are only examples of the preparation methods of some compounds of the present invention, and the skilled person can synthesize the compounds of the present invention by similar methods based on the above synthetic schemes according to the well-known techniques in the art.
The compounds of the invention or their salts can be administered alone as the active substance, preferably in the form of their pharmaceutical compositions.
In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I, II, III, IV or V, or a pharmaceutically acceptable salt, solvate, polymorph or metabolite thereof, as an active ingredient, in combination with one or more pharmaceutically acceptable carriers.
"pharmaceutical composition" refers to a formulation of one or more compounds of the present invention or salts thereof with carriers generally accepted in the art for delivery of biologically active compounds to an organism (e.g., a human). The purpose of the pharmaceutical composition is to facilitate administration of the compounds of the invention to an organism.
The term "pharmaceutically acceptable carriers" refers to those carriers which do not significantly stimulate the organism and do not impair the biological activity and performance of the active compound. "pharmaceutically acceptable carrier" means an inert substance useful for administering an active ingredient in conjunction with the active ingredient, including, but not limited to, any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that is 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 types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Administration of a compound of the invention or a pharmaceutically acceptable salt thereof, in pure form or in a suitable pharmaceutical composition, may be carried out by any acceptable mode of administration providing agents of similar use. The pharmaceutical compositions of the invention may be prepared by combining a compound of the invention with a suitable pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres, aerosols, and the like.
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, intravenous administration. The preferred route of administration is oral.
The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.
In a preferred embodiment, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, slurries, suspensions and the like, for oral administration to a patient.
Solid oral pharmaceutical compositions can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: the active compounds are mixed with solid excipients, the resulting mixture is optionally milled, if desired with further suitable auxiliaries, and the mixture is then processed to granules, to give tablets or dragee cores. Suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like. Such as microcrystalline cellulose, glucose solutions, gum arabic syrups, gelatin solutions, sucrose and starch pastes; talc, starch, magnesium stearate, calcium stearate or stearic acid; lactose, sucrose, starch, mannitol, sorbitol, or dicalcium phosphate; silicon dioxide; croscarmellose sodium, pregelatinized starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methylcellulose, agar, carboxymethylcellulose, crospovidone, and the like. The dragee cores may optionally be coated, in particular with enteric coatings, according to methods well known in normal pharmaceutical practice.
The pharmaceutical compositions may also be adapted for parenteral administration, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Suitable excipients, such as fillers, buffers or surfactants can be used.
Yet another aspect of the invention relates to the use of a compound of formula I through formula VI, or a pharmaceutically acceptable salt, solvate, polymorph, metabolite, etc. thereof, in the manufacture of a medicament for the treatment of a disease benefiting from the inhibition of SHP 2. The disease benefiting from the inhibition of SHP2 is selected from cancer.
The substituted alkynyl heterocyclic compound provided by the invention has very good SHP2 inhibitory activity, and is expected to become a high-efficiency SHP2 inhibitor drug.
Detailed Description
The following specific examples are included to provide those skilled in the art with a clear understanding of the invention and are included to provide a further understanding of the invention. They should not be considered as limiting the scope of the invention but merely as being exemplary illustrations and representative of the invention. Those skilled in the art will understand that: there are other synthetic routes to the compounds of the present invention, and the following non-limiting examples are provided.
All operations involving easily oxidizable or hydrolyzable raw materials were carried out under nitrogen protection. Unless otherwise indicated, the starting materials used in the present invention were commercially available and used without further purification.
The column chromatography adopts silica gel (200-300 mesh) produced by Qingdao chemical industry Co. The thin layer chromatography was carried out using a precast slab (silica gel 60 PF) manufactured by Merck, Inc2540.25 mm). Chiral compound separation and determination of enantiomeric excess (ee) using Agilent LC 1200 series (column: CHIRALPAK AD-H,
Figure BSA0000197904800000113
Figure BSA0000197904800000115
mm, 5 μm, 30 ℃). Nuclear magnetic resonance chromatography (NMR) was measured using a Varian VNMRS-400 nuclear magnetic resonance instrument; liquid chromatography-Mass Spectrometry (LC/MS) the use of FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18,
Figure BSA0000197904800000114
mm, 5 μm, 35 ℃), using ESI (+) ion mode.
Experimental part
Intermediate 1:(3S, 4S) -3-methyl-2-oxa-8-azaspiro [4.5]Decane-4-amine dihydrochloride
Figure BSA0000197904800000111
(3S, 4S) -3-methyl-2-oxa-8-azaspiro [4.5] decan-4-amine dihydrochloride was synthesized following the procedure of intermediate 14 in patent WO 2017216706.
Intermediate 2:6-chloro-5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d]Pyrimidine-4 (5H) - Ketones
Figure BSA0000197904800000112
Step 1: 4, 6-dichloro-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d ] pyrimidine
4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine (2.7g), 3, 4-dihydro-2H-pyran (2.4g), p-toluenesulfonic acid (0.25g) were added to tetrahydrofuran (25mL), 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 ].
Step 2: 6-chloro-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d ] pyrimidin-4 (5H) -one
4, 6-dichloro-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d]Pyrimidine (3.0g) and 20% sodium hydroxide solution (4mL) were added to acetonitrile (40mL), stirred at room temperature overnight, concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 6: 1) to give 6-chloro-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d]Pyrimidin-4 (5H) -one (2.4 g). MS m/z [ LC-MS]：255.07[M+1]。1HNMR(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)。
And step 3: 6-chloro-5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d ] pyrimidin-4 (5H) -one
Reacting 6-chloro-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d]Pyrimidin-4 (5H) -one (2.4g) and potassium carbonate (3.8g) were added to N, N-dimethylformamide (40mL), and methyl iodide (1.0mL) was added dropwise thereto and the mixture was stirred at room temperature for 4 hours. Pouring the reaction solution into 200mL of water, extracting with dichloromethane, washing the extract with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain 6-chloro-5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [3, 4-d]Pyrimidin-4 (5H) -one (1.8 g). MS m/z [ LC-MS]：269.08[M+1]。1HNMR(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)。
Intermediate 3:9-iodo-5-chloro-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c]Pyrazolo [4, 3-e]Pyrimidines
Figure BSA0000197904800000121
Step 1: 3-iodo-4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine
4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine (4.0g) and N-iodosuccinimide (5.72g) were added to acetonitrile (25mL), and the mixture was heated to 100 ℃ for microwave reaction for 25 minutes, cooled to room temperature, concentrated under reduced pressure and subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to give 3-iodo-4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine (5.4 g). MS m/z [ LC-MS ]: 314.87[ M +1 ].
Step 2: 3-iodo-6-chloro-1H-pyrazolo [3, 4-d ] pyrimidin-4-amine
3-iodo-4, 6-dichloro-1H-pyrazolo [3, 4-d ] pyrimidine (1.2g) and concentrated aqueous ammonia (25% -28%, 2mL) were added to acetonitrile (20mL), stirred overnight at room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 3: 1) to give 3-iodo-6-chloro-1H-pyrazolo [3, 4-d ] pyrimidin-4-amine (1.1 g). MS m/z [ LC-MS ]: 295.92[ M +1 ].
And step 3: 3-iodo-6-chloro-1- (4-methoxybenzyl) -1H-pyrazolo [3, 4-d ] pyrimidin-4-amine
3-iodo-6-chloro-1H-pyrazolo [3, 4-d ] pyrimidin-4-amine (1.1g) and potassium carbonate (1.3g) were added to N, N-dimethylformamide (20mL), followed by dropwise addition of 4-methoxybenzyl chloride (0.5mL), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into 150mL of water, 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 then subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to give 3-iodo-6-chloro-1- (4-methoxybenzyl) -1H-pyrazolo [3, 4-d ] pyrimidin-4-amine (0.90 g). MS m/z [ LC-MS ]: 415.98[ M +1 ].
And 4, step 4: 9-iodo-5-chloro-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c ] pyrazolo [4, 3-e ] pyrimidine
3-iodine-6-chlorine-1- (4-methoxybenzyl) -1H-pyrazolo [3, 4-d]Adding pyrimidine-4-amine (700mg) and chloroacetaldehyde (2mL) into acetonitrile (20mL), heating to 100 ℃, sealing and reacting for 5 hours, pouring into water, adjusting the pH value to 9-10 with saturated sodium carbonate aqueous solution, extracting with dichloromethane, washing extract with saturated saline, drying with anhydrous sodium sulfate, filtering, concentrating filtrate under reduced pressure, and separating by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain 9-iodine-5-chlorine-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c ] (1, 2-c)]Pyrazolo [4, 3-e]Pyrimidine (460 mg). MS m/z [ LC-MS]：439.98[M+1]。1HNMR(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)。
Intermediate 4:6-chloro-3-iodo-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [4, 3-b]Pyrazine esters
Figure BSA0000197904800000131
6-chloro-3-iodo-1- (tetrahydro-2H-pyran-2-yl) -1H-pyrazolo [4, 3-b ] pyrazine is synthesized following the same intermediate as in example 29 on page 83 of patent WO 2018057884.
Intermediate 5:1- (3-iodo-5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d)]Pyrimidin-6-yl) -4- Methylpiperidin-4-ylcarbamic acid tert-butyl ester
Figure BSA0000197904800000132
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-ylcarbamic acid tert-butyl ester
Intermediate 2(538mg), tert-butyl 4-methylpiperidin-4-ylcarbamate (514mg) and diisopropylethylamine (1mL) were added to tetrahydrofuran (20mL), and the mixture was heated to 120 ℃ and stirred for 2 hours, then the solvent was removed by rotary evaporation under reduced pressure, and the mixture was subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 5: 1) to give tert-butyl 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 (800 mg). MS m/z [ LC-MS ]: 447.27[ M +1 ].
Step 2: 4-methyl-1- (5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d ] pyrimidin-6-yl) piperidin-4-ylcarbamic acid tert-butyl ester
Adding tert-butyl 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 (445mg) into methanol (10mL), adding 1M hydrochloric acid solution (2mL), stirring overnight at room temperature, adjusting the pH value to 8-9 with saturated aqueous sodium bicarbonate solution, removing the organic solvent by rotary evaporation, extracting with ethyl acetate, drying and concentrating the extract, separating by silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to obtain 4-methyl-1- (5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [ 3), 4-d ] pyrimidin-6-yl) piperidin-4-ylcarbamic acid tert-butyl ester (305 mg). MS m/z [ LC-MS ]: 363.22[ M +1 ].
And step 3: 1- (3-iodo-5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d ] pyrimidin-6-yl) -4-methylpiperidin-4-ylcarbamic acid tert-butyl ester
Mixing 4-methyl-1- (5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d)]Pyrimidine-6-yl) piperidin-4-ylcarbamic acid tert-butyl ester (300mg) and N-iodosuccinimide (177mg) were added to acetonitrile (5mL), heated to 100 ℃ for microwave reaction for 25 minutes, cooled to room temperature, concentrated under reduced pressure, and then separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to give 1- (3-iodo-5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d ]]Pyrimidin-6-yl) -4-methylpiperidin-4-ylcarbamic acid tert-butyl ester (320 mg). 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)。
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]Decan-4-yl) carbamic acid tert-butyl ester
Figure BSA0000197904800000141
Step 1: 6- (((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-8-yl) -5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d ] pyrimidin-4-one
Referring to the procedure of step 1 in intermediate 5, intermediate 1 was used instead of tert-butyl 4-methylpiperidin-4-ylcarbamate to give 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-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 ].
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] decan-4-yl) carbamic acid tert-butyl ester
Adding 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-8-yl) -5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d ] pyrimidin-4-one (400mg) and triethylamine (500mg) to dichloromethane (10mL), dropwise adding di-tert-butyl dicarbonate (260mg) under cooling in an ice water bath, heating to room temperature after completion of dropwise addition, stirring for 4 hours, washing with water and saturated common salt in order, drying and concentrating the organic phase, and separating by silica gel column chromatography (petroleum ether: ethyl acetate, 4: 1) to obtain ((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] decan-4-yl) carbamic acid tert-butyl ester (420 mg). MS m/z [ LC-MS ]: 503.3[ M +1 ].
And 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] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 2 in intermediate 5, tert-butyl 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] decan-4-yl) carbamate was substituted with tert-butyl ((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) piperidin-4-ylcarbamate, to obtain ((3S, 4S) -3-methyl-8- (5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d ] pyrimidine-6-yl) -2-oxa-8-azaspiro [4.5] decane-4-yl) carbamic acid tert-butyl ester. MS m/z [ LC-MS ]: 419.24[ M +1 ].
And 4, 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] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 3 of intermediate 5, using ((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]Decan-4-yl) carbamic acid tert-butyl ester instead of 4-methyl-1- (5-methyl-4-oxo-4, 5-dihydro-1H-pyrazolo [3, 4-d]Pyrimidin-6-yl) piperidin-4-ylcarbamic acid tert-butyl ester to give ((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]Decan-4-yl) carbamic acid tert-butyl ester. MS m/z [ LC-MS]：545.14[M+1]。1H NMR(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)。
Intermediate 7:((3S, 4S) -8- (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c)]Pyrazolo [4, 3- 4]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-yl) carbamic acid tert-butyl ester
Figure BSA0000197904800000151
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] decan-4-amine
Referring to the procedure of step 1 in intermediate 5, intermediate 3 was used instead of intermediate 2 and intermediate 1 was used instead of tert-butyl 4-methylpiperidin-4-ylcarbamate to give (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] decan-4-amine. MSm/z [ LC-MS ]: 574.14[ M +1 ].
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] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 2 in intermediate 6, the intermediate was prepared using (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]Decan-4-amine substituted 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5%]Decan-8-yl) -5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Obtaining ((3S, 4S) -8- (9-iodine-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c)]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-yl) carbamic acid tert-butyl ester. MS m/z [ LC-MS]：674.2[M+1]。1H 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)。
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]Decan-4-yl) carbamic acid tert-butyl ester
Figure BSA0000197904800000152
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] decan-4-amine
Referring to the procedure of step 1 in intermediate 5, intermediate 4 was used instead of intermediate 2 and intermediate 1 was used instead of tert-butyl 4-methylpiperidin-4-ylcarbamate to give (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] decan-4-amine. MS m/z [ LC-MS ]: 499.13[ M +1 ].
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] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure for step 2 in intermediate 6, the starting material was (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]Decan-4-amine substituted 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5%]Decan-8-yl) -5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidine-4-one 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]Decan-4-yl) carbamic acid tert-butyl ester. MS m/z [ LC-MS]：599.19[M+1]。1H 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＝8.4Hz)，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)。
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]Decan-4-yl) carbamic acid tert-butyl ester
Figure BSA0000197904800000161
Step 1: 8-bromo-7-chloro-5- (methylthio) imidazo [1, 2-c ] pyrimidine
5-bromo-6-chloro-2- (methylthio) pyrimidin-4-amine (1.0g) and 40% chloroacetaldehyde in water (1.2mL) were dissolved in dioxane (6mL), refluxed for 14 hours under nitrogen, 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 ].
Step 2: 8-bromo-7-chloro-5- (methylsulfinyl) imidazo [1, 2-c ] pyrimidine
8-bromo-7-chloro-5- (methylthio) imidazo [1, 2-c ] pyrimidine (557mg) was dissolved in methylene chloride (20mL), m-chloroperoxybenzoic acid (85%, 487mg) was added thereto, the mixture was stirred at room temperature for 6 hours, the reaction mixture was washed with a saturated aqueous sodium bicarbonate solution, water and a saturated aqueous sodium chloride solution in this order, dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation of the filtrate, and 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 ].
And step 3: (3S, 4S) -8- (8-bromo-7-chloroimidazo [1, 2-c ] pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5] decan-4-amine
Referring to the procedure of step 1 in intermediate 5, intermediate 2 was replaced with 8-bromo-7-chloro-5- (methylsulfinyl) imidazo [1, 2-c ] pyrimidine and intermediate 1 was used instead of tert-butyl 4-methylpiperidin-4-ylcarbamate to give (3S, 4S) -8- (8-bromo-7-chloroimidazo [1, 2-c ] pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5] decan-4-amine. MS m/z [ LC-MS ]: 400.06[ M +1 ].
And 4, step 4: ((3S, 4S) -8- (8-bromo-7-chloroimidazo [1, 2-c ] pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 2 in intermediate 6, the reaction product was prepared from (3S, 4S) -8- (8-bromo-7-chloroimidazo [1, 2-c)]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amine substituted 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5%]Decan-8-yl) -5-methyl-1- (tetrahydro-2H-pyran-2-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Obtaining (3S, 4S) -8- (8-bromo-7-chloroimidazo [1, 2-c) by using pyrimidine-4-one]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-yl) carbamic acid tert-butyl ester. MS m/z [ LC-MS]：500.11[M+1]。1HNMR(400MHz，DMSO-d6)：δ＝7.79(1H，d，J＝1.6Hz)，7.62(1H，d，J＝1.6Hz)，
Intermediate 10：2-ethynyl-1, 1-difluorocyclopropane
Figure BSA0000197904800000171
2-ethynyl-1, 1-difluorocyclopropane was synthesized according to the procedure of intermediate 3.1.35d in patent WO 2015066413.
Intermediate 11:1-ethynyl-1-methylcyclopropane
Figure BSA0000197904800000172
1-ethynyl-1-methylcyclopropane was synthesized according to the procedure of example 4 in patent WO 2011059784.
Intermediate 12:(2- (2-chloro-3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) ethane Alkynyl) trimethylsilane
Figure BSA0000197904800000173
Step 1: (2- (3-bromo-2-chlorophenyl) ethynyl) trimethylsilane
1-bromo-2-chloro-3-iodobenzene (560mg), ethynyltrimethylsilane (196mg), cuprous iodide (34mg), palladium (121mg) bis (triphenylphosphine) dichloride and triethylamine (533mg) were added to tetrahydrofuran (10mL), the air in the reaction system was replaced with high-purity nitrogen three times, refluxed for 12 hours, cooled to room temperature, concentrated under reduced pressure and separated by silica gel column chromatography (using n-hexane as an eluent) to give (2- (3-bromo-2-chlorophenyl) ethynyl) trimethylsilane (424 mg).1HNMR(400MHz，CDCl3)：δ＝7.55(1H，m)，7.43(1H，m)，7.03(1H，m)，0.25(9H，m)。
Step 2: (2- (2-chloro-3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) ethynyl) trimethylsilane
(2- (3-bromo-2-chlorophenyl) ethynyl) trimethylsilane (232mg), pinacol ester diboronate (244mg), (1, 1' -bis (diphenylphosphino) ferrocene) dichloropalladium (56mg) and anhydrous potassium acetate (157mg) were added to dioxane (2mL), the air in the reaction system was replaced with high-purity nitrogen three times, and the mixture was heated to 90 ℃ to react for 12 hours, and the reaction mixture was allowed to proceed with the next coupling reaction without treatment. MS m/z [ LC-MS ]: 335.14[ M +1 ].
Intermediate 13:5- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3H-spiro [ benzofuran- 2, 1' -cyclopropane]-3-ketones
Figure BSA0000197904800000174
Starting material 5-bromo-3H-spiro [ benzofuran-2, 1' -cyclopropane ] -3-one reference Chemical and Pharmaceutical Bulletin; vol.32; nb.9; (1984) (ii) a p.3532-3550.
5-bromo-3H-spiro [ benzofuran-2, 1' -cyclopropane ] -3-one is reacted according to the method of step 2 of the intermediate 12 to obtain corresponding borate, and the reaction mixture can be directly subjected to the next coupling reaction without treatment. MS m/z [ LC-MS ]: 287.15[ M +1 ].
Intermediate 14:2- (3-chloro- [1, 1' -biphenyl)]-4-yl) -4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborole Alkane (I) and its preparation method
Figure BSA0000197904800000181
Starting material 4-bromo-3-chloro-1, 1' -biphenyl according to the Journal of the Chemical Society of the literature; (1964) (ii) a p.3786-3790.
Reacting 4-bromo-3-chloro-1, 1' -biphenyl according to the method of the step 2 of the intermediate 12 to obtain corresponding borate, and directly carrying out the next coupling reaction on the reaction mixture without treatment. MS m/z [ LC-MS ]: 315.13[ M +1 ].
Example 1:6- (4-amino-4-methylpiperidin-1-yl) -3- (2- (2-chlorophenyl) ethynyl) -5-methyl-1H- Pyrazolo [3, 4-d]Pyrimidin-4 (5H) -ones
Figure BSA0000197904800000182
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) carbamic acid tert-butyl ester
Intermediate 5(150mg), 1-chloro-2-ethynylbenzene (50mg), cuprous iodide (12mg), triethylamine (100mg), bis (triphenylphosphine) palladium dichloride (21mg) and tetrahydrofuran (10mL) were added to a sealed tube, and the mixture was heated to 80 ℃ with nitrogen substitution and stirred overnight. Cooled to room temperature, poured into water, extracted with dichloromethane, the extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 6: 1) to give 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 ].
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
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) carbamic acid tert-butyl ester (70mg) was added to a 4M dioxane solution of hydrogen chloride (3mL), stirred at room temperature for 1 hour, spin-dried, added with a 10% aqueous sodium carbonate solution (10mL), extracted with dichloromethane, the extract was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and chromatographed on thin layer silica gel (dichloromethane: methanol, 10: 1) to give 6- (4-amino-4-methylpiperidin-1-yl) -3- (2- (2-chlorophenyl) ethynyl) -5-methyl-1H-pyrazolo [3, 4-d ]]Pyrimidin-4 (5H) -one (35 mg). MS m/z [ LC-MS]：397.16[M+1]。1HNMR(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＝8.0Hz，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)。
Example 2:6- ((3S, 4S) -4-Ammonia3-methyl-2-oxa-8-azaspiro [4.5] yl]Decan-8-yl) -3- ((2, 4-difluorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000191
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：455.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.2-13.9(1H，brs)，7.69(1H，td，J＝8.4Hz，6.4Hz)，7.45(1H，td，J＝9.6Hz，2.8Hz)，7.19(1H，td，J＝8.4Hz，2.8Hz)，6.71-6.84(2H，brs)，4.13-4.17(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)。
Example 3:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-chlorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000192
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：453.18[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.66(1H，dd，J＝7.6Hz，2.0Hz)，7.59(1H，dd，J＝8.0Hz，1.6Hz)，7.46(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)。
Example 4:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8-yl) -3- ((3-Azophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000193
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：453.18[M+1]。1H 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)。
Example 5:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-methoxyphenyl) ethynyl) -5-methyl-1, 5-dioxo-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000201
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：449.23[M+1]。1H 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)。
Example 6:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (Phenylethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000202
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：419.22[M+1]。1H 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)。
Example 7:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-fluorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000203
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：437.21[M+1]。1H NMR(400MHz，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)。
Example 8:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((3-aminophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000211
Reference example 1The target compound can be synthesized by adopting proper starting materials and intermediates. MS m/z [ LC-MS]：434.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.3-13.8(1H，brs)，7.05(1H，t，J＝8.0Hz)，6.73(1H，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)。
Example 9:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (pyridin-3-ylethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000212
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)，7.96-8.04(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)。
Example 10:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (pyridin-4-ylethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000213
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：420.22[M+1]。1H 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)。
Example 11:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((3, 5-Dimethoxyphenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000221
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)。
Example 12:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- (3-hydroxy-3-methylbut-1-yn-1-yl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000222
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：401.23[M+1]。1H 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)。
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
Figure BSA0000197904800000223
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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(3H，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)。
Example 14:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((4-chlorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000231
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：453.18[M+1]。1H 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(3H，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)。
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]Decan-4-amines
Figure BSA0000197904800000232
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)，3.01(1H，d，J＝4.0Hz)，1.88-2.03(2H，m)，1.67-1.79(2H，m)，1.24(3H，d，J＝6.8Hz)。
Example 16:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- (3-amino-3-methylbut-1-yn-1-yl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000233
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：400.25[M+1]。1H 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)。
Example 17:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- ((thien-2-yl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000241
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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，0.8Hz)，7.14(1H，dd，J＝5.2Hz，3.6Hz)，4.07-4.13(1H，m)，3.72(1H，d，J＝8.8Hz)，3.53(1H，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)。
Example 18:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-aminophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000242
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：434.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.21(1H，d，J＝8.0Hz)，7.09(1H，t，J＝8.0Hz)，6.71(1H，d，J＝8.4Hz)，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)。
Example 19:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- (cyclopropylethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000243
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)，1.50-1.62(2H，m)，1.08(3H，d，J＝6.4Hz)，0.91-0.94(2H，m)，0.75-0.83(2H，m)。
Example 20:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((3-amino-4-fluorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000251
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：452.22[M+1]。1H 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)。
Example 21:2- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-cyanophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000252
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)。
Example 22:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- ((2- (trifluoromethyl) phenyl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000253
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：487.21[M+1]。1H 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)。
Example 23:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2, 6-difluorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000261
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)，4.04-4.11(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)。
Example 24:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (prop-1-yn-1-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000262
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：357.21[M+1]。1H 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)。
Example 25:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- (imidazo [1, 2-b ]]Pyridazin-3-ylethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000263
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)。
Example 26:(3S, 4S) -8- (9- ((2-fluorophenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo [4, 3- e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000271
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：446.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.71(1H，m)，7.68(1H，t，J＝8.0Hz)，7.50-7.55(2H，m)，7.37(1H，t，J＝8.0Hz)，7.31(1H，t，J＝8.0Hz)，4.11-4.16(1H，m)，3.76(1H，d，J＝8.0Hz)，3.58-3.64(2H，m)，3.14-3.32(4H，m)，1.89-1.99(2H，m)，1.72-1.77(1H，m)，1.62-1.68(1H，m)，1.14(3H，d，J＝6.8Hz)。
Example 27:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3-ethane Alkynyl-5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000272
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] decan-4-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 1 in example 1, intermediate 6 was used instead of intermediate 5 and trimethylsilylacetylene was used instead of 1-chloro-2-ethynylbenzene to obtain the objective compound. MS m/z [ LC-MS ]: 515.28[ M +1 ].
Step 2: 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-8-yl) -3-ethynyl-5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d ] pyrimidin-4-one
((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]Decen-4-yl) carbamic acid tert-butyl ester (40mg) was dissolved in methylene chloride (1mL), and 1M tetrabutylammonium fluoride in tetrahydrofuran (0.5mL) was added and the mixture was stirred at room temperature for 0.5 hour. Then 4M hydrogen chloride in dioxane (3mL) was added, the mixture was stirred at room temperature for 1 hour, spin-dried, and 10% aqueous sodium carbonate (10mL) was added, followed by extraction with dichloromethane, and the extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and subjected to thin layer silica gel chromatography to give the objective compound (15 mg). MS m/z [ LC-MS]：343.19[M+1]。1H 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)。
Example 28:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (3, 3, 3-trifluoroprop-1-yn-1-yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000273
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：452.22[M+1]。1H 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)。
Example 29:(3S, 4S) -8- (8- ((3-aminophenyl) ethynyl) -7-chloroimidazo [1, 2-c)]Pyrimidine-5- Yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000281
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：437.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.75(1H，d，J＝1.6Hz)，7.59(1H，d，J＝1.6Hz)，7.10(1H，t，J＝8.0Hz)，6.98(1H，t，J＝1.6Hz)，6.95(1H，d，J＝7.6Hz)，6.73(1H，dd，J＝8.0Hz，1.6Hz)，4.56(1H，s)，4.23-4.29(1H，m)，3.84-3.93(3H，m)，3.79(1H，d，J＝9.2Hz)，3.35-3.39(1H，m)，3.23(1H，d，J＝4.8Hz)，1.94-2.05(2H，m)，1.83-1.87(1H，m)，1.74-1.78(1H，m)，1.26(3H，d，J＝6.4Hz)。
Example 30:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-aminopyridin-3-yl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000282
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：435.23[M+1]。1H 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)。
Example 31:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((1-hydroxycyclopropyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000283
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：399.22[M+1]。1H 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)。
Example 32:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- ((1-methyl-1H-imidazol-4-yl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000291
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：423.23[M+1]。1H 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)。
Example 33:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- (pyridin-2-ylethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000292
The title compound was synthesized according to the procedure described in example 1 using the appropriate starting materials and intermediates]：420.22[M+1]。1H 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(3H，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)。
Example 34:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- ((1-methyl-1H-pyrazol-4-yl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000293
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)。
Example 35:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -5-carboxylic acid methyl ester 3- ((1-methyl-1H-pyrazol-3-yl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d)]Pyrimidin-4-ones
Figure BSA0000197904800000301
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：423.23[M+1]。1H 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＝8.8Hz)，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)。
Example 36:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- (2-chloro-3-ethynylphenyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000302
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] decan-4-yl) carbamic acid tert-butyl ester
Intermediate 6(160mg), the reaction solution containing intermediate 12 (0.8mL), tetrakis (triphenylphosphine) palladium (18mg), and potassium phosphate (185mg) were added to a mixed solvent of dioxane and water (10: 1, 5mL), and the air in the reaction system was replaced with nitrogen three times, heated to 80 ℃ and stirred overnight. Cooled to room temperature, poured into water, extracted with dichloromethane, the extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate, 3: 1) to give tert-butyl ((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] decan-4-yl) carbamate (80 mg). MS m/z [ LC-MS ]: 625.27[ M +1 ].
Step 2: 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [4.5] decan-8-yl) -3- (2-chloro-3-ethynylphenyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d ] pyrimidin-4-one
((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]Decan-4-yl) carbamic acid tert-butyl ester (80mg) was added to a 4M solution of hydrogen chloride in dioxane (3mL), stirred at room temperature for 1 hour, spin-dried, addedAdding 10% sodium carbonate aqueous solution (10mL), extracting with dichloromethane, drying the extractive solution with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating with thin layer silica gel chromatography (dichloromethane: methanol, 10: 1) to obtain 6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-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)。
Example 37:6- (4-amino-4-methylpiperidin-1-yl) -3- ((2-fluoro-3, 5-dimethoxyphenyl) acetylene Yl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000311
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：441.21[M+1]。1H 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)。
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-ones
Figure BSA0000197904800000312
With reference to the procedure of example 1, usingThe target compound can be synthesized by the starting materials and the intermediate. 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)。
Example 39:(S) -1- (4- (9- (2, 3-dichlorophenyl) -7H-imidazo [1, 2-c)]Pyrazolo [4, 3-e]Pyrimidine as one kind of food Pyridin-5-yl) -1H-pyrazol-1-yl) propan-2-amine
Figure BSA0000197904800000313
The title compound was synthesized by the method described in example 36 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：427.10[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.78-8.92(1H，brs)，8.28-8.42(1H，brs)，8.24(1H，s)，7.77(1H，dd，J＝8.0Hz，1.6Hz)，7.69(1H，dd，J＝7.6Hz，1.6Hz)，7.48-7.51(2H，m)，4.22-4.54(2H，m)，3.50-3.85(1H，m)，1.02-1.32(3H，m)。
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-ones
Figure BSA0000197904800000321
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：353.19[M+1]。1H 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)。
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-ones
Figure BSA0000197904800000322
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：365.18[M+1]。1H 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)。
Example 42:6- (4-amino-4-methylpiperidin-1-yl) -3- ((3, 5-bis (trifluoromethyl) phenyl) ethynyl) - 5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000323
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：499.17[M+1]。1H 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)。
Example 43:6- (4-amino-4-methylpiperidin-1-yl) -5-methyl-3- ((3- (trifluoromethyl) phenyl) acetylene Yl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000324
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：431.18[M+1]。1H 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)。
Example 44:6- (4-amino-4-methylpiperidin-1-yl) -5-methyl-3- (thien-3-ylethynyl) -1, 5-di hydrogen-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000331
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：369.14[M+1]。1H 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)。
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-ones
Figure BSA0000197904800000332
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：381.19[M+1]。1H 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)。
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-ones
Figure BSA0000197904800000333
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：399.18[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.43-7.47(1H，m)，7.30-7.37(1H，m)，7.17-7.23(1H，m)，3.51-3.59(5H，m)，3.21-3.29(2H，m)，1.99-2.06(2H，m)，1.88-1.94(2H，m)，1.48(3H，s)。
Example 47:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2, 3-difluorophenyl) ethynyl) -5-methyl-1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000334
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. 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)。
Example 48:6- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decan-8-yl) -3- ((2-fluorophenyl) ethynyl) -1, 5-dihydro-4H-pyrazolo [3, 4-d]Pyrimidin-4-ones
Figure BSA0000197904800000341
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：423.20[M+1]。1H 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)。
Example 49:(3S, 4S) -8- (9- ((2, 3-difluorophenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000342
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：464.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.48-7.59(3H，m)，7.28-7.33(1H，m)，4.13-4.21(1H，m)，3.81(1H，d，J＝8.4Hz)，3.60-3.72(3H，m)，3.13-3.25(3H，m)，1.90-2.01(2H，m)，1.62-1.80(2H，m)，1.17(3H，d，J＝5.6Hz)。
Example 50:(3S, 4S) -8- (9- ((3-chloro-2-fluorophenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000343
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：480.17[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.62-7.72(3H，m)，7.52(1H，d，J＝1.6Hz)，7.33(1H，t，J＝7.6Hz)，4.14-4.20(1H，m)，3.82(1H，d，J＝8.8Hz)，3.62-3.72(3H，m)，3.12-3.24(3H，m)，1.91-1.99(2H，m)，1.74-1.81(1H，m)，1.64-1.71(1H，m)，1.17(3H，d，J＝6.0Hz)。
Example 51：(3S, 4S) -8- (9- ((2-fluoro-3, 5-dimethoxyphenyl) ethynyl) -7H-imidazo [1, 2- c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000351
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：506.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.69(1H，s)，7.50(1H，s)，6.84(1H，dd，J＝6.8Hz，2.8Hz)，6.70-6.69(1H，m)，4.14-4.19(1H，m)，3.85(3H，s)，3.77-3.81(4H，m)，3.60-3.69(3H，m)，3.12-3.22(4H，m)，1.89-1.99(2H，m)，1.63-1.68(1H，m)，1.64-1.68(1H，m)，1.16(3H，d，J＝6.8Hz)。
Example 52:(3S, 4S) -8- (9- ((2-fluoro-5- (trifluoromethyl) phenyl) ethynyl) -7H-imidazo [1, 2- c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000352
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：514.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝8.04-8.07(1H，m)，7.89-7.94(1H，m)，7.70(1H，s)，7.64(1H，t，J＝8.8Hz)，7.52(1H，s)，4.15-4.21(1H，m)，3.81(1H，d，J＝8.0Hz)，3.61-3.70(3H，m)，3.13-3.23(3H，m)，1.90-2.01(2H，m)，1.73-1.80(1H，m)，1.62-1.70(1H，m)，1.17(3H，d，J＝6.4Hz)。
Example 53:(3S, 4S) -3-methyl-8- (9- ((3- (trifluoromethyl) phenyl) ethynyl) -7H-imidazo [1, 2-c]pyrazolo [4, 3-e]Pyrimidin-5-yl) -2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000353
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：496.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.90-7.93(2H，m)，7.83(1H，d，J＝7.6Hz)，7.69-7.74(2H，m)，7.53(1H，s)，4.15-4.21(1H，m)，3.83(1H，d，J＝9.2Hz)，3.63-3.71(3H，m)，3.34(1H，d，J＝5.2Hz)，3.10-3.19(2H，m)，1.92-2.01(2H，m)，1.75-1.79(1H，m)，1.65-1.70(1H，m)，1.18(3H，d，J＝6.4Hz)。
Example 54:(3S, 4S) -8- (9- ((2, 5-difluorophenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000361
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：464.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.48-7.59(2H，m)，7.35-7.44(2H，m)，4.13-4.21(1H，m)，3.82(1H，d，J＝8.4Hz)，3.61-3.72(3H，m)，3.13-3.25(3H，m)，1.91-2.01(2H，m)，1.62-1.81(2H，m)，1.17(3H，d，J＝5.6Hz)。。
Example 55:(3S, 4S) -8- (9- ((2-fluoro-3- (trifluoromethyl) phenyl) ethynyl) -7H-imidazo [1, 2- c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000362
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：514.20[M+1]。1H NMR(400MHz，CD3OD)：δ＝8.11(1H，t，J＝6.8Hz)，7.72-7.77(2H，m)，7.52(1H，d，J＝1.2Hz)，7.42(1H，t，J＝8.0Hz)，4.27-4.32(1H，m)，3.96(1H，d，J＝8.8Hz)，3.79-3.89(3H，m)，3.41(1H，d，J＝4.0Hz)，3.17-3.28(2H，m)，1.95-2.09(3H，m)，1.78-1.82(1H，m)，1.30(3H，d，J＝6.8Hz)。
Example 56:3- (5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8-yl) - 7H-imidazo [1, 2-c ]]Pyrazolo [4, 3-e]Pyrimidin-9-yl) -2-chlorophenyldimethylcarbamates
Figure BSA0000197904800000363
The title compound was synthesized by the method described in example 36 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：525.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.85(1H，s)，7.64(1H，d，J＝1.6Hz)，7.60(1H，dd，J＝7.2Hz，1.6Hz)，7.45(1H，t，J＝7.6Hz)，7.36-7.39(2H，m)，4.15-4.21(1H，m)，3.84(1H，d，J＝9.2Hz)，3.60-3.72(3H，m)，3.06-3.20(6H，m)，2.91(3H，s)，1.90-1.99(2H，m)，1.74-1.81(1H，m)，1.64-1.70(1H，m)，1.19(3H，d，J＝6.4Hz)。
Example 57:3- ((5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8-yl) - 7H-imidazo [1, 2-c ]]Pyrazolo [4, 3-e]Pyrimidin-9-yl) ethynyl) -2-fluorobenzonitrile
Figure BSA0000197904800000371
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：471.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.99-8.06(2H，m)，7.72(1H，s)，7.48-7.52(2H，m)，4.09-4.15(1H，m)，3.46(1H，d，J＝8.4Hz)，3.55-3.66(3H，m)，3.12-3.25(3H，m)，1.87-1.99(2H，m)，1.62-1.74(2H，m)，1.13(3H，d，J＝6.4Hz)。
Example 58:(3S, 4S) -8- (9- (cyclopropylethynyl) -7H-imidazo [1, 2-c)]Pyrazolo [4, 3-e]Pyrimidine as one kind of food Pyridin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000372
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：392.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.56-13.84(1H，brs)，7.65(1H，d，J＝0.8Hz)，7.45(1H，d，J＝0.8Hz)，4.05-4.13(1H，m)，3.94-4.02(1H，m)，3.71(1H，d，J＝8.4Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.74(1H，m)，1.10(3H，d，J＝6.4Hz)，0.90-0.94(2H，m)，0.75-0.82(2H，m)。
Example 59:n- (3- ((5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8- Yl) -7H-imidazo [1, 2-c]Pyrazolo [4, 3-e]Pyrimidin-9-yl) ethynyl) -2-fluorophenyl) acetamide
Figure BSA0000197904800000373
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：503.23[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.98(1H，t，J＝7.6Hz)，7.75(1H，d，J＝1.6Hz)，7.59(1H，t，J＝7.2Hz)，7.50(1H，d，J＝1.6Hz)，7.19(1H，t，J＝8.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝9.2Hz)，3.77-3.85(3H，m)，3.33-3.36(1H，m)，3.14-3.24(2H，m)，2.18(3H，s)，1.99-2.10(2H，m)，1.89-1.95(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。
Example 60:(3S, 4S) -8- (9- ((3-Ammonia)Yl-2-fluorophenyl) ethynyl) -7H-imidazo [1, 2-c]Pyrazoles And [4 ]，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000381
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：461.22[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.73(1H，d，J＝2.0Hz)，7.49(1H，d，J＝2.0Hz)，7.03-7.07(1H，m)，6.93(1H，t，J＝7.6Hz)，6.88(1H，td，J＝8.0Hz，2.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝9.2Hz)，3.76-3.85(3H，m)，3.35(1H，d，J＝4.4Hz)，3.17-3.25(2H，m)，1.99-2.10(2H，m)，1.88-1.96(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。
Example 61:5- (5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8-yl) - 7H-imidazo [1, 2-c ]]Pyrazolo [4, 3-e]Pyrimidin-9-yl) -3H-spiro [ benzofuran-2, 1' -cyclopropane]-3-ketones
Figure BSA0000197904800000382
The title compound was synthesized by the method described in example 36 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：486.23[M+1]。1H NMR(400MHz，CD3OD)：δ＝8.93(1H，s)，7.87(1H，d，J＝8.8Hz)，7.73(1H，s)，7.49(1H，s)，7.38(1H，d，J＝8.4Hz)，4.25-4.32(1H，m)，3.98(1H，d，J＝9.2Hz)，3.76-3.88(3H，m)，3.44(1H，d，J＝4.4Hz)，3.11-3.29(2H，m)，2.04-2.11(2H，m)，1.93-1.98(1H，m)，1.74-1.84(3H，m)，1.55-1.58(2H，m)，1.31(3H，d，J＝6.4Hz)。
Example 62:(3S, 4S) -8- (9- ((2, 3-dichlorophenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-nitrogenHetero spiro [4.5]]Decan-4-amines
Figure BSA0000197904800000383
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：496.14[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.61-7.74(3H，m)，7.51(1H，d，J＝1.6Hz)，7.31(1H，t，J＝7.6Hz)，4.11-4.20(1H，m)，3.78(1H，d，J＝8.4Hz)，3.61-3.74(3H，m)，3.10-3.24(3H，m)，1.89-1.99(2H，m)，1.73-1.85(1H，m)，1.64-1.72(1H，m)，1.16(3H，d，J＝6.4Hz)。
Example 63:(3S, 4S) -8- (9- ((2-fluoro-3-methoxyphenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyridine (II) Azolo [4, 3-e ] s]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000391
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：476.22[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，m)，7.50(1H，m)，7.16-7.32(3H，m)，4.10-4.22(1H，m)，3.87(3H，s)，3.78(1H，d，J＝8.4Hz)，3.57-3.69(3H，m)，3.12-3.26(3H，m)，1.87-2.01(2H，m)，1.71-1.79(1H，m)，1.62-1.69(1H，m)，1.15(3H，d，J＝5.6Hz)。
Example 64:(3S, 4S) -8- (9- (3-chloro- [1, 1' -biphenyl)]-4-yl) -7H-imidazo [1, 2-c]Pyrazoles And [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000392
With reference to the procedure of example 36, using appropriate starting materialsThe target compound can be synthesized from the starting materials and the intermediates. MS m/z [ LC-MS]：514.21[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.84(1H，s)，7.70-7.72(5H，m)，7.49(2H，t，J＝7.6Hz)，7.38-7.42(1H，m)，7.35(1H，d，J＝1.6Hz)，4.26-4.32(1H，m)，3.97(1H，d，J＝8.8Hz)，3.78-3.87(3H，m)，3.39(1H，d，J＝3.6Hz)，3.17-3.27(2H，m)，1.99-2.12(2H，m)，1.91-1.98(1H，m)，1.78-1.84(1H，m)，1.30(3H，d，J＝6.8Hz)。
Example 65:2- ((3- (5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8- Yl) -7H-imidazo [1, 2-c]Pyrazolo [43-e]Pyrimidin-9-yl) -2-chlorophenyl) thio) acetamide
Figure BSA0000197904800000393
The title compound was synthesized by the method described in example 36 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：527.18[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.71-7.77(1H，m)，7.53(1H，d，J＝6.8Hz)，7.36-7.46(3H，m)，4.29-4.35(1H，m)，4.01(1H，d，J＝10.0Hz)，3.81-3.91(3H，m)，3.76(2H，s)，3.52-3.54(1H，m)，3.17-3.24(2H，m)，2.03-2.22(2H，m)，1.95-2.04(1H，m)，1.80-1.86(1H，m)，1.33(3H，d，J＝6.4Hz)。
Example 66:3- ((5- ((3S, 4S) -4-amino-3-methyl-2-oxa-8-azaspiro [ 4.5)]Decane-8-yl) - 7H-imidazo [1, 2-c ]]Pyrazolo [4, 3-e]Pyrimidin-9-yl) ethynyl) -2-fluorophenol
Figure BSA0000197904800000401
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：462.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝10.14-10.29(1H，brs)，7.68(1H，d，J＝1.2Hz)，7.50(1H，d，J＝1.2Hz)，7.01-7.08(3H，m)，4.16-4.23(1H，m)，3.84(1H，d，J＝9.2Hz)，3.63-3.73(3H，m)，3.40(1H，d，J＝4.4Hz)，3.08-3.19(2H，m)，1.89-2.01(2H，m)，1.76-1.82(1H，m)，1.64-1.72(1H，m)，1.19(3H，d，J＝6.8Hz)。
Example 67:(3S, 4S) -8- (9- ((2, 2-difluorobenzo [ d ]][1，3]Dioxol-4-yl) acetylene Yl) -7H-imidazo [1, 2-c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decane- 4-amines
Figure BSA0000197904800000402
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：508.19[M+1]。1H NMR(400MHz，CD3OD)：δ＝7.74(1H，d，J＝1.6Hz)，7.55(1H，d，J＝8.0Hz)，7.51(1H，d，J＝1.6Hz)，7.26(1H，d，J＝8.0Hz)，7.20(1H，t，J＝8.0Hz)，4.26-4.32(1H，m)，3.95(1H，d，J＝8.8Hz)，3.77-3.88(3H，m)，3.39(1H，d，J＝4.4Hz)，3.16-3.25(2H，m)，2.01-2.02(2H，m)，1.89-1.96(1H，m)，1.76-1.83(1H，m)，1.29(3H，d，J＝6.4Hz)。
Example 68:(3S, 4S) -8- (9- ((2-fluoro-3-methylphenyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazoles And [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000403
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：460.23[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.80(1H，m)，7.62-7.70(2H，m)，7.52(1H，d，J＝1.6Hz)，7.41-7.20(1H，m)，4.13-4.20(1H，m)，3.81(1H，d，J＝8.8Hz)，3.60-3.72(3H，m)，3.27-3.29(1H，m)，3.12-3.21(2H，m)，2.35(3H，s)，1.89-1.97(2H，m)，1.61-1.79(2H，m)，1.18(3H，d，J＝6.4Hz)。
Example 69:(3S, 4S) -8- (9- ((2-fluoro-3-isopropylphenyl) ethynyl) -7H-imidazo [1, 2-c]Pyridine (II) Azolo [4, 3-e ] s]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000411
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：504.25[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，d，J＝1.4Hz)，7.51(1H，d，J＝1.4Hz)，7.18-7.32(3H，m)，4.55(1H，m)，4.10-4.21(1H，m)，3.77(1H，d，J＝8.4Hz)，3.57-3.68(3H，m)，3.10-3.25(3H，m)，1.87-2.02(2H，m)，1.70-1.79(1H，m)，1.60-1.70(1H，m)，1.32(6H，d，J＝6.8Hz)，1.16(3H，d，J＝5.6Hz)。
Example 70:(3S, 4S) -8- (9- ((3- (difluoromethoxy) -2-fluorophenyl) ethynyl) -7H-imidazo [1, 2-c]pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000412
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：512.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.76(1H，d，J＝1.6Hz)，7.51(1H，d，J＝1.6Hz)，7.38(1H，t，J＝56.0Hz)，7.21-7.35(3H，m)，4.12-4.22(1H，m)，3.79(1H，d，J＝8.4Hz)，3.58-3.68(3H，m)，3.12-3.26(3H，m)，1.88-2.01(2H，m)，1.70-1.80(1H，m)，1.61-1.70(1H，m)，1.18(3H，d，J＝5.6Hz)。
Example 71:(3S, 4S) -8- (9- ((3- (difluoromethyl) -2-fluorophenyl) ethynyl) -7H-imidazo[1，2- c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000413
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：496.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.84-7.88(1H，m)，7.70-7.74(2H，m)，7.51(1H，d，J＝1.6Hz)，7.41-7.18(1H，m)，7.28(1H，t，J＝54.0Hz)，4.13-4.19(1H，m)，3.80(1H，d，J＝8.8Hz)，3.60-3.71(3H，m)，3.27-3.28(1H，m)，3.12-3.21(2H，m)，1.90-1.99(2H，m)，1.61-1.78(2H，m)，1.17(3H，d，J＝6.8Hz)。
Example 72:(3S, 4S) -3-methyl-8- (9- ((2, 3, 4-trifluorophenyl) ethynyl) -7H-imidazo [1, 2- c]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000421
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：482.19[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.70(1H，d，J＝1.2Hz)，7.52-7.59(1H，m)，7.51(1H，d，J＝1.2Hz)，7.40-7.47(1H，m)，4.13-4.19(1H，m)，3.80(1H，d，J＝8.8Hz)，3.60-3.71(3H，m)，3.28-3.29(1H，m)，3.12-3.21(2H，m)，1.90-1.99(2H，m)，1.72-1.79(1H，m)，1.64-1.70(1H，m)，1.16(3H，d，J＝6.8Hz)。
Example 73:(3S, 4S) -8- (9- ((3-chloro-2, 5-difluorophenyl) ethynyl) -7H-imidazo [1, 2-c]Pyridine (II) Azolo [4, 3-e ] s]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000422
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：498.16[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.85-8.11(1H，brs)，7.77-7.83(1H，m)，7.74(0.5H，d，J＝1.6Hz)，7.70(0.5H，d，J＝1.6Hz)，7.59-7.63(1H，m)，7.52(0.5H，d，J＝1.6Hz)，7.49(0.5H，d，J＝1.6Hz)，4.18-4.28(1H，m)，3.66-3.88(4H，m)，3.43(1H，d，J＝4.4Hz)，3.11-3.21(2H，m)，1.91-2.02(2H，m)，1.66-1.86(2H，m)，1.14-1.28(3H，m)。
Example 74:(3S, 4S) -8- (9- ((2-fluoro-3- (2, 2, 2-trifluoroethoxy) phenyl) ethynyl) -7H-imidazole And [1, 2-c ]]Pyrazolo [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000423
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：544.21[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.73(1H，d，J＝1.6Hz)，7.50(1H，d，J＝1.6Hz)，7.20-7.34(3H，m)，4.46(2H，m)，4.10-4.22(1H，m)，3.78(1H，d，J＝8.4Hz)，3.57-3.68(3H，m)，3.12-3.25(3H，m)，1.87-2.01(2H，m)，1.70-1.79(1H，m)，1.62-1.70(1H，m)，1.17(3H，d，J＝5.6Hz)。
Example 75:(3S, 4S) -8- (9- (33-dimethylbut-1-yn-1-yl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4.3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000431
With reference to the procedure in example 1, using appropriate starting materials andthe intermediate can be synthesized to obtain the target compound. MS m/z [ LC-MS]：408.25[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.65(1H，d，J＝1.2Hz)，7.45(1H，d，J＝1.2Hz)，4.04-4.16(1H，m)，3.70(1H，d，J＝8.6Hz)，3.40-3.60(3H，m)，3.00-3.24(3H，m)，1.81-1.99(3H，m)，1.64-1.75(1H，d，J＝6.8Hz)，1.27(9H，s)，1.08(3H，d，J＝6.4Hz)。
Example 76:(3S, 4S) -8- (9- (3-chloro-3-methylbut-1-yn-1-yl) -7H-imidazo [1, 2-c)]Pyrazoles And [4, 3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000432
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：428.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.46-13.81(1H，brs)，7.68(1H，d，J＝1.2Hz)，7.50(1H，d，J＝1.2Hz)，4.02-4.17(1H，m)，3.71(1H，m)，3.42-3.61(3H，m)，2.99-3.25(3H，m)，1.80-2.00(9H，m)，1.64-1.79(1H，d，J＝6.8Hz)，1.10(3H，d，J＝6.8Hz)。
Example 77:(3S, 4S) -3-methyl-8- (9- (3-methylbut-1-yn-1-yl) -7H-imidazo [1, 2-c)]Pyridine (II) Azolo [4, 3-e ] s]Pyrimidin-5-yl) -2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000433
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：394.24[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.65(1H，d，J＝1.2Hz)，7.45(1H，d，J＝1.2Hz)，4.04-4.16(1H，m)，3.70(1H，d，J＝8.6Hz)，3.40-3.60(3H，m)，2.98-3.24(4H，m)，1.81-1.99(3H，m)，1.64-1.75(1H，m)，1.28(6H，d，J＝6.8Hz)，1.07(3H，d，J＝6.4Hz)。
Example 78:(3S, 4S) -3-methyl-8- (9- ((1-methylcyclopropyl) ethynyl) -7H-imidazo [1, 2-c] Pyrazolo [4, 3-e]Pyrimidin-5-yl) -2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000441
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：406.24[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝13.58-13.84(1H，brs)，7.63(1H，d，J＝0.8Hz)，7.43(1H，d，J＝0.8Hz)，4.05-4.14(1H，m)，3.72(1H，d，J＝8.6Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.74(1H，m)，1.26(3H，s)，1.10(3H，d，J＝6.4Hz)，0.92-0.95(2H，m)，0.72-0.80(2H，m)。
Example 79:(3S, 4S) -8- (9- ((2, 2-Difluorocyclopropyl) ethynyl) -7H-imidazo [1, 2-c)]Pyrazolo ring [4，3-e]Pyrimidin-5-yl) -3-methyl-2-oxa-8-azaspiro [4.5]Decan-4-amines
Figure BSA0000197904800000442
The title compound can be synthesized by following the procedure of example 1 using appropriate starting materials and intermediates. MS m/z [ LC-MS]：428.20[M+1]。1H NMR(400MHz，DMSO-d6)：δ＝7.68(1H，d，J＝1.2Hz)，7.48(1H，d，J＝1.2Hz)，4.05-4.17(2H，m)，3.71(1H，d，J＝8.6Hz)，3.46-3.60(3H，m)，3.02-3.23(3H，m)，1.80-1.99(3H，m)，1.64-1.78(2H，m)，1.52(1H，m)，1.12(3H，d，J＝6.4Hz)。
Determination of the inhibitory Effect of Compounds on the in vitro enzymatic Activity of SHP2
The enzymatic activity of SHP2 in this patent was detected by rapid fluorescence using DiFMUP as a surrogate substrateThe reaction and optimization establish a high throughput screening platform. The detection of the inhibitory activity of compounds on SHP2 was performed on this platform. The specific method comprises the following steps: a final concentration of 1nM SHP2 was preincubated with 2.5. mu.M of a mixture of the peptide fragment of diphosphorylated IRS1 (SEQ ID NO: H2N-LN (pY) IDLDLV (dPEG8) LST (pY) ASINFQK-amide) at 23 ℃ for 30 min. Compounds were diluted 5-fold in 100% DMSO (7 concentrations) starting at 0.2mM, and 2. mu.L of each compound was added to 48. mu.L of reaction buffer (60mM HEPES, pH 7.2, 75mM NaCl, 75mM KCl, 1mM EDTA, 0.05% Tween 20, 5mM DTT) for dilution and mixing. mu.L of the diluted compound was added to a black 384 well plate (OptiPlate-384, cat # 6007270, purchased from Perkinelmer), followed by 10. mu.L of the pre-incubated peptide mixture of SHP2 and IRS1, centrifuged and mixed, and incubated at 23 ℃ for 30 minutes. mu.L of the surrogate substrate DiFMUP (final concentration 50. mu.M, cat # D6567, purchased from Invitrogen) was added to the reaction and incubated for 60 min at 23 ℃. The reaction was then stopped by adding 5. mu.L of 160. mu. MbpV (Phen) solution (SC-22137, purchased from Santa). Immediately after the reaction is terminated, fluorescence signals are detected and measured by using a microplate reader (Perkin-Elmer) at excitation and emission wavelengths of 340nm and 450nm respectively, and the data are calculated by using GraphPad Prism software to obtain the IC of the compound50The value is obtained.
Determination of the inhibitory Effect of Compounds on the proliferation of SHP 2-Positive cells
Human non-small cell lung cancer cell line NCI-H358 cells were cultured using RPMI-1640 medium (cat # C11875500BT, purchased from Biological Industries) plus 10% fetal bovine serum (FBS, cat # 04-001-ACS, purchased from Biological Industries, BI) and 1% penicillin/streptomycin diabody (P/S, cat # 15070-063, purchased from Cibco) at 37 ℃ with 5% CO 2. The day before compound detection, NCI-H358 cells were plated in a 196-well plate (cat. No. 3917, purchased from Corning) at a concentration of 2000 cells/195. mu.L/well. After 24 hours, compounds were diluted in 100% DMSO in a 3-fold gradient starting at 10mM (10 concentrations total), and then 2. mu.L of each compound was added to 48. mu.L of the serum-poor and double-antibody medium. The diluted compounds were added to the plated cell suspension in 5. mu.L/concentrationIn (1), the compound and cells were co-incubated in a Cell incubator for 72 hours (3 days), the medium was aspirated and then incubated again for 5-10 minutes with 25. mu.L of Cell-Titer Glo (G7570, purchased from Promega) reagent. Fluorescence values were then read on Envision and the data were calculated using GraphPad Prism software to obtain the IC of the compound for inhibition of cell proliferation50The value is obtained.
Human acute myeloblastic leukemia cell line Kasumi-1 cells were cultured using RPMI-1640 medium (cat # C11875500BT, purchased from Biological Industries) plus 20% fetal bovine serum (FBS, cat # 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin/streptomycin diabody (P/S, cat # 15070-063, purchased from Gibco) at 37 ℃ with 5% CO 2. The day before compound detection, Kasumi-1 cells were plated in a 196-well plate (cat # 3599, purchased from Corning) at a concentration of 3000 cells/195. mu.L/well. After 24 hours the compounds were diluted in 100% DMSO in a 3-fold gradient starting at 10mM (10 concentrations in total), and then 2. mu.L of each compound was added to 48. mu.L of medium without serum and double antibody. mu.L of each diluted compound was added to the plated Cell suspension, the compound and cells were co-incubated in a Cell incubator for 72 hours (3 days), 35. mu.L of Cell-Titer Blue (G8082, purchased from Promega) reagent was added and the incubation was repeated for 4 hours. Fluorescence values were then read on Flexstation III (560nm excitation, 590nm detection) and the data were calculated using GraphPad Prism software to obtain the IC for inhibition of cell proliferation by this compound50The value is obtained.
TABLE 1 inhibitory Activity of the Compounds of the examples on SHP2 enzyme and cell proliferation
Figure BSA0000197904800000451
The test data in table 1 show that the compound provided by the invention has good SHP2 kinase inhibitory activity, and also has good inhibitory activity on the proliferation of SHP2 positive expression cells.

Claims (10)

1. A compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof,
Figure FSA0000197904790000011
wherein the content of the first and second substances,
a is selected from the following two-or three-heterocyclic ring;
Figure FSA0000197904790000012
R1and R2Each independently selected from hydrogen, halogen and C1-6An alkyl group;
x is selected from hydrogen and C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl groups may be fused to unsaturated alicyclic, heteroalicyclic, spirocyclic rings, and may optionally be substituted with one or more halogens, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C6-10Heteroaryl substitution;
R3and R4Each independently selected from hydrogen and C1-6An alkyl group;
y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more halogens, -OH, -O-C1-6Alkyl, -NH2、C1-6Alkyl, or C3-6Cycloalkyl optionally substituted by halogen, -OH, -O-C1-6Alkyl, or-NH2Substitution;
z is selected from the group consisting of a single bond, -S-and-C.ident.C-.
2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, having the following formulas II-X:
Figure FSA0000197904790000013
Figure FSA0000197904790000021
wherein R is1And R2Each independently selected from hydrogen, halogen and C1-6An alkyl group;
x is selected from hydrogen and C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl groups may be fused to unsaturated alicyclic, heteroalicyclic, spirocyclic rings, and may optionally be substituted with one or more halogens, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C6-10Heteroaryl substitution;
R3and R4Each independently selected from hydrogen and C1-6An alkyl group;
y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more halogens, -OH, -O-C1-6Alkyl, -NH2、C1-6Alkyl, or C3-6Cycloalkyl substituted, said alkyl or cycloalkylOptionally substituted by halogen, -OH, -O-C1-6Alkyl, or-NH2And (4) substitution.
3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein R1And R2Each independently selected from hydrogen and C1-6An alkyl group.
4. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein X is selected from hydrogen, C1-6Alkyl radical, C3-8Cycloalkyl radical, C6-10Aryl and C5-10Heteroaryl of said C1-6Alkyl and C3-8Cycloalkyl groups may optionally be substituted by one or more halogens, -OH, -O-C1-6Alkyl, -NH2Or C1-6Alkyl substitution, said C6-10Aryl and C5-10Heteroaryl may optionally be substituted by one or more halogen, -CF3、-OH、-CN、-O-C1-6Alkyl, -NR3R4、-O-C(O)NR3R4、-NH-(CO)-C1-6Alkyl, -S-CH2-CONH2、C1-6Alkyl radical, C3-6Cycloalkyl radical, C6-10Aryl, or C5-10A heteroaryl group is substituted by a heteroaryl group,
R3and R4Each independently selected from hydrogen and C1-6An alkyl group.
5. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein Y is selected from C6-10Aryl radical, C5-10Heteroaryl and C3-12Heteroalicyclic, said aryl, heteroaryl and heteroalicyclic optionally substituted with one or more-OH, -NH2Or C1-6Alkyl substitution.
6. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt, solvate, polymorph or tautomer thereofIsomers, wherein Y is selected from C3-12Heteroalicyclic optionally substituted with one or more-OH, -NH2Or C1-6Alkyl substitution.
7. The following compounds, or pharmaceutically acceptable salts, solvates, polymorphs, or tautomers thereof:
Figure FSA0000197904790000031
Figure FSA0000197904790000041
Figure FSA0000197904790000051
8. a pharmaceutical composition comprising a compound according to any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, and optionally a pharmaceutically acceptable adjuvant.
9. Use of a compound of any one of claims 1-7, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition according to claim 8, in the manufacture of a medicament for the treatment of a disease associated with SHP 2.
10. The use of claim 9, wherein the disease associated with SHP2 is leukemia, melanoma, glioblastoma, lung cancer, breast cancer, or knoop syndrome.
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