Substituted heterocyclic compounds

Abstract

The invention relates to a substituted alkynyl heterocyclic compound shown in a formula I. The invention relates to a substituted alkynyl heterocyclic compound with inhibitory activity, a preparation method and a pharmaceutical composition thereof, and also relates to application of the compound and the pharmaceutical composition thereof, such as cancer treatment. In the preparation process, the compound shown in the formula I is obtained through reactions such as iodination, amination, upper protection, coupling, deprotection and ring-closure reaction. # imgabs0 #

Description

Technical Field

[0001] The present invention generally relates to novel substituted alkenyl heterocyclic compounds having SHP2 inhibitory activity, methods for preparing the same, pharmaceutical compositions thereof, and uses of the compounds and pharmaceutical compositions thereof for treating diseases that benefit from SHP2 enzyme inhibition, such as cancer. Background Art

[0002] Cancer is a serious disease that threatens human health and life. In particular, the incidence and mortality of cancer have been rising rapidly in recent years, surpassing cardiovascular disease to become the number one killer of human health. Tumor proliferation, apoptosis, metastasis, etc. are closely related to the abnormality of a certain link in a series of signal transduction pathways inside and outside the cell. In these signal transduction pathways, protein phosphorylation and dephosphorylation are crucial. This reversible process is jointly regulated by kinases and phosphatases. Phosphorylation of protein tyrosine kinase (PTK) and dephosphorylation of protein tyrosine phosphatase (PTP) are such a pair of reversible processes, and they maintain a dynamic balance to maintain the normal physiological function of cells. On the contrary, abnormal phosphorylation can lead to cancer, inflammation, diabetes and other diseases.

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

[0004] The SHP2 protein consists of two tandem SH2 domains (N-SH2 and C-SH2) at the N-terminus, a PTP catalytic domain, and a C-terminal tail with a regulatory function. The SH2 domain is a conformational switch that mediates the interaction of the SHP2 protein with phosphotyrosine-containing activators (such as insulin receptor substrate 1-IRS1 and GRB2-associated binding protein 1-GAB1) and the intramolecular interaction between the SH2 domain and the PTP catalytic domain. In the absence of stimulation, the SHP2 domain binds to the PTP domain, blocking the catalytic active site and placing the SHP2 phosphatase activity in an autoinhibitory state. When the SH2 domain binds to the activator, the inhibitory intramolecular interaction is released, and the SHP2 phosphatase is in an open conformation, allowing the SHP2 substrate to locate to the catalytic active site and perform the phosphatase function. This activity conversion characteristic of SHP2 makes it possible for various mutations in SHP2 to destroy the SHP2 autoinhibitory state, leading to overactivation of the SHP2 protein phosphatase activity, and then inducing carcinogenesis. Both experimental and clinical data have confirmed that SHP2 plays a promoting role in most cancers. As the first tyrosine phosphatase discovered to promote cancer development, it has received great attention in the field of cancer. Its phosphatase activity plays an important role in intracellular signal regulation.

[0005] SHP2 is involved in regulating cell signal transduction pathways activated by cytokines, growth factors and hormones, including RAS / ERK, JAK / STAT, PI3K / AKT and NF-κB signaling pathways, thereby regulating physiological functions such as cell proliferation, differentiation, cell cycle maintenance and migration. At the same time, SHP2 also mediates compensatory activation pathways after kinases such as MEK are inhibited, thereby promoting the occurrence of tumor resistance. As a downstream molecule of the PD-1 receptor, SHP2 is also involved in the conduction of T cell inhibitory signals. Studies have shown that SHP2 is a downstream molecule of PD-1 signaling, which not only inhibits T cell activation but also promotes T cell dysfunction. Therefore, targeting SHP2 can restore or enhance T cell-mediated anti-tumor immune function. In addition, SHP2 can inhibit IFN-γ-mediated immune responses by inactivating the signal transducer and transcription activator STAT1.

[0006] In recent years, SHP2 activation mutations and high expression have been found in leukemia, solid tumors, melanoma, malignant glioma, lung cancer, breast cancer and Nunan syndrome, and are closely related to the occurrence, development and prognosis of tumors. At present, SHP2 has been studied as a target molecule for clinical tumors. The mechanism of action of traditional SHP2 inhibitors (such as II-B08 and PHPS1) is to bind to the PTP catalytic domain of SHP2, prevent tyrosine phosphorylated substrates from entering the catalytic site, and thus inhibit the phosphatase activity of SHP2. However, due to the highly conserved, polar and charged environment of the PTP catalytic domains of various phosphatases, traditional SHP2 inhibitors have great defects in specificity and bioavailability, which limits their clinical application. Therefore, the development of SHP2 inhibitors with high specificity, high safety and strong cell membrane permeability is the key to determining whether SHP2 can become a new tumor intervention target, and SHP2 protein allosteric inhibitors have become the main direction of current research. Summary of the invention

[0007] In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof,

[0008]

[0009] in,

[0010] Ring A is a benzene ring or a 5-6 membered heteroaromatic ring, and the benzene ring and the heteroaromatic ring may be optionally substituted with halogen, -CN, -OH, -NH2, C 1-6 Alkyl, or -OC 1-6 Alkyl Substitution

[0011] X1 and X2 are each independently -CHR1-, -NR2-, -O-, or -S-,

[0012] R1 is H, halogen, -CN, -OH, -NH2, C 1-6 Alkyl, or -OC 1-6 alkyl,

[0013] R2 is selected from H or C 1-6 alkyl.

[0014] In some embodiments, X1 and X2 are each independently -CHR1-, -NR2-, or -O-, and R1 and R2 are as described above.

[0015] In some embodiments, R1 is H or NH2.

[0016] In some embodiments, the present invention provides the following compounds or pharmaceutically acceptable salts, solvates, polymorphs, or tautomers thereof:

[0017]

[0018]

[0019] 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, and optionally comprising a pharmaceutically acceptable excipient.

[0020] In another aspect, the present invention provides a method for treating a disease associated with SHP2, comprising administering 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 to a mammal, preferably a human, in need of such treatment.

[0021] On the other hand, the present invention provides a compound of the present invention or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition thereof, or use of any of the foregoing in combination with a SHP2 inhibitor or a KRAS inhibitor or an EGFR inhibitor in the preparation of a medicament for treating diseases associated with SHP2 and / or KRAS and / or EGFR.

[0022] In some embodiments of the present invention, the disease associated with SHP2 and / or KRAS and / or EGFR is leukemia, melanoma, malignant glioma, lung cancer, breast cancer or Noonan syndrome.

[0023] On the other hand, SHP2 acts upstream of KRAS. Hana Algul's team (Mutant KRAS-driven cancers depend on PTPN11 / SHP2phosphatase, Nature Medicine 2018) confirmed that SHP2 small molecule inhibitors have significant therapeutic effects on aggressive KRAS tumors such as pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). Protein tyrosine phosphatase SHP2 has become a key drug target for aggressive KRAS tumors. In addition, Schneeberger, VE's team (Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcmoma. Onco target 2015) confirmed that in EGFR-mutated non-small cell lung cancer, SHP2 can promote RAS-RAF-MEK-ERK signaling. Therefore, the use of the SHP2 small molecule inhibitor of the present invention alone or in combination with KRAS inhibitors (AMG510, etc.) and EGFR inhibitors (Iressa, Tarceva, etc.) in the prior art can effectively inhibit the occurrence and progression of tumors.

[0024] On the other hand, similarities in biological activity are often found in functional groups with similar spatial or electronic properties. Structures that play the same biological role in drug molecules are called bioisosteres. The isostere structure replaces the original functional group in the drug with another functional group without affecting the main biological activity of the drug. For example, the replacement of monovalent atoms or groups such as OH, NH2, F, SH, etc., the carboxyl group can be replaced by phosphate, tetrazole, etc.

[0025] Some chemical terms

[0026] The "compounds" of the present invention include all stereoisomers, geometric isomers, tautomers and isotopes. The compounds of the present invention may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present invention containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0027] The compounds of the present invention also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond accompanied by the migration of a proton.

[0028] The present invention also includes all isotopes of atoms, whether in the intermediates or the final compounds. Isotopes of atoms include atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

[0029]

[0063] The term "fused" or "fused ring," as used herein, alone or in combination, refers to a cyclic structure in which two or more rings share one or more bonds.

[0030]

[0063] The term "spiro" or "spirocycle," as used herein, alone or in combination, refers to a cyclic structure having two or more rings sharing one or more atoms.

[0031] The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

[0032] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group consisting of carbon atoms and hydrogen atoms, such as C 1-20 Alkyl, preferably C 1-6 Alkyl, such as methyl, ethyl, propyl (such as n-propyl and isopropyl), butyl (such as n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (such as n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylhexyl, etc. The alkyl group may be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, cyano, carboxyl, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphoryl and hydroxyl.

[0033] The term "C 1-6 "Alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond and has 1-6 carbon atoms. The alkyl group can be unsubstituted or substituted by one or more substituents selected from alkyl, alkoxy, amino, halogen 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, etc.

[0034] The term "aryl" refers to a fully conjugated π electron system of all-carbon monocyclic or fused rings having 6-14 carbon atoms, preferably 6-12 carbon atoms, and most preferably 6 carbon atoms. Aryl can be unsubstituted or substituted with one or more substituents, examples of which include but are not limited to alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, sulfonyl, sulfinyl, phosphoryl and heteroalicyclic. Non-limiting examples of unsubstituted aryl include but are not limited to phenyl, naphthyl and anthracenyl.

[0035] The term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic or fused ring of 5-12 ring atoms, having 5, 6, 7, 8, 9, 10, 11 or 12 ring atoms, of which 1, 2, 3 or 4 ring atoms are selected from N, O, S, and the remaining ring atoms are C, and have a completely conjugated π-electron system. The heteroaryl group can be unsubstituted or substituted, and the substituents include, but are not limited to, alkyl, alkyloxy, aryl, aralkyl, amino, halogen, hydroxyl, cyano, nitro, carbonyl and heteroalicyclic groups. Non-limiting examples of unsubstituted heteroaryl groups include, but are not limited to, pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, triazinyl.

[0036] The compound of the present invention or a salt thereof can be administered alone as an active substance, or preferably in the form of a pharmaceutical composition thereof.

[0037] "Pharmaceutical composition" refers to a preparation of one or more compounds of the present invention or their salts with carriers generally accepted in the art for delivering biologically active compounds to organisms (eg, humans). The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present invention to an organism.

[0038] The term "pharmaceutically acceptable carrier" refers to those carriers that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. "Pharmaceutically acceptable carrier" refers to an inert substance that is administered together with the active ingredient and is conducive to the administration of 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 approved by the U.S. Food and Drug Administration as acceptable for use in humans or animals (e.g., livestock). Non-limiting examples of the carrier include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycol.

[0039] The administration of the compounds of the present invention or their pharmaceutically acceptable salts in pure form or in the form of suitable pharmaceutical compositions can be carried out by any acceptable mode of administration of medicaments of similar use. The pharmaceutical composition of the present invention can be prepared by combining the compounds of the present invention with suitable pharmaceutically acceptable carriers, diluents or excipients. 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 and aerosols, etc.

[0040] Typical routes of administration of the compounds of the invention or their pharmaceutically acceptable salts or their pharmaceutical compositions include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. The preferred route of administration is oral administration.

[0041] The pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulating methods, making sugar-coated pills methods, grinding methods, emulsifying methods, freeze-drying methods, and the like.

[0042] In a preferred embodiment, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with a pharmaceutically acceptable carrier well known in the art. These carriers enable the compounds of the present invention to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.

[0043] Solid oral pharmaceutical compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by the following method: the active compound is mixed with a solid excipient, the mixture is optionally ground, other suitable adjuvants are added if necessary, and then the mixture is processed into particles to obtain the core of a tablet or a sugar-coated agent. Suitable adjuvants include, but are not limited to, adhesives, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, etc. Such as microcrystalline cellulose, glucose solution, acacia gum, gelatin solution, sucrose and starch paste; talc, starch, magnesium stearate, calcium stearate or stearic acid; lactose, sucrose, starch, mannitol, sorbitol or dicalcium phosphate; silicon dioxide; cross-linked sodium carboxymethylcellulose, pre-cross-linked starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methylcellulose, agar, carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, etc. The core of the sugar-coated agent can be optionally coated according to methods known in common pharmaceutical practice, especially using enteric coatings.

[0044] The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Appropriate excipients such as fillers, buffers or surfactants can be used.

[0045] Another aspect of the present invention relates to the use of compounds of Formula I to Formula VI or pharmaceutically acceptable salts, solvates, polymorphs, metabolites, etc., in drugs for treating diseases that benefit from SHP2 inhibition. The diseases that benefit from SHP2 inhibition are selected from cancer.

[0046] The substituted alkynyl heterocyclic compounds provided by the present invention have very good SHP2 inhibitory activity and are expected to become highly effective SHP2 inhibitor drugs. DETAILED DESCRIPTION

[0047] The following specific examples are intended to enable those skilled in the art to more clearly understand and implement the present invention. They should not be considered as limiting the scope of the present invention, but are merely exemplary and typical representations of the present invention. It will be appreciated by those skilled in the art that there are other synthetic pathways for forming the compounds of the present invention, and non-limiting examples are provided below.

[0048] All operations involving raw materials that are easily oxidized or hydrolyzed are carried out under nitrogen protection. Unless otherwise specified, the raw materials used in the present invention are directly purchased from the market and used directly without further purification.

[0049] Column chromatography used silica gel (200-300 mesh) produced by Qingdao Chemical Co., Ltd. Thin layer chromatography used prefabricated plates (silica gel 60PF 254 Chiral compound separation and enantiomeric excess (ee) determination were performed using an Agilent LC 1200 series (column: CHIRALPAK AD-H, x 250 mm, 5 μm, 30°C). Nuclear magnetic resonance chromatography (NMR) was measured using a Varian VNMRS-400 nuclear magnetic resonance instrument; liquid chromatography-mass spectrometry (LC / MS) was performed using a FINNIGAN Thermo LCQ Advantage MAX, Agilent LC 1200 series (column: Waters Symmetry C18, x 50 mm, 5 μm, 35°C), using ESI (+) ion mode.

[0050] Experimental Section

[0051] Experimental Section

[0052] Intermediate 1: (R)-N-((S)-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidinyl]-6-yl)-2-methyl 2-Propanesulfenamide trifluoroacetate

[0053]

[0054] The target compound was synthesized according to the method in Example 8 of patent CN113493440A.

[0055] Intermediate 2: 6-Chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazine

[0056]

[0057] The target compound was synthesized according to the same method as Preparation 178 on page 237 of patent WO2019167000.

[0058] Intermediate 3: (R)-N-((S)-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[iota] [4,3-b]pyrazin-6-yl)-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidin]-6-yl)-2-methylpropane-2- Sulfenamide

[0059]

[0060] Intermediate 1 (412 mg), diisopropylethylamine (1 mL), intermediate 2 (411 mg) and n-butanol (5 mL) were added to a sealed tube in sequence, filled with nitrogen, heated to 120°C and stirred for 2 hours, cooled to room temperature, concentrated under reduced pressure to remove the organic solvent, and the residue was separated by silica gel column chromatography (dichloromethane: methanol, 30: 1) to obtain the target product (2.82 g). MS m / z [LC-MS]: 688.14 [M+1].

[0061] Intermediate 4: 9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine

[0062]

[0063] Step 1: 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine

[0064] 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (4.0 g) and N-iodosuccinimide (5.72 g) were added to acetonitrile (25 mL), heated to 100°C for microwave reaction for 25 minutes, cooled to room temperature, concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain the target product (5.4 g). MS m / z [LC-MS]: 314.87 [M+1].

[0065] Step 2: 3-dian-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine

[0066] 3-iodo-4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (1.2 g) and concentrated aqueous ammonia (25%-28%, 2 mL) were added to acetonitrile (20 mL), stirred at room temperature overnight, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1) to obtain the target product (1.1 g). MS m / z [LC-MS]: 295.92 [M+1].

[0067] Step 3: 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

[0068] 3-iodo-6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1.1 g) and potassium carbonate (1.3 g) were added to N,N-dimethylformamide (20 mL), and then 4-methoxybenzyl chloride (0.5 mL) was added dropwise and stirred at room temperature for 2 hours. The reaction solution was poured into 150 mL of water and extracted with dichloromethane. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to obtain the target product (0.90 g). MS m / z[LC-MS]: 415.98[M+1].

[0069] Step 4: 9-iodo-5-chloro-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidine

[0070] 3-iodo-6-chloro-1-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (700 mg) and chloroacetaldehyde (2 mL) were added to acetonitrile (20 mL), heated to 100°C and sealed for reaction for 5 hours, poured into water, adjusted the pH value to 9-10 with saturated sodium carbonate aqueous solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain the target product (460 mg). MS m / z[LC-MS]: 439.98[M+1]. 1 H NMR (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).

[0071] Intermediate 5: (S)-5-Fluoro-1,3-dihydrospiro[indene-2,4′-piperidinyl]-1-amine dihydrochloride

[0072]

[0073] The target compound was synthesized according to the method of intermediate BX in patent WO2019183367A1.

[0074] Intermediate 6: (S)-(5-Fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4, tert-Butyl 3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidinyl]-1-yl)carbamate

[0075]

[0076] Step 1: (S)-5-Fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0077] Referring to the method of intermediate 3, intermediate 4 and intermediate 5 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 624.14 [M+1].

[0078] Step 2: (S)-tert-butyl (5-fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0079] (S)-5-fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine (187 mg) was added to dichloromethane (3 mL), and then diisopropylethylamine (0.2 mL) was added. Di-tert-butyl dicarbonate (130 mg) was added dropwise under ice-water cooling. After the addition was completed, the temperature was raised to room temperature and stirred for 4 hours. The organic solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to obtain the target product (200 mg). MS m / z[LC-MS]: 724.19[M+1].

[0080] Intermediate 7: (S)-(5-Fluoro-1′-(3-iodo-1-(((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[ ... tert-Butyl [3,4-b]pyrazin-6-yl)-1,2-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0081]

[0082] Step 1: (S)-5-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0083] Referring to the method of intermediate 3, intermediate 2 and intermediate 5 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 595.15 [M+1].

[0084] Step 2: (S)-tert-butyl(5-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0085] Referring to the method of step 2 in intermediate 6, (S)-5-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine was used as a raw material to obtain the target product. MS m / z [LC-MS]: 695.20 [M+1].

[0086] Intermediate 8: (S)-(5-Fluoro-1′-(3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)oxy)methyl) 1-yl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)amino Tert-Butyl Formate

[0087]

[0088] The starting intermediate (6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazine-5-yl)methanol was synthesized according to the same method as Preparation 179 on page 237 of patent WO2019167000.

[0089] Step 1: 6-Chloro-3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazine

[0090] The intermediate (6-chloro-3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-5-yl)methanol (2.28 g), triisopropylsilyl chloride (1.49 g) and imidazole (693 mg) were added to dichloromethane (40 mL) and stirred at room temperature overnight. The reaction solution was washed with water, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 5: 1) to obtain the target product (2.75 g). MS m / z [LC-MS]: 597.14 [M+1].

[0091] Step 2: (S)-5-Fluoro-1′-(3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0092] 6-Chloro-3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazine (596 mg) was added to n-butanol (10 mL), and then intermediate 5 (293 mg) and diisopropylethylamine (1 mL) were added, heated to 120° C. and stirred for 2 hours, cooled to room temperature, and concentrated under reduced pressure to remove the organic solvent. The residue was separated by silica gel column chromatography (dichloromethane: methanol, 15: 1) to obtain the target product (475 mg). MS m / z [LC-MS]: 781.3 [M+1].

[0093] Step 3: (S)-tert-butyl(5-fluoro-1′-(3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0094] (S)-5-fluoro-1′-(3-iodo-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine (470 mg) was added to dichloromethane (5 mL), and diisopropylethylamine (0.5 mL) was added. Di-tert-butyl dicarbonate (262 mg) was added dropwise under ice-water cooling. After the addition was completed, the temperature was raised to room temperature and stirred for 4 hours. The organic solvent was removed by concentration under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8: 1) to obtain the target product (490 mg). MS m / z[LC-MS]: 881.35[M+1].

[0095] Intermediate 9: (R)-3H-Spiro[benzofuran-2,4′-piperidinyl]-3-amine dihydrochloride

[0096]

[0097] The target compound was synthesized according to the method of intermediate CB in patent WO2019183367A1.

[0098] Intermediate 10: (R)-(1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3- b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin-3-yl)carbamic acid tert-butyl ester

[0099]

[0100] Step 1: (R)-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0101] Referring to the method of intermediate 3, intermediate 2 and intermediate 9 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 579.14 [M+1].

[0102] Step 2: (R)-tert-butyl(1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0103] Referring to the method of step 2 in intermediate 6, (R)-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine was used as the starting material to obtain the target product. MS m / z [LC-MS]: 679.19 [M+1].

[0104] Intermediate 11: (S)-6-Fluoro-13-dihydrospiro[indene-2,4′-piperidinyl]-1-amine dihydrochloride

[0105]

[0106] The target compound was synthesized according to the method of intermediate Y in patent WO2019183367A1.

[0107] Intermediate 12: (S)-(5-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[1,2-dihydro-1,2-dihydro-2-(2-(trimethylsilyl)ethoxy)methyl) ... tert-Butyl [3,4-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamate

[0108]

[0109] Step 1: (S)-6-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0110] Referring to the method of intermediate 3, intermediate 2 and intermediate 11 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 595.15 [M+1].

[0111] Step 2: (S)-tert-butyl(5-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamate

[0112] Referring to the method of step 2 in intermediate 6, (S)-6-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine was used as a raw material to obtain the target product. MS m / z [LC-MS]: 695.20 [M+1].

[0113] Intermediate 13: (R)-(1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e] tert-Butyl pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin-3-yl)carbamate

[0114]

[0115] Step 1: (R)-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0116] Referring to the method of intermediate 3, intermediate 4 and intermediate 9 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 608.13 [M+1].

[0117] Step 2: (R)-tert-butyl(1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0118] Referring to the method of step 2 in intermediate 6, (R)-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine was used as the starting material to obtain the target product. MS m / z [LC-MS]: 708.18 [M+1].

[0119] Intermediate 14: (S)-(5-Fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo ... tert-Butyl [4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidinyl]-3-yl)carbamate

[0120]

[0121] Step 1: (S)-6-Fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0122] Referring to the method of intermediate 3, intermediate 4 and intermediate 11 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 624.14 [M+1].

[0123] Step 2: (S)-tert-butyl (5-fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamate

[0124] Referring to the method of step 2 in intermediate 6, (S)-6-fluoro-1′-(9-iodo-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine was used as a raw material to obtain the target product. MS m / z [LC-MS]: 724.19 [M+1].

[0125] Intermediate 15: (R)-6-Fluoro-3H-spiro[benzofuran-2,4′-piperidinyl]-3-amine dihydrochloride

[0126]

[0127] The target compound was synthesized according to the method of intermediate FI in patent WO2019183367A1.

[0128] Intermediate 16: (R)-(6-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[1,2-dihydro-1,2-dihydro-2-(2-(trimethylsilyl)ethoxy)methyl) ... tert-Butyl [4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0129]

[0130] Step 1: (R)-6-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0131] Referring to the method of Intermediate 3, Intermediate 2 and Intermediate 15 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 597.13 [M+1].

[0132] Step 2: (R)-tert-butyl(6-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0133] Referring to the method of step 2 in intermediate 6, (R)-6-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine was used as the starting material to obtain the target product. MS m / z [LC-MS]: 697.18 [M+1].

[0134] Intermediate 17: (R)-5-Fluoro-3H-spiro[benzofuran-2,4′-piperidinyl]-3-amine dihydrochloride

[0135]

[0136] The target compound was synthesized according to the method of intermediate FD in patent WO2019183367A1.

[0137] Intermediate 18: (R)-(5-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[1,2-dihydro-1,2-dihydro-2-(2 ... [4.3-b] tert-butyl [pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0138]

[0139] Step 1: (R)-5-Fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0140] Referring to the method of Intermediate 3, Intermediate 2 and Intermediate 17 were used as raw materials to obtain the target product. MS m / z [LC-MS]: 597.13 [M+1].

[0141] Step 2: (R)-tert-butyl(5-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0142] Referring to the method of step 2 in intermediate 6, (R)-5-fluoro-1′-(3-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine was used as the starting material to obtain the target product. MS m / z [LC-MS]: 697.18 [M+1].

[0143] Embodiment 1: (S)-1′-(2-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)- 4,6-Dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidin]-6-amine

[0144]

[0145] Step 1: (R)-N-((S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidin]-6-yl)-2-methylpropane-2-sulfenamide

[0146] Under nitrogen protection, a mixture of intermediate 3 (137 mg), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (50 mg), sodium carbonate (64 mg), tetrakis(triphenylphosphine)palladium (12 mg) and a 10:1 mixed solvent of dioxane / water (10 mL) was heated to 80°C and stirred overnight. The mixture was cooled to room temperature, poured into water, extracted with dichloromethane, and the extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane: methanol, 30: 1) to obtain the target product (88 mg). MS m / z [LC-MS]: 644.29 [M+1].

[0147] Step 2: (S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidin]-6-amine

[0148] (R)-N-((S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4′-piperidin]-6-yl)-2-methylpropane-2-sulfinamide (85 mg) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (1 mL) was added, the mixture was stirred at room temperature for 1 hour, the solvent was removed by concentration under reduced pressure, 10% aqueous sodium carbonate solution (10 mL) was added, the mixture was extracted with dichloromethane, the extract was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was separated by thin layer silica gel chromatography (dichloromethane: methanol, 10: 1) to obtain the target product (35 mg). MS m / z [LC-MS]: 410.18 [M+1]. 1 HNMR (400MHz, CD3OD): δ = 9.15 (s, 1H), 8.33 (s, 1H), 7.19 (s, 1H), 4.54-4.62 (m, 1H), 4.41-4.51 (m, 2H), 4.34-4.38 (m, 2H), 3. 94 (t, J=5.6Hz, 2H), 3.34-3.47 (m, 2H), 3.21 (d, J=16.0Hz, 1H), 3.05 (d, J=16.0Hz, 1H), 2.64-2.70 (m, 2H), 1.76-2.02 (m, 4H).

[0149] Embodiment 2: (S)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3- e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0150]

[0151] Step 1: (S)-tert-butyl (1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0152] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 6 as a raw material. MS m / z [LC-MS]: 680.34 [M+1].

[0153] Step 2: (S)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0154] Referring to the method of step 2 in Example 1, the target product was obtained using (S)-(1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamic acid tert-butyl ester as a raw material. MS m / z [LC-MS]: 460.23 [M+1]. 1 HNMR (400MHz, CD3OD): δ=7.74-7.78 (m, 1H), 7.71 (d, J=1.6Hz, 1H), 7.47 (dd, J=8.8Hz, 4.8Hz, 1H ), 7.44 (d, J=1.6Hz, 1H), 7.08 (dd, J=8.8Hz, 2.4Hz, 1H), 7.02 (td, J=8.8Hz, 2.4Hz, 1H), 4.44 (q, J =2.4Hz, 2H), 4.30 (s, 1H), 3.97 (t, J = 5.6Hz, 2H), 3.76-3.88 (m, 2H), 3.29-3.37 (m, 2H), 3.17 (d, J=16.0Hz, 1H), 3.07 (d, J=16.0Hz, 1H), 2.70-2.74 (m, 2H), 1.99-2.13 (m, 2H), 1.66-1.78 (m, 2H).

[0155] Embodiment 3: (S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)- 5-Fluoro-1,3-dihydrospiro[indene-2,4′-piperidinyl]-1-amine

[0156]

[0157] Step 1: (S)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0158] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 7 as a raw material. MS m / z [LC-MS]: 651.35 [M+1].

[0159] Step 2: (S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0160] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (S)-(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate as a raw material. MS m / z [LC-MS]: 421.22 [M+1]. 1 HNMR (400MHz, CD3OD): δ=8.32 (s, 1H), 7.47 (dd, J=8.8Hz, 4.8Hz, 1H), 7.18 (s, 1H), 7. 10 (dd, J=8.8Hz, 2.0Hz, 1H), 7.04 (td, J=8.8Hz, 2.0Hz, 1H), 4.42-4.49 (m, 1H), 4.32-4 .39 (m, 3H), 4.28 (s, 1H), 3.94 (t, J = 5.6Hz, 2H), 3.31-3.42 (m, 2H), 3.19 (d, J = 16.8Hz, 1H), 3.13 (d, J=16.8Hz, 1H), 2.64-2.69 (m, 2H), 1.76-1.91 (m, 2H), 1.60-1.74 (m, 2H).

[0161] Embodiment 4: (S)-(6-(1-amino-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidinyl]-1′-yl)-3-(3,6-dihydro- Hydrogen-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-5-yl)methanol

[0162]

[0163] Step 1: (S)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate

[0164] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 8 as a raw material. MS m / z [LC-MS]: 837.49 [M+1].

[0165] Step 2: (S)-(6-(1-amino-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1′-yl)-3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-5-yl)methanol

[0166] (S)-tert-butyl (1′-(3-(3,6-dihydro-2H-pyran-4-yl)-5-(((triisopropylsilyl)oxy)methyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-yl)carbamate was dissolved in tetrahydrofuran solution (0.1 M), and tetrabutylammonium fluoride (2.5 eq) was added and stirred at room temperature for 2 hours. After the solvent was removed by concentration under reduced pressure, the reaction and treatment were carried out according to the method of step 2 in Example 1 to obtain the target product. MS m / z [LC-MS]: 451.23 [M+1]. 1 HNMR (400MHz, CD3OD): δ=7.49 (dd, J=8.8Hz, 4.8Hz, 1H), 7.40 (s, 1H), 7.11 (dd, J=8.8H z, 2.0Hz, 1H), 7.05 (td, J=8.8Hz, 2.0Hz, 1H), 4.78 (s, 2H), 4.60 (s, 1H), 4.36-4.40 (m, 2 H), 3.96 (t, J=5.6Hz, 2H), 3.75-3.82 (m, 1H), 3.66-3.74 (m, 1H), 3.20-3.28 (m, 2H), 3. 14(s, 2H), 2.67-2.73(m, 2H), 1.90-2.07(m, 2H), 1.73-1.80(m, 1H), 1.62-1.70(m, 1H).

[0167] Embodiment 5: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)- 3H-Spiro[benzofuran-2,4′-piperidinyl]-3-amine

[0168]

[0169] Step 1: (R)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0170] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 10 as a raw material. MS m / z [LC-MS]: 635.34 [M+1].

[0171] Step 2: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0172] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (R)-(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate as a raw material. MS m / z [LC-MS]: 405.20 [M+1]. 1 HNMR (400MHz, CD3OD): δ=8.24 (s, 1H), 7.32 (d, J=7.2Hz, 1H), 7.23-7.26 (m, 1H), 7.21 (t, J=7.6Hz, 1H), 6.92 (t, J=7.6Hz, 1H), 6.82 (d, J=7.6Hz, 1H), 4 .36-4.44(m, 3H), 4.25-4.32(m, 1H), 4.12(s, 1H), 3.98(t, J=6.0Hz, 2H), 3 .53-3.61(m, 2H), 2.70-2.77(m, 2H), 1.87-2.02(m, 3H), 1.75-1.82(m, 1H).

[0173] Embodiment 6: (S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)- 6-Fluoro-1,3-dihydrospiro[indene-2,4′-piperidinyl]-1-amine

[0174]

[0175] Step 1: (S)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamate

[0176] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 12 as a raw material. MS m / z [LC-MS]: 651.35 [M+1].

[0177] Step 2: (S)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-6-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0178] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (S)-(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamate as a raw material. MS m / z [LC-MS]: 421.22 [M+1]. 1 HNMR (400MHz, CD3OD): δ=8.28 (s, 1H), 7.15-7.21 (m, 2H), 7.08 (dd, J=8.8Hz, 2.0Hz, 1H), 6.88-6.93 (m, 1H), 4.34-4.42 (m, 4H), 3.92-3.95 (m, 3H), 3.24-3. 34(m, 2H), 3.13(d, J=15.6Hz, 1H), 2.76(d, J=15.6Hz, 1H), 2.64-2.68(m, 2H) , 1.86-1.94(m, 1H), 1.74-1.82(m, 1H), 1.59-1.65(m, 1H), 1.37-1.44(m, 1H).

[0179] Embodiment 7: (R)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[43- e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0180]

[0181] Step 1: (R)-tert-butyl (1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0182] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 13 as a raw material. MS m / z [LC-MS]: 664.32 [M+1].

[0183] Step 2: (R)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0184] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (R)-(1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate as a raw material. MS m / z [LC-MS]: 444.21 [M+1]. 1 HNMR (400MHz, CD3OD): δ=7.74-7.80 (m, 2H), 7.44 (s, 1H), 7.41 (d, J=7.2Hz, 1H), 7.20 (t, J=8.0Hz, 1H), 6.91 (t, J=7.2Hz, 1H), 6.81 (d, J=8.0Hz, 1H), 4. 44(q, J=2.8Hz, 2H), 4.22(s, 1H), 3.97(t, J=5.6Hz, 2H), 3.77-3.86(m, 2H), 3.44-3.58 (m, 2H), 2.70-2.75 (m, 2H), 2.00-2.30 (m, 3H), 1.87-1.93 (m, 1H).

[0185] Embodiment 8: (S)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3- e]pyrimidin-5-yl)-6-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0186]

[0187] Step 1: (S)-tert-butyl (1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]3-yl)carbamate

[0188] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 14 as a raw material. MS m / z [LC-MS]: 680.34 [M+1].

[0189] Step 2: (S)-1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-6-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-1-amine

[0190] Referring to the method of step 2 in Example 1, the target product was obtained using (S)-(1′-(9-(3,6-dihydro-2H-pyran-4-yl)-7-(4-methoxybenzyl)-7H-imidazo[1,2-c]pyrazolo[4,3-e]pyrimidin-5-yl)-5-fluoro-1,3-dihydrospiro[indene-2,4′-piperidin]-3-yl)carbamic acid tert-butyl ester as a raw material. MS m / z [LC-MS]: 460.23 [M+1]. 1 HNMR (400MHz, CD3OD): δ=7.75 (s, 1H), 7.71 (d, J=1.6Hz, 1H), 7.42 (d, J=1.6Hz, 1H), 7.22 (dd, J= 8.4Hz, 5.2Hz, 1H), 7.13 (dd, J=8.8Hz, 2.4Hz, 1H), 6.91-6.96 (m, 1H), 4.43 (q, J=2.4Hz, 2H), 4.07 (s, 1H), 3.96 (t, J=6.0Hz, 2H), 3.78-3.84 (m, 2H), 3.22-3.32 (m, 2H), 3.13 (d, J=16.0Hz, 1H), 2.8 1(d, J=16.0Hz, 1H), 2.69-2.75(m, 2H), 1.99-2.14(m, 2H), 1.67-1.73(m, 1H), 1.50-1.56(m, 1H).

[0191] Embodiment 9: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)- 6-Fluoro-3H-spiro[benzofuran-2,4′-piperidinyl]-3-amine

[0192]

[0193] Step 1: (R)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-6-fluoro-3H-spiro[benzofuran-2,4′-piperidin]3-yl)carbamate

[0194] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 16 as a raw material. MS m / z [LC-MS]: 653.33 [M+1].

[0195] Step 2: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-6-fluoro-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0196] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (R)-(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-6-fluoro-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate as a raw material. MS m / z [LC-MS]: 423.19 [M+1].

[0197] Embodiment 10: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazine-6- 1-[4-(4-(4-piperidinyl)-5-fluoro-3H-spiro[benzofuran-2,4′-piperidinyl]3-amine

[0198]

[0199] Step 1: (R)-tert-butyl(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate

[0200] Referring to the method of step 1 in Example 1, the target product was obtained using intermediate 18 as a raw material. MS m / z [LC-MS]: 653.33 [M+1].

[0201] Step 2: (R)-1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-3H-spiro[benzofuran-2,4′-piperidin]-3-amine

[0202] Referring to the method of step 2 in Example 1, the target product was obtained using tert-butyl (R)-(1′-(3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-5-fluoro-3H-spiro[benzofuran-2,4′-piperidin]-3-yl)carbamate as a raw material. MS m / z [LC-MS]: 423.19 [M+1].

[0203] Biological Testing

[0204] Determination of the inhibitory effect of compounds on SHP2 in vitro enzyme activity

[0205] In this patent, the enzymatic activity detection of SHP2 is carried out by rapid fluorescence method, DiFMUP is used as an alternative substrate for reaction and a high-throughput screening platform is optimized and established. The detection of the inhibitory activity of the compound on SHP2 is operated on this platform. The specific method is as follows: a mixture of SHP2 with a final concentration of 1nM and a diphosphorylated IRS 1 peptide (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) is pre-incubated at 23°C for 30 minutes. The compound is diluted 5 times with 100% DMSO starting from 0.2mM (7 concentrations in total), and 2μL of the compound is added to 48μL of reaction buffer (60mM HEPES, pH 7.2, 75mMNaCl, 75mM KCl, 1mM EDTA, 0.05% Tween 20, 5mMDTT) for dilution and mixing. Take 5 μL of the diluted compound and add it to a black 384-well plate (OptiPlate-384, product number 6007270, purchased from PerkinElmer), then add 10 μL of the pre-incubated SHP2 and IRS1 peptide mixture, centrifuge to mix, and incubate at 23°C for 30 minutes. Add 5 μL of the alternative substrate DiFMUP (final concentration 50 μM, product number D6567, purchased from Invitrogen) to the reaction and incubate at 23°C for 60 minutes. The reaction was then terminated by adding 5 μL of 160 μM bpV (Phen) solution (SC-22137, purchased from Santa). Immediately after the reaction was terminated, an ELISA reader (Perkin-Elmer) was used to detect the fluorescence signal at excitation and emission wavelengths of 340 nm and 450 nm, respectively. The data was calculated using GraphPad Prism software to obtain the IC50 value of the compound. It was found that the specific compounds in the embodiments of the present invention all have strong SHP2 in vitro enzymatic inhibitory activity, IC 50 The values ​​are in the range of 1 nM to 10 nM. The activities of some compounds are listed in Table 1:

[0206] Table 1. Inhibitory activity of some compounds in the examples on SHP2 enzyme

[0207] Example <![CDATA[IC 50 (nM)]]> 1 7.56 2 2.56 3 2.69 4 4.60 5 6.14 6 5.28 7 9.82 8 4.24

[0208] Determination of the inhibitory effect of compounds on the proliferation of SHP2-positive cells

[0209] Human non-small cell lung cancer cell line NCI-H358 cells were cultured using RPMI-1640 medium (Cat. No. C11875500BT, purchased from Biological Industries) plus 10% fetal bovine serum (FBS, Cat. No. 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin dual antibody (P / S, Cat. No. 15070-063, purchased from Gibco), and the culture conditions were 37°C, 5% CO2. One day before the 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 μL / well. After 24 hours, the compound was diluted 3 times from 10 mM with 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was taken at each concentration and added to 48 μL of serum-free and dual antibody-free culture medium for dilution. 5 μL of each diluted compound concentration was added to the cell suspension, and the compound and cells were incubated in a cell culture incubator for 72 hours (3 days). After the culture medium was removed, 25 μL of Cell-Titer Glo (G7570, purchased from Promega) reagent was added and incubated again for 5-10 minutes. The fluorescence value was then read on Envision, and the data were calculated using GraphPad Prism software to obtain the IC value of the compound's inhibition of cell proliferation. 50Value. Kasumi-1 cells, a human acute myeloblastic leukemia cell line, were cultured using RPMI-1640 medium (Cat. No. C11875500BT, purchased from Biological Industries) plus 20% fetal bovine serum (FBS, Cat. No. 04-001-1ACS, purchased from Biological Industries, BI) and 1% penicillin / streptomycin dual antibody (P / S, Cat. No. 15070-063, purchased from Gibco) at 37°C and 5% CO2. One day before compound detection, Kasumi-1 cells were plated in a 196-well plate (Cat. No. 3599, purchased from Corning) at a concentration of 3000 cells / 195 μL / well. After 24 hours, the compound was diluted 3-fold from 10 mM using 100% DMSO (a total of 10 concentrations), and then 2 μL of the compound was taken at each concentration and diluted in 48 μL of culture medium without serum and dual antibody. 5 μL of each diluted compound concentration was added to the cell suspension, and the compound and cells were incubated in a cell culture incubator for 72 hours (3 days), and 35 μL of Cell-Titer Blue (G8082, purchased from Promega) reagent was added and incubated again for 4 hours. After that, the fluorescence value was read on Flexstation III (560 nm excitation, 590 nm detection), and the data were calculated using GraphPad Prism software to obtain the IC value of the compound's inhibition of cell proliferation. 50 It was found through testing that the specific compounds in the examples of the present invention all have strong inhibitory activity on cell proliferation, with IC50 values ​​ranging from 10 nM to 1 μM. The test results of some compounds are shown in Table 2.

[0210] Table 2. Inhibitory activity of the example compounds on cell (NCI-H358) proliferation

[0211] Example IC50(nM) 1 54.0 2 19.4 3 46.2 4 20.5 5 51.5 6 48.6 8 12.9

[0212] Determination of pharmacokinetic data of compounds in SD rats

[0213] Male SD rats were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The rats were divided into groups, 3 in each group, and the suspension of the sample to be tested (5 mg / kg, the suspension was 20% EtOH, 40% PEG400, 40% H2O) was orally administered. The animals were fasted overnight before the experiment, and the fasting time was from 10 hours before administration to 4 hours after administration. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. After anesthesia with isoflurane using a small animal anesthesia machine, 0.3 mL of whole blood was collected through the fundus venous plexus and placed in a heparin anticoagulant tube. The sample was centrifuged at 4°C and 4000 rpm for 5 minutes, and the plasma was transferred to a centrifuge tube and stored at -80°C until analysis. The sample in the plasma was extracted using a protein precipitation method, and the extract was analyzed by LC / MS. The detection results of some compounds are shown in Table 2.

[0214] Table 3. Pharmacokinetic parameters of the compounds of the examples

[0215]

[0216] Table 3 lists the pharmacokinetic data of the compounds of the present invention in SD rats. The data show that the compounds provided by the present invention have relatively good in vivo metabolism levels.

[0217] Determination of the inhibitory activity of compounds on CYP450 isoform 3A4

[0218] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis was used to detect the relative activities of the test substances and positive controls on the metabolites of the major subtype CYP3A4 of mixed human liver microsomal CYP450 enzymes, and the IC50 values ​​of the inhibitory effects of the positive control and the test substances on mixed human liver microsomal cytochrome P450 enzymes were calculated to evaluate the in vitro inhibitory effects of the test substances on CYP3A4, the major subtype of CYP450 enzymes.

[0219] The experiment was divided into a positive control group and a test group. The positive control or test was incubated with human liver microsomes and the probe substrate of the CYP3A4 enzyme, including human liver microsomes (0.05 mg / mL), NADPH (1.5 mM), PBS buffer (100 mM, pH = 7.4), probe substrate (midazolam 4 μM or testosterone 40 μM) and inhibitor (the final concentration of the test substance was 0, 1, 2.5, 5.0, 10.0, 25.0 μM; the final concentration of the positive control ketoconazole was 0, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 μM.), and the total incubation volume was 100 μL. The specific steps are as follows:

[0220] 2.1 Preparation of liver microsome dilution

[0221] A 20 mg / mL human liver microsome stock solution was thawed on ice and diluted 40 times with PBS buffer (100 mM, pH=7.4) to prepare a 0.5 mg / mL liver microsome dilution solution.

[0222] 2.2 Preparation of mixed incubation solution

[0223] A mixed incubation solution containing the liver microsome dilution and substrate solution (midazolam or testosterone) was prepared using PBS buffer (100 mM, pH=7.4).

[0224] 2.3 Pre-incubate the mixed incubation solution in a constant temperature oscillator at 37°C and 100 rpm for 5 minutes.

[0225] 2.4 After pre-incubation, different concentrations of test substance working solution or positive control ketoconazole working solution were added (the final concentration of the test substance was 0, 1, 2.5, 5.0, 10.0, 25.0 μM; the final concentration of positive control ketoconazole was 0, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 μM.), vortexed and mixed, and NADPH (final concentration 1.5 mM) was added to start the reaction, and the mixture was kept at 37°C for 10 min. 0rpm constant temperature oscillator, the total incubation volume is 100μL, the concentration of organic solvent in the incubation system is within 1%, after a certain period of incubation (the probe substrate is midazolam solution incubation for 10 minutes, the probe substrate is testosterone solution incubation for 15 minutes), 150μL of ice internal standard solution is added to terminate the reaction, vortexed and centrifuged at 12000rpm for 10 minutes, 200μL of supernatant is taken for UPLC-MS / MS injection analysis, and the peak area of ​​each metabolite and internal standard propranolol is calculated by MassLynx V4.1 SCN962 software, and then the peak area ratio of each metabolite (metabolite peak area / internal standard peak area) is calculated using Excel software. The inhibition IC50 values ​​of the positive control group and the test substance are obtained by directly calculating two concentration points in Excel, or calculated by Graphpad Prism (version 6.01) software.

[0226] Table 4. Inhibition of the major isoform of CYP450 enzymes by the example compounds on isoform 3A4

[0227]

[0228]

[0229] Table 4 lists the IC50 values ​​of the inhibitory effects of the compounds of the present invention on the major subtype 3A4 of the CYP450 enzyme (testosterone as substrate). The data show that the inhibitory effects of the compounds provided by the present invention on the major subtype 3A4 of the CYP450 enzyme are very weak, which is beneficial to avoid the potential risk of drug interactions and improve the safety of clinical medication.

[0230] Determination of hERG inhibition by compounds

[0231] A patch clamp amplifier system (Multiclamp 700B Amplifier) ​​(AXON), an Olympus (Olympus IX51 / 71 / 73) microscope, and an MP285 micromanipulator were used.

[0232] The HEK293 cell line stably transfected with hERG was purchased from Invitrogen, Inc. The cells were grown in a medium containing 85% DMEM, 10% fetal bovine serum, 0.1 mM non-essential amino acids, 25 mM HEPES buffer, 100 U / mL penicillin-streptomycin, 5 μg / mL blasticidin, and 400 μg / mL G418 geneticin.

[0233] Cells were passaged three times a week using TrypLE Express to maintain approximately 40%-80% confluency. Before testing, 5 × 10 cells were plated per 6 cm diameter dish. 5 The cells were seeded at a density of 10 cells / mL and induced with 1 μg / mL doxycycline for 48 hours.

[0234] The compound solutions were tested at concentrations of 10, 1, and 0.1 μM, each diluted 1000-fold, and the final DMSO concentration was in the range of 0.1%.

[0235] Adjust the manipulator to move the electrode tip toward the cell surface to form a high-position blockade. Compensate for the liquid interface potential and fast capacitance, and aspirate the cell membrane to form a whole-cell recording mode. Set the membrane potential to -60 mV to ensure that the hERG channel is not open. Use the C slow To eliminate the peak of the capacity current. Set the holding voltage to -90mV for 500ms. The leakage current test is performed at -80mV for 500ms. Depolarize to +30mV for 4.8 seconds to activate the hERG channel, then restore the voltage to -50mV for 5.2 seconds and observe the tail current.

[0236] Peak current suppression = [1-(peak tail current 抑制剂 - Peak tail current 阳性对照 ) / (peak tail current 空白 - Peak tail current 阳性对照 )]×100%.

[0237] Table 5. Inhibitory activity of the example compounds on hERG

[0238] Example Inhibition rate@1μM Inhibition rate@3μM Inhibition rate@10μM 2 4.44％±4.53％ / 63.59％±1.28％ 3 27.23％±1.88％ / 87.82％±0.43％

[0239] Table 5 lists the inhibition rate data of the compounds of the present invention at different concentrations on hERG. This indicates that the compounds provided by the present invention have relatively low inhibitory activity on hERG and may have relatively low cardiac toxicity.

[0240] Industrial Applicability

[0241] The compounds of the present invention can inhibit SHP2 protease activity to provide anti-tumor effects.

[0242] Join by reference

[0243] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, in, Ring A is a benzene ring or a 5-6 membered heteroaromatic ring, and the benzene ring and the heteroaromatic ring may be optionally substituted with halogen, -CN, -OH, -NH2, C 1-6 Alkyl, or -OC 1-6 Alkyl Substitution X1 and X2 are each independently -CHR1-, -NR2-, -O-, or -S-, R1 is H, halogen, -CN, -OH, -NH2, C 1-6 Alkyl, or -OC 1-6 alkyl, R2 is selected from H or C 1-6 alkyl. In some embodiments, X1 and X2 are each independently -CHR1-, -NR2-, or -O-, and R1 and R2 are as described above.

2. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein X1 and X2 are each independently -CHR1-, -NR2-, or -O-, and R1 and R2 are as described in claim 1.

3. The compound according to claim 1 or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein R1 is H or NH2.

4. The following compound or its pharmaceutically acceptable salt, solvate, polymorph, or tautomer:

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, and optionally comprising a pharmaceutically acceptable excipient.

6. Use of a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition according to claim 5, or a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition according to claim 5 in combination with a SHP2 inhibitor or a KRAS inhibitor or an EGFR inhibitor in the preparation of a medicament for treating diseases associated with SHP2 and / or KRAS and / or EGFR.

7. The use according to claim 6, wherein the disease associated with SHP2 and / or KRAS and / or EGFR is leukemia, melanoma, malignant glioma, lung cancer, breast cancer or Noonan syndrome.

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