CN117327101A - Substituted heterocyclic compounds - Google Patents

Substituted heterocyclic compounds Download PDF

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Publication number
CN117327101A
CN117327101A CN202210728176.3A CN202210728176A CN117327101A CN 117327101 A CN117327101 A CN 117327101A CN 202210728176 A CN202210728176 A CN 202210728176A CN 117327101 A CN117327101 A CN 117327101A
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China
Prior art keywords
pyrazolo
shp2
compound
fluoro
dihydro
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Inventor
段小伟
张凯
孙永亮
陈曦
李海州
崔先杰
许新合
刘希杰
孙颖慧
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Capital Pharmaceutical Holdings Beijing Co ltd
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Capital Pharmaceutical Holdings Beijing Co ltd
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    • 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
    • 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
    • C07D487/02—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
    • 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

Abstract

The present application relates to substituted alkynyl heterocyclic compounds of formula I. The present invention relates to substituted alkynyl heterocyclic compounds having inhibitory activity, processes for their preparation, pharmaceutical compositions thereof, and to the use of such compounds and pharmaceutical compositions thereof, for example in the treatment of cancer. In the preparation process, the compound of the formula I is obtained through the reactions of iodination, amination, upper protection, coupling, deprotection, ring closure reaction and the like.

Description

Substituted heterocyclic compounds
Technical Field
The present invention relates generally to novel substituted alkenyl heterocyclic compounds having SHP2 inhibitory activity, processes for their preparation, pharmaceutical compositions thereof, and to the use of such compounds and pharmaceutical compositions thereof for the treatment of diseases benefiting from SHP2 enzyme inhibition, such as for the treatment of cancer.
Background
Cancer is a serious disease which seriously threatens human health and life, especially the incidence and death rate of cancer in recent years are rapidly rising, and cardiovascular diseases are the first killers of human health. Tumor proliferation, apoptosis, metastasis, etc. are closely related to abnormalities in a link in a series of intracellular and extracellular signaling pathways. In these signaling pathways, protein phosphorylation and dephosphorylation are critical, 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 balance 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 a non-receptor protein tyrosine phosphatase coded by the ptpn11 gene, is widely expressed in various tissues, and participates in important physiological and pathological processes such as embryo development, metabolism, immune response, tumorigenesis and the like.
SHP2 proteins consist of two tandem SH2 domains at the N-terminus (N-SH 2 and C-SH 2), a PTP catalytic domain and a C-terminal tail with regulatory effects. 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-associated binding protein 1-GAB 1) 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 the SHP2 phosphatase activity in a self-inhibiting state. When the SH2 domain binds to the activator, the 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 perform phosphatase functions. The nature of this shift in activity of SHP2 makes it possible for various mutations in SHP2 to disrupt SHP2 self-inhibition, resulting in overactivation of SHP2 protein phosphatase activity and thus the initiation of cancerous changes. Both experimental and clinical data confirm that SHP2 plays a promoting role in most cancers, as the first tyrosine phosphatase discovered to promote cancer progression, which has received great attention in the cancer field, its phosphatase activity plays an important role in intracellular signal regulation.
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 signal pathways, thereby regulating physiological functions such as cell proliferation, differentiation, cell cycle maintenance and migration. Meanwhile, SHP2 also mediates compensatory activation pathways after MEK and other kinases are inhibited, so that the occurrence of tumor drug resistance is promoted. SHP2 is also involved in T cell inhibitory signaling as a downstream molecule of the PD-1 receptor. Studies have 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 may restore or enhance T cell mediated anti-tumor immune function. In addition, SHP2 can inhibit IFN-gamma mediated immune responses by inactivating signal transduction and transcriptional activator STAT 1.
In recent years, SHP2 activating mutations and high expression have been continuously found in leukemia, solid tumor, melanoma, glioblastoma, lung cancer, breast cancer and knoop-male 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 mechanism of action of conventional SHP2 inhibitors (e.g., II-B08, PHPS 1) is to bind to the PTP catalytic domain of SHP2, preventing tyrosine phosphorylated substrates from entering the catalytic site, thereby inhibiting the phosphatase activity of SHP 2. However, due to the highly conserved, polar and charged environments of the various phosphatase PTP catalytic domains, traditional inhibitors of SHP2 have major drawbacks in terms of specificity and bioavailability, limiting their clinical applications. Therefore, the development of SHP2 inhibitors with high specificity, high safety and strong cell membrane permeability is a key for determining whether SHP2 can become a novel tumor intervention target, and SHP2 protein allosteric inhibitors are the main direction of current research.
Disclosure of Invention
In one aspect, the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof,
wherein,
the A ring is a benzene ring or a 5-6 membered heteroaromatic ring, which may optionally be substituted with halogen, -CN, -OH, -NH 2 、C 1-6 Alkyl, or-O-C 1-6 Alkyl substitution
X 1 And X 2 Each independently is-CHR 1 -、-NR 2 -, -O-, or-S-,
R 1 is H, halogen, -CN, -OH, -NH 2 、C 1-6 Alkyl, or-O-C 1-6 An alkyl group, a hydroxyl group,
R 2 selected from H or C 1-6 An alkyl group.
In some embodiments, X 1 And X 2 Each independently is-CHR 1 -、-NR 2 -, or-O-, R 1 And R is 2 As described above.
In some embodiments, R 1 Is H or NH 2 。
In some embodiments, the invention provides the following compounds, or pharmaceutically acceptable salts, solvates, polymorphs, or tautomers thereof:
in another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, and optionally a pharmaceutically acceptable adjuvant.
In another aspect, the invention provides a method of treating a disorder 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 invention, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, or a pharmaceutical composition thereof.
In another aspect, the invention provides the use of a compound of the invention, or a pharmaceutically acceptable salt, solvate, polymorph or tautomer thereof, or a pharmaceutical composition thereof, or any of the foregoing, in combination with an inhibitor of SHP2 or an inhibitor of KRAS or an EGFR inhibitor, for the manufacture of a medicament for the treatment of a disease associated with SHP2 and/or KRAS and/or EGFR.
In some embodiments of the invention, the SHP2 and/or KRAS and/or EGFR-associated disease is leukemia, melanoma, glioblastoma, lung cancer, breast cancer, or knoop-male syndrome.
On the other hand, SHP2 acts upstream of KRAS, a Hana Algul team (Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphotase, nature Medicine 2018) demonstrated that SHP2 small molecule inhibitors have significant efficacy against invasive KRAS tumors such as Pancreatic Ductal Adenocarcinoma (PDAC) and non-small cell lung carcinoma (NSCLC), protein tyrosine phosphatase SHP2 has been a key drug target for invasive KRAS tumors, and furthermore, schneeberger, v.e. team (Inhibition of Shp2 suppresses Mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma. Oncotarget 2015) demonstrated that SHP2 can promote RAS-RAF-MEK-ERK signaling in EGFR mutated non-small cell lung carcinoma. Therefore, the SHP2 small molecule inhibitor of the present invention can be used alone or in combination with KRAS inhibitors (AMG 510, etc.) and EGFR inhibitors (Iressa, tarceva, etc.) in the prior art to effectively inhibit the occurrence and progression of tumors.
On the other hand, similarity in biological activity is often found in functional groups with similar steric or electronic properties. Structures that perform the same biological function in a drug molecule are called bioisosteres, which do not affect the primary biological activity of the drug by replacing an original functional group in the drug with another functional group. Substitution of monovalent atoms or groups such as OH, NH2, F, SH, etc., the carboxyl groups may be replaced with phosphate esters, tetrazoles, etc.
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., have 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 pure or racemic form. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.
The compounds of the invention also include tautomeric forms. Tautomers originate from the exchange of one single bond with an adjacent double bond and accompany the migration of one proton.
The invention also includes all isotopically-substituted atoms, whether in intermediate or final compounds. The atoms of the isotope include atoms having the same atomic number but different mass numbers. Isotopes of hydrogen include, for example, tritium and deuterium.
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.
The term "spiro" or "spiro" as used herein, alone or in combination, refers to a cyclic structure in which two or more rings share one or more atoms.
The term "halogen" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
The term "alkyl" refers to a straight or branched saturated hydrocarbon group consisting of carbon and hydrogen atoms, e.g. C 1-20 Alkyl, preferably C 1-6 Alkyl groups such as methyl, ethyl, propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl or tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), n-hexyl, 2-methylhexyl, and the like. 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 hydroxy.
The term "C 1-6 Alkyl "refers to a straight or branched saturated aliphatic hydrocarbon group consisting of carbon and hydrogen atoms, which is attached to the rest of the molecule by a single bond, having 1-6 carbon atoms. The alkyl group may be unsubstituted or substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, amino, halogen and hydroxy. Non-limiting examples of unsubstituted alkyl groups include, but are not limited to, alkyl groups such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, t-butyl, n-pentyl, 2-methylbutyl, neopentyl, n-hexyl, 2-methylhexyl, and the like.
The term "aryl" refers to an all-carbon monocyclic or fused ring having a fully conjugated pi-electron system, which has 6 to 14 carbon atoms, preferably 6 to 12 carbon atoms, and 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, halogen, 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", "heteroaromatic ring" 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, halogen, 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 compounds of the present invention or salts thereof may be administered alone as the active substance, preferably in the form of a pharmaceutical composition thereof.
"pharmaceutical composition" refers to a formulation of one or more compounds of the invention or salts thereof with a carrier commonly 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 the administration of the compounds of the invention to an organism.
The term "pharmaceutically acceptable carrier" refers to those carriers that have no significant irritating effects on the organism and do not impair the biological activity and properties of the active compound. By "pharmaceutically acceptable carrier" is meant an inert substance administered with the active ingredient that facilitates administration of the active ingredient, including but not limited to any glidants, sweeteners, diluents, preservatives, dyes/colorants, flavoring enhancers, surfactants, wetting agents, dispersing agents, disintegrants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers approved by the U.S. food and drug administration for use in humans or animals (e.g., livestock). Non-limiting examples of such carriers include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
Administration of the compounds of the invention, or pharmaceutically acceptable salts thereof, in pure form or in the form of a suitable pharmaceutical composition may be carried out by any acceptable mode of administration that provides for a similarly useful agent. The pharmaceutical compositions of the present invention may be prepared by combining a compound of the present invention with a suitable pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical compositions of the present invention may be formulated into solid, semi-solid, liquid or gaseous formulations such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, gels, microspheres, aerosols and the like.
Typical routes of administration of the compounds of the invention or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, transmucosal, enteral administration, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. The preferred route of administration is oral.
The pharmaceutical composition of the present invention can be manufactured by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar coating pill method, a milling method, an emulsification method, a freeze-drying method, etc.
In a preferred embodiment, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical compositions may be formulated by mixing the active compound with pharmaceutically acceptable carriers well known in the art. These 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 may be prepared by conventional mixing, filling or tabletting methods. For example, it can be obtained by the following method: the active compound is admixed with solid excipients, the resulting mixture is optionally milled, if desired with other suitable auxiliaries, and the mixture is then processed into granules, giving 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, dextrose solution, acacia syrup, 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; crosslinked sodium carboxymethyl cellulose, pre-crosslinked starch, sodium starch glycolate, alginic acid, corn starch, potato starch, methyl cellulose, agar, carboxymethyl cellulose, crosslinked polyvinylpyrrolidone, and the like. The cores of the drags may optionally be coated according to methods well known in the usual pharmaceutical practice, in particular with enteric coatings.
The pharmaceutical compositions may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Suitable excipients, for example fillers, buffers or surfactants, can be used.
A further aspect of the invention relates to the use of a compound of formula I to formula VI, or a pharmaceutically acceptable salt, solvate, polymorph, metabolite etc. thereof, in a medicament for the treatment of a disease benefiting from SHP2 inhibition. The disease that benefits from SHP2 inhibition is selected from cancers.
The substituted alkynyl heterocyclic compound provided by the invention has very good SHP2 inhibition activity, and is expected to become an efficient SHP2 inhibitor drug.
Detailed Description
The following specific examples are put forth so as to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof. Those skilled in the art will appreciate that: there are other synthetic routes to form the compounds of the present invention, and the following non-limiting examples are provided.
All operations involving readily oxidizable or hydrolyzable feedstocks are performed under nitrogen protection. Unless otherwise indicated, the starting materials used in the present invention are all commercially available and used without further purification.
Column chromatography was performed on silica gel (200-300 mesh) produced by Qingdao chemical Co., ltd. Thin layer chromatography was performed using prefabricated plates (silica gel 60 PF) manufactured by E.Merck company 254 0.25 mm). Chiral compound isolation and enantiomeric excess value (ee) determination Agilent LC 1200 series (column: CHIRALPAK AD-H,4.6 x 250 mm, 5 microns, 30 ℃). Nuclear magnetic resonance chromatography (NMR)) Measured using a Varian VNMS-400 Nuclear magnetic resonance apparatus; liquid chromatography-mass spectrometry (LC/MS) using FINNIGAN Thermo LCQ Advantage MAX Agilent LC 1200 series (column: waters Symmetry C, 18,)>4.6 x 50 mm, 5 microns, 35 ℃) using ESI (+) ion mode.
Experimental part
Experimental part
Intermediate 1:(R) -N- ((S) -4, 6-dihydrospiro [ cyclopenta [ d ]]Thiazole-5, 4' -piperidines]-6-yl) -2-methyl Ylpropane-2-sulfinamide trifluoroacetate
The target compound was synthesized according to the procedure in example 8 of patent CN113493440 a.
Intermediate 2:6-chloro-3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b]Pyrazine compounds
The target compound was synthesized according to the same method as in preparation 178 at page 237 of patent WO 2019167000.
Intermediate 3:(R) -N- ((S) -1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4，3-b]Pyrazin-6-yl) -4, 6-dihydro-spiro [ cyclopenta [ d ]]Thiazole-5, 4' -piperidines]-6-yl) -2-methylpropan-2- Sulfenamides
Intermediate 1 (412 mg), diisopropylethylamine (1 mL), intermediate 2 (411 mg) and n-butanol (5 mL) were sequentially added to a sealed tube, the mixture was heated to 120℃for 2 hours after charging nitrogen gas, 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 give the objective product (2.82 g). MS m/z [ LC-MS ]:688.14[ M+1].
Intermediate 4:9-iodo-5-chloro-7- (4-methoxybenzyl) -7H-imidazo [1,2-c]Pyrazolo [4,3-e]Pyrimidine
Step 1: 3-iodo-4, 6-dichloro-1H-pyrazolo [3,4-d ] pyridine
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 100deg.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 give the desired product (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 (1.2 g) and concentrated aqueous ammonia (25% -28%,2 mL) were added to acetonitrile (20 mL), stirred overnight at room temperature, concentrated under reduced pressure, and separated by silica gel column chromatography (petroleum ether: ethyl acetate, 3:1) to give the desired product (1.1 g). MS m/z [ LC-MS ]:295.92[ M+1].
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 (1.1 g) and potassium carbonate (1.3 g) were added to N, N-dimethylformamide (20 mL), followed by dropwise addition of 4-methoxybenzyl chloride (0.5 mL), and stirring at room temperature for 2 hours. The reaction mixture was poured into 150mL of water, extracted with methylene chloride, the extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and then separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to give the objective product (0.90 g). MS m/z [ LC-MS ]:415.98[ M+1].
Step 4: 9-iodo-5-chloro-7- (4-methoxybenzyl) -7H-imidazo [1,2-c ] pyrazolo [4,3-e ] pyrimidine
3-iodo-6-chloro-1- (4-methyl)Oxybenzyl) -1H-pyrazolo [3,4-d]Pyrimidine-4-amine (700 mg) and chloroacetaldehyde (2 mL) were added to acetonitrile (20 mL), heated to 100deg.C, tube-sealed for reaction for 5 hours, poured into water, pH adjusted to 9-10 with saturated aqueous sodium carbonate, 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 separated by silica gel column chromatography (petroleum ether: ethyl acetate, 8:1) to give the objective product (460 mg). MS m/z [ LC-MS ]：439.98[M+1]。 1 H NMR(400MHz，DMSO-d 6 )：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 5:(S) -5-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-1-amine dihydrochloride
The target compound is synthesized according to the method of an intermediate BX in the patent WO2019183367A 1.
Intermediate 6:(S) - (5-fluoro-1' - (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c)]Pyrazolo [4 ] is selected from the group consisting of, 3-e]pyrimidin-5-yl) -1, 3-dihydro-spiro [ indene-2, 4' -piperidines]-1-yl) carbamic acid tert-butyl ester
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 [ inden-2, 4' -piperidin ] -1-amine
Referring to the method of intermediate 3, intermediate 4 and intermediate 5 are used as raw materials to obtain the target product. MS m/z [ LC-MS ]:624.14[ M+1].
Step 2: (S) - (5-fluoro-1 '- (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1,2-c ] pyrazolo [4,3-e ] pyrimidin-5-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
(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), diisopropylethylamine (0.2 mL) was added, di-tert-butyl dicarbonate (130 mg) was added dropwise under ice-water bath cooling, and after the dropwise addition was stirred at room temperature for 4 hours, the organic solvent was removed by vacuum concentration, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 4:1) to give the objective product (200 mg). MS m/z [ LC-MS ]:724.19[ M+1].
Intermediate 7:(S) - (5-fluoro-1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3，4-b]Pyrazin-6-yl) -1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-1-yl) carbamic acid tert-butyl ester
Step 1: (S) -5-fluoro-1 '- (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b ] pyrazin-6-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-amine
Referring to the method of the intermediate 3, the target product is obtained by using the intermediate 2 and the intermediate 5 as raw materials. MS m/z [ LC-MS ]:595.15[ M+1].
Step 2: (S) - (5-fluoro-1 '- (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
Referring to the method of step 2 in intermediate 6, the (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 starting material to afford the target product. MS m/z [ LC-MS ]:695.20[ M+1].
Intermediate 8:(S) - (5-fluoro-1' - (3-iodo-5- (((triisopropylsilyl) oxy) methyl) -1- ((2- (trimethylsilicon) Group) ethoxy) methyl) -1H-pyrazolo [4,3-b]Pyrazin-6-yl) -1, 3-dihydro-spiro [ indene-2, 4' -piperidine ]-1-yl) amino Formic acid tert-butyl ester
The starting intermediate (6-chloro-3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b ] pyrazin-5-yl) methanol was synthesized following the same procedure as in preparation 179 at page 237 of patent WO 2019167000.
Step 1: 6-chloro-3-iodo-5- (((triisopropylsilyl) oxy) methyl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b ] pyrazine
Intermediate (6-chloro-3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b ] pyrazin-5-yl) methanol (2.28 g), triisopropylsilicon chloride (1.49 g) and imidazole (693 mg) were added to dichloromethane (40 mL), stirred overnight at room temperature, the reaction solution was washed with water, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (petroleum ether: ethyl acetate, 5:1) to give the desired product (2.75 g). MS m/z [ LC-MS ]:597.14[ M+1].
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 [ inden-2, 4' -piperidin ] -1-amine
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), intermediate 5 (293 mg) and diisopropylethylamine (1 mL) were added, heated to 120℃for 2 hours, cooled to room temperature, concentrated under reduced pressure to remove organic solvents, and the residue was separated by silica gel column chromatography (dichloromethane: methanol, 15:1) to give the title compound (475 mg). MS m/z [ LC-MS ]:781.3[ M+1].
Step 3: (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 [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
(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), diisopropylethylamine (0.5 mL) was added, di-tert-butyl dicarbonate (262 mg) was added dropwise under ice water bath cooling, and after the dropwise addition was completed, the mixture was stirred at room temperature 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 give the objective product (490 mg). MS m/z [ LC-MS ]:881.35[ M+1].
Intermediate 9:(R) -3H-spiro [ benzofuran-2, 4' -piperidine]-3-amine dihydrochloride
The target compound is synthesized according to the method of intermediate CB in patent WO2019183367A 1.
Intermediate 10: :(R) - (1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3- ] b]Pyrazin-6-yl) -3H-spiro [ benzofuran-2, 4' -piperidine]-3-yl) carbamic acid tert-butyl ester
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
With reference to the method of the intermediate 3, the target product is obtained by using the intermediate 2 and the intermediate 9 as raw materials. MS m/z [ LC-MS ]:579.14[ M+1].
Step 2: (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
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 starting material to afford the target product. MS m/z [ LC-MS ]:679.19[ M+1].
Intermediate 11:(S) -6-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-1-amine dihydrochloride
The target compound was synthesized according to the method of intermediate Y in patent WO2019183367 A1.
Intermediate 12:(S) - (5-fluoro-1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3，4-b]Pyrazin-6-yl) -1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-3-yl) carbamic acid tert-butyl ester
Step 1: (S) -6-fluoro-1 '- (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4,3-b ] pyrazin-6-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-amine
Referring to the method of intermediate 3, the target product is obtained by using intermediate 2 and intermediate 11 as raw materials. MS m/z [ LC-MS ]:595.15[ M+1].
Step 2: (S) - (5-fluoro-1 '- (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -3-yl) carbamic acid tert-butyl ester
Referring to the method of step 2 in intermediate 6, the (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 starting material to afford the target product. MS m/z [ LC-MS ]:695.20[ M+1].
Intermediate 13:(R) - (1' - (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c)]Pyrazolo [4,3-e] Pyrimidin-5-yl) -3H-spiro [ benzofuran-2, 4' -piperidines]-3-yl) carbamic acid tert-butyl ester
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
Referring to the method of intermediate 3, the target product is obtained by using intermediate 4 and intermediate 9 as raw materials. MS m/z [ LC-MS ]:608.13[ M+1].
Step 2: (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-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 2 in intermediate 6, starting from (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, the desired product is obtained. MS m/z [ LC-MS ]:708.18[ M+1].
Intermediate 14:(S) - (5-fluoro-1' - (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1, 2-c)]Pyrazolo compounds [4，3-e]Pyrimidin-5-yl) -1, 3-dihydro-spiro [ indene-2, 4' -piperidines]-3-yl) carbamic acid tert-butyl ester
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 [ inden-2, 4' -piperidin ] -1-amine
Referring to the method of intermediate 3, the target product is obtained by using intermediate 4 and intermediate 11 as raw materials. MS m/z [ LC-MS ]:624.14[ M+1].
Step 2: (S) - (5-fluoro-1 '- (9-iodo-7- (4-methoxybenzyl) -7H-imidazo [1,2-c ] pyrazolo [4,3-e ] pyrimidin-5-yl) -1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -3-yl) carbamic acid tert-butyl ester
Referring to the method of step 2 in intermediate 6, the target product is obtained using (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 as starting material. MS m/z [ LC-MS ]:724.19[ M+1].
Intermediate partBody 15:(R) -6-fluoro-3H-spiro [ benzofuran-2, 4' -piperidine]-3-amine dihydrochloride
The target compound was synthesized according to the method of intermediate FI in patent WO2019183367 A1.
Intermediate 16: :(R) - (6-fluoro-1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4，3-b]Pyrazin-6-yl) -3H-spiro [ benzofuran-2, 4' -piperidine]-3-yl) carbamic acid tert-butyl ester
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
Referring to the method of intermediate 3, the target product is obtained by using intermediate 2 and intermediate 15 as raw materials. MS m/z [ LC-MS ]:597.13[ M+1].
Step 2: (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-yl) carbamic acid tert-butyl ester
Referring to the method of step 2 in intermediate 6, the target product is obtained using (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 as starting material. MS m/z [ LC-MS ]:697.18[ M+1].
Intermediate 17: (R) -5-fluoro-3H-spiro [ benzofuran-2, 4' -piperidine]-3-amine dihydrochloride
The target compound was synthesized according to the method of intermediate FD in patent WO2019183367 A1.
Intermediate 18: :(R) - (5-fluoro-1' - (3-iodo-1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [4，3-b]Pyrazin-6-yl) -3H-spiro [ benzofuran-2, 4' -piperidine]-3-yl) carbamic acid tert-butyl ester
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
Referring to the method of intermediate 3, the target product is obtained by using intermediate 2 and intermediate 17 as raw materials. MS m/z [ LC-MS ]:597.13[ M+1].
Step 2: (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-yl) carbamic acid tert-butyl ester
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 starting material to afford the target product. MS m/z [ LC-MS ]:697.18[ M+1].
Example 1:(S) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ]Pyrazin-6-yl) 4, 6-dihydro-spiro [ cyclopenta [ d ]]Thiazole-5, 4' -piperidines]-6-amine
Step 1: (R) -N- ((S) -1'- (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 ] thiazol-5, 4' -piperidin ] -6-yl) -2-methylpropan-2-sulfinamide
A mixture of intermediate 3 (137 mg), 2- (3, 6-dihydro-2H-pyran-4-yl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan (50 mg), sodium carbonate (64 mg), tetrakis (triphenylphosphine) palladium (12 mg) and dioxane/water 10:1 mixed solvent (10 mL) was heated to 80℃under nitrogen 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 the residue was separated by silica gel column chromatography (dichloromethane: methanol, 30:1) to give the objective product (88 mg). MS m/z [ LC-MS ]:644.29[ M+1].
Step 2: (S) -1'- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -4, 6-dihydrospiro [ cyclopenta [ d ] thiazol-5, 4' -piperidin ] -6-amine
(R) -N- ((S) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b ]Pyrazin-6-yl) -4, 6-dihydro-spiro [ cyclopenta [ d ]]Thiazole-5, 4' -piperidines]-6-yl) -2-methylpropan-2-sulfinamide (85 mg) was dissolved in methylene chloride (1 mL), trifluoroacetic acid (1 mL) was added, stirred at room temperature for 1 hour, concentrated under reduced pressure to remove the solvent, 10% aqueous sodium carbonate (10 mL) was added, extracted with difluoromethane, the extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the residue was chromatographed on thin layer silica gel (methylene chloride: methanol, 10:1) to give the objective product (35 mg). MS m/z [ LC-MS]：410.18[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 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' -piperidines]-1-amine
Step 1: (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 [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 6 as starting material to give the desired product. MS m/z [ LC-MS ]:680.34[ M+1].
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 [ inden-2, 4' -piperidin ] -1-amine
Reference is made to step 2 of example 1 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' -piperidines]-1-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：460.23[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 3:(S) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) 5-fluoro-1, 3-dihydrospiro [ indene-2, 4' -piperidine]-1-amine
Step 1: (S) - (1 '- (3, 6-dihydro-2H-pyran-4-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -5-fluoro-1, 3- & dihydro-spiro [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 7 as starting material to give the desired product. MS m/z [ LC-MS ]:651.35[ M+1].
Step 2: (S) -1'- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -5-fluoro-1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-amine
Referring to the procedure of step 2 of example 1, using (S) - (1' - (3, 6-dihydro-2H-pyran-4-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) -5-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine ]-1-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：421.22[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 4:(S) - (6- (1-amino-5-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine)]-1' -yl) -3- (3, 6-di hydrogen-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazin-5-yl) methanol
Step 1: (S) - (1 '- (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 [ inden-2, 4' -piperidin ] -1-yl) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 8 as starting material to give the desired product. MS m/z [ LC-MS ]:837.49[ M+1].
Step 2: (S) - (6- (1-amino-5-fluoro-1, 3-dihydrospiro [ inden-2, 4 '-piperidin ] -1' -yl) -3- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ] pyrazin-5-yl) methanol
(S) - (1' - (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' -piperidine]The tert-butyl-1-yl) carbamate was dissolved in tetrahydrofuran (0.1M) and stirred with tetrabutylammonium fluoride (2.5 eq) at room temperature for 2 hours, concentrated under reduced pressure to remove the solvent, and then reacted and treated in the same manner as in step 2 of example 1 to give the desired product. MS m/z [ LC-MS ]：451.23[M+1]。 1 HNMR(400MHz，CD 3 OD)：δ＝7.49(dd，J＝8.8Hz，4.8Hz，1H)，7.40(s，1H)，7.11(dd，J＝8.8Hz，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，2H)，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)。
Example 5:(R) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) 3H-spiro [ benzofuran-2, 4' -piperidines]-3-amine
Step 1: (R) - (1 '- (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) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 10 as starting material to give the desired product. MS m/z [ LC-MS ]:635.34[ M+1].
Step 2: (R) -1'- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -3H-spiro [ benzofuran-2, 4' -piperidin ] -3-amine
Referring to step 2 of example 1, the method of (R) - (1' - (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' -piperidine]-3-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：405.20[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 6:(S) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) 6-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-1-amine
Step 1: (S) - (1 '- (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 [ inden-2, 4' -piperidin ] -3-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 1 in example 1, the intermediate 12 was used as a starting material to obtain the desired product. MS m/z [ LC-MS ]:651.35[ M+1].
Step 2: (S) -1'- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b ] pyrazin-6-yl) -6-fluoro-1, 3-dihydrospiro [ inden-2, 4' -piperidin ] -1-amine
Referring to the procedure of step 2 of example 1, using (S) - (1' - (3, 6-dihydro-2H-pyran-4-yl) -1- ((2- (trimethylsilyl) ethoxy) methyl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) -5-fluoro-1, 3-dihydro-spiro [ indene-2, 4' -piperidine]-3-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：421.22[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 7:(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' ] sPiperidine compounds]-3-amine
Step 1: (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) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 13 as starting material to give the desired product. MS m/z [ LC-MS ]:664.32[ M+1].
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
Reference is made to step 2 of example 1 using (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' -piperidines]-3-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：444.21[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)。
Example 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' -piperidines]-1-amine
Step 1: (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 [ inden-2, 4' -piperidin ] -3-yl) carbamic acid tert-butyl ester
Referring to the procedure of step 1 in example 1, the intermediate 14 was used as a starting material to obtain the desired product. MS m/z [ LC-MS ]:680.34[ M+1].
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 [ inden-2, 4' -piperidin ] -1-amine
Reference is made to step 2 of example 1 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' -piperidines]-3-yl) carbamic acid tert-butyl ester is used as a raw material to obtain a target product. MS m/z [ LC-MS]：460.23[M+1]。 1 HNMR(400MHz，CD 3 OD)：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)，2.81(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)。
Example 9:(R) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazin-6-yl) 6-fluoro-3H-spiro [ benzofuran-2, 4' -piperidine]-3-amine
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Step 1: (R) - (1 '- (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) carbamic acid tert-butyl ester
Referring to the procedure of step 1 in example 1, the intermediate 16 was used as a starting material to obtain the desired product. MS m/z [ LC-MS ]:653.33[ M+1].
Step 2: (R) -1'- (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
The procedure of step 2 of example 1 was followed using tert-butyl (R) - (1 '- (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 starting material to give the title product. MS m/z [ LC-MS ]:423.19[ M+1].
Example 10: (R) -1' - (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b]Pyrazine-6-17-doped Phenyl) -5-fluoro-3H-spiro [ benzofuran-2, 4' -piperidines]-3-amine
Step 1: (R) - (1 '- (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) carbamic acid tert-butyl ester
The procedure of step 1 of example 1 was followed using intermediate 18 as starting material to give the desired product. MS m/z [ LC-MS ]:653.33[ M+1].
Step 2: (R) -1'- (3, 6-dihydro-2H-pyran-4-yl) -1H-pyrazolo [3,4-b1 pyrazin-6-yl) -5-fluoro-3H-spiro [ benzofuran-2, 4' -piperidin ] -3-amine
The procedure of step 2 of example 1 was followed using tert-butyl (R) - (1 '- (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 starting material to give the title product. MS m/z [ LC-MS ]:423.19[ M+1].
Biological testing
Determination of the inhibition of the in vitro enzymatic Activity of SHP2 by Compounds
The enzymatic activity detection of SHP2 in this patent is performed using a rapid fluorescence method, using DiFMUP as an alternative substrate for reaction and optimizing the establishment of a high throughput screening platform. Inhibition of SHP2 by Compounds Detection of activity was performed on this platform. The specific method comprises the following steps: SHP2 at a final concentration of 1nM was pre-incubated with 2.5. Mu.M of a mixture of phosphorylated IRS1 peptide fragments (sequence: H2N-LN (pY) IDLDLV (dPEG 8) LST (pY) ASINFQK-amide) for 30 min at 23 ℃. The compound was diluted 5-fold with 100% dmso from 0.2mM (7 total concentration), and 2 μl of the compound was added to 48 μl 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. No. 6007270, purchased from Perkinelmer), followed by 10. Mu.L of a pre-incubated mixture of SHP2 and IRS1 peptide fragments, centrifuged and mixed, and incubated at 23℃for 30 minutes. 5. Mu.L of the alternative substrate DiFMUP (final concentration 50. Mu.M, cat. D6567, purchased from Invitrogen) was added to the reaction and incubated for 60 minutes at 23 ℃. The reaction was then terminated by adding 5. Mu.L of 160. Mu. MbpV (Phen) solution (SC-22137, purchased from Santa). Immediately after termination of the reaction, fluorescence signals were detected using a microplate reader (Perkin-Elmer) at excitation and emission wavelengths of 340nm and 450nm, respectively, and the data were calculated using GraphPad Prism software to obtain the IC50 values for the compounds. The detection shows that the specific compounds in the embodiment of the invention have strong in vitro enzyme inhibitory activity of SHP2 and IC 50 Values lie in the interval 1nM to 10nM, the activity of exemplary partial compounds is shown in Table 1:
TABLE 1 inhibitory Activity of the mid-fraction differentiation Compounds on SHP2 enzyme
Examples 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
Determination of the inhibition of SHP 2-positive cell proliferation by Compounds
Human non-small cell lung cancer cell line NCI-H358 cells were cultured using RPMI-1640 medium (cat# C11875500BT, purchased at Biological Industries) plus 10% fetal bovine serum (FBS, cat# 04-001-1ACS, purchased at Biological Industries, BI) and 1% penicillin/streptomycin diabodies (P/S, cat# 15070-063, purchased at Gibco) at 37℃under 5% CO2. The day before compound detection, NCI-H358 cells were plated in 196 well plates (cat No. 3917, purchased from Corning) at a concentration of 2000 cells/195. Mu.L/well. After 24 hours, the compounds were diluted 3-fold in a gradient from 10mM with 100% DMSO (total 10 concentrations), and then 2. Mu.L of each concentration was added to 48. Mu.L of serum-free and double antibody-free medium for dilution. mu.L of the diluted compound was added to the spread Cell suspension at each concentration, the compound was incubated with the cells in a Cell culture tank for 72 hours (3 days), and 25. Mu.L of Cell-Titer Glo (G7) was added after the medium was drained 570, purchased from Promega) reagent was incubated for a further 5-10 minutes. The fluorescence values were then read on Envision and the data were calculated using GraphPad Prism software to obtain ICs for inhibition of cell proliferation by the compound 50 Values. Human acute myeloblastic leukemia cell line Kasumi-1 cells were cultured using RPMI-1640 medium (cat# C11875500BT, purchased at Biological Industries) plus 20% fetal bovine serum (FBS, cat# 04-001-1ACS, purchased at Biological Industries, BI) and 1% penicillin/streptomycin diabodies (P/S, cat# 15070-063, purchased at Gibco) at 37℃under 5% CO2. The day before compound detection, kasumi-1 cells were plated in 196 well plates (cat No. 3599, purchased from Corning) at a concentration of 3000 cells/195. Mu.L/well. After 24 hours, the compounds were diluted 3-fold in a gradient from 10mM with 100% DMSO (total 10 concentrations), and then 2. Mu.L of each concentration was added to 48. Mu.L of serum-free and double antibody-free medium for dilution. mu.L of the diluted compound was added to the plated Cell suspension at each concentration, the compound was incubated with the cells in a Cell incubator for 72 hours (3 days), and 35. Mu.L of Cell-Titer Blue (G8082, purchased from Promega) reagent was added for another 4 hours. The fluorescence values were then read on Flexstation III (560 nm excitation, 590nm detection) and the data were calculated using GraphPad Prism software to obtain IC's for inhibition of cell proliferation by the compound 50 Values. The specific compounds in the examples of the present invention all have strong inhibitory activity on cell proliferation, the IC50 value is in the range of 10nM to 1. Mu.M, and the results of partial compound detection are shown in Table 2.
TABLE 2 inhibitory Activity of example Compounds against cell (NCI-H358) proliferation
Examples IC50(nM)
1 54.0
2 19.4
3 46.2
4 20.5
5 51.5
6 48.6
8 12.9
Determination of pharmacokinetic data of Compounds in SD rats
Male SD rats were derived from Peking Vitolihua laboratory animal technologies Co., ltd, rats were grouped into groups of 3, each group was orally infused with suspensions of the samples to be tested (5 mg/kg, suspension 20% EtOH,40% PEG400, 40% H, respectively 2 O). Animals were fasted overnight prior to the experiment, with a time of fasting ranging from 10 hours prior to dosing to 4 hours post dosing. Blood was collected at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after dosing, respectively. After isoflurane anesthesia using a small animal anesthesia machine, 0.3mL of whole blood was collected through the fundus venous plexus, placed in a heparin anticoagulation tube, the sample was centrifuged at 4000rpm for 5min at 4 ℃, and the plasma was transferred to a centrifuge tube and stored at-80 ℃ until analysis. Samples from plasma were extracted using protein precipitation and the extracts were analyzed by LC/MS. The results of partial compound detection are shown in Table 2.
TABLE 3 pharmacokinetic parameters of the example Compounds
Table 3 lists the pharmacokinetic data of the compounds of the examples of the present invention in SD rats. The data indicate that the compounds provided by the invention have relatively good in vivo metabolic levels.
Assay of compounds for CYP450 subtype 3A4 inhibition
Detecting the relative activity of the test substance and the positive control on the main subtype CYP3A4 metabolite of the mixed human liver microsome CYP450 enzyme by a liquid chromatography-tandem mass spectrometer (LC-MS/MS) analysis method, calculating the IC50 value of the inhibition effect of the positive control substance and the test substance on the mixed human liver microsome cytochrome P450 enzyme, and evaluating the in vitro inhibition effect of the test substance on the main subtype CYP3A4 of the CYP450 enzyme.
The experiments were divided into positive control and test groups. Positive controls or test substances were incubated with human liver microsomes and probe substrates for CYP3A4 enzyme, including human liver microsomes (0.05 mg/mL), NADPH (1.5 mM), PBS buffer (100 mM, ph=7.4), probe substrates (midazolam 4 μm or testosterone 40 μm) and inhibitors (final test substance concentrations of 0, 1, 2.5, 5.0, 10.0, 25.0 μm; final positive control ketoconazole concentrations of 0, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 μm.) in a total incubation volume of 100 μl. The specific operation steps are as follows:
2.1 Preparation of liver microsome dilution
A stock solution of 20mg/mL human liver microsomes was thawed on ice, and diluted 40-fold with PBS buffer (100 mM, pH=7.4) to prepare a dilution of 0.5mg/mL liver microsomes.
2.2 Preparing mixed incubation liquid
A mixed incubation solution containing a dilution of liver microsomes and a substrate solution (midazolam or testosterone) was prepared using PBS buffer (100 mm, ph=7.4).
2.3 The mixed incubation was pre-incubated in a thermostated shaker at 37℃and 100rpm for 5 minutes.
2.4 After pre-incubation, respectively adding test object working solutions or positive control ketoconazole working solutions with different concentrations (the final concentration of the test object is 0, 1, 2.5, 5.0, 10.0 and 25.0 mu M; the final concentration of the positive control ketoconazole is 0, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1 and 0.25 mu M), adding NADPH (final concentration of 1.5 mM) after vortex mixing uniformly, starting reaction, continuously placing in a constant-temperature oscillator at 37 ℃ and 100rpm, incubating for a total volume of 100 mu L, incubating for a certain time (the solution of a probe substrate of midazolam is incubated for 10 minutes, and the solution of testosterone is incubated for 15 minutes), adding 150 mu L of ice internal standard solution, centrifuging for 10 minutes at 12000rpm after vortex oscillation, taking 200 mu L of supernatant, analyzing by UPLC-MS/MS, calculating the metabolic products and the internal standard naphthalene peak by Massnx V4.1 SCN962 software, and calculating the metabolic product area ratio of the metabolic products per area of the metabolic products by using the software. The inhibition IC50 values for the positive control and test subjects were calculated from Excel directly at the two concentration points or by Graphpad Prism (version 6.01) software.
TABLE 4 inhibition of CYP450 enzyme major subtype 3A4 by example compounds
Table 4 shows the IC50 values (testosterone as substrate) of the inhibition of the CYP450 enzyme major subtype 3A4 by the compounds of the present invention. The data show that the compound provided by the invention has very weak inhibition effect on CYP450 enzyme main subtype 3A4, is beneficial to avoiding potential drug interaction risks and improves the safety of clinical medication.
Assay of hERG inhibition by Compounds
An Olympus IX51/71/73 microscope, MP285 micromanipulator was used with a patch clamp Amplifier system (Multiclamp 700B Amplifier) (AXON).
HEK293 cell lines stably transformed with hERG were purchased from Invitrogen. Cells were grown in medium containing 85% DMEM,10% fetal bovine serum, 0.1mM nonessential amino acids, 25mM HEPES buffer, 100U/mL penicillin-streptomycin, 5. Mu.g/mL blasticidin, 400. Mu.g/mL G418 geneticin.
Three passages per week, the cells were digested with TrypLE Express, maintaining about 40% -80% confluency. Before detection, 5X 10 plates with each diameter of 6 cm were used 5 The cells were seeded at a density of individual cells and 1 μg/mL doxycycline was induced for 48 hours.
The compound solutions were tested at 10,1,0.1 μm and diluted 1000-fold, respectively, with a final DMSO concentration in the range of 0.1%.
The manipulator is adjusted to move the electrode tip towards the cell surface to form a high-level seal. Compensating the liquid boundary potential and the fast capacitance, and sucking and breaking the cell membrane to form a whole cell recording mode. The membrane potential was set to-60 mV to ensure that the hERG channel was not opened. Using C on an amplifier slow To eliminate the peak value of the capacity current. The holding voltage was set at-90 mV for 500ms. The leakage current test was performed at-80 mV for 500ms. The hERG channel was activated by depolarizing +30mV for 4.8 seconds, and the voltage was restored to-50 mV for 5.2 seconds, and tail current was observed.
Peak current suppression= [1- (tail current peak value) Inhibitors Tail current peak value Positive control ) /(tail current peak value) Blank space Tail current peak value Positive control )]×100％。
TABLE 5 inhibitory Activity of example Compounds against hERG
Examples Inhibition ratio @ 1. Mu.M Inhibition ratio @3 muM Inhibition ratio @ 10. Mu.M
2 4.44％±4.53％ / 63.59％±1.28％
3 27.23％±1.88％ / 87.82％±0.43％
Table 5 lists the inhibition data for hERG at various concentrations for the compounds of the examples of the present invention. This suggests that the compounds provided by the present invention have relatively low inhibitory activity against hERG, and may have relatively low cardiotoxicity.
Industrial applicability
The compounds of the present invention can inhibit SHP2 protease activity to provide anti-tumor effects.
Incorporation by reference
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 (7)

1. A compound of formula (I) or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof,
wherein,
the A ring is a benzene ring or a 5-6 membered heteroaromatic ring, which may optionally be substituted with halogen, -CN, -OH, -NH 2 、C 1-6 Alkyl, or-O-C 1-6 Alkyl substitution
X 1 And X 2 Each independently is-CHR 1 -、-NR 2 -, -O-, or-S-,
R 1 is H, halogen, -CN, -OH, -NH 2 、C 1-6 Alkyl, or-O-C 1-6 An alkyl group, a hydroxyl group,
R 2 selected from H or C 1-6 An alkyl group.
In some embodiments, X 1 And X 2 Each independently is-CHR 1 -、-NR 2 -, or-O-, R 1 And R is 2 As described above.
2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein X 1 And X 2 Each independently is-CHR 1 -、-NR 2 -, or-O-, R 1 And R is 2 The method of claim 1.
3. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, wherein R 1 Is H or NH 2 。
4. The following compounds, or pharmaceutically acceptable salts, solvates, polymorphs, or tautomers thereof:
5. a pharmaceutical composition comprising a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, polymorph, or tautomer thereof, and optionally a pharmaceutically acceptable adjuvant.
6. Use of a compound of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, polymorph or tautomer thereof, or a pharmaceutical composition of claim 5, or a compound of any one of claims 1-4, or a pharmaceutically acceptable salt, solvate, polymorph or tautomer thereof, or a pharmaceutical composition of claim 5 in combination with an inhibitor of SHP2 or KRAS or an EGFR inhibitor for the manufacture of a medicament for the treatment of a disorder associated with SHP2 and/or KRAS and/or EGFR.
7. The use according to claim 6, wherein the SHP2 and/or KRAS and/or EGFR related disease is leukemia, melanoma, glioblastoma, lung cancer, breast cancer or knoop-male syndrome.
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