Pyrazolo[1,5-a]pyridine compounds, processes for their preparation and uses thereof

Abstract

The present application relates to a kind of pyrazolo [1, 5-a] pyridine compound and its preparation method and application, including the pharmaceutical composition of active ingredient or its pharmaceutically acceptable salt of the compound described herein.The present application further relates to the compound of formula (I) in for treating and preventing the disease that can be treated with wild type, gene fusion type and mutant (including but not limited to G804 and G810) RET kinase inhibitor, including the disease or condition mediated by RET kinase.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to pyrazolo[1,5-a]pyridine compounds, their preparation methods, and applications. Background Technology

[0002] Transfection rearrangement (RET) kinase is a single-transmembrane receptor tyrosine kinase that plays a crucial role in the development of the kidneys and enteric nervous system, as well as in maintaining homeostasis in the nervous, endocrine, hematopoietic, and male reproductive systems. The structure of RET is divided into an extracellular domain, a transmembrane domain, and an intracellular kinase domain. Its ligand, the neurotrophic factor (GDNF) family, does not directly bind to RET. Instead, it first forms a complex, GFL–GFRα, with GDNF family receptor α, which then catalyzes RET homodimerization. This leads to autophosphorylation of RET in the intracellular domain, subsequently recruiting adaptor proteins and pathway proteins to activate various signaling pathways, including MAPK, PI3K, JAK-STAT, PKA, and PKC, thereby participating in cell proliferation, nerve conduction, cell migration, and cell differentiation (Alexander Drilon, Nature Reviews Clinical Oncology, 2018, 15:151–167).

[0003] The gene encoding the RET protein is located on the long arm of human chromosome 10. Abnormalities in the RET protein (gene fusion, mutation, etc.) can cause a variety of diseases, including papillary thyroid carcinoma (PTC), medullary thyroid carcinoma (MTC), congenital megacolon, lung adenocarcinoma, irritable bowel syndrome, etc.

[0004] Chromosomal rearrangement of the RET gene can lead to RET gene breakage. After breakage, the 3' end of the RET gene can fuse with different genes such as KIF5B, TRIM33, CCDC6, or NCOA4, forming fusion genes. The expressed fusion protein is persistently activated, driving tumorigenesis. It has been reported that RET gene fusions exist in approximately 10-20% of PTC patients, primarily CCDC6-RET and NCOA4-RET fusions. Approximately 1%–2% of lung adenocarcinoma patients have RET fusion genes, mainly KIF5B-RET, CCDC6-RET, TRIM33-RET, and NCOA4-RET, with KIF5B-RET being the most common (Rosell R, and Karachaliou N, Lancet Oncol., 2016, 17:1623-1625).

[0005] Point mutations leading to RET gene activation can cause multiple endocrine adenoma type 2 (MEN2), manifested as proliferation or tumors of neuroendocrine cells in the thyroid, adrenal medulla, and parathyroid glands (Mulligan LM, Nat RevCancer., 2014, 14:173-86). Approximately 60% of MTC patients have RET mutations.

[0006] Therefore, compounds that can inhibit gene fusion or mutated RET kinases are very useful for the prevention and treatment of RET-driven tumors.

[0007] Several multi-target kinase inhibitors exhibit some inhibitory activity against RET, such as cabozantinib, vandetanib, lenvatini, and ponatinib, but these are all non-specific RET inhibitors. Furthermore, due to the significant homology between RET and VEGFR2's kinase domains, these compounds, in addition to inhibiting RET, also have inhibitory effects on multiple targets, including VEGFR2. This leads to a high risk of off-target toxicity, making it difficult to achieve satisfactory therapeutic effects.

[0008] Currently, two RET-targeted therapies are on the market: LOXO-292 (selpercatinib / LY3527723) from Loxo Oncology and BLU-667 (pralsetinib / Gavreto) from Blurprint. These two targeted therapies have demonstrated ideal efficacy and safety in patients with RET fusions or mutations, particularly in RET fusion-positive non-small cell lung cancer (NSCLC) and RET mutation-positive medullary thyroid cancer (MTC).

[0009] In the treatment of RET fusion-positive NSCLC and RET mutation-positive MTC with selpercatinib, some patients developed resistance due to solvent-front mutations in RET G810R, G810S, and G810C before circulating tumor DNA (ctDNA) was detected. In phase 1 and 2 clinical trials of selpercatinib, acquired mutations in RET G810 were found in tumor tissue from one CCDC6-RET fusion-positive NSCLC patient and in plasma from another RET fusion-positive NSCLC patient. Preclinical studies have reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft model that has acquired resistance to selpercatinib. Structural modeling predicted that mutations in G810 would spatially impede the binding of selpercatinib. In vitro assays confirmed that multi-kinase inhibitors against RET and selective RET inhibitors lost RET activity against G810 mutations (Solomon BJ, Tan L, Lin JJ et al., J Thorac Oncol. 2020 Apr; 15(4): 541-549.).

[0010] Therefore, there is an urgent need in this field to develop drugs that are highly specific and efficient at inhibiting wild-type, fusion-type, and mutant (including but not limited to G804 and G810) RET kinases. Summary of the Invention

[0011] The purpose of this invention is to provide a novel class of compounds with RET kinase inhibitory activity and / or good pharmacodynamic / pharmacokinetic properties and their uses.

[0012] In a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug thereof.

[0013]

[0014] In the formula,

[0015] R m and R n Each group is independently selected from the following group: H, C1-C3 alkyl; wherein the substitution refers to substitution by 1-2 halogen atoms.

[0016] In another preferred embodiment, the compound or its pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug, R m and R nEach is independently selected from: H, methyl, ethyl, n-propyl, -CH2F, CHF2, -CH2CH2F, -CH2CHF2, -CHFCH3, -CHFCH2F, -CH2CH2CH2F or -CH2CHFCH2F.

[0017] In another preferred embodiment, the compound or its pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug, R n Selected from: H or methyl; R m Selected from: H, methyl, ethyl, n-propyl, -CH2F, CHF2, -CH2CH2F, -CH2CHF2, -CHFCH3, -CHFCH2F, -CH2CH2CH2F or -CH2CHFCH2F.

[0018] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound, or prodrug thereof, wherein the compound has the structure shown in Formula II.

[0019]

[0020] R m Selected from: H, methyl, ethyl, propyl, -CH2F, -CHF2, -CH2CH2F, -CH2CHF2, -CHFCH3, -CHFCH2F, -CH2CH2CH2F or -CH2CHFCH2F.

[0021] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound, or prodrug thereof, wherein the compound has the structure shown in Formula III.

[0022]

[0023] In the formula,

[0024] R A and R B Each is independently selected from: H, F, and methyl.

[0025] In another preferred embodiment, the compound, or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound, or prodrug thereof, is selected from the group consisting of:

[0026]

[0027]

[0028] In another preferred embodiment, the compound or its pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug, wherein the pharmaceutically acceptable salt is acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphor, camphor sulfonate, cyclopentanepropionate, diethylene glycolate, dodecyl sulfate, ethanesulfonate, fumarate, glucono-heptyl sulfate, glycerol phosphate, hemisulfate, heptaate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, dodecanoate.

[0029] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound described in the first aspect or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug thereof; and a pharmaceutical carrier or diluent.

[0030] In another preferred embodiment, the pharmaceutical composition further includes a second cancer therapeutic agent.

[0031] In another preferred embodiment, the second cancer therapeutic agent includes a radioactive agent, a cytotoxic agent, a kinase inhibitor, an immune-targeting inhibitor, and an angiogenesis inhibitor.

[0032] In another preferred embodiment, the second cancer therapeutic agent is selected from one or more of the following groups:

[0033] PD-1 inhibitors (such as nivolumab, pembrolizumab, JS-001, SHR-120, BGB-A317, IBI-308, GLS-010, GB-226, STW204, HX008, HLX10, BAT1306, AK105, LZM 009, or biosimilars of the above drugs), PD-L1 inhibitors (such as durvalumab, atezolizumab, CS1001, KN035, HLX20, SHR-1316, BGB-A333, JS003, CS1003, KL-A167, F...) 520, GR1405, MSB2311 or biosimilars of the above drugs, CD20 antibodies (such as rituximab, olibutuzumab, oflamumab, tosimomumab, teimomab, etc.), CD47 antibodies (such as Hu5F9-G4, CC-90002, TTI-621, TTI-622, OSE-172, SRF-231, ALX-148, NI-1701, SHR-1603, IBI188, IMM01), ALK inhibitors (such as ceritinib, alectinib) (e.g., brigatinib, lorlatinib, oxcalinib), PI3K inhibitors (e.g., ederaris, dactolisib, taselisib, buparlisib), BTK inhibitors (e.g., ibrutinib, tirabrutinib, acalabrutinib), EGFR inhibitors (e.g., afatinib, gefitinib, erlotinib, lapatinib, dacomitinib, icotinib, cannatinib), VEGFR inhibitors (e.g., sorafenib, pazopanib, rivatinib, cabozantinib, sunitinib) Donafenib, etc.), HDAC inhibitors (such as Givinostat, Droxinostat, Entenolol, Dacistar, Tetracycline, etc.), CDK inhibitors (such as Palbociclib, Ribociclib, Abemaciclib, Lerociclib, etc.), MEK inhibitors (such as Selmetinib (AZD6244), Trametinib (GSK1120212), PD0325901, U0126, AS-703026, PD184352 (CI-1040), etc.), Ak t inhibitors (such as MK-2206, Ipatasertib, Capivasertib, Afuresertib, Uprosertib, etc.), mTOR inhibitors (such as Vistusertib, etc.), SHP2 inhibitors (such as RMC-4630, JAB-3068, TNO155, etc.), IGF-1R inhibitors (such as Ceritinib, Ocalatinib, Linsitinib, BMS-754807, GSK1838705A, etc.) or combinations thereof.

[0034] In a third aspect, the present invention provides the use of the compound described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a medicament for inhibiting RET kinase activity in cells or subjects.

[0035] In another preferred embodiment, the RET kinase is wild-type, gene fusion type, or mutant type.

[0036] In another preferred embodiment, the RET kinase is a mutant, preferably G804 or G810.

[0037] In another preferred embodiment, the drug is used to treat RET-related diseases and disorders of expression, activity, or level of the RET gene, RET kinase, or any of them.

[0038] In another preferred embodiment, the disease is selected from the group consisting of: eye diseases, rheumatoid arthritis, pulmonary fibrosis, liver fibrosis, and tumors, including: bladder cancer, ovarian cancer, adenocarcinoma, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neurocytoma, rectal cancer, colon cancer, familial adenomatous polyposis carcinoma, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urinary tract cancer, melanoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, and multiple myeloma.

[0039] In a fourth aspect, the present invention provides a method for preparing the compound described in the first aspect or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound or prodrug thereof, comprising the following steps:

[0040]

[0041] (i-1) In an inert solvent under basic conditions (e.g., Cs2CO3, etc.), compound 2-1 reacts with a hydroxyl protecting agent (e.g., benzyl bromide) to give compound 2-2;

[0042] (i-2) In an inert solvent, under basic conditions (e.g., K3PO4) and in the presence of a catalyst (e.g., cuprous iodide and L-proline), compound 2-2 reacts with the amine compound NHR. m R n The reaction yields compounds 2-3;

[0043] (i-3) In an inert solvent and under acidic conditions (e.g., HBr), compound 2-3 is deprotected to give compound 2-4;

[0044] (i-4) In an inert solvent, compound 2-4 reacts with PhNTf2 to give compound 2-5;

[0045] (i-5) In an inert solvent and in the presence of a catalyst (e.g., palladium catalyst and cuprous halide), compounds 2-5 react with compounds 1-8 to give compounds of formula I;

[0046] In the formula, R m R n The definition is as described above.

[0047] In another preferred embodiment, the preparation of the compound of formula I further includes the following steps:

[0048]

[0049] (i) Compound 1-1 and Compound 1-2 react in an inert solvent (e.g., DMSO) under alkaline conditions (e.g., K2CO3, Na2CO3, Cs2CO3, etc.) to give Compound 1-3;

[0050] (ii) In an inert solvent, under acidic conditions (e.g., trifluoroacetic acid), compounds 1-3 are deamined to give compounds 1-4;

[0051] (iii) In an inert solvent, in the presence of a reducing agent (e.g., NaBH(OAc)3), compounds 1-4 and 1-5 are reductively aminationd to give compounds 1-6;

[0052] (iv) In an inert solvent (e.g., DMF) and under the action of a catalyst (e.g., palladium catalyst), compounds 1-6 react with diboronpinaol ester to give compounds 1-8.

[0053] In a fifth aspect, the present invention provides a method for treating RET-related diseases, the method comprising administering to a subject identified or diagnosed with RET-related diseases a therapeutically effective amount of the compound described in the first aspect above or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition described in the second aspect above.

[0054] In a sixth aspect, the present invention provides a method for inhibiting RET kinase activity in cells or a subject, the method comprising the steps of contacting the cells with or administering to the subject the compound or pharmaceutical composition as described in the first aspect above or the pharmaceutical composition of the second aspect above.

[0055] In another preferred embodiment, the cell is a mammalian cell.

[0056] In another preferred embodiment, the subject is a mammal, preferably a human.

[0057] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0058] Through extensive and in-depth research, the inventors have discovered a class of compounds with good RET kinase activity (wild-type, gene fusion, and various mutant RET kinases), especially mutant RET kinases, and good selectivity for VEGFR2 kinase. Furthermore, these compounds exhibit excellent inhibitory activity against RET kinase-sensitive cells and possess favorable pharmacodynamic / pharmacokinetic properties. Based on these findings, this invention was completed.

[0059] the term

[0060] In this invention, unless otherwise specified, the terms used have the general meanings known to those skilled in the art.

[0061] When a substituent is described using a conventional chemical formula written from left to right, it also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH2O- is equivalent to -OCH2-.

[0062] The term "alkyl" itself, or as part of another substituent, refers to a straight-chain or branched hydrocarbon group having a specified number of carbon atoms (i.e., C1-C6 refers to one to six carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, and similar alkyl groups. One or more positions of the alkyl group are substituted, particularly 1-4 substituents, which can be substituted at any position.

[0063] The term "haloalkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group having a specified number of carbon atoms and being substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Examples of haloalkyl groups also include "fluoroalkyl" groups having a specified number of carbon atoms and being substituted with one or more fluorine atoms in a branched or straight-chain saturated aliphatic hydrocarbon group.

[0064] In this invention, the term "substitution" refers to the substitution of one or more hydrogen atoms on a specific group by a specific substituent. The specific substituent is the substituent described accordingly above, or the substituent appearing in the various embodiments. Unless otherwise specified, a substituted group may have a substituent selected from a specific group at any substituted site of that group, and the substituents may be the same or different at each position. Those skilled in the art will understand that the combinations of substituents contemplated in this invention are those that are stable or chemically feasible. Typical substitutions include, but are not limited to, one or more of the following groups: such as hydrogen, deuterium, halogen (e.g., monohalogen substituents or polyhalogen substituents, the latter such as trifluoromethyl or alkyl containing Cl3), nitrile, nitro, oxo (e.g., =O), trifluoromethyl, trifluoromethoxy, cycloalkyl, alkenyl, alkynyl, heterocyclic, aromatic, OR a SR a S(=O)R e S(=O)2R e P(=O)2R e S(=O)2OR e P(=O)2OR e NR b R c NR b S(=O)2R e NR b P(=O)2R e S(=O)2NR b R c P(=O)2NR b R c C(=O)OR d C(=O)R a C(=O)NR b R c OC(=O)R a OC (=O)NR b R c NR b C(=O)OR e NR d C(=O)NR b R c NR d S(=O)2NR b R c NR d P(=O)2NR b R c NR b C(=O)R a , or NR b P(=O)2R e , where Ra It can independently represent hydrogen, deuterium, alkyl, cycloalkyl, alkenyl, ynyl, heterocyclic, or aromatic rings, R b R c and R d It can independently represent hydrogen, deuterium, alkyl, cycloalkyl, heterocyclic or aromatic ring, or R b and R c It can form heterocycles together with N atoms; R e It can independently represent hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic, or aromatic ring. The above-mentioned typical substituents, such as alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocyclic, or aromatic ring, can be optionally substituted. Such substituents include (but are not limited to): halogen, hydroxyl, cyano, carboxyl (-COOH), C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, 3-12 membered heterocyclic, aryl, heteroaryl, C1-C8 aldehyde, C2-C10 acyl, C2-C10 ester, amino, C1-C6 alkoxy, C1-C10 sulfonyl, and C1-C6 urea, etc.

[0065] Unless otherwise stated, it is assumed that any heteroatom in a suboptimal valence state has enough hydrogen atoms to compensate for its valence state.

[0066] The term "halogenated" or "halogen" includes fluorine, chlorine, bromine, and iodine.

[0067] Active ingredients

[0068] As used herein, the terms “compound of the invention” or “active ingredient of the invention” are used interchangeably to refer to a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, isotopic compound (such as a deuterated compound), or prodrug thereof. The term also includes racemic mixtures and optical isomers.

[0069] Salts that may form from the compounds of this invention are also within the scope of this invention. Unless otherwise stated, compounds of this invention are understood to include their salts. The term "salt" as used herein refers to a salt formed from an inorganic or organic acid and a base in an acidic or basic form. Furthermore, when a compound of this invention contains a basic segment, it includes, but is not limited to, pyridine or imidazole; when it contains an acidic segment, it includes, but is not limited to, carboxylic acids; and any zwitterions ("internal salts") that may form are included within the scope of the term "salt." Pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salts are preferred, although other salts are also useful, for example, for separation or purification steps in the preparation process. Compounds of this invention may form salts, for example, by reacting compound I with a certain amount, such as an equimolar amount, of an acid or base, precipitating it in a medium, or by freeze-drying it in an aqueous solution.

[0070] The compounds of this invention contain basic fragments, including but not limited to amines, pyridines, or imidazole rings, which may form salts with organic or inorganic acids. Typical acids that can form salts include acetates (such as acetic acid or trihaloacetic acids, such as trifluoroacetic acid), adipates, alginates, ascorbic acid salts, aspartate salts, benzoates, benzenesulfonates, hydrogen sulfates, borates, butyrates, citrates, camphor salts, camphor sulfonates, cyclopentanepropionate, diethylene glycol salts, dodecyl sulfates, ethanesulfonates, fumarates, glucono-2-phosphates, glycerol phosphates, hemisulfates, heptarates, hexanoates, hydrochlorides, hydrobromide, and hydroiodide. Salts, hydroxyethanesulfonates (e.g., 2-hydroxyethanesulfonate), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g., 2-naphthalenesulfonate), nicotinates, nitrates, oxalates, pectates, persulfates, phenylpropionates (e.g., 3-phenylpropionates), phosphates, picrates, neopentanoates, propionates, salicylates, succinates, sulfates (e.g., those formed with sulfuric acid), sulfonates, tartrates, thiocyanates, toluenesulfonates such as p-toluenesulfonate, dodecanoates, etc.

[0071] The prodrugs and solvates of the compounds in this invention are also included within the scope of this invention. The term "prodrug" here refers to a compound that, in the course of treatment of a related disease, undergoes a chemical transformation through metabolism or a chemical process to produce the compounds, salts, or solvates of this invention. The compounds of this invention include solvates, such as hydrates.

[0072] The compounds, salts, or solvates of this invention may exist in tautomer forms (e.g., amides and imine ethers). All such tautomers are part of this invention.

[0073] All stereoisomers of compounds (e.g., those with asymmetric carbon atoms due to various substitutions), including their enantiomers and diastereomeric forms, are within the scope of this invention. The independent stereoisomers of the compounds in this invention may not coexist with other isomers (e.g., possessing special activity as a pure or substantially pure optical isomer), or may be mixtures, such as racemates, or mixtures formed with all other stereoisomers or a portion thereof. The chiral center of this invention has two configurations, S or R, as defined by the International Union of Theoretical and Applied Chemistry (IUPAC) in 1974. Racemic forms can be resolved by physical methods, such as stepwise crystallization, or by derivatization into diastereomers followed by crystallization, or by chiral column chromatography. Individual optical isomers can be obtained from racemates by suitable methods, including but not limited to conventional methods, such as recrystallization after salting with an optically active acid.

[0074] The compounds of this invention, obtained sequentially through preparation, separation, and purification, have a weight content equal to or greater than 90%, for example, equal to or greater than 95%, or equal to or greater than 99% (“very pure” compounds), as listed in the text description. Such “very pure” compounds of this invention are also included as part of this invention.

[0075] All configurational isomers of the compounds of this invention are included within the scope of this invention, whether in mixtures, pure or very pure forms. The definition of compounds in this invention includes both cis (Z) and trans (E) olefin isomers, as well as cis and trans isomers of carbocyclic and heterocyclic compounds.

[0076] Throughout the specification, groups and substituents can be selected to provide stable fragments and compounds.

[0077] Specific functional groups and chemical terminology definitions are detailed below. For the purposes of this invention, chemical elements are defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75. th The definitions in Ed. are consistent. The definitions of specific functional groups are also described there. In addition, the basic principles of organic chemistry, as well as specific functional groups and reactivity, are explained in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, the full contents of which are included in the references.

[0078] Some compounds of this invention may exist in specific geometric or stereoisomeric forms. This invention covers all compounds, including their cis and trans isomers, R and S enantiomers, diastereomers, (D) isomers, (L) isomers, racemic mixtures, and other mixtures. Additionally, the asymmetric carbon atom may represent a substituent, such as an alkyl group. All isomers and mixtures thereof are included in this invention.

[0079] According to the present invention, the ratio of isomers in a mixture of isomers can be varied. For example, a mixture containing only two isomers can have the following combinations: 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0. All ratios of isomers are within the scope of the present invention. Similar ratios readily understood by those skilled in the art, as well as ratios for mixtures of more complex isomers, are also within the scope of the present invention.

[0080] This invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. However, in practice, it is common for one or more atoms to be replaced by atoms with different atomic weights or mass numbers. Examples of isotopes that can be included in the compounds of this invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine isotopes, respectively as follows: 2 H, 3 H, 13 C 11 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl. The compounds of this invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates, wherein the isotopes or other isotopic atoms of the aforementioned compounds are all within the scope of this invention. Certain isotopically labeled compounds of this invention, for example... 3 H and 14 Radioactive isotopes of carbon are also included, and are useful in tissue distribution experiments of drugs and substrates. Tritium, i.e. 3 H and carbon-14, i.e. 14 C, their preparation and detection are relatively easy. They are the preferred isotopes. In addition, heavier isotopes such as deuterium are used for substitution. 2 H, due to its excellent metabolic stability, offers advantages in certain therapies, such as increasing half-life or reducing dosage in vivo, and therefore may be preferred in some cases. Isotopically labeled compounds can be prepared using general methods, by replacing the non-isotopic reagent with an readily available isotopically labeled reagent, according to the scheme described in the examples.

[0081] To design the synthesis of a specific enantiomer of the compound of this invention, it can be prepared asymmetrically or derivatized with a chiral auxiliary. The resulting diastereomeric mixture is then separated, and the chiral auxiliary is removed to obtain the pure enantiomer. Alternatively, if the molecule contains a basic functional group, such as an amino acid, or an acidic functional group, such as a carboxyl group, it can be formed with a suitable optically active acid or base to form a diastereomer salt, which is then separated by conventional methods such as separation crystallization or chromatography to obtain the pure enantiomer.

[0082] As described herein, the compounds of this invention can be expanded with any number of substituents or functional groups. Generally, whether the term "substitution" appears before or after the term "optional," the general formula for substituents in the formulations of this invention refers to replacing a hydrogen radical with a substituent of a specified structure. When multiple positions in a particular structure are replaced by multiple specific substituents, each position of the substituent can be the same or different. The term "substitution" as used herein includes all permissible substitutions in organic compounds. In a broad sense, permissible substituents include acyclic, cyclic, branched-unbranched, carbocyclic, and heterocyclic, aromatic and non-aromatic organic compounds. In this invention, heteroatomic nitrogen may be supplemented with hydrogen substituents or any permissible organic compound described above to complete its valence state. Furthermore, this invention is not intended to limit permissible substituted organic compounds in any way. This invention considers the combination of substituents and variable groups to be beneficial in the treatment of diseases in the form of stable compounds. The term "stable" here refers to a compound that is stable enough to maintain the integrity of its structure when tested over a sufficiently long period of time, preferably remaining effective over a sufficiently long period of time, and is used here for the purposes described above.

[0083] The compounds involved in this application and their pharmaceutically acceptable salt metabolites, as well as prodrugs that can be converted in vivo into structures of the compounds involved in this application and their pharmaceutically acceptable salts, are also included in the claims of this application.

[0084] Preparation method

[0085] The preparation method of the compound of formula (I) of the present invention is described in more detail below, but these specific methods do not constitute any limitation on the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, such combinations can be easily performed by those skilled in the art.

[0086] Typically, in the preparation process, each reaction is carried out under inert gas protection, in a suitable solvent, at room temperature to 90°C, and the reaction time is usually 2-24 hours.

[0087] Preferably, the compound of formula I is prepared by the following method:

[0088]

[0089] In the formula, R m R n It has the definition described in this invention;

[0090] (i) Compound 1-1 and Compound 1-2 react in an inert solvent (e.g., DMSO) under alkaline conditions (e.g., K2CO3, Na2CO3, Cs2CO3, etc.) to give Compound 1-3;

[0091] (ii) In an inert solvent, under acidic conditions (e.g., trifluoroacetic acid), compounds 1-3 are deamined to give compounds 1-4;

[0092] (iii) In an inert solvent, in the presence of a reducing agent (e.g., NaBH(OAc)3), compounds 1-4 and 1-5 are reductively aminationd to give compounds 1-6;

[0093] (iv) In an inert solvent (e.g., DMF) and under the action of a catalyst (e.g., palladium catalyst), compounds 1-6 react with diboronpinaol ester to give compounds 1-8;

[0094] (v) Compound 2-1 is reacted with a hydroxyl protecting agent (e.g., benzyl bromide) in an inert solvent under basic conditions (e.g., Cs2CO3, etc.) to give compound 2-2;

[0095] (vi) In an inert solvent, under basic conditions (e.g., K3PO4) and with a catalyst (e.g., cuprous iodide and L-proline), compound 2-2 reacts with the amine compound NHR. m R n The reaction yields compounds 2-3;

[0096] (vii) In an inert solvent and under acidic conditions (e.g., HBr), compound 2-3 is deprotected to give compound 2-4;

[0097] (viii) In an inert solvent, compound 2-4 reacts with PhNTf2 to give compound 2-5;

[0098] (ix) In an inert solvent, under the action of a catalyst (e.g., palladium catalyst and cuprous halide), compounds 2-5 react with compounds 1-8 to give compound I.

[0099] In the above reaction steps, the reaction solvent, reaction temperature, reaction time, catalyst, etc., can be selected according to the specific reactants.

[0100] Pharmaceutical Compositions and Administration

[0101] The pharmaceutical compositions described in this invention are used to prevent and / or treat the following diseases: inflammation, cancer, cardiovascular disease, infection, immune disease, and metabolic disease.

[0102] The compound of formula (I) can be used in combination with other known drugs for treating or improving similar symptoms. When administered in combination, the original drug's administration method and dosage can remain unchanged, while the compound of formula I is taken simultaneously or subsequently. When the compound of formula I is taken concurrently with one or more other drugs, a pharmaceutical composition containing one or more known drugs and the compound of formula I is preferred. Drug combination also includes taking the compound of formula I with one or more other known drugs during overlapping time periods. When the compound of formula I is used in combination with one or more other drugs, the dosage of the compound of formula I or the known drug may be lower than the dosage of either drug alone.

[0103] Drugs or active ingredients that can be used in combination with compounds of general formula (I) include, but are not limited to: PD-1 inhibitors (such as nivolumab, pembrolizumab, JS-001, SHR-120, BGB-A317, IBI-308, GLS-010, GB-226, STW204, HX008, HLX10, BAT1306, AK105, LZM 009 or biosimilars of the above drugs), PD-L1 inhibitors (such as durvalumab, atezolizumab, CS1001, KN035, HLX20, SHR-1316, BGB-A333, JS003, CS1003, KL-A167, F... 520, GR1405, MSB2311 or biosimilars of the above drugs, CD20 antibodies (such as rituximab, olibutuzumab, oflamumab, tosimomumab, teimomab, etc.), CD47 antibodies (such as Hu5F9-G4, CC-90002, TTI-621, TTI-622, OSE-172, SRF-231, ALX-148, NI-1701, SHR-1603, IBI188, IMM01), ALK inhibitors (such as ceritinib, alectinib) (e.g., brigatinib, lorlatinib, oxcalinib), PI3K inhibitors (e.g., ederaris, dactolisib, taselisib, buparlisib), BTK inhibitors (e.g., ibrutinib, tirabrutinib, acalabrutinib), EGFR inhibitors (e.g., afatinib, gefitinib, erlotinib, lapatinib, dacomitinib, icotinib, cannatinib), VEGFR inhibitors (e.g., sorafenib, pazopanib, rivatinib, cabozantinib, sunitinib) Donafenib, etc.), HDAC inhibitors (such as Givinostat, Droxinostat, Entenolol, Dacistar, Tetracycline, etc.), CDK inhibitors (such as Palbociclib, Ribociclib, Abemaciclib, Lerociclib, etc.), MEK inhibitors (such as Selmetinib (AZD6244), Trametinib (GSK1120212), PD0325901, U0126, AS-703026, PD184352 (CI-1040), etc.), Ak t inhibitors (such as MK-2206, Ipatasertib, Capivasertib, Afuresertib, Uprosertib, etc.), mTOR inhibitors (such as Vistusertib, etc.), SHP2 inhibitors (such as RMC-4630, JAB-3068, TNO155, etc.), IGF-1R inhibitors (such as Ceritinib, Ocalatinib, Linsitinib, BMS-754807, GSK1838705A, etc.) or combinations thereof.

[0104] The dosage forms of the pharmaceutical compositions of the present invention include (but are not limited to): injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, controlled-release or sustained-release or nano-formulations.

[0105] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0106] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0107] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0108] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0109] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0110] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0111] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0112] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0113] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0114] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0115] The treatment method of the present invention can be used alone or in combination with other treatment methods or drugs.

[0116] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 50–1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0117] The present invention also provides a method for preparing a pharmaceutical composition, comprising the steps of: mixing a pharmaceutically acceptable carrier with a compound of general formula (I) or its crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate of the present invention, thereby forming a pharmaceutical composition.

[0118] The present invention also provides a treatment method comprising the steps of: administering to a subject requiring treatment a compound of general formula (I) as described in the present invention, or a crystal form thereof, a pharmaceutically acceptable salt, hydrate or solvate thereof, or administering a pharmaceutical composition as described in the present invention for selectively inhibiting RET, particularly mutant RET.

[0119] The present invention has the following main advantages:

[0120] (1) The compounds of the present invention have excellent inhibitory ability against RET kinase and excellent selectivity against RET kinase, and low inhibitory activity against other kinases such as VEGFR2.

[0121] (2) The compounds of the present invention have lower toxicity and side effects.

[0122] (3) The compounds of the present invention have better pharmacodynamic and pharmacokinetic properties.

[0123] (4) The compounds of the present invention have ideal inhibitory activity against wild-type, gene fusion and mutant (including but not limited to G804 and G810 mutations) RET kinases.

[0124] (5) The structure-activity relationship data of the compounds in this invention show that R m It is an ethyl or fluoroethyl (preferably CH2CH2F), R n When the methyl group is H or methyl group (preferably H), the compounds of the present invention exhibit good inhibitory activity against RET mutations of G804 and G810.

[0125] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0126] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0127] The structures of the compounds of the present invention were determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

[0128] NMR was performed using Bruker AVANCE-400 and Bruker AVANCE-500 NMR spectrometers. The solvents used for the determination included deuterated dimethyl sulfoxide (DMSO-d6), deuterated acetone (CD3COCD3), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). Tetramethylsilane (TMS) was used as the internal standard. Chemical shifts were measured in parts per million (ppm).

[0129] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1260 mass spectrometer. HPLC determinations were performed using an Agilent 1100 high-performance chromatograph (Microsorb 5micron C18 100x 3.0 mm column).

[0130] Thin-layer chromatography (TLC) uses Qingdao GF254 silica gel plates, with a thickness of 0.15-0.20 mm for TLC and 0.4-0.5 mm for preparative TLC. Column chromatography typically uses Qingdao 200-300 mesh silica gel as the support.

[0131] The starting materials used in the embodiments of the present invention are all known and commercially available, or can be synthesized using or in accordance with literature reported in the art.

[0132] Unless otherwise specified, all reactions in this invention are carried out under the protection of a dry inert gas (such as nitrogen or argon) by continuous magnetic stirring, and the reaction temperature is [degrees Celsius].

[0133] The following abbreviations are used throughout this invention.

[0134] THF: Tetrahydrofuran

[0135] MeOH: Methanol

[0136] HCl: hydrochloric acid

[0137] Pd(PPh3)4: Tetraphenylphosphine palladium

[0138] K2CO3: Potassium carbonate

[0139] AcOK: Potassium acetate

[0140] NaOH: Sodium hydroxide

[0141] H2O: water

[0142] TEA: Triethylamine

[0143] DIEA: N,N-Diisopropylethylamine

[0144] DMF: N,N-dimethylformamide

[0145] DMA: N,N-dimethylacetamide

[0146] Py: Pyridine

[0147] DCE: 1,2-Dichloroethane

[0148] DMSO: Dimethyl sulfoxide

[0149] TFA: Trifluoroacetic acid

[0150] NaBH(AcO)3: Sodium triacetylborohydride

[0151] Sn2(Bu-n)6: Hexahedral ditin

[0152] AlCl3: Aluminum trichloride

[0153] CuI: Cuprous iodide

[0154] DPPA: Diphenyl azidophosphate

[0155] BuOH: tert-Butanol

[0156] Cs2CO3: Cesium carbonate

[0157] K3PO4: Potassium phosphate

[0158] BnBr: Benzyl Bromide

[0159] Pd2(dba)3: Tris(dibenzylacetone)dipalladium

[0160] X-Phos: 2-Dicyclohexylphosphine-2,4,6-triisopropylbiphenyl

[0161] EA: Ethyl acetate

[0162] NaHCO3: Sodium bicarbonate

[0163] DIPEA: N,N-Diisopropylethylamine

[0164] HBr: Hydrogen bromide

[0165] Example

[0166] Synthesis of intermediate 1:

[0167]

[0168] Step 1: Synthesis of 4-(5-bromopyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester

[0169] 5-Bromo-2-fluoropyridine (10 g, 56.8 mmol), potassium carbonate (31 g, 227.3 mmol), and tert-butylpiperazine-1-carboxylic acid tert-butyl ester (10.6 g, 56.8 mmol) were dissolved in DMF. ( The reaction mixture was stirred at 120℃ for 16 h in 50 mL of water. After the reaction was complete, it was extracted with 30 mL of EA (30 mL x 2). The organic phase was washed with water, dried, concentrated, and subjected to column chromatography to obtain 15 g of 4-(5-bromopyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester. MS m / z (ESI): 342.5 [M + H] + .

[0170] Step 2: Synthesis of 1-(5-bromopyridin-2-yl)piperazine

[0171] 15 g (43.99 mmol) of 4-(5-bromopyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester was dissolved in 20 mL of dichloromethane at room temperature, followed by the addition of 80 mL of dioxane hydrochloride. The reaction was allowed to proceed for 1 hour at room temperature until completion. The reaction solution was concentrated, adjusted to pH 9 with potassium carbonate solution, extracted with ethyl acetate, washed with water, dried, concentrated, and subjected to column chromatography to give 8.5 g of 1-(5-bromopyridin-2-yl)piperazine. MS m / z (ESI): 242.2 [M+H] + .

[0172] Step 3: Synthesis of 1-(5-bromopyridin-2-yl)-4-((6-methoxypyridin-3-yl)methyl)piperazine

[0173] 1-(5-bromopyridin-2-yl)piperazine (8.5 g, 35.3 mmol) was dissolved in dichloromethane (150 mL), and sodium triacetylborohydride (22.5 g, 105.9 mmol) and 6-methoxynicotinaldehyde (9.7 g, 70.6 mmol) were added. The mixture was stirred at room temperature for 2 h. After the reaction was monitored for completeness, it was diluted with dichloromethane, and the reaction was quenched with ammonium chloride solution (50 mL). The mixture was extracted with dichloromethane (50 mL * 2.), the organic phase was washed with water, dried, concentrated, and subjected to column chromatography to give 8.3 g of 1-(5-bromopyridin-2-yl)-4-((6-methoxypyridin-3-yl)methyl)piperazine. MS m / z (ESI): 363.3 [M + H] + .

[0174] Step 4: Synthesis of 1-((6-methoxypyridin-3-yl)methyl)-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxolane-2-yl)pyridin-2-yl)piperazine

[0175] 1-(5-bromopyridin-2-yl)-4-((6-methoxypyridin-3-yl)methyl)piperazine (8.3 g, 22.93 mmol) was dissolved in DMF (40 mL), and pinacol diboronate (11.65 g, 45.86 mmol), palladium acetate (0.26 g, 1.15 mmol), triphenylphosphine (1.2 g, 4.586 mmol), and potassium acetate (6.74 g, 68.78 mmol) were added. The mixture was then stirred at 80 °C for 16 h. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate (30 mL * 2), washed with water, dried, concentrated, and subjected to column chromatography to obtain 4.98 g of 1-((6-methoxypyridin-3-yl)methyl)-4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxolane-2-yl)pyridin-2-yl)piperazine (intermediate 1). MS m / z (ESI): 411.2 [M+H] + .

[0176] Example 1: Synthesis of Compound 1

[0177]

[0178] Step 1: Synthesis of 4-((4-methoxybenzyl)oxy)-6-(methylamino)pyrazolo[1,5-a]pyridine-3-carboxynitrile

[0179] At room temperature, 6-bromo-4-((4-methoxybenzyl)oxy)pyrazoline[1,5-a]pyridine-3-carboxylonitrile (500 mg, 1.4 mmol), cuprous iodide (40 mg, 0.21 mmol), L-proline (32 mg, 0.28 mmol), and potassium carbonate (1.9 g, 14 mmol) were dissolved in anhydrous DMSO (15 mL) under nitrogen protection, and methylamine hydrochloride (940 mg, 14 mmol) was added. The reaction was carried out at 100 °C for 10 hours until complete. After cooling to room temperature, the mixture was extracted with EA (50 mL * 2) and diluted with water (50 mL). The mixture was stirred and filtered. After separation, extraction, drying, filtration, concentration, and column chromatography (PE:EA = 3:1), 4-((4-methoxybenzyl)oxy)-6-(methylamino)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (150 mg) was obtained. MS m / z(ESI): 309.1 [M+H] + .

[0180] Step 2: Synthesis of 4-hydroxy-6-(methylamino)pyrazolo[1,5-a]pyridine-3-carboxynitrile

[0181] At room temperature, 150 mg (0.48 mmol) of 4-((4-methoxybenzyl)oxy)-6-(methylamino)pyrazolo[1,5-a]pyridine-3-carboxynitrile (DCM) was dissolved in 3 mL of DCM. 1 mL of trifluoroacetic acid was added at 0 °C, and the reaction was allowed to proceed for 0.5 h. After the reaction was complete, the product was concentrated to dryness at room temperature and used directly in the next step without further purification. MS m / z (ESI): 189.1 [M+H] + .

[0182] Step 3: Synthesis of 3-cyano-6-(methylamino)pyrazolo[1,5-a]pyridin-4-yltrifluoromethanesulfonate

[0183] 4-Hydroxy-6-(methylamino)pyrazolo[1,5-a]pyridine-3-carboxynitrile (150 mg, 0.8 mmol) and DIEA (307 mg, 2.4 mmol) were dissolved in 5 mL of DMF. N-phenylbis(trifluoromethanesulfonyl)imide (286 mg, 0.8 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 0.5 h until complete. The solution was extracted, dried, filtered, concentrated, and subjected to column chromatography (PE:EA = 3:1) to obtain 3-cyano-6-(methylamino)pyrazolo[1,5-a]pyridine-4-yltrifluoromethanesulfonate (180 mg). MS m / z (ESI): 321.3 [M+H] + .

[0184] Step 4: Synthesis of 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)-6-(methylamino)pyrazolo[1,5-a]pyridin-3-carboxynitrile

[0185] At room temperature, 3-cyano-6-(methylamino)pyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (0.15 g, 0.46 mmol), 6-((6-methoxypyridin-3-yl)methyl)-3-(5-(4,4,5,5-tetramethyl-1,3-dioxane-2-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane (0.20 g, 0.46 mmol), Pd2(dba)3 (46 mg, 0.05 mmol), and x-phos (24 mg) were added. 0.05 mmol) was dissolved in Dioxane / H₂O = 20 mL / 4 mL, purged with nitrogen three times, and reacted at 100 °C for 6 h until complete. The mixture was cooled, concentrated, extracted with DCM, dried, filtered, concentrated, and then subjected to column chromatography (DCM:MeOH = 10:1) to give 60 mg of 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)-6-(methylamino)pyrazolo[1,5-a]pyridin-3-carboxynitrile (compound 1). MS m / z (ESI): 467.2 [M+H] + .

[0186] 1 H NMR (500MHz, DMSO) δ8.28(s,1H),8.13(d,J=1.4Hz,1H),8.07(s,1H),7.92(d,J=2.7Hz,1H),7.79(dd,J=8.8,2.5Hz,1H),7.67(d,J=9.8Hz,1H),7 .05(d,J=1.8Hz,1H),6.76(t,J=8.1Hz,2H),5.92(t,J=5.4Hz,1H),3.85 (d,J=3.4Hz,4H),3.76–3.62(m,5H),3.50(s,4H),1.03(t,J=7.4Hz,3H).

[0187] Example 2: Synthesis of Compound 2

[0188]

[0189] Step 1: Synthesis of 6-bromo-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile

[0190] 6-Bromo-3-cyanopyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (1.0 g, 2.7 mmol) was dissolved in 1,4-dioxane (15 mL) and water (2 mL), and then 6-((6-methoxypyridin-3-yl)methyl)-3-(5-(4,4,5,5-tetramethyl-1,3-dioxane-2-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane (1.1 g, 2.7 mmol), Pd(dppf)2Cl2 (0.22 g, 0.27 mmol), and KOAc (0.8 g, 8.1 mmol) were added, followed by nitrogen purging. The reaction was then stirred at room temperature for 16 h. After the reaction was monitored to be complete, water was added for dilution, followed by extraction with ethyl acetate (30 mL * 2). The organic phase was washed with water, dried, concentrated, and subjected to column chromatography to obtain 0.7 g of 6-bromo-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile, in 54% yield. MS m / z (ESI): 517.2 [M + H] + .

[0191] Step 2: Synthesis of 6-((2-fluoroethyl)amino)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile

[0192] Under nitrogen protection at room temperature, 6-bromo-4-(6-(6-((((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carboxylonitrile (600 mg, 1.2 mmol), cuprous iodide (22 mg, 0.12 mmol), L-proline (14 mg, 0.12 mmol), and potassium carbonate (1.6 g, 12 mmol) were dissolved in anhydrous DMSO (15 mL), and 2-fluoroethylamine hydrochloride (1.2 g) was added. g, 12 mmol). The reaction was carried out at 100℃ for 10 h until complete. After cooling to room temperature, the mixture was diluted with water (30 mL), extracted with EA (50 mL * 2), stirred and filtered, separated, dried, filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 10:1) to give 50 mg of 6-((2-fluoroethyl)amino)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile (compound 2). MS m / z (ESI): 499.3 [M + H) + .

[0193] 1 H NMR (400MHz, DMSO) δ8.40 (s, 1H), 8.34 (d, J = 2.4Hz, 1H), 8.09 (t, J = 3.2Hz, 2H), 7 .77(dd,J=8.8,2.5Hz,1H),7.68(d,J=7.6Hz,1H),7.11(d,J=1.8Hz,1H),6.77(t ,J=7.7Hz,2H),6.25(t,J=5.8Hz,1H),5.74(s,1H),4.68(t,J=4.7Hz,1H),4.56( t,J=4.7Hz,1H),3.81(s,3H),3.78–3.62(m,4H),3.50(dd,J=25.0,11.4Hz,6H).

[0194] Example 3: Synthesis of Compound 3

[0195]

[0196] Step 1: Synthesis of 6-bromo-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile

[0197] 6-Bromo-3-cyanopyrazolo[1,5-a]pyridin-4-yl trifluoromethanesulfonate (1.0 g, 2.7 mmol) was dissolved in 1,4-dioxane (15 mL) and water (2 mL), and then 6-((6-methoxypyridin-3-yl)methyl)-3-(5-(4,4,5,5-tetramethyl-1,3-dioxane-2-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane (1.1 g, 2.7 mmol), Pd(dppf)2Cl2 (0.22 g, 0.27 mmol), and KOAc (0.8 g, 8.1 mmol) were added, followed by nitrogen purging. The reaction was then stirred at room temperature for 16 h. After the reaction was monitored to be complete, water was added for dilution, followed by extraction with ethyl acetate (30 mL * 2). The organic phase was washed with water, dried, concentrated, and subjected to column chromatography to obtain 0.7 g of 6-bromo-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile, in 54% yield. MS m / z (ESI): 517.2 [M + H] + .

[0198] Step 2: Synthesis of 6-(((2,2-difluoroethyl)amino)-4-(6-(6-(((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile

[0199] At room temperature, 6-bromo-4-(6-(6-((((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptyl-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxylonitrile (600 mg, 1.2 mmol), cuprous iodide (22 mg, 0.12 mmol), L-proline (14 mg, 0.12 mmol), and potassium carbonate (1.6 g, 12 mmol) were dissolved in anhydrous DMSO (15 mL) under nitrogen protection, and 2-fluoroethylamine hydrochloride (1.2 g, 12 mmol). The reaction was carried out at 100℃ for 10 hours until complete. After cooling to room temperature, the mixture was diluted with water (30 mL), extracted with EA (50 mL * 2), stirred, filtered, separated, dried, filtered, concentrated, and subjected to column chromatography (DCM:MeOH = 10:1) to obtain 80 mg of 6-(((2,2-difluoroethyl)amino)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridin-3-carboxynitrile (compound 3). MS m / z (ESI): 517.3 [M + H) + .

[0200] 1 H NMR (500MHz, DMSO) δ8.43 (s, 1H), 8.37–8.32 (m, 1H), 8.24 (d, J = 1.9Hz, 1H), 8.07(s,1H),7.78(dd,J=8.8,2.5Hz,1H),7.68(dd,J=8.5,2.3Hz,1H),7.14( d,J=2.0Hz,1H),6.77(dd,J=10.8,8.8Hz,2H),6.32(t,J=6.4Hz,1H),3.81( s,3H),3.76–3.47(m,10H),2.53(s,1H),1.58(d,J=8.4Hz,1H),1.22(s,1H).

[0201] Example 4: Synthesis of Compound 4

[0202]

[0203] Step 1: 4-((4-methoxybenzyl)oxy)-6-(propylamino)pyrazoline[1,5-a]pyridine-3-carbamate

[0204] At room temperature, 6-bromo-4-((4-methoxybenzyl)oxy)pyrazoline[1,5-a]pyridine-3-carbamate (500 mg, 1.4 mmol), cuprous iodide (40 mg, 0.21 mmol), L-proline (32 mg, 0.28 mmol), and potassium carbonate (966 mg, 7.0 mmol) were dissolved in 10 mL of anhydrous DMSO under nitrogen protection, and n-propylamine (827 mg, 14 mmol) was added. The reaction was carried out overnight at 120 °C until complete. After cooling to room temperature, the mixture was diluted with EA (50 mL) and water (50 mL), stirred, filtered, extracted, dried, filtered, concentrated, and subjected to column chromatography (PE:EA = 3:1) to obtain 4-((4-methoxybenzyl)oxy)-6-(propylamino)pyrazoline[1,5-a]pyridine-3-carbamate (170 mg), yield 36%. MS m / z (ESI): 337.1 [M+H] + .

[0205] Step 2: 4-Hydroxy-6-(propylamino)pyrazole[1,5-a]pyridine-3-carbamate

[0206] At room temperature, 170 mg (0.5 mmol) of 4-((4-methoxybenzyl)oxy)-6-(propylamino)pyrazoline[1,5-a]pyridine-3-carbamate (DCM) was dissolved in 1 mL of DCM, and 1 mL of trifluoroacetic acid was added at 0 °C. The reaction was allowed to proceed for 0.5 h. After the reaction was complete, the product was concentrated to dryness at room temperature and used directly in the next step without further purification. MS m / z (ESI): 217.1 [M+H] + .

[0207] Step 3: 3-Cyano-6-(propylamino)pyrazole[1,5-a]pyridine-4-trifluoromethanesulfonate

[0208] 4-Hydroxy-6-(propylamino)pyrazole[1,5-a]pyridine-3-carbamate (109 mg, 0.5 mmol) and DIEA (193 mg, 1.5 mmol) were dissolved in 5 mL of DMF. N-phenylbis(trifluoromethanesulfonyl)imide (178 mg, 0.55 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 0.5 h until complete. The solution was extracted, dried, filtered, concentrated, and subjected to column chromatography (PE:EA = 3:1) to obtain 3-cyano-6-(propylamino)pyrazole[1,5-a]pyridine-4-yltrifluoromethanesulfonic acid (170 mg), yield 96%. MS m / z (ESI): 349.1 [M+H] + .

[0209] Step 4: 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)-6-(propylamino)pyrazoline[1,5-a]pyridin-3-carbamate

[0210] At room temperature, 3-cyano-6-(propylamino)pyrazole[1,5-a]pyridine-4-trifluoromethanesulfonate (170 mg, 0.48 mmol), 6-((6-methoxypyridin-3-yl)methyl)-3-(5-(tributyltinyl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane (281 mg, 0.48 mmol), Pd(PPh3)4 (55 mg, 0.048 mmol), and cuprous iodide (9 mg, 0.048 mmol) were dissolved in water. The xylene was dissolved in 10 mL, purged three times with nitrogen, and reacted at 130 °C for 3 h until complete. The reaction was then cooled, concentrated to remove xylene, diluted, extracted, dried, filtered, and concentrated. Column chromatography (DCM:MeOH = 10:1) yielded 35 mg of 4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)-6-(propylamino)pyrazoline[1,5-a]pyridine-3-carbamate (compound 4), yield: 14%. MS m / z (ESI): 494.1 [M+H] + .

[0211] 1 H NMR(400MHz,DMSO)δ8.38(s,1H),8.32(d,1H),8.06(s,1H),7.92(s,1H),7.77(d,1H),7.66(d,1H),7.04(s,1H),6.74 -6.78(m,2H),5.95(m,1H),3.80(s,3H),3.50-3.74(m,9H),2.97-3.02(m,2H),1.55-1.63(m,3H),0.93-0.98(m,3H).

[0212] Example 5: Synthesis of Compound 5

[0213]

[0214] Step 1: 6-(ethylamino)-4-((4-methoxybenzyl)oxy)pyrazole[1,5-a]pyridine-3-carbamate

[0215] At room temperature, 6-bromo-4-((4-methoxybenzyl)oxy)pyrazoline[1,5-a]pyridine-3-carbamate (1.0 g, 2.8 mmol), cuprous iodide (53 mg, 0.28 mmol), L-proline (48 mg, 0.42 mmol), and potassium carbonate (3.8 g, 28 mmol) were dissolved in 15 mL of anhydrous DMSO under nitrogen protection, and ethylamine hydrochloride (2.28 g, 28 mmol) was added. The reaction was carried out overnight at 100 °C until complete. After cooling to room temperature, the mixture was diluted with EA (100 mL) and water (50 mL), stirred, filtered, extracted, dried, filtered, concentrated, and subjected to column chromatography (PE:EA = 3:1) to obtain 6-(ethylamino)-4-((4-methoxybenzyl)oxy)pyrazoline[1,5-a]pyridine-3-carbamate (470 mg), yield 52%. MS m / z(ESI): 323.1 [M+H] + .

[0216] Step 2: 6-(ethylamino)-4-hydroxypyrazoline[1,5-a]pyridine-3-carbamate

[0217] At room temperature, 2.28 g (7 mmol) of 6-(ethylamino)-4-((4-methoxybenzyl)oxy)pyrazole[1,5-a]pyridine-3-carbamate was dissolved in 10 mL of DCM, and 10 mL of trifluoroacetic acid was added at 0 °C. The reaction was allowed to proceed for 0.5 h. After the reaction was complete, the product was concentrated to dryness at room temperature and used directly in the next step without further purification. MS m / z (ESI): 203.1 [M+H] + .

[0218] Step 3: 3-Cyano-6-(ethylamino)pyrazoline[1,5-a]pyridin-4-yltrifluoromethanesulfonate

[0219] At room temperature, 1.43 g (7 mmol) of 6-(ethylamino)-4-hydroxypyrazoline[1,5-a]pyridine-3-carbamate and DIEA (2.7 g, 21 mmol) were dissolved in 10 mL of DMF. N-phenylbis(trifluoromethanesulfonyl)imide (2.5 g, 7 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 0.5 h until complete. The solution was extracted, dried, filtered, concentrated, and subjected to column chromatography (PE:EA = 3:1) to obtain 1.74 g (cyano-6-(ethylamino)pyrazoline[1,5-a]pyridine-4-yltrifluoromethanesulfonate), yield 76%. MS m / z (ESI): 335.1 [M+H] + .

[0220] Step 4: 6-(ethylamino)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazoline[1,5-a]pyridin-3-carbamate

[0221] At room temperature, 6-(ethylamino)pyrazole[1,5-a]pyridin-4-yl trifluoromethanesulfonate (0.9 g, 2.69 mmol), 6-((6-methoxypyridin-3-yl)methyl)-3-(5-(4,4,5,5-tetramethyl-1,3-dioxane-2-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane (1.47 g, 3.5 mmol), Pd2(dba)3 (246 mg, 0.269 mmol), and x-phos (256 mg, 0.053 mmol) were dissolved in Di In a solution of oxane / H₂O (20 mL / 4 mL), nitrogen was purged three times, and the reaction was carried out at 100 °C for 16 h until complete. The mixture was cooled, concentrated, diluted, extracted, dried, filtered, and concentrated. Column chromatography (DCM:MeOH = 10:1) yielded 1.0 g of 6-(ethylamino)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptane-3-yl)pyridin-3-yl)pyrazoline[1,5-a]pyridine-3-carbamate (compound 5). The 1.0 g was then purified again by slurrying with diethyl ether to obtain 400 mg of the compound. Yield: 31%. MS m / z (ESI): 481.1 [M+H] + .

[0222] 1 H NMR(400MHz,DMSO)δ8.38(s,1H),8.32(d,1H),8.06(s,1H),7.92(s,1H),7.77(d,1H),7.66(d,1H),7.04(s, 1H),6.74-6.78(m,2H),5.95(m,1H),3.80(s,3H),3.50-3.74(m,9H),1.55-1.63(m,3H),0.93-0.98(m,3H).

[0223] Physicochemical property tests:

[0224] (a) Solubility test

[0225] The experimental method is as follows:

[0226] 1. Sample preparation

[0227] Take 5 μL of 20 mM compound stock solution prepared with DMSO and add it to 495 μL of phosphate buffer (pH = 7.4) (final concentration 200 μM). Mix well and incubate at 25°C with shaking at 1000 rpm for 1.5 h (n = 2). After complete dissolution, filter the sample solution. Take 2 μL of each filtrate and add 98 μL of acetonitrile aqueous solution containing internal standard (200 nM tolbutamide) (v / v = 1 / 1). Mix well for 1 min and analyze by LC-MS / MS.

[0228] 2. External Standard Point Method

[0229] Preparation of standard solutions: Take 2 μL of 20 mM compound stock solution prepared with DMSO and add it to 198 μL of acetonitrile (final concentration 200 μM), (n=2). After mixing, incubate at 25℃ and shake at 1000 rpm for 10 min (n=2) until completely dissolved. Take 2 μL of each filtrate and add 198 μL of acetonitrile aqueous solution containing internal standard (v / v=1 / 1). Mix for 1 min and analyze by LC-MS / MS.

[0230] The concentration of the sample solution is calculated by detecting the peak area of ​​the standard solution and the peak area of ​​the sample solution with known concentrations.

[0231]

[0232] 3. Standard Curve Method

[0233] Preparation of standard solutions: Take 2 μL of 20 mM compound stock solution prepared with DMSO and add it to 198 μL of acetonitrile (final concentration 200 μM) (n=2). After mixing, incubate at 25℃ with shaking at 1000 rpm for 10 min (n=2) until completely dissolved. Take 4 μL of each filtrate and add 196 μL of acetonitrile aqueous solution containing internal standard (v / v=1 / 1), i.e., concentration 4000 nM. Then, dilute successively with acetonitrile aqueous solution containing internal standard (v / v=1 / 1) to prepare standard solutions with concentrations of 2000, 500, 100, 20, 4 and 2 nM, respectively, and analyze by LC-MS / MS.

[0234] The solubility of the compounds is summarized below:

[0235] The solubility of compounds 2 and 5 was obtained using the standard curve method, while the solubility of the remaining compounds was obtained using the external standard point method.

[0236] Example 1 of bioactivity test:

[0237] (a) In vitro screening experiment - HTRF method to detect the inhibitory activity of compounds on RET

[0238] The experimental method is as follows:

[0239] 1.1x kinase buffer preparation: Mix 5x enzyme buffer with distilled water at a ratio of 1:4 to achieve a final concentration of 5mM magnesium chloride and 1mM dithiothreitol.

[0240] 2. Dilute the test compound (5 mM stock solution) 5 times to 1 mM with 100% dimethyl sulfoxide, and perform 10 serial dilutions at a ratio of 1:3 in a 384-well dilution plate.

[0241] 3. Using an Echo 550 pipette system, 0.2 μL of serially diluted compounds were added to 384-well plates, with two replicates per concentration. The final concentration of dimethyl sulfoxide was 0.5% (v / v). The test compound concentration gradients were 5000, 1666.7, 555.5, 185.18, 61.72, 20.57, 6.858, 2.286, 0.764, and 0.254 nM.

[0242] 4. Prepare 2x RET (0.1 ng / μL) using 1x kinase buffer.

[0243] 5. Add 5 μL of 2x RET to a 384-well plate, centrifuge at 1000g for 30s, and incubate at room temperature for 10min.

[0244] 6. Prepare a mixture of 2x tyrosine kinase-biotin-labeled substrate (2 μM) and adenine nucleoside triphosphate (20 μM) using 1x kinase buffer.

[0245] 7. Add 5 μL of a mixture of tyrosine kinase-biotin-labeled substrate and adenine triphosphate to initiate the reaction. Centrifuge at 1000g for 30 s, seal the plate, and incubate at room temperature for 30 min.

[0246] 8. Prepare a mixture of 2x Sa-XL 665 (Note: a reagent) (125 μM) and tyrosine kinase-antibody-catheter compound using homogeneous time-resolved fluorescence detection buffer.

[0247] 9. Add 10 μl of Sa-XL 665 and tyrosine kinase-antibody-catheter compound mixture to each blank, centrifuge at 1000g for 30s, and incubate at room temperature for 1h.

[0248] 10. Use an Envision 2104 microplate reader to read plates at 615nm and 665nm and calculate the ratio (665 / 615nm).

[0249] The 11% inhibition rate was calculated as follows:

[0250]

[0251] In the formula,

[0252] R0 is the average ratio of the solvent blank group on the microplate reader.

[0253] R1 is the ratio of the test compound to the microplate.

[0254] R2 is the average ratio of microplates that inhibit RET enzyme activity by 100%.

[0255] IC was calculated using GraphPad 6.0 software by fitting the inhibition rate values ​​and logarithms of compound concentrations into a nonlinear regression (dose-response-variable slope). 50 .

[0256] (b) In vitro screening experiment - HTRF method for detecting the VEGFR2 inhibitory activity of compounds

[0257] The experimental method is as follows:

[0258] 1.1x Kinase Buffer Preparation: Mix 5x enzyme buffer with distilled water at a ratio of 1:4 to achieve a final concentration of 5mM magnesium chloride, 1mM dithiothreitol, and 1mM manganese chloride.

[0259] 2. Dilute the test compound (5 mM stock solution) 5 times to 1 mM with 100% dimethyl sulfoxide, and perform 10 serial dilutions at a ratio of 1:3 in a 384-well dilution plate.

[0260] 3. Using an Echo 550 pipetting system, 0.2 μL of serially diluted compounds were added to 384-well cell culture plates (Corning, 3570), with two replicates per concentration. The final dimethyl sulfoxide concentration was 0.5% (v / v). The test compound concentration gradients were 5000, 1666.7, 555.5, 185.18, 61.72, 20.57, 6.858, 2.286, 0.764, and 0.254 nM.

[0261] 4. Prepare 2x VEGFR2 (0.02 ng / μL) using 1x kinase buffer.

[0262] 5. Add 5 μL of 2x VEGFR2 to a 384-well plate, centrifuge at 1000g for 30s, and incubate at room temperature for 10min.

[0263] 6. Prepare a mixture of 2x tyrosine kinase-biotin-labeled substrate (2 μM) and ATP (8 μM) using 1x kinase buffer.

[0264] 7. Add 5 μL of a mixture of tyrosine kinase-biotin-labeled substrate and adenine triphosphate to initiate the reaction. Centrifuge at 1000g for 30s, seal the plate, and incubate at room temperature for 40min.

[0265] 8. Prepare a 2x Sa-XL 665 (125 μM) and tyrosine kinase-antibody-catheter compound mixture using homogeneous time-resolved fluorescence detection buffer.

[0266] 9. Add 10 μl of Sa-XL 665 and tyrosine kinase-antibody-catheter compound mixture to each well, centrifuge at 1000g for 30s, and incubate at room temperature for 1h.

[0267] 10. Use an Envision 2104 microplate reader to read plates at 615nm and 665nm and calculate the ratio (665 / 615nm).

[0268] The 11% inhibition rate was calculated as follows:

[0269]

[0270] In the formula,

[0271] R0 is the average ratio of the solvent blank group on the microplate reader.

[0272] R1 is the ratio of the test compound to the microplate.

[0273] R2 is the average ratio of microplates that inhibit RET enzyme activity by 100%.

[0274] 12. By fitting the inhibition rate values ​​and logarithms of compound concentrations to a nonlinear regression (dose-response-variable slope).

[0275] In the middle, GraphPad 6.0 is used to calculate IC. 50 .

[0276] (c) In vitro screening assay - CellTiter-Glo luminescence assay for detecting compounds inhibiting Ba / F3-KIF5B-RET cell viability

[0277] Experimental steps:

[0278] 1. Utilize The transfection system method introduced a mammalian cell expression vector containing human KIF5B-RET cDNA into Ba / F3 cells. After selection with puromycin, the surviving clones were subjected to cell growth inhibition function experiments and Western blotting to verify the cell line with stable high RET expression.

[0279] 2. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 μg / mL puromycin, and incubated at 37°C in a 5% carbon dioxide cell culture incubator.

[0280] 3. Dilute the test compound (5 mM stock solution) 2.5 times to 2 mM with 100% dimethyl sulfoxide, and perform 10 serial dilutions at a ratio of 1:3 in a 384-well dilution plate.

[0281] 4. Using an Echo 550 pipetting system, 0.2 μL of serially diluted compounds were added to 384-well cell culture plates (Corning, 3570), with two replicates per concentration. The final dimethyl sulfoxide concentration was 0.5% (v / v). The test compound concentration gradients were 5000, 1666.7, 555.5, 185.18, 61.72, 20.57, 6.858, 2.286, 0.764, and 0.254 nM.

[0282] 5. Add 40 μL of a suspension containing 800 Ba / F3-KIF5B-RET cells to each well and incubate in a 5% CO2 cell culture incubator for 72 h.

[0283] 6. Add 20 μL of Cell Titer-Glo reagent to each well of the cell culture plate, vortex and mix for 2 min to lyse the cells, then incubate at room temperature for 30 min, and read the fluorescence signal value using an Envision 2104 microplate reader.

[0284] 7. Data is generated by XLFit 5.0 using a 4-parameter formula:

[0285] Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)) (Calculate IC using fitting) 50 value.

[0286] (d) In vitro screening experiment - HTRF method to detect the inhibitory activity of the compound on RET G810R / RET G810S

[0287] The experimental method is as follows:

[0288] 1.1x kinase buffer preparation: Mix 5x enzyme buffer with distilled water at a ratio of 1:4 to achieve a final concentration of 5mM magnesium chloride and 1mM dithiothreitol.

[0289] 2. Dilute the test compound (5 mM stock solution) 5 times to 1 mM with 100% dimethyl sulfoxide, and perform 10 serial dilutions at a ratio of 1:3 in a 384-well dilution plate.

[0290] 3. Using an Echo 550 pipette system, 0.2 μL of serially diluted compounds were added to 384-well plates, with two replicates per concentration. The final concentration of dimethyl sulfoxide was 0.5% (v / v). The test compound concentration gradients were 5000, 1666.7, 555.5, 185.18, 61.72, 20.57, 6.858, 2.286, 0.764, and 0.254 nM.

[0291] 4. Prepare 2x RET G810R / RET G810S (0.1 ng / μL) using 1x kinase buffer.

[0292] 5. Add 5 μL of 2x RET G810R / RET G810S to a 384-well plate, centrifuge at 1000g for 30s, and incubate at room temperature for 10min.

[0293] 6. Prepare a mixture of 2x tyrosine kinase-biotin-labeled substrate (2 μM) and adenine nucleoside triphosphate (20 μM) using 1x kinase buffer.

[0294] 7. Add 5 μL of a mixture of tyrosine kinase-biotin-labeled substrate and adenine triphosphate to initiate the reaction. Centrifuge at 1000g for 30s, seal the plate, and incubate at room temperature for 40min.

[0295] 8. Prepare 4x Sa-XL 665 (Note: one reagent) (125 μM) for detection buffer using homogeneous time-resolved fluorescence technology.

[0296] 9. Add 5 μL of Sa-XL 665 and 5 μL of tyrosine kinase-antibody-catheter compound mixture to each blank, centrifuge at 1000g for 30s, and incubate at room temperature for 1h.

[0297] 10. Use an Envision 2104 microplate reader to read plates at 615nm and 665nm and calculate the ratio (665 / 615nm).

[0298] The 11% inhibition rate was calculated as follows:

[0299]

[0300] In the formula,

[0301] R0 is the average ratio of the solvent blank group on the microplate reader.

[0302] R1 is the ratio of the test compound to the microplate.

[0303] R2 is the average ratio of microplates that inhibit RET enzyme activity by 100%.

[0304] IC was calculated using GraphPad 6.0 software by fitting the inhibition rate values ​​and logarithms of compound concentrations into a nonlinear regression (dose-response-variable slope). 50 .

[0305] The activities of the compounds are summarized in Table 1 below.

[0306] Table 1

[0307]

[0308] The results showed that the compounds of the present invention all possessed good RET kinase activity. Furthermore, the compounds exhibited excellent inhibitory activity against the mutant strains G810S and G810R, especially compound 2 / 5, whose inhibitory activity against the two RET kinase mutants G810S and G810R was as low as nM. Simultaneously, the compounds of the present invention showed good selectivity for VEGFR2 kinase. In addition, all the compounds exhibited good RET kinase-sensitive cell inhibitory activity.

[0309] discuss

[0310] Structure-activity relationship studies showed that, compared with the existing positive control (LOXO-292), when compound R of formula I of the present invention... m and R n When the ethyl group is hydrogen atom and the ethyl group is ethyl, the compounds of the present invention have good inhibitory activity against G810 mutant RET, especially when the ethyl group is replaced by a fluorine atom, the compounds have better inhibitory activity against G810 mutant RET.

[0311] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt or isotope thereof, In the formula, R m Selected from the following group, substituted or unsubstituted: H, C1-C3 alkyl; wherein, The substitution refers to the substitution by 1-2 halogen atoms, R n It is a methyl group.

2. The compound of claim 1 or a pharmaceutically acceptable salt or isotope thereof, characterized in that, R m Selected from: H, methyl, ethyl, n-propyl, -CH2F, -CHF2, -CH2CH2F, -CH2CHF2, -CHFCH3, -CHFCH2F, -CH2CH2CH2F or -CH2CHFCH2F.

3. A compound or a pharmaceutically acceptable salt or isotope thereof, characterized in that, The compound has the structure shown in Formula II. R m Selected from: methyl, -CH2F, -CHF2, -CHFCH3, -CHFCH2F or -CH2CHFCH2F.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or isotopic compound thereof, characterized in that, The pharmaceutically acceptable salts are selected from acetate, adipic acid salt, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphor salt, camphor sulfonate, cyclopentanepropionate, diethylene glycolate, dodecyl sulfate, ethanesulfonate, fumarate, glucono-heptahydrate, glyceryl phosphate, hemisulfate, heptahydrate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, and dodecanoate.

5. Use of a compound or a pharmaceutically acceptable salt or isotope thereof in the preparation of a medicament for inhibiting RET kinase activity in cells or subjects; characterized in that, The compounds are selected from the group consisting of: The RET kinase mentioned is the mutant G810S.

6. The use as described in claim 5, characterized in that, The drug is used to treat diseases related to RET kinase, wherein the RET kinase is a mutant G810S, and the diseases are selected from the group consisting of: eye diseases, rheumatoid arthritis, pulmonary fibrosis, liver fibrosis, and tumors, including: bladder cancer, ovarian cancer, adenocarcinoma, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neurocytoma, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, and laryngeal cancer. Cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urinary tract cancer, melanoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma.

7. Use of a compound or a pharmaceutically acceptable salt or isotope thereof in the preparation of a medicament for inhibiting RET kinase activity in cells or subjects; characterized in that, The compounds are selected from the group consisting of: The RET kinase mentioned is the mutant G810.

8. The use as described in claim 7, characterized in that, The drug is used to treat diseases related to RET kinase, wherein the RET kinase is a mutant G810, and the diseases are selected from the group consisting of: eye diseases, rheumatoid arthritis, pulmonary fibrosis, liver fibrosis, and tumors, including: bladder cancer, ovarian cancer, adenocarcinoma, gastric cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neurocytoma, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, and laryngeal cancer. Cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urinary tract cancer, melanoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma.

9. The use as described in any one of claims 5 to 8, characterized in that, The pharmaceutically acceptable salts are selected from acetate, adipic acid salt, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphor salt, camphor sulfonate, cyclopentanepropionate, diethylene glycolate, dodecyl sulfate, ethanesulfonate, fumarate, glucono-heptahydrate, glyceryl phosphate, hemisulfate, heptahydrate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxyethanesulfonate, lactate, maleate, methanesulfonate, naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, and dodecanoate.

10. A pharmaceutical composition, characterized in that, It comprises the compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt or isotopic compound thereof; and also comprises a pharmaceutical carrier or diluent.

11. Use of a compound as described in any one of claims 1 to 4 or a pharmaceutical composition as described in claim 10 in the preparation of a medicament for inhibiting RET kinase activity in cells or subjects.

12. The use as described in claim 11, wherein the RET kinase is selected from wild type, gene fusion type and mutant type.

13. The use as described in claim 12, wherein the RET kinase is a mutant, specifically G804 or G810.

14. The use as described in any one of claims 11-13, wherein the medicament is used to treat RET-related diseases and disorders of expression, activity, or level of the RET gene, RET kinase, or any of them.

15. The use as described in claim 14, wherein the disease is selected from the group consisting of: eye diseases, rheumatoid arthritis, pulmonary fibrosis, liver fibrosis, and tumors, wherein the tumors include: Bladder cancer, ovarian cancer, adenocarcinoma, stomach cancer, pancreatic cancer, prostate cancer, colon cancer, lung cancer, bone cancer, brain cancer, neurocytoma, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parenchymal carcinoma, ovarian cancer, cervical cancer, uterine endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, urinary tract cancer, melanoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma.

16. A method for preparing the compound according to any one of claims 1-4 or its pharmaceutically acceptable salt or isotopic compound, characterized in that, Includes the following steps: (i-1) In an inert solvent under alkaline conditions, compound 2-1 reacts with a hydroxyl protecting agent to give compound 2-2; (i-2) In an inert solvent, under alkaline conditions and in the presence of a catalyst, compound 2-2 reacts with the amine compound NHR. m R n The reaction yields compounds 2-3; (i-3) In an inert solvent under acidic conditions, compound 2-3 is deprotected to give compound 2-4; (i-4) In an inert solvent, compound 2-4 reacts with PhNTf2 to give compound 2-5; (i-5) In an inert solvent and in the presence of a catalyst, compounds 2-5 react with compounds 1-8 to give compound I; In the formula, R m R n The definition is as described in any one of claims 1-3.

Images