CN112851664A - Pyrazolo [1,5-a ] pyridine-3-nitrile compound and application thereof in medicine - Google Patents

Abstract

The invention provides a compound shown in a formula (I) or a pharmaceutically acceptable salt thereof, a preparation method thereof, and application thereof as a therapeutic agent, in particular as a Rearrangement (RET) kinase inhibitor during selective transfection, wherein the definition of each substituent in the formula (I) is the same as that in the specification.

Description

Pyrazolo [1,5-a ] pyridine-3-nitrile compound and application thereof in medicine

Technical Field

The invention relates to the technical field of medicines, in particular to a novel pyrazolo [1,5-a ] pyridine-3-nitrile compound, a preparation method thereof, a pharmaceutical composition containing the derivative and application thereof as a therapeutic agent, especially as a rearrangement during selective transfection (RET) kinase inhibitor.

Background

RET is a single transmembrane receptor belonging to the superfamily of tyrosine kinases and is required for normal development, maturation and maintenance of several tissues and cells, unlike other receptor tyrosine kinases, RET is linked to the cell surface via a glycosylphosphoinositide linkage with the glial cell derived neurotrophic factor (GDNF) family of receptors-alpha (GFR α). Glial cell derived neurotrophic factor family ligand (GFL) and glial cell derived neurotrophic factor (GDNF) family receptor-alpha (GFR α) family members form a binary complex that binds to RET and recruits it to the cholesterol-rich membrane pressure domain of lipid rafts. In which RET signaling occurs.

Aberrant expression of the RET gene is associated with a variety of cancer diseases. The gene is fused with other genes through chromosome rearrangement or is in a continuous activation state through site-directed variation, and the gene is independent of a ligand, so that a signal path is abnormal, and the cell is over-proliferated and the cancer is generated.

In recent years, there has been increasing evidence that RET gene fusion and mutation are the driving forces for some cancer induction, and do not coincide with other driving genes, with significant specificity. RET gene fusion is most common in papillary thyroid carcinomas and non-small cell lung carcinomas, such as 30% sporadic papillary thyroid carcinomas and 70% radiation-induced papillary thyroid carcinomas and about 2% non-small cell lung carcinomas are driven by fusion of RET. RET gene mutations are most found in medullary thyroid cancers, such as more than 50% of medullary thyroid cancers, and nearly all congenital medullary cancers and multiple endocrine adenomatosis are caused by site-directed mutations in the RET gene.

Current therapeutic approaches mainly employ multi-targeted kinase inhibitors with RET inhibitory activity to treat cancer patients with RET fusions or mutations. However, under these conditions, the dose of drug is insufficient to achieve a level sufficient to inhibit abnormal RET gene expression due to off-target effects and drug toxicity. In addition, cancer cells develop resistance through mutation during the course of cancer treatment. Once resistance develops, patient treatment options become very limited. Therefore, there is a great need for a selective inhibitor of RET to treat patients with RET gene fusions or mutations.

There are no drugs on the market that are selective for RET targets, and RET positive patients can be treated with multi-target kinase inhibitors. A series of selective RET kinase inhibitor patents have been disclosed including WO2016127074, WO2017079140, WO2017011776, WO2017161269, WO2018022761, WO2018136661, WO2018136663, etc., and the drugs currently in clinical phase I include Blu-667, Loxo-292, GSK-3352589, etc. However, these are far from sufficient for antitumor studies, and there is still a need to study and develop new rearrangement-during-selective-transfection (RET) kinase inhibitors to address the unmet medical need.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a compound shown as a formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof:

wherein:

X₁、X₂each is independently selected from CH or N;

l is selected from:

wherein L is optionally joined at both ends with A and R₂Connecting;

a is selected from 4-6 membered monocyclic heterocyclic group, -NH-4-6 membered heterocyclic group, 7-11 membered bridgeA heterocyclic group, a 7-to 11-membered spiroheterocyclic group or a 7-to 11-membered fused ring heterocyclic group, wherein said monocyclic heterocyclic group, -NH-4-to 6-membered heterocyclic group, bridged heterocyclic group, spiroheterocyclic group or fused ring heterocyclic group is optionally further substituted with one or more groups selected from C₁-C₃Alkyl, hydroxyalkyl, halogeno C₁-C₃Alkyl, hydroxy, C₃-C₆Cycloalkyl or substituted with a substituent of ═ O;

R₁selected from: c₃-C₆Cycloalkyl, 3-6 membered heterocyclic group, 6-7 membered heteroaryl or 7-11 membered fused heterocyclic group, wherein the cycloalkyl and heterocyclic groups are optionally further substituted by one or more groups selected from hydroxy, hydroxyalkyl, amino, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Substituted by a substituent of alkoxy; said heteroaryl and fused heterocyclyl is optionally further substituted by one or more groups selected from halogen, hydroxy, amino, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy radical, C₃-C₆Cycloalkyl, -NHR₃、-NR₃R₄Or substituted with a substituent of ═ O;

R₂is selected from C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, 3-6 membered heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy radical, C₃-C₆Cycloalkyl, -NHR₃、-NR₃R₄Or substituted with a substituent of ═ O;

R₃、R₄each independently selected from C₁-C₆An alkyl group;

wherein said halogen is C₁-C₃Alkyl or halo C₁-C₃Alkoxy radicalPreferably 1 to 3 fluoro C₁-C₃Alkyl or C₁-C₃An alkoxy group.

In some preferred embodiments of the present invention, the compound of formula (I) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof is a compound of formula (II) or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof:

wherein: A. l, R₁And R₂As defined in formula (I).

In some preferred embodiments of the invention, the compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein a is selected from the group consisting of:

wherein: the 1 endpoint and the 2 endpoint are optionally connected with the L.

In some preferred embodiments of the invention, the compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R is₁Selected from:

in some preferred embodiments of the invention, the compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R is₂Is selected from C₁-C₆Alkyl radical, C₄-C₆Cycloalkyl, 4-6 membered heterocyclic group, phenyl, 5-6 membered heteroaryl or 10 membered heteroaryl, wherein said C₄-C₆Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl or 10 membered heteroaryl optionally further substituted with one or more substituents selected from cyano,Halogen, hydroxy, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy or ═ O.

In some preferred embodiments of the invention, the compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R is₂Selected from:

in a preferred embodiment of the present invention, there is provided a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.

Further, the present invention provides a method of treating a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, comprising:

the method comprises the following steps:

reacting compound (IA) and compound (IB) under basic conditions in the presence of a condensation agent to give a compound of formula (I);

wherein:

the condensation reagent is selected from 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N '-dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1-hydroxy-7-azobenzotriazole, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, pentafluorophenyl diphenyl phosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate or benzotriazol-1-yloxytripyrrolidinyl phosphonium hexafluorophosphate; preferably 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate;

the reagent for providing the alkaline condition is an organic base, and the organic base is selected from N, N-diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine and 4-dimethylaminopyridine, and is preferably N, N-diisopropylethylamine or triethylamine;

A、L、X₁、X₂、R₁and R₂As defined in formula (I); or

The method 2 comprises the following steps:

reacting compound (IA) and compound (IB) under basic conditions to obtain a compound of formula (I);

wherein:

the reagent for providing the alkaline condition is an inorganic base, wherein the inorganic base is selected from the group consisting of potassium phosphate, potassium phosphate trihydrate, potassium acetate, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, and potassium hydride, preferably sodium carbonate or potassium carbonate;

g is selected from a leaving group, preferably halogen;

A、L、X₁、X₂、R₁and R₂As defined in formula (I).

Still further, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

The invention provides an application of a compound shown as a formula (I) or (II) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof in preparing medicines.

The invention also provides the use of a compound of formula (I) or (II) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a Rearrangement (RET) kinase inhibitor during transfection.

The present invention further provides the use of a compound of formula (I) or (II) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disease driven by rearrangement during transfection (RET) genes, wherein said disease is preferably cancer, wherein said cancer is preferably lung cancer, thyroid cancer, colon cancer, breast cancer or pancreatic cancer.

The present invention provides compounds of formula (I) or (II) or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, which are inhibitors of Rearrangement (RET) kinase during selective transfection. Accordingly, the present invention provides a method of selectively inhibiting Rearrangement (RET) kinase during transfection, comprising contacting the Rearrangement (RET) kinase during transfection with a compound of formula (I) or (II) of the present invention, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. Accordingly, the present invention also provides a method for treating a disease driven by rearrangement during transfection (RET) gene, comprising administering to a subject in need thereof a compound of formula (I) or (II) of the present invention or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, wherein the disease is preferably cancer, wherein the cancer is preferably lung cancer, thyroid cancer, colon cancer, breast cancer or pancreatic cancer.

Detailed description of the invention

Some of the terms used in the specification and claims of the present invention are defined as follows:

"alkyl" when taken as a group or part of a group means including C₁-C₂₀Straight-chain or branched aliphatic hydrocarbon groups. Preferably C₁-C₁₀Alkyl, more preferably C₁-C₆An alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, and the like. Alkyl groups may be substituted or unsubstituted.

"cycloalkyl" refers to saturated or partially saturated monocyclic, fused, bridged, and spiro carbocyclic rings. Preferably C₃-C₁₂Cycloalkyl, more preferably C₃-C₈Cycloalkyl, most preferably C₃-C₆A cycloalkyl group. Examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like, with cyclopropyl, cyclohexenyl being preferred.

"Heterocyclyl", "heterocycle" or "heterocyclic" are used interchangeably herein and all refer to non-aromatic heterocyclic groups in which one or more of the ring-forming atoms is a heteroatom, such as oxygen, nitrogen, sulfur, and the like, including monocyclic, fused, bridged, and spiro rings. Preferably having a 5-7 membered monocyclic ring or a 7-10 membered bicyclic or tricyclic ring which may contain 1,2 or 3 atoms selected from nitrogen, oxygen or sulfur. Examples of "heterocyclyl" include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1-dioxo-thiomorpholinyl, piperidinyl, alkenylpiperidinyl, 3, 6-dihydro-2H-pyranyl, 1-methyl-2-oxo-1, 2-dihydropyridine, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo [3.2.1] octyl, and piperazinyl. The heterocyclic group may be substituted or unsubstituted.

"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C₆-C₁₀Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. The aryl group may be substituted or unsubstituted. The "aryl" may be fused to a heteroaryl, heterocyclyl or cycloalkyl group, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples include, but are not limited to:

"heteroaryl" refers to an aromatic 5-7 membered monocyclic or 9-10 membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, 1,2, 3-thiadiazolyl, benzodioxolyl, benzimidazolyl, indolyl, isoindolyl, 1, 3-dioxo-isoindolyl, quinolinyl, indazolyl, benzisothiazolyl, benzoxazolyl, and benzisoxazolyl. Heteroaryl groups may be substituted or unsubstituted. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples include, but are not limited to:

"alkoxy" refers to a radical of (alkyl-O-). Wherein alkyl is as defined herein. C₁-C₆Alkoxy groups of (4) are preferred. Examples include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.

"hydroxy" means-OH.

"halogen" means fluorine, chlorine, bromine and iodine, preferably chlorine, bromine and fluorine.

"amino" means-NH₂。

"cyano" means-CN.

"nitro" means-NO₂。

"benzyl" means-CH₂-phenyl.

"carboxy" refers to-C (O) OH.

"carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.

"DMSO" refers to dimethyl sulfoxide.

"Boc" refers to tert-butoxycarbonyl.

"Ms" refers to sulfonyl.

"Ts" refers to 4-methylbenzenesulfonyl.

A "leaving group", or leaving group, an atom or functional group that is removed from a larger molecule in a chemical reaction, is a term used in nucleophilic substitution and elimination reactions. In nucleophilic substitution reactions, the reactant attacked by the nucleophile is called the substrate (substrate), and the atom or group of atoms cleaved from the substrate molecule with a pair of electrons is called the leaving group. Groups that accept electrons easily and have a strong ability to bear negative charges are good leaving groups. The lower the pKa of the conjugate acid of the leaving group, the easier it is for the leaving group to be cleaved from other molecules. The reason is that the tendency to exist as an anion (or an electrically neutral leaving group) is enhanced when the pKa of its conjugate acid is smaller, and the corresponding leaving group does not need to be bound to another atom. Common leaving groups include, but are not limited to, halogen, -OTs, or-OH.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in a group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

As used herein, "substituted" or "substituted," unless otherwise specified, means that the group may be substituted with one or more groups selected from: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, amino, haloalkyl, hydroxyalkyl, carboxyl, carboxylate, NH-alkyl, N' N-dialkyl, or ═ O.

The definition and convention of stereochemistry in the present invention is generally used with reference to the following documents: S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-HillBook company, New York; and Eliel, E.and Wilen, S., "stereoschemistry of Organic Compounds", John Wiley & Sons, Inc., New York,1994. All stereoisomeric forms of the compounds of the present invention, including but in no way limited to diastereomers, enantiomers, atropisomers and mixtures thereof, such as racemic mixtures, form part of the present invention. Diastereomers may be separated into individual diastereomers on the basis of their physicochemical differences by chromatography, crystallization, distillation, sublimation, or the like. Enantiomers can be separated, such that a chiral isomeric mixture is converted into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., a chiral auxiliary, such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers, and converting the individual diastereomers to the corresponding pure enantiomers. The intermediates and compounds of the invention may also exist in different tautomeric forms and all such forms are included within the scope of the invention. Many organic compounds exist in optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefix D, L or R, S is used to indicate the absolute configuration of the chiral center of the molecule. The prefixes d, l or (+), (-) are used to designate the sign of the rotation of plane polarized light of the compound, with (-) or l indicating that the compound is left-handed and the prefix (+) or d indicating that the compound is right-handed. The atoms or groups of these stereoisomers are attached to each other in the same order, but they differ in their steric structure. A particular stereoisomer may be an enantiomer, and a mixture of isomers is commonly referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during the chemical reaction. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

"tautomer" or "tautomeric form" means that isomers of structures of different energies can be interconverted through a low energy barrier. For example, proton tautomers (i.e., prototropic tautomers) include tautomers that move through protons, such as keto-enol and imine-enamine isomerizations. Valence (valence) tautomers include tautomers that recombine into bond electrons. Unless otherwise indicated, the structural formulae depicted herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers): such as the R, S configuration containing an asymmetric center, the (Z), (E) isomers of the double bond, and the conformational isomers of (Z), (E). Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers, or geometric isomers thereof are intended to be within the scope of the present invention.

"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain their biological activity and are suitable for pharmaceutical use. The pharmaceutically acceptable salt of the compound represented by the formula (I) may be a metal salt, a salt with a suitable acid.

"pharmaceutical composition" means a mixture containing one or more compounds described herein, or a pharmaceutically acceptable salt or prodrug thereof, in admixture with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

Detailed Description

The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention.

Examples

The examples show the preparation of representative compounds represented by formula (I) and the associated structural identification data. It must be noted that the following examples are intended to illustrate the invention and are not intended to limit the invention. The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS).¹The H NMR spectra were obtained using a Bruker instrument (400MHz) and the chemical shifts are expressed in ppm using tetramethylsilane internal standard (0.00 ppm).¹Method for H NMR expression: s is singlet, d is doublet, m is multiplet, br is broadened, dd is doublet of doublet, dt is doublet of triplet. If a coupling constant is provided, it is in Hz. FI for Mass SpectrometryNNIGAN LCQad (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of Taiwan yellow sea as a carrier.

Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, and the like.

In the examples, unless otherwise specified, the reaction was carried out under an air atmosphere in the open air.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L. The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

In the examples, the reaction temperature was room temperature unless otherwise specified.

The room temperature is the optimum reaction temperature, and the temperature range is 20-30 ℃.

The progress of the reaction in the examples was monitored by Thin Layer Chromatography (TLC) using a developing solvent system of: a: dichloromethane and methanol systems; b: n-hexane and ethyl acetate, and the volume ratio of the solvent is adjusted according to the polarity of the compound.

The system of eluents for column chromatography and developing agents for thin layer chromatography used for purifying compounds include: a: dichloromethane and methanol systems; b: the volume ratio of the n-hexane and the ethyl acetate is adjusted according to the polarity of the compound, and a small amount of acidic or basic reagents such as triethylamine and the like can be added for adjustment.

Preparation of intermediates

Intermediates 1-d

The first step is as follows: boc-mesitylsulfonylhydroxylamine

2,4, 6-Trimethylbenzenesulfonyl chloride (a) (200g,914.5mmol), tert-butyl N-hydroxycarbamate (b) (133.0g,1000mmol) and methyl tert-butyl ether (1.5L) were added to a 3L single-neck flask, stirred in an ice bath for 30min, then triethylamine (170mL,1371.7mmol) was slowly added dropwise, reacted at 0 ℃ for 30min after the addition was complete, and then stirred at room temperature for 4 h. The reaction solution was added with diluted HCl (2N) to adjust the pH to about 4, the solution was separated, the organic phase was washed with water three times (30 mL. times.3) and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain 290g of Boc-mesitylsulfonylhydroxylamine (1-a) as a white solid. Yield: 74.2 percent.

MS m/z(ESI):316.2[M+1]

The second step is that: synthesis of mesitylenesulfonylhydroxylamine

Trifluoroacetic acid (500mL) is added into a 2L single-mouth bottle, the temperature of an ice salt bath is reduced to-15 to-20 ℃, Boc-s-trimethyl sulfonyl hydroxylamine (1-a) (290g,919.5mmol) is slowly added in batches, after the addition is finished, the reaction is carried out for 6 hours in the ice salt bath, after the reaction is finished, reaction liquid is poured into 6L ice water, a large amount of white solid is separated out, the filtration is carried out, and a filter cake is washed by water until the filtrate is neutral. Dissolving the filter cake in dichloromethane, separating liquid, drying an organic phase by using anhydrous sodium sulfate, filtering, and directly carrying out the next reaction on the obtained organic phase.

MS m/z(ESI):216.2[M+1]

The third step: synthesis of (3, 5-dichloropyridin-onium-1-yl) ((trimelsulfonyl) hydroxylamine

A solution of mesitylenesulfonylhydroxylamine (1-b) in dichloromethane (197g,915.1mmol) was added to a 2L single-neck flask, the ice bath was cooled to 0 deg.C, 3, 5-dichloropyrimidine (1-b) (135.4g,915.1mmol) was added, and the mixture was stirred at room temperature overnight. After the reaction was completed, filtration was carried out, and the filter cake was washed with dichloromethane and dried to obtain 210g of a white solid (1-c), yield: and (3.2).

MS m/z(ESI):362.02[M+1]

The fourth step: synthesis of 4, 6-dichloropyrazoline [1,5-a ] pyridine-3-carbonitrile

Adding (1-c) (105g,290.7mmol), triethylamine (84mL,481.4mmol) and 300mL of ethanol into a 1L single-neck bottle, stirring, adding 3-methoxyacrylonitrile (d) (25g,290.7mmol), reacting at 80 ℃ for 4h, cooling the reaction liquid to room temperature, pouring into 3L of ice water, filtering, and purifying a filter cake by silica gel column chromatography (n-hexane: ethyl acetate ═ 3:1) to obtain 25g of a brown yellow product, namely 4, 6-dichloropyrazoline [1,5-a ] pyridine-3-nitrile (1-d), wherein the yield is as follows: 40.6 percent.

MS m/z(ESI):213.2[M+1]

Intermediates 1-f

The first step is as follows: synthesis of 4- (5-bromopyridin-2-yl) piperazine-1-carboxylic acid tert-butyl ester

In a 200mL single-neck flask was added 2-fluoro-5-bromopyridine (e) (20g,113.6mmol), Boc-piperazine (f) (32g,170.5mmol), K₂CO₃(37g,113.6mmol), 100mL of N, N-dimethylacetamide, reacted at 110 ℃ for 4h, after the reaction was completed and the reaction solution was cooled to room temperature, poured into 1L of water, extracted three times (300mL × 3) with an organic solvent (MeOH/DCM ═ 10:1), the organic phase was washed with water (300mL × 3) and a saturated aqueous solution of sodium chloride (300mL), dried over anhydrous sodium sulfate, concentrated over a column, and purified by silica gel column chromatography (N-hexane: ethyl acetate ═ 2:1) to obtain 29.9g of the product, tert-butyl 4- (5-bromopyridin-2-yl) piperazine-1-carbonate (1-e), in yield: 77.3 percent.

MS m/z(ESI):342.2[M+1]

The second step is that: 6- (4-Boc-1-piperazinyl) pyridine-3-boronic acid pinacol ester

In a 100mL single vial was added 4- (5-bromopyridin-2-yl) piperazine-1-carbon tert-butyl ester (1-e) (29.9g,87.3mmol), diboron pinacol ester (33.3g,131.0mmol), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (3.2g,4.37mmol), potassium acetate (17.2g,174.5mmol) and 400mL1, 4-dioxane, under nitrogen, at 100 deg.C overnight. The reaction solution was cooled to room temperature, concentrated, and purified by silica gel column chromatography (n-hexane: ethyl acetate ═ 2:1) to give 24.2g of the product 6- (4-Boc-1-piperazinyl) pyridine-3-boronic acid pinacol ester (1-f), yield: 71.2 percent.

Intermediate 2-f

The first step is as follows: 3- (5-Bromopyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester

The first step of the synthesis of intermediate 1-f was repeated except that starting material 2-a was substituted for f to give intermediate 3- (5-bromopyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 2-e.

MS m/z(ESI):354.07[M+1]

The second step is that: tert-butyl-3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carbonate

The synthesis procedure of the second step of intermediate 1-f was repeated except that 1-e was replaced with the starting material 2-e to give the intermediate tert-butyl-3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carbonate 2-f.

MS m/z(ESI):402.25[M+1]

Intermediate 3-f

The first step is as follows: synthesis of 3- (5-bromopyridin-2-yl) -3, 6-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

The first step of the synthesis of intermediate 1-f was repeated except that f was replaced with starting material 3-a to give intermediate 3- (5-bromopyridin-2-yl) -3, 6-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester 3-e.

MS m/z(ESI):368.09[M+1]

The second step is that: 3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

The synthesis procedure of the second step of intermediate 1-f was repeated except that 1-e was replaced with the starting material 3-e to give intermediate 3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carbonic acid tert-butyl ester 3-f.

MS m/z(ESI):416.25[M+1]

Intermediate 4-f

The first step is as follows: synthesis of tert-butyl 3- (5-bromopyridin-2-yl) piperidin-4-yl) carbonate

The first step of synthesis of intermediate 1-f was repeated except that f was replaced with starting material 4-a to give intermediate 3- (5-bromopyridin-2-yl) piperidin-4-yl) carbonic acid tert-butyl ester 4-e.

MS m/z(ESI):368.09[M+1]

The second step is that: 3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) piperidin-4-yl) carbonic acid tert-butyl ester

The synthesis procedure of the second step of intermediate 1-f was repeated except that 1-e was replaced with 4-e as a starting material to give intermediate 3- (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridin-2-yl) piperidin-4-yl) carbonic acid tert-butyl ester 4-f.

MS m/z(ESI):416.25[M+1]

Intermediates 1-i

The first step is as follows: synthesis of tert-butyl 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-carbonate

In a 100mL single neck flask was added 4, 6-dichloropyrazoline [1,5-a ] pyridine-3-carbonitrile (1-d) (2.0g,9.4mmol), 6- (4-Boc-1-piperazinyl) pyridine-3-boronic acid pinacol ester (1-f) (4.04g,10.3mmol), potassium phosphate (4.0g,18.8mmol), tris (dibenzylideneacetone) dipalladium (0.8g,0.94mmol), tricyclohexylphosphine (0.53g,1.88mmol) and 60mL of solvent (1, 4-dioxane: water ═ 10:1), reacted at 70 ℃ for 5H under nitrogen protection, and a solution of 3, 6-dihydro-2H-pyran-4-boronic acid pinacol ester (H) (2.96g,14.1mmol) in 1, 4-dioxane was added and refluxed overnight. After the reaction was completed, the reaction solution was cooled to room temperature, concentrated, and purified by silica gel column chromatography (n-hexane: ethyl acetate ═ 1:1) to obtain 1.4g of tert-butyl 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-carbonate (1-H) as a product, in a yield: 30.7 percent.

MS m/z(ESI):486.2[M+1]

The second step is that: synthesis of 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine.

Adding 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl-pyrazoline [1, 5-a) ] into a 100mL single-mouth bottle]Pyridin-4-yl) pyridin-2-yl) piperazine-1-carboxylic acid tert-butyl ester (1-h) (1.4g,2.88mmol) and 25mL of ethanol, 5mL of concentrated hydrochloric acid, were reacted at room temperature overnight. After the reaction was complete, the reaction was concentrated, and the residue was dissolved in water (100mL) and saturated NaHCO was used₃Adjusting pH to 8-9 with water solution, extracting with dichloromethane three times (30mL × 3), combining organic phases, washing the organic phase with water (30mL × 3) and saturated aqueous sodium chloride solution (30mL), drying with anhydrous sodium sulfate, filtering, and concentrating to obtain the product 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazole [1, 5-a-]Pyridin-4-yl) pyridin-2-yl) piperazine (1-i)1.1g, yield: 99.1 percent.

MS m/z(ESI):386.2[M+1]

Intermediate 2-i

The first step is as follows: synthesis of 3- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester

The first step of the synthesis of intermediate 1-i was repeated except that starting material 2-f was substituted for 1-f to give intermediate 3- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carbonic acid tert-butyl ester 2-H.

MS m/z(ESI):499.2[M+1]

The second step is that: synthesis of 4- (6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-H was replaced with 2-H starting material to give 4- (6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 2-i as an intermediate.

MS m/z(ESI):399.2[M+1]

Intermediate 3-i

The first step is as follows: synthesis of 3- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

The first step of the synthesis of intermediate 1-i was repeated except that starting material 3-f was used instead of 1-f to give intermediate 3- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carbonic acid tert-butyl ester 3-H.

MS m/z(ESI):513.2[M+1]

The second step is that: synthesis of 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-H was replaced with 3-H as the starting material to give 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 3-i as an intermediate.

MS m/z(ESI):413.2[M+1]

Intermediate 4-i

The first step is as follows: synthesis of tert-butyl (1- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperidin-4-amino) carbonate

The first step of the synthesis of intermediate 1-i was repeated except that 1-f was replaced with the starting material 4-f to give the intermediate tert-butyl (1- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperidin-4-amino) carbonate 4-H.

MS m/z(ESI):501.2[M+1]

The second step is that: synthesis of 4- (6- (4-aminopiperidin-1-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-H was replaced with 4-H as the starting material to give 4- (6- (4-aminopiperidin-1-yl) pyridin-3-yl) -6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 4-i as an intermediate. MS M/z (ESI) 401.2[ M +1]

Intermediate 5-i

The first step is as follows: synthesis of tert-butyl 4- (5- (3-cyano-6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-carbonate

The first step of the synthesis of intermediate 1-i was repeated except that starting material j was used instead of starting material h to give intermediate 4- (5- (3-cyano-6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-tert-butyl carbonate 5-h.

MS m/z(ESI):496.2[M+1]

The second step is that: synthesis of 6- (2-methylpyridin-4-yl) -4- (6- (piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-h was replaced with 5-h of starting material to give intermediate 6- (2-methylpyridin-4-yl) -4- (6- (piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 5-i.

MS m/z(ESI):396.2[M+1]

Intermediate 6-i

The first step is as follows: synthesis of 3- (5- (3-cyano-6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

The first step of the synthesis of intermediate 1-i was repeated except that starting material 3-f was substituted for 1-f and starting material j was substituted for h to give intermediate 3- (5- (3-cyano-6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carbonic acid tert-butyl ester 6-h.

MS m/z(ESI):522.2[M+1]

The second step is that: synthesis of 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-h was replaced with 6-h of the starting material to give 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (2-methylpyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 6-i as an intermediate.

MS m/z(ESI):422.2[M+1]

Intermediate 7-i

The first step is as follows: synthesis of tert-butyl 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-carbonate

The first step of the synthesis of intermediate 1-i was repeated except that starting material k was used instead of starting material h to give intermediate 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine-1-tert-butyl carbonate 7-h.

The synthesis method is shown in the first step of the intermediate 1-i.

MS m/z(ESI):512.2[M+1]

The second step is that: synthesis of 6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) -4- (6- (piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-h was replaced with the starting material 7-h to give intermediate 6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) -4- (6- (piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 7-i.

MS m/z(ESI):412.2[M+1]

Intermediate 8-i

The first step is as follows: synthesis of tert-butyl 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carbonate

The first step of the synthesis of intermediate 1-i was repeated except starting material 2-f instead of 1-f and starting material k instead of h to give intermediate 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester 8-h.

MS m/z(ESI):524.2[M+1]

The second step is that: synthesis of 4- (6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The synthesis procedure of the second step of intermediate 1-i was repeated except that 1-h was replaced with 8-h of starting material to give intermediate 4- (6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 8-i.

MS m/z(ESI):424.2[M+1]

Intermediate 9-i

The first step is as follows: synthesis of tert-butyl 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carbonate

The first step of the synthesis of intermediate 1-i was repeated except that starting material 3-f was substituted for 1-f and starting material k was substituted for h to give intermediate 3- (5- (3-cyano-6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester 9-h.

MS m/z(ESI):538.2[M+1]

The second step is that: synthesis of 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile.

The second step of the synthesis of intermediate 1-i was repeated except that 1-h was replaced with 9-h starting material to give intermediate 4- (6- (3, 8-diazabicyclo [3.2.1] octan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile 9-i.

MS m/z(ESI):438.2[M+1]

Intermediate 1-m

The first step is as follows: synthesis of (6-methoxypyridin-3-yl) methanol

6-methoxynicotinic acid (1-j) (15g,97.95mmol) and 150mL tetrahydrofuran were added to a 100mL single-neck flask, stirred in an ice bath for 30min, lithium aluminum hydride (5.58g,146.93mmol) was added slowly in portions, reacted for 1h in an ice bath, the ice bath was removed, followed by reaction at room temperature for 2h, sodium sulfate pentahydrate was added slowly after the reaction was over until no bubbles emerged, filtered, the filter cake was washed with dichloromethane (200mL), and the filtrate was concentrated to give 13g of product, yield: 93.4 percent. The second step is that: synthesis of 5- (chloromethyl) -2-methoxypyridine

(6-methoxypyridin-3-yl) methanol (1-k) (13g,93.42mmol) and 150mL of dichloromethane were added to a 100mL single-necked flask, stirred in an ice bath for 30min, thionyl chloride (13.5mL,186.84mmol) was added dropwise, the ice bath was removed, and the reaction was allowed to proceed overnight at room temperature. After the reaction, a saturated aqueous solution of sodium bicarbonate was added to the reaction mixture to adjust the PH to 7 to 8, liquid separation was performed, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography (n-hexane: ethyl acetate: 10:1) to obtain 2.7g of a product, yield: 18.3 percent.

Example 1

Preparation of Compound I-1

Synthesis of (R) -6- (3, 6-dihydro-2H-pyran-4-yl) -4- (6- (4- (2-hydroxy-3-methylbutyryl) piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile

In a 100mL single-necked flask were added 4- (5- (3-cyano-6- (3, 6-dihydro-2H-pyran-4-yl) pyrazolo [1,5-a ] pyridin-4-yl) pyridin-2-yl) piperazine (1-i) (100mg,0.26mmol), (R) -2-hydroxy-3-methylbutyric acid (61.3mg,0.52mmol), N, N-diisopropylethylamine (134mg,1.03mmol), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (536.0mg,1.03mmol), and 10mL of N, N-dimethylformamide, and reacted at room temperature overnight. After the reaction was completed, the reaction solution was poured into water (100mL), extracted three times with dichloromethane (30mL × 3), the organic phases were combined, the organic phase was washed with water (30mL × 3), a saturated aqueous solution of sodium chloride (30mL), dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (dichloromethane: methanol ═ 30:1) to give the product (R) -6- (3, 6-dihydro-2H-pyran-4-yl) -4- (6- (4- (2-hydroxy-3-methylbutyryl) piperazin-1-yl) pyridin-3-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile (I-1)42mg, yield: 33.3 percent.

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.68(s,1H),8.40(d,J＝2.4Hz,1H),7.86(dd,J＝8.9,2.4Hz,1H),7.72(s,1H),7.01(d,J＝8.9Hz,1H),6.61(s,1H),4.77(d,J＝7.0Hz,1H),4.26(d,J＝3.3Hz,2H),4.10(d,J＝6.1Hz,1H),3.85(t,J＝5.5Hz,2H),3.74-3.55(m,8H),2.55(s,2H),1.89(q,J＝6.6Hz,1H),0.91(d,J＝6.7Hz,3H),0.85(d,J＝6.7Hz,3H).

MS m/z(ESI):487.6[M+1]

Example 2 example 36

Preparation of Compounds I-2 to I-36

Referring to example 1, the procedure for the preparation of compound I-1, using 1I, 2I, 3I, 4I, 5I, 6I, 7I, 8I and 9I, respectively, and reacting with a suitable carboxylic acid, gives compounds I-2 to I-36, whose structures and characterization data are as follows:

I-2

1H NMR(400MHz,Chloroform-d)δ8.49(s,1H),8.35(s,1H),8.27(s,1H),7.78(s,1H),7.57(s,1H),7.42(s,1H),6.90(d,J＝32.1Hz,2H),6.66(s,1H),6.31(s,1H),4.80(s,1H),4.54(s,1H),4.34(d,J＝25.8Hz,3H),3.98(s,2H),3.70(d,J＝43.6Hz,3H),2.92(s,1H),2.81(d,J＝4.6Hz,1H),2.54(s,2H),1.77(s,1H).

MS m/z(ESI):539.2[M+1]

I-3

¹H NMR(400MHz,Chloroform-d)δ8.84(s,1H),8.65(d,J＝5.2Hz,1H),8.43(d,J＝2.4Hz,1H),8.37-8.29(m,2H),7.82(dd,J＝8.7,2.4Hz,1H),7.74(dd,J＝8.6,2.2Hz,1H),7.56(s,1H),7.43(s,1H),7.37(d,J＝5.3Hz,1H),6.83(dd,J＝8.7,5.4Hz,2H),4.00(s,3H),3.78(d,J＝23.8Hz,8H),2.68(s,3H).

MS m/z(ESI):531.2[M+1]

I-4

¹H NMR(400MHz,Chloroform-d)δ8.83(s,1H),8.64(s,1H),8.35(d,J＝12.7Hz,2H),7.76(d,J＝8.5Hz,1H),7.58-7.18(m,8H),6.72(d,J＝8.7Hz,1H),4.76(s,1H),4.25-3.16(m,9H),2.97(d,J＝11.9Hz,1H),2.67(d,J＝5.5Hz,3H).

MS m/z(ESI):530.2[M+1]

I-5

¹H NMR(400MHz,Chloroform-d)δ8.52(d,J＝1.6Hz,1H),8.38(d,J＝2.5Hz,1H),8.29(s,1H),7.81(dd,J＝8.9,2.5Hz,1H),7.45(d,J＝1.6Hz,1H),7.28-7.23(m,1H),7.19-7.07(m,2H),6.69(d,J＝8.9Hz,1H),6.34(dd,J＝3.2,1.8Hz,1H),4.85(s,1H),4.58(s,1H),4.40(q,J＝2.8Hz,2H),4.31(d,J＝11.3Hz,1H),4.01(t,J＝5.4Hz,2H),3.81(t,J＝14.2Hz,2H),3.69(d,J＝12.0Hz,1H),2.95(q,J＝7.0Hz,1H),2.61-2.53(m,2H),1.82(d,J＝8.8Hz,1H).

MS m/z(ESI):539.2[M+1].

I-6

¹H NMR(400MHz,DMSO-d₆)δ8.88(d,J＝1.5Hz,1H),8.67(s,1H),8.39(d,J＝2.5Hz,1H),8.02(d,J＝2.4Hz,1H),7.84(dd,J＝8.8,2.6Hz,1H),7.71(d,J＝1.6Hz,1H),7.56(dd,J＝8.5,2.5Hz,1H),7.00(d,J＝8.8Hz,1H),6.77(d,J＝8.5Hz,1H),6.62-6.55(m,1H),4.25(q,J＝2.8Hz,2H),3.83(d,J＝8.9Hz,5H),3.74(s,2H),3.69-3.56(m,8H),2.54(s,2H).

MS m/z(ESI):536.2[M+1]

I-7

¹H NMR(400MHz,Chloroform-d)δ8.52(d,J＝1.6Hz,1H),8.38(d,J＝2.5Hz,1H),8.29(s,1H),7.80(d,J＝8.7Hz,1H),7.73-7.66(m,2H),7.44(d,J＝1.6Hz,1H),7.13(t,J＝8.6Hz,2H),6.70(d,J＝9.1Hz,1H),6.33(dd,J＝3.5,1.9Hz,1H),4.77(s,2H),4.40(q,J＝2.8Hz,2H),4.01(t,J＝5.4Hz,2H),3.75(s,4H),2.96(q,J＝7.1Hz,1H),2.57(s,2H),1.80(d,J＝8.8Hz,1H).

MS m/z(ESI):521.2[M+1]

I-8

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.68(s,1H),8.37(d,J＝2.4Hz,1H),8.17(s,2H),7.87-7.77(m,1H),7.70(s,1H),7.45-7.35(m,2H),7.32(d,J＝7.1Hz,1H),6.95(d,J＝8.9Hz,1H),6.61(s,1H),4.26(s,2H),3.86(t,J＝5.5Hz,2H),3.70–3.54(m,6H),3.15(dt,J＝12.2,5.7Hz,4H),2.55(s,2H).

MS m/z(ESI):521.2[M+1]

I-9

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.68(d,J＝1.6Hz,1H),8.40(d,J＝2.5Hz,1H),8.34(d,J＝2.3Hz,1H),7.85(td,J＝8.1,2.3Hz,2H),7.72(s,1H),7.00(d,J＝8.9Hz,1H),6.90(s,1H),6.61(s,1H),4.26(d,J＝3.1Hz,2H),3.91(s,3H),3.85(t,J＝5.5Hz,2H),3.70(s,4H),3.34(s,4H),2.55(s,2H).

MS m/z(ESI):522.2[M+1]

I-10

¹H NMR(400MHz,DMSO-d₆)δ9.50(d,J＝1.6Hz,1H),8.77(d,J＝1.7Hz,1H),8.55(d,J＝5.3Hz,1H),8.45(t,J＝2.5Hz,3H),7.96–7.84(m,3H),7.77(d,J＝5.4Hz,1H),7.62(d,J＝8.1Hz,1H),7.41–7.33(m,1H),6.97(d,J＝8.9Hz,1H),3.50(d,J＝81.3Hz,8H),2.55(s,3H),1.42(d,J＝5.2Hz,2H),1.32(s,2H).

MS m/z(ESI):541.2[M+1]

I-11

¹H NMR(400MHz,DMSO-d₆)δ8.90(s,1H),8.69(s,1H),8.42(dd,J＝16.1,2.4Hz,2H),7.98-7.89(m,1H),7.86(dd,J＝8.8,2.5Hz,1H),7.73(s,1H),6.92(dd,J＝8.9,3.2Hz,2H),6.62(s,1H),4.81(s,1H),4.23(d,J＝31.4Hz,4H),3.94(d,J＝5.8Hz,4H),3.86(t,J＝5.6Hz,2H),3.15(d,J＝12.2Hz,2H),2.56(s,2H),1.94(s,2H),1.74(d,J＝8.9Hz,2H).

MS m/z(ESI):548.2[M+1]

I-12

¹H NMR(400MHz,DMSO-d₆)δ9.44(s,1H),8.78(s,1H),8.48(d,J＝2.5Hz,1H),8.35(d,J＝2.3Hz,1H),7.93(d,J＝8.9Hz,1H),7.85(d,J＝8.8Hz,3H),7.02(d,J＝8.3Hz,2H),6.93(d,J＝8.6Hz,1H),6.85(d,J＝7.2Hz,1H),3.92(s,3H),3.72(s,8H),3.48(s,3H).

MS m/z(ESI):547.2[M+1]

I-13

¹H NMR(400MHz,DMSO-d₆)δ9.50(s,1H),8.78(s,1H),8.56(d,J＝5.3Hz,1H),8.45(d,J＝2.4Hz,1H),7.94(s,1H),7.90(d,J＝7.6Hz,2H),7.78(d,J＝5.3Hz,1H),7.26(t,J＝8.0Hz,1H),6.98(d,J＝8.9Hz,1H),6.81(d,J＝7.6Hz,2H),6.70(t,J＝2.0Hz,1H),3.75(s,3H),3.59(d,J＝37.7Hz,8H),2.56(s,3H),1.36(d,J＝2.4Hz,2H),1.25-1.20(m,2H).

MS m/z(ESI):570.2[M+1]

I-14

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.69(s,1H),8.37(d,J＝2.5Hz,1H),7.81(dd,J＝8.8,2.5Hz,1H),7.71(s,1H),7.64(d,J＝8.2Hz,1H),6.99(d,J＝9.0Hz,1H),6.62(s,1H),5.28(d,J＝5.7Hz,1H),4.42–4.31(m,2H),4.27(d,J＝3.2Hz,2H),3.98-3.76(m,3H),3.66(t,J＝4.8Hz,1H),3.02(t,J＝12.4Hz,2H),2.56(s,2H),2.09-1.91(m,1H),1.79(d,J＝12.6Hz,2H),1.52(p,J＝13.6,13.2Hz,2H),1.27(dd,J＝13.6,6.7Hz,1H),0.91(d,J＝6.9Hz,3H),0.79(d,J＝6.8Hz,3H).

MS m/z(ESI):501.2[M+1]

I-15

¹H NMR(400MHz,DMSO-d₆)δ8.91(s,1H),8.71(s,1H),8.41(d,J＝2.4Hz,1H),8.34(d,J＝7.7Hz,1H),7.84(dd,J＝8.8,2.5Hz,1H),7.72(s,1H),7.46(d,J＝7.6Hz,1H),7.40(dd,J＝14.2,5.8Hz,2H),7.10(dd,J＝8.0,2.5Hz,1H),7.04(d,J＝8.9Hz,1H),6.64(s,1H),4.47(d,J＝13.2Hz,2H),4.29(d,J＝3.3Hz,2H),4.14(s,1H),3.88(t,J＝5.5Hz,2H),3.82(s,3H),3.07(t,J＝12.6Hz,2H),2.58(s,2H),1.98-1.82(m,2H),1.60(q,J＝12.0Hz,2H).

MS m/z(ESI):535.2[M+1]

I-16

¹H NMR(400MHz,DMSO-d₆)δ8.91(s,1H),8.70(s,1H),8.40(d,J＝2.5Hz,1H),7.86(dd,J＝8.8,2.4Hz,1H),7.74(s,1H),6.93(d,J＝8.8Hz,1H),6.63(s,1H),4.75(d,J＝32.4Hz,2H),4.28(d,J＝3.2Hz,2H),4.18(t,J＝11.9Hz,2H),3.98(d,J＝6.4Hz,1H),3.90-3.82(m,2H),3.12-2.88(m,2H),2.57(s,2H),1.83(dd,J＝57.4,28.5Hz,6H),0.90(d,J＝7.0Hz,6H).

MS m/z(ESI):513.2[M+1]

I-17

¹H NMR(400MHz,DMSO-d₆)δ9.52(d,J＝1.7Hz,1H),8.79(s,1H),8.53(dd,J＝28.9,3.9Hz,2H),8.01–7.84(m,2H),7.79(d,J＝5.3Hz,1H),7.04(d,J＝8.9Hz,1H),4.79(d,J＝7.1Hz,1H),4.13(t,J＝6.5Hz,1H),3.77–3.53(m,8H),2.56(s,3H),1.91(q,J＝6.6Hz,1H),0.89(dd,J＝22.5,6.6Hz,6H).

MS m/z(ESI):496.2[M+1]

I-18

¹H NMR(400MHz,DMSO-d₆)δ9.53(d,J＝1.7Hz,1H),8.80(s,1H),8.58(d,J＝5.3Hz,1H),8.50(d,J＝2.5Hz,1H),8.00-7.89(m,3H),7.81(d,J＝5.3Hz,1H),7.05(d,J＝8.8Hz,1H),4.82(d,J＝7.0Hz,1H),4.15(s,1H),3.67(dt,J＝28.8,8.5Hz,7H),2.58(s,3H),1.65(dd,J＝50.6,12.3Hz,7H),1.17(ddt,J＝35.9,23.9,12.2Hz,2H).

MS m/z(ESI):536.2[M+1]

I-19

¹H NMR(400MHz,DMSO-d₆)δ9.51(s,1H),8.78(s,1H),8.55(d,J＝5.3Hz,1H),8.44(d,J＝2.5Hz,1H),7.97-7.82(m,3H),7.78(d,J＝5.3Hz,1H),7.35(d,J＝4.4Hz,4H),7.27(q,J＝4.3Hz,1H),6.97(d,J＝8.9Hz,1H),4.76(t,J＝5.4Hz,1H),4.18(t,J＝6.9Hz,1H),4.02(dt,J＝14.5,7.1Hz,1H),3.78-3.38(m,8H),3.09(s,1H),2.56(s,3H).

MS m/z(ESI):544.2[M+1]

I-20

1H NMR(400MHz,DMSO-d₆)δ9.81(s,1H),8.87(d,J＝6.2Hz,2H),8.60-8.47(m,1H),8.47-8.36(m,1H),8.12(s,1H),8.02(dd,J＝8.8,2.4Hz,1H),7.94(t,J＝6.8Hz,1H),7.80(d,J＝7.7Hz,1H),7.44(t,J＝6.5Hz,2H),7.14(d,J＝8.9Hz,1H),4.46(s,2H),3.54(d,J＝62.9Hz,8H),2.77(s,3H).

MS m/z(ESI):522.2[M+1]

I-21

¹H NMR(400MHz,DMSO-d₆)δ8.90(s,1H),8.69(s,1H),8.39(d,J＝2.4Hz,1H),7.85(dd,J＝8.9,2.5Hz,1H),7.73(s,1H),7.61-7.43(m,4H),6.92(d,J＝8.9Hz,1H),6.62(s,1H),4.84(s,1H),4.32-4.07(m,5H),3.86(t,J＝5.5Hz,2H),3.13(s,2H),2.56(s,2H),1.93(s,2H),1.75(s,2H).

MS m/z(ESI):517.2[M+1]

I-22

¹H NMR(400MHz,DMSO-d₆)δ8.90(s,1H),8.69(s,1H),8.39(d,J＝2.4Hz,1H),7.89-7.78(m,2H),7.72(s,1H),6.91(d,J＝8.9Hz,1H),6.62(s,1H),6.47(s,1H),6.31(dd,J＝6.9,1.8Hz,1H),4.78(d,J＝5.8Hz,1H),4.32-4.06(m,5H),3.86(t,J＝5.5Hz,2H),3.47(s,3H),3.09(dd,J＝20.1,12.1Hz,2H),2.56(s,2H),1.94(d,J＝9.7Hz,2H),1.73(dd,J＝10.5,4.4Hz,2H).

MS m/z(ESI):548.2[M+1]

I-23

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.68(s,1H),8.36(d,J＝2.4Hz,1H),7.83(dd,J＝8.8,2.5Hz,1H),7.72(s,1H),7.62(q,J＝7.5Hz,5H),6.87(d,J＝8.9Hz,1H),6.62(s,1H),4.85-4.76(m,1H),4.42(s,1H),4.30-4.18(m,3H),4.08(d,J＝12.5Hz,1H),3.86(t,J＝5.6Hz,2H),3.00(d,J＝12.3Hz,1H),2.59(d,J＝29.0Hz,4H),1.85(s,1H),1.71(d,J＝9.1Hz,3H).

MS m/z(ESI):567.2[M+1]

I-24

¹H NMR(400MHz,DMSO-d₆)δ8.89(s,1H),8.69(s,1H),8.39(d,J＝2.4Hz,1H),7.85(dd,J＝8.6,2.4Hz,1H),7.72(s,1H),7.41(t,J＝8.2Hz,1H),7.16-7.04(m,3H),6.91(d,J＝8.9Hz,1H),6.62(s,1H),4.83(s,1H),4.23(d,J＝34.3Hz,5H),3.84(d,J＝16.4Hz,4H),3.13(s,2H),2.70(s,3H),2.56(s,2H),1.93(s,2H),1.74(s,2H).

MS m/z(ESI):547.2[M+1]

I-25

¹H NMR(400MHz,DMSO-d₆)δ9.43(s,1H),8.78(s,1H),8.46(s,1H),7.97-7.78(m,3H),7.03(s,1H),6.97-6.89(m,1H),6.87-6.81(m,1H),4.71(d,J＝22.1Hz,2H),4.16(t,J＝12.8Hz,2H),3.99(s,1H),3.48(s,3H),3.09-2.87(m,2H),1.96-1.46(m,10H),1.28-0.97(m,6H).

MS m/z(ESI):578.2[M+1]

I-26

¹H NMR(400MHz,DMSO-d₆)δ9.44(s,1H),8.79(s,1H),8.51-8.41(m,2H),7.92(d,J＝8.7Hz,2H),7.85(d,J＝8.0Hz,2H),7.03(s,1H),6.93(d,J＝8.7Hz,2H),6.85(d,J＝7.2Hz,1H),4.21(s,4H),3.93(s,3H),3.48(s,3H),3.16(d,J＝12.1Hz,2H),1.94(s,2H),1.75(d,J＝9.0Hz,2H).

MS m/z(ESI):573.2[M+1]

I-27

¹H NMR(400MHz,Chloroform-d)δ8.50(s,1H),8.37(d,J＝2.4Hz,1H),8.27(s,1H),7.93(s,2H),7.76(dd,J＝8.8,2.4Hz,1H),7.42(s,1H),6.75(d,J＝8.9Hz,1H),6.31(s,1H),5.03(s,1H),4.65(s,1H),4.37(d,J＝3.6Hz,2H),4.14(d,J＝45.4Hz,2H),3.98(t,J＝5.5Hz,2H),3.29(d,J＝12.0Hz,2H),2.55(d,J＝5.2Hz,2H),2.09-1.84(m,4H).

MS m/z(ESI):507.2[M+1]

I-28

¹H NMR(400MHz,Chloroform-d)δ8.47(s,1H),8.34(s,1H),8.25(s,1H),7.74(s,2H),7.65(s,1H),7.40(s,1H),7.24(d,J＝7.4Hz,1H),6.72(s,1H),6.35-6.16(m,1H),4.35(s,2H),4.14(s,2H),3.96(s,2H),3.31(s,2H),2.54(s,2H),1.99(d,J＝46.3Hz,4H),1.24(s,2H).

MS m/z(ESI):507.2[M+1]

I-29

¹H NMR(400MHz,Chloroform-d)δ8.88(s,1H),8.68(d,J＝5.3Hz,1H),8.41(d,J＝18.9Hz,2H),7.83(t,J＝8.6Hz,1H),7.58(s,1H),7.47(d,J＝18.0Hz,2H),6.78(dd,J＝16.5,8.9Hz,1H),5.05(s,0.5H),4.88(s,0.5H),4.32(q,J＝12.5Hz,2H),4.21-4.02(m,2H),3.37-3.07(m,2H),2.74(s,3H),2.10-1.13(m,15H).

MS m/z(ESI):562.2[M+1]

I-30

¹H NMR(400MHz,Chloroform-d)δ8.87(s,1H),8.68(d,J＝5.3Hz,1H),8.45(s,2H),8.38(s,1H),7.84(t,J＝8.1Hz,2H),7.58(s,1H),7.50-7.38(m,2H),6.82(dd,J＝24.4,8.7Hz,2H),5.03(s,1H),4.40(s,1H),4.18(s,2H),4.03(s,3H),3.32(s,2H),2.72(s,3H),2.13-1.85(m,4H).

MS m/z(ESI):557.2[M+1]

I-31

¹HNMR(400MHz,Chloroform-d)δ8.84(d,J＝1.6Hz,1H),8.65(d,J＝5.3Hz,1H),8.42(d,J＝2.5Hz,1H),8.36(s,1H),7.80(dd,J＝8.8,2.6Hz,1H),7.56(d,J＝1.6Hz,1H),7.44(d,J＝1.8Hz,1H),7.38(dd,J＝5.2,1.8Hz,1H),6.75(d,J＝8.9Hz,1H),4.40(d,J＝6.7Hz,1H),4.31-4.26(m,1H),4.11-3.93(m,2H),3.72-3.47(m,4H),3.37(s,3H),3.32(dd,J＝11.9,2.4Hz,1H),3.22(dd,J＝12.0,2.4Hz,1H),2.69(s,3H),1.99(tdd,J＝21.1,9.7,4.4Hz,4H),1.84(t,J＝8.9Hz,2H).

MS m/z(ESI):549.2[M+1]

I-32

¹H NMR(400MHz,Chloroform-d)δ8.53(d,J＝1.6Hz,1H),8.40(d,J＝2.5Hz,1H),8.30(s,1H),7.80(dd,J＝8.9,2.5Hz,1H),7.46(d,J＝1.6Hz,1H),7.23(t,J＝7.8Hz,1H),6.90-6.78(m,3H),6.65(d,J＝8.8Hz,1H),6.35(p,J＝1.6Hz,1H),4.67(d,J＝5.9Hz,1H),4.54(s,1H),4.41(q,J＝2.8Hz,2H),4.22-4.14(m,1H),4.02(t,J＝5.4Hz,2H),3.84(s,2H),3.77(s,3H),3.68(d,J＝11.3Hz,1H),3.55(d,J＝6.8Hz,2H),2.80(p,J＝7.0Hz,1H),2.58(dt,J＝7.0,3.7Hz,2H),1.71(d,J＝8.8Hz,1H).

MS m/z(ESI):547.2[M+1]

I-33

¹H NMR(400MHz,Chloroform-d)δ8.78(d,J＝1.6Hz,1H),8.41-8.34(m,2H),8.13(d,J＝2.3Hz,1H),7.75(dd,J＝8.9,2.6Hz,1H),7.51-7.41(m,2H),6.87(d,J＝2.1

Hz,1H),6.79(d,J＝8.5Hz,1H),6.70(d,J＝8.8Hz,1H),6.44(dd,J＝7.1,2.1Hz,1H),3.97(s,5H),3.64(s,5H),3.38(s,2H),2.13-2.03(m,2H),1.86-1.67(m,2H).

MS m/z(ESI):556.2[M+1]

I-34

¹H NMR(400MHz,Chloroform-d)δ8.52(d,J＝1.5Hz,1H),8.38(d,J＝2.5Hz,1H),8.29(s,1H),7.81(d,J＝8.8Hz,1H),7.44(d,J＝1.5Hz,1H),7.35(t,J＝7.9Hz,1H),7.25-7.20(m,2H),7.08-6.99(m,1H),6.70(d,J＝9.1Hz,1H),6.34(s,1H),4.78(s,2H),4.40(q,J＝2.8Hz,2H),4.01(t,J＝5.5Hz,2H),3.86(s,3H),3.76(s,4H),2.96(q,J＝7.1Hz,1H),2.57(s,2H),1.80(d,J＝8.8Hz,1H).

MS m/z(ESI):533.2[M+1]

I-35

¹H NMR(400MHz,Chloroform-d)δ8.52(s,1H),8.38(d,J＝2.2Hz,1H),8.29(s,1H),7.80(s,0H),7.66(d,J＝8.7Hz,2H),7.44(s,1H),6.94(d,J＝8.6Hz,2H),6.70(s,1H),6.33(s,1H),4.77(d,J＝6.2Hz,2H),4.40(q,J＝2.7Hz,2H),4.01(t,J＝5.4Hz,2H),3.88(s,3H),3.73(d,J＝27.9Hz,4H),2.95(d,J＝7.7Hz,1H),2.57(s,2H),1.78(d,J＝8.8Hz,1H).

MS m/z(ESI):533.2[M+1]

I-36

¹H NMR(400MHz,Chloroform-d)δ8.55-8.49(m,2H),8.38(d,J＝2.5Hz,1H),8.29(s,1H),7.92(dd,J＝8.6,2.4Hz,1H),7.81(d,J＝8.7Hz,1H),7.44(d,J＝1.6Hz,1H),6.80(dd,J＝8.6,0.7Hz,1H),6.70(d,J＝9.0Hz,1H),6.34(s,1H),4.79(d,J＝6.2Hz,2H),4.40(q,J＝2.8Hz,2H),4.01(s,5H),3.81(d,J＝15.1Hz,4H),2.98(q,J＝7.2Hz,1H),2.57(d,J＝1.8Hz,2H),1.81(d,J＝8.9Hz,1H).

MS m/z(ESI):534.2[M+1]

example 37

Preparation of Compound I-37

4- (6- (8- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile

In a 100mL single neck flask was added 4- (6- (3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile (8-i) (100mg,0.24mmol), 5- (chloromethyl) -2-methoxypyridine 1-m (55.8mg,0.35mmol), potassium carbonate (65.3mg,0.47mmol) and 10mL acetonitrile, and reacted at 60 ℃ overnight. After the reaction was completed, the reaction solution was poured into water (100mL), extracted three times with dichloromethane (30mL × 3), the organic phases were combined, the organic phase was washed with water (30mL × 3) and a saturated aqueous sodium chloride solution (30mL), dried over anhydrous sodium sulfate, the filtrate was concentrated and purified by silica gel column chromatography (dichloromethane: methanol ═ 30:1) to give the product 4- (6- (8- ((6-methoxypyridin-3-yl) methyl) -3, 6-diazabicyclo [3.1.1] heptan-3-yl) pyridin-3-yl) -6- (1-methyl-2-oxo-1, 2-dihydropyridin-4-yl) pyrazolo [1,5-a ] pyridine-3-carbonitrile (I-37)67mg, yield: 52.1 percent.

¹H NMR(400MHz,Chloroform-d)δ8.80(d,J＝1.6Hz,1H),8.47(d,J＝2.4Hz,1H),8.37(s,1H),8.14(s,1H),7.84(dd,J＝8.9,2.4Hz,1H),7.72(s,1H),7.52(d,J＝1.7Hz,1H),7.47(d,J＝7.1Hz,1H),6.89(d,J＝2.1Hz,1H),6.74(dd,J＝8.6,5.9Hz,2H),6.45(dd,J＝7.0,2.1Hz,1H),3.91(d,J＝27.9Hz,7H),3.64(s,6H),2.79(s,1H)1.52(m,2H).

MS m/z(ESI):545.2[M+1]

Example 38 example 40

Preparation of Compounds I-38 to I-40

Referring to the preparation procedure of compound I-37, compound I-38, compound I-39 and compound I-40 were obtained by reacting 3I, 5I and 9I, respectively, with the appropriate halide, and the structures and characterization data are as follows:

I-38

¹H NMR(400MHz,Chloroform-d)δ8.83(d,J＝1.6Hz,1H),8.65(d,J＝5.2Hz,1H),8.41(d,J＝2.5Hz,1H),8.35(s,1H),8.10(d,J＝2.3Hz,1H),7.78(dd,J＝8.8,2.6Hz,1H),7.64(dd,J＝8.5,2.4Hz,1H),7.55(d,J＝1.6Hz,1H),7.42(d,J＝1.7Hz,1H),7.36(dd,J＝5.3,1.8Hz,1H),6.78(dd,J＝12.0,8.6Hz,2H),3.96(s,3H),3.68(t,J＝5.1Hz,4H),3.52(s,2H),2.68(s,3H),2.58(t,J＝5.1Hz,4H).

MS m/z(ESI):517.2[M+1]

I-39

¹H NMR(400MHz,Chloroform-d)δ8.51(d,J＝1.5Hz,1H),8.37–8.34(m,1H),8.29(s,1H),7.75(dd,J＝8.8,2.6Hz,1H),7.51(s,1H),7.43(d,J＝1.6Hz,1H),6.70(d,J＝8.8Hz,1H),6.33(d,J＝3.4Hz,1H),4.40(q,J＝2.8Hz,2H),4.06–3.88(m,6H),3.72–3.29(m,3H),2.57(d,J＝6.2Hz,2H),2.10(s,2H),1.79(d,J＝70.8Hz,6H).

MS m/z(ESI):507.2[M+1]

I-40

1H NMR(400MHz,Chloroform-d)δ8.78(d,J＝1.6Hz,0H),8.41–8.36(m,0H),8.36(s,0H),8.13(d,J＝2.3Hz,0H),7.84–7.78(m,1H),7.75(dd,J＝8.9,2.6Hz,0H),7.51–7.40(m,1H),6.87(d,J＝2.1Hz,0H),6.79(d,J＝8.5Hz,0H),6.70(d,J＝8.8Hz,0H),6.44(dd,J＝7.1,2.1Hz,0H),3.64(s,1H),3.60(s,1H),3.38(s,1H),3.26(s,1H),2.15–2.01(m,1H),1.82(d,J＝8.1Hz,1H).

biological evaluation

Test example 1 determination of RET kinase Activity by Compounds of the present invention

The method uses Cisbio

The KinEASE-TK tyrosine kinase kit (cat # 62TK0PEB) was assayed by time-resolved fluorescence energy resonance transfer (TR-FRET) by determining the degree of phosphorylation of biotinylated polypeptide substrates. Human RET protein (RET kinase) was purchased from Carna bioscience (Japan, cat # 08-159-5. mu.g).

The experimental procedure was as follows:

(1) test compounds were dissolved in 100% DMSO to a final concentration of 10 mM.

(2) Dissolving 4uL of the test compound solution prepared in the step (1) in 46uL of 100% DMSO, and numbering the solution obtained in this step as No. 2.

(3) Solution No. 2 was subjected to subsequent gradient dilutions 5-fold (i.e., 20 μ L of 100% DMSO plus 5 μ L compound) for a total of 9 gradients numbered 3 to 11.

Note: no. 2 was not used for the dilution in step (4).

(unless otherwise specified, the following steps are carried out on ice)

(4) The solutions No. 3 to No. 11 were further diluted by 20-fold (i.e., 1uL of the buffer solution 3 to No. 11 was added with 19. mu.L of the buffer solution) in the buffer provided in the kit (Cisbio, cat # 62TK0PEB) in 12-20 serial numbers. In this case, the final concentration of the test compound in system 12-20 was 3200nM to 0.008nM (9 gradients) and the final concentration of DMSO was 2%.

(5) And (4) sequentially adding the 9 gradient concentration compound solutions to be detected in the step (4) into a 384-well plate according to the concentration, wherein each well is 4 mu L, and two multiple wells are arranged.

(6) mu.L of human RET protein was added to each well and incubated on ice for 10 min.

(7) Phosphorylation was initiated by the addition of 2. mu.L of ATP (Sigma # A7699) and 2. mu.L of biotinylated polypeptide substrate (Cisbio, cat # 62TK0PEB) per well. Incubate at 37 ℃ for half an hour.

(8) mu.L of an anti-phosphotyrosine antibody conjugated with a europium-based element compound (supplied in the kit, cat # 62TK0PEB) and 5. mu.L of streptavidin conjugated with a modified allophycocyanin XL665 (Cisbio, cat # 62TK0PEB) were added to each well.

(9) Incubation was continued for 1 hour at room temperature. After the incubation was completed, the excitation wavelength of each well was measured at 304nM using TF-FRET mode of a microplate reader (BMG Labtech, model: FLUOStar Omega), the fluorescence intensities of each well at the emission wavelengths of 615nM and 665nM were read, and the ratio was automatically calculated.

(10) The inhibition of the compound at each concentration was calculated by comparison with the fluorescence intensity ratio of the control group, and the IC of the compound was calculated by curve fitting with GraphPad Prism5 as logarithmic concentration-inhibition₅₀Values, see table 1 below.

The control kinase chosen was another receptor tyrosine kinase KDR with a similar structure to RET kinase. Purchased from Carna bioscience (Japan, cat # 08-191-5. mu.g). The step of gradient dilution is the same as RET kinase, so that the final concentration of the test compound in the reaction system is 16000nM to 0.04nM, (step 4 of gradient dilution with the test compound) the other reaction conditions are the same as above, and the final concentration of DMSO is 2%. IC of test compound for KDR kinase inhibition₅₀Value calculation method and IC for RET kinase inhibition₅₀The value calculation method is the same.

TABLE 1 IC inhibition of RET kinase and KDR kinase by Compounds₅₀Value of

Remarking:

the structure of LOXO292 is shown below, and its preparation method is described in example 163 of WO 2018071447.

As can be seen from the above table, the compounds of the present invention have significant inhibitory effects on RET kinase activity. The compounds of the invention have superior inhibitory activity against RET kinase over KDR kinase. Therefore, the compounds of the invention can be used as a type of effective selective RET kinase inhibitor.

Test example 2 determination of hERG inhibition ratio by Compounds of the present invention

TABLE 2

Abbreviations	Full scale
		CHO	Chinese hamster ovary cells
DMSO	Dimethyl sulfoxide
		ECG	Electrocardiogram
EGTA	Ethylene glycol bis (2-aminoethylether) -N, N, N ', N' -tetraacetic acid
		FBS	Fetal bovine serum
HEPES	N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid)
		hERG	Human ether-a-go-go-related gene
QT	Time between Q-wave and T-wave in Electrocardiogram (ECG)

2.1 cell culture

2.1.1 the cells used in this experiment were CHO cell lines (supplied by Sophion Bioscience, Denmark) transfected with hERG cDNA and stably expressing the hERG channel, and the cell generation number was P13-P14. Cells were cultured in media containing the following components (all from Invitrogen): ham's F12 medium, 10% (v/v) inactivated fetal bovine serum, 100. mu.g/ml hygromycin B, 100. mu.g/ml Geneticin.

2.1.2CHO hERG cells were grown in dishes containing the above-mentioned culture medium and cultured in an incubator containing 5% CO2 at 37 ℃.24 to 48 hours before the electrophysiological experiment, CHO hERG cells were transferred to a round glass slide placed in a petri dish and grown in the same culture medium and culture conditions as above. The density of CHO hERG cells on each circular slide is required to achieve the requirement that most cells are independent and individual.

2.2 test solutions

The following solutions (recommended by Sophion) were used for electrophysiological recording.

TABLE 3 composition of intracellular and extracellular fluids

TABLE 4 reagent details

Name of reagent	Goods number	Batch number	Molecular weight	Suppliers of goods
					NaCl	S1679-1KG	WXBC1368V	58.44	Sigma
KCl	31248-100G	WXBC2571V	74.55	Sigma
					CaCl2(1M solution)	21114-1L	BCBM6063V	110.98	Sigma
MgCl2·6H2O	M7304-100G	V900020-500G	203.30	Sigma
					HEPES	H3375-1KG	SLBP2246V	238.30	Sigma
Glucose	G8270-1KG	WXBC2393V	180.16	Sigma
					EGTA	03777-50G	SLBP2807V	380.15	Sigma
Na2-ATP	A-7699-5G	SLBJ8915V	551.14	Sigma
					NaOH(2M solution)	35254-1L	BCBG6297V	40.00	Sigma
KOH	232041-50G	SLBK9251V	149.91	Sigma

2.3 electrophysiological recording system

The whole cell current was recorded using a manual patch clamp system (HEKA EPC-10 signal amplifier and digital conversion system, available from HEKA Electronics, Germany). The round slide with the CHO hERG cells grown on the surface was placed in an electrophysiological recording chamber under an inverted microscope. Continuous perfusion with extracellular fluid (approximately 1 ml per minute) was recorded in the wells. The experimental process adopts a conventional whole-cell patch clamp current recording technology. Unless otherwise stated, the experiments were carried out at normal room temperature (25 ℃ C. + -2 ℃ C.). The cells were clamped at-80 mV. The cell clamp voltage depolarized to +20mV to activate the hERG potassium channel, and 5 seconds later clamped to-50 mV to eliminate inactivation and generate a tail current. The tail current peak value was used as a value for the magnitude of the hERG current. And (3) after the hERG potassium current recorded in the step is stable in the continuous extracellular fluid perfusion in the recording groove, overlaying and perfusing the drug to be tested until the inhibition effect of the drug on the hERG current reaches a stable state. The most recent overlapping of consecutive 3 current recording lines is generally used as a criterion for determining whether the current recording lines are in a steady state. After reaching a stable state, the hERG current is flushed by perfusion with extracellular fluid until it returns to the level before the drug is added. One or more drugs, or multiple concentrations of the same drug, can be tested on one cell, but with extra-cellular fluid washes between different drugs. Cisapride (purchased from Sigma) was used in the experiments as a positive control to ensure that the cell quality used was normal.

2.4 Compound treatment and dilution

To obtain the IC50 of the compound, we selected the following concentrations (30,10,3,1,0.3 and 0.1 μ M) to test. Prior to the assay, 10,3,1,0.3 and 0.1mM stock solutions were diluted in gradient dilution with DMSO (Cat #: V900090-500ML, Lot # WXBC6681V, Sigma) and extracellular fluid to the final 0.1. mu.M assay concentration. The final concentration of DMSO in each concentration of compound solution was 0.1. mu.M, and the test concentration of the positive control, Cisapride (Cisapride), was 0.1. mu.M. All compound solutions were subjected to conventional 5 to 10 minutes sonication and shaking to ensure complete dissolution of the compound.

2.5 data analysis

The experimental data were analyzed by data analysis software supplied by HEKA Patchmaster (V2x73.2), Microsoft Excel, and Graphpad Prism 5.0.

2.6 quality control

The test data in the report need to meet the following criteria:

recording parameters

Membrane resistance Rm >500M omega

Access resistance (Ra) <10M omega

Tail current amplitude >300pA

Current rundown (spontaneous decrease) < 2% per minute

Leakage current <200pA or 10% of hERG current peak (within 90% of the recording time)

Pharmacological parameters

Sisapari (C4740-10mg, Sigma) at 0.1. mu.M blocked more than 50% of hERG current as a positive control.

Table 5 hERG inhibition rates of the compounds of the invention.

As can be seen from the above table, the compounds of the present invention blocked hERG by less than fifty percent at a concentration of 10 μ M and by far more than fifty percent under the same conditions as the control LOXO-292 compound, the compounds of the present invention have good hERG safety. Therefore, the compounds of the invention can be used as a safe RET kinase inhibitor.

In conclusion, the compound has a remarkable inhibitory effect on the activity of RET kinase, has good RET/KDR selectivity and good hERG safety, so that the compound can be used as a selective transfection period Rearrangement (RET) kinase inhibitor and can solve the unmet medical requirements.

Claims (11)

1. A compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:

wherein:

X₁、X₂each is independently selected from CH or N;

l is selected from:

wherein L is optionally joined at both ends with A and R₂Connecting;

a is selected from 4-6 membered monocyclic heterocyclic group, -NH-4-6 membered heterocyclic group, 7-11 membered bridged heterocyclic group, 7-11 membered spiro heterocyclic group or 7-11 membered fused ring heterocyclic group, wherein the monocyclic heterocyclic group, -NH-4-6 membered heterocyclic group, bridged heterocyclic group, spiro heterocyclic group or fused ring heterocyclic group is optionally further substituted by one or more groups selected from C₁-C₃Alkyl, hydroxyalkyl, halogeno C₁-C₃Alkyl, hydroxy, C₃-C₆Cycloalkyl or substituted with a substituent of ═ O;

R₁selected from: c₃-C₆Cycloalkyl, 3-6 membered heterocyclic group, 6-7 membered heteroaryl or 7-11 membered fused heterocyclic group, wherein the cycloalkyl and heterocyclic groups are optionally further substituted by one or more groups selected from hydroxy, hydroxyalkyl, amino, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Substituted by a substituent of alkoxy; said heteroaryl and fused heterocyclyl is optionally further substituted by one or more groups selected from halogen, hydroxy, amino, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy radical, C₃-C₆Cycloalkyl, -NHR₃、-NR₃R₄Or substituted with a substituent of ═ O;

R₂is selected from C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl, 3-6 membered heterocyclyl, aryl or heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxy, cyano, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy radical, C₃-C₆Cycloalkyl, -NHR₃、-NR₃R₄Or substituted with a substituent of ═ O;

R₃、R₄each independently selected from C₁-C₆An alkyl group;

wherein said halogen is C₁-C₃Alkyl or halo C₁-C₃The alkoxy is preferably 1 to 3 fluorinated C₁-C₃Alkyl or C₁-C₃An alkoxy group.

2. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, which is a compound of formula (II):

wherein: A. l, R₁And R₂Is as defined in claim 1.

3. A compound according to claim 1 or 2, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein a is selected from:

wherein: the 1 endpoint and the 2 endpoint are optionally connected with the L.

4. The compound of claim 1 or 2, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R₁Selected from:

5. the compound of claim 1 or 2, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R₂Is selected from C₁-C₆Alkyl radical, C₄-C₆Cycloalkyl, 4-6 membered heterocyclic group, phenyl, 5-6 membered heteroaryl or 10 membered heteroaryl, wherein said C₄-C₆Cycloalkyl, 4-6 membered heterocyclyl, phenyl, 5-6 membered heteroaryl or 10 membered heteroaryl optionally further substituted with one or more substituents selected from cyano, halogen, hydroxy, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, halo C₁-C₃Alkyl, halo C₁-C₃Alkoxy or ═ O.

6. A compound according to claim 5, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein R₂Selected from:

7. a compound according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein said compound is selected from the group consisting of:

8. a process for preparing a compound of formula (I) according to claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, which comprises:

the method comprises the following steps:

reacting compound (IA) and compound (IB) under basic conditions in the presence of a condensation agent to give a compound of formula (I); wherein:

the condensation reagent is selected from 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N, N '-dicyclohexylcarbodiimide, N, N' -diisopropylcarbodiimide, 1-hydroxy-7-azobenzotriazole, 1H-benzotriazole-1-oxytripyrrolidinyl hexafluorophosphate, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, pentafluorophenyl diphenyl phosphate, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate or benzotriazol-1-yloxytripyrrolidinyl phosphonium hexafluorophosphate; preferably 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate;

the reagent for providing the alkaline condition is an organic base, and the organic base is selected from N, N-diisopropylethylamine, pyridine, triethylamine, piperidine, N-methylpiperazine and 4-dimethylaminopyridine, and is preferably N, N-diisopropylethylamine or triethylamine;

A、L、X₁、X₂、R₁and R₂As defined in claim 1; or

The method 2 comprises the following steps:

reacting compound (IA) and compound (IB) under basic conditions to obtain a compound of formula (I);

wherein:

the reagent for providing the alkaline condition is an inorganic base, wherein the inorganic base is selected from the group consisting of potassium phosphate, potassium phosphate trihydrate, potassium acetate, sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydride, and potassium hydride, preferably sodium carbonate or potassium carbonate;

g is selected from a leaving group, preferably halogen;

A、L、X₁、X₂、R₁and R₂Is as defined in claim 1.

9. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1-7, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.

10. Use of a compound according to any one of claims 1 to 7, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9, for the preparation of a rearrangement kinase inhibitor during transfection.

11. Use of a compound according to any one of claims 1 to 7, or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 9, for the manufacture of a medicament for the treatment of a disease driven by gene rearrangement during transfection, wherein the disease is preferably a cancer, wherein the cancer is preferably lung, thyroid, colon, breast or pancreatic cancer.