CN111499613B - N-carboxamide derivatives, method for the production thereof and their use in medicine - Google Patents

N-carboxamide derivatives, method for the production thereof and their use in medicine Download PDF

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CN111499613B
CN111499613B CN201910097765.4A CN201910097765A CN111499613B CN 111499613 B CN111499613 B CN 111499613B CN 201910097765 A CN201910097765 A CN 201910097765A CN 111499613 B CN111499613 B CN 111499613B
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pharmaceutically acceptable
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CN111499613A (en
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张盼盼
颜孙力
李英
椰得杜拉·H·瑞迪
郭陈莉
叶成
钱文建
胡泰山
陈磊
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Zhejiang Hisun Pharmaceutical Co Ltd
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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Abstract

The invention provides an N-carboxamide derivative shown in a formula (I) as a selective RET (rearrangement during transfection) kinase inhibitor, a preparation method thereof and application thereof in medicine. Wherein D ring, X, R in formula (I) 1 ，R 1’ ，R 2 ，R 2’ ，R 3 ，R 3’ ，R 4 And R is 4’ The definition of (2) is the same as that in the specification.

Description

N-carboxamide derivatives, method for the production thereof and their use in medicine
Technical Field
The invention relates to the technical field of medicines, in particular to an N-formamide derivative serving as a selective RET kinase inhibitor, a preparation method thereof and application thereof in medicines.
Background
The rearrangement gene (RET gene) is a protooncogene that encodes a tyrosine kinase receptor in humans and regulates the proliferation and survival of cells during transfection. Activation of this gene requires the formation of dimers by co-action with receptors of the glial cell-derived neurotrophic factor family and alpha receptors of this family, and functions to regulate signaling and regulate vital activity by phosphorylation. Abnormal expression of RET gene is associated with various cancer diseases. The gene is fused with other genes through chromosome rearrangement or mutated at fixed points, and can be in a continuous activation state independent of ligands, so that abnormal signal paths are caused, and cell hyperproliferation and cancer are caused.
In recent years, there is increasing evidence that RET gene fusion and mutation are driving forces for the induction of certain cancers, and are not coincident 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% of non-small cell lung carcinomas are driven by RET fusion genes. RET gene mutations are most common 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 of the RET gene.
The current treatment approaches mainly employ multi-target kinase inhibitors with RET kinase inhibitory activity to treat patients with RET gene fusion or mutation. However, under these conditions, the dosage of the drug is insufficient to reach a level sufficient to inhibit aberrant RET gene expression due to off-target effects and drug toxicity. In addition, cancer cells develop resistance through mutation during the course of treatment of cancer. Once resistance develops, the patient's treatment options become very limited. Thus, there is a great need for selective RET kinase inhibitors for the treatment of patients with RET gene fusions or mutations.
There are no drugs on the market that are selectively targeted to RET kinase targets, and a series of patents for selective RET kinase inhibitors have been disclosed, including WO2016127074, WO2017079140, WO2017011776, WO2017161269, WO2018017983, WO2018022761, WO2018071454, WO2018136661, WO2018136663, etc., and drugs currently in clinical stage I are Blu-667, loxo-292, GSK-3352589, etc. However, these are far from adequate for anti-tumor studies, and there is still a need to research and develop new selective RET kinase inhibitors to address unmet medical needs.
Disclosure of Invention
In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to disclose a compound represented by formula (I):
Figure BDA0001964875480000021
wherein:
x is selected from CH or N;
R 1 ，R 1’ ，R 2 ，R 2’ ，R 3 ，R 3’ ，R 4 ，R 4’ independently selected from hydrogen, C 1 -C 6 Alkyl or C 1 -C 3 Is used for the preparation of a composition for treating a bacterial infection,
or R is 1 And R is 1’ ，R 2 And R is 2’ ，R 3 And R is 3’ ，R 4 And R is 4’ Represents one or more groups of (a) for =o,
or R is 1 And R is 1’ ，R 2 And R is 2’ ，R 3 And R is 3’ Or R is 4 And R is 4’ Linked together to form cyclopropyl, cyclobutyl or oxetanyl,
or R is 1 And R is 2 ，R 1 And R is 3 ，R 1 And R is 4 ，R 1’ And R is 2’ ，R 1’ And R is 3’ ，R 1’ And R is 4’ ，R 2 And R is 3 ，R 2 And R is 4 ，R 2’ And R is 3’ ，R 2’ And R is 4’ ，R 3 And R is 4 Or R is 3’ And R is 4’ Are linked together to form- (CH) 2 ) q-or- (CH) 2 OCH 2 )-，
q is 1,2 or 3;
the D ring is selected from aryl or heteroaryl, said aryl or heteroaryl optionally being further substituted with one or more R 5 Substituted, the R 5 Each independently selected from halogen, cyano, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl or C 1 -C 3 Halogen-containing alkyl groups.
In a preferred embodiment of the present invention, a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein the B ring is selected from the following structures:
Figure BDA0001964875480000031
wherein the 1 and 2 endpoints are optionally connected to the A ring.
In a preferred embodiment of the present invention, a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein the B ring is selected from the following structures:
Figure BDA0001964875480000032
wherein the 1 and 2 endpoints are optionally connected to the A ring.
In a preferred embodiment of the present invention, a compound represented by formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein the D ring is selected from phenyl,
Figure BDA0001964875480000033
or a six membered heteroaryl group containing 1 to 3N atoms, said phenyl group,
Figure BDA0001964875480000034
or a six membered heteroaryl group containing 1 to 3N atoms optionally further substituted with one or more R 5 Substituted, the R 5 Independently selected from halogen, cyano, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl or C 1 -C 3 Halogen-containing alkyl, R 5 Preferably halogen, more preferably fluorine.
Specific compounds of the present application include, but are not limited to:
Figure BDA0001964875480000041
/>
Figure BDA0001964875480000051
or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof.
The invention provides a preparation method of a compound shown in a formula (I) or a stereoisomer, a tautomer or pharmaceutically acceptable salt thereof, which comprises the following steps: in an organic solvent, under alkaline conditions, the compound of the formula (I-B) or salt thereof, the compound of the formula (I-A) or salt thereof and triphosgene react to prepare the compound of the formula (I),
Figure BDA0001964875480000061
wherein D ring, X, R 1 ，R 1’ ，R 2 ，R 2’ ，R 3 ，R 3’ ，R 4 And R is 4’ The definition of (a) is as described in formula (I).
In a preferred embodiment of the invention, the organic solvent is dichloromethane, acetonitrile, tetrahydrofuran or N, N-dimethylformamide, preferably dichloromethane; the base used in the alkaline condition is diisopropylethylamine or triethylamine, preferably diisopropylethylamine; the reaction temperature of the reaction is-60-0 ℃.
The present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
The present invention provides the use of a compound of formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the preparation of a rearrangement kinase inhibitor during transfection.
The present invention provides the use of a compound of formula (I) or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the manufacture of a medicament for the treatment of a disease driven by a rearranged gene during transfection, 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 description and claims of the present invention are defined as follows:
"alkyl" when taken as a group or part of a group is meant to include C 1 -C 20 Straight chain or branched aliphatic hydrocarbon groups. Preferably C 1 -C 10 Alkyl, more preferably C 1 -C 6 An 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 carbocycles. Preferably C 3 -C 12 Cycloalkyl, more preferably C 3 -C 6 Cycloalkyl groups. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like, with cyclopropyl, cyclohexenyl being preferred.
"aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be linked together in a fused manner. The term "aryl" includes aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl. Preferably aryl is C 6 -C 10 Aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl. Aryl groups may be substituted or unsubstituted.
"heteroaryl" refers to an aromatic 5-to 6-membered monocyclic or 9-to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur. Examples of "heteroaryl" include, but are not limited to, furyl, pyridyl, 2-oxo-1, 2-dihydropyridyl, 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.
"hydroxy" refers to-OH.
"halogen" means fluorine, chlorine, bromine and iodine, preferably chlorine, bromine and fluorine, more preferably fluorine.
"cyano" refers to-CN.
"benzyl" means-CH 2 -phenyl.
"ester" means-C (O) O (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
"DMSO" refers to dimethyl sulfoxide.
"Boc" refers to tert-butoxycarbonyl.
"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.
"substituted" or "substituted" as used herein, unless otherwise indicated, means that the group may be substituted with one or more groups selected from the group consisting of: halogen, cyano, alkyl, cycloalkyl, ester, hydroxy, or alkoxy, and the like;
the definition and use of stereochemistry in the present invention is generally referred to in 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., "Stereochemistry of Organic Compounds", john Wiley & Sons, inc., new York,1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist as different stereoisomers. All stereoisomeric forms of the compounds of the invention, including but in no way limited to diastereomers, enantiomers, atropisomers and mixtures thereof, such as racemic mixtures, form part of the invention. Diastereomers can be separated into the individual diastereomers by chromatography, crystallization, distillation, or sublimation, based on their physical-chemical differences. Enantiomers may be converted into diastereomeric mixtures by separation 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 encompassed 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 denote the absolute configuration of the chiral center of the molecule. The prefix d, l or (+), (-) is used to name the sign of the compound plane polarization rotation, where (-) or l means that the compound is left-handed and the prefix (+) or d means that the compound is right-handed. The atoms or groups of atoms of these stereoisomers are connected in the same order but in different steric structures. The particular stereoisomer may be an enantiomer, and the mixture of isomers is commonly referred to as an enantiomeric mixture. The 50:50 enantiomeric mixture is known as a racemic mixture or racemate, which may result in the absence of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.
"tautomer" or "tautomeric form" refers to isomers of structures of different energies that can be interconverted by a low energy barrier. For example, proton tautomers (i.e., proton-shifted tautomers) include tautomerism by proton shift, such as keto-enol and imine-enamine isomerisation. Valency (valence) tautomers include tautomers that reorganize into bond electrons. Unless otherwise indicated, the structural formulae described herein include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers): for example, R, S configuration containing asymmetric centers, the (Z), (E) isomers of double bonds, and the conformational isomers of (Z), (E). Thus, individual stereochemical isomers of the compounds of the invention, or enantiomers, diastereomers, or mixtures of geometric isomers thereof, are all within the scope of the invention.
"pharmaceutically acceptable salts" refers to certain salts of the above compounds which retain the original biological activity and are suitable for pharmaceutical use. Pharmaceutically acceptable salts of the compounds represented by formula (I) may be formed with amine salts of suitable acids including inorganic and organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, malic acid, maleic acid, mandelic acid, methanesulfonic acid, nitric acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonic acid and the like.
"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
"DIPEA" means: diisopropylethylamine.
"triphosgene" means: trichloromethyl carbonate, english is called BTC for short.
“Pd 2 (dba) 3 "means: tris (dibenzylideneacetone) dipalladium.
"t-BuXPhos" means: 2-di-tert-butyl phosphino-2, 4, 6-triisopropyl biphenyl.
"KOAc" means: potassium acetate.
"DMA" means: n, N-dimethylacetamide.
“PCy 3 "means: tricyclohexylphosphine.
Detailed Description
The invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention.
Examples
The preparation of representative compounds represented by formula (I) and related structural identification data are presented in the examples. It must be noted that the following examples are given by way of illustration and not by way of limitation. The structure of the compound is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). 1 H NMR was determined using a Bruker instrument (400 MHz) and chemical shifts were expressed in ppm using tetramethylsilane internal standard (0.00 ppm). 1 H NMR representation method: s=singlet, d=doublet, m=multiplet, br=broadened, dd=doublet of doublet, dt=doublet of triplet. If a coupling constant is provided, it is in Hz. The mass spectrum was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: thermo, model: finnigan LCQ advantage MAX).
The thin layer chromatography silica gel plate uses a smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate, the specification of the silica gel plate used by the Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product used by the TLC is 0.4 mm-0.5 mm silica gel preparation plate.
Column chromatography generally uses 200-300 mesh silica gel of yellow sea as a carrier.
The known starting materials of the present invention may be synthesized using or following methods known in the art, or may be purchased from the companies ABCR GmbH & Co.KG, acros organics, aldrich Chemical Company, shaoshima chemical technology (Accela ChemBio Inc), darui chemical, and the like.
In the examples, unless otherwise specified, the reactions were carried out in an open atmosphere.
The examples are not particularly described, and the solution in the reaction is an aqueous solution.
In the examples, the reaction temperature was room temperature unless otherwise specified.
The temperature range of the room temperature is 20-30 ℃.
The reaction progress in the examples was monitored by Thin Layer Chromatography (TLC) using the following system of developing agents: the volume ratio of the developing agent is adjusted according to the polarity of the compound by a methylene dichloride and methanol system or an n-hexane and ethyl acetate system, and can be adjusted by adding a small amount of triethylamine, an acidic or alkaline reagent and the like.
The intermediate compound (A) is prepared by referring to the process of patent WO2016127074A 1.
Example 1
(S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- (5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide
Figure BDA0001964875480000101
The first step: synthesis of 2-chloro-6-methyl-N- (5-methyl-1H-pyrazol-3-yl) pyrimidin-4-amine
Figure BDA0001964875480000111
In a 100mL single-necked flask, 6-methyl-2, 4-dichloropyrimidine (1 a) (9.72 g,60 mmol), 5-methyl-1H-3-aminopyrazole (a) (7.0 g,72 mmol), DMSO (30 mL) and DIPEA (11.6 g,72 mmol) were added and reacted at 60℃for 24 hours. TLC monitors the disappearance of the raw materials, reduces the temperature to room temperature, adds 200mL ethyl acetate into the reaction liquid for extraction, washes the organic phase three times (30 mL multiplied by 3) with water respectively, washes the organic phase with saturated common salt once, dries the organic phase with anhydrous sodium sulfate, filters the organic phase, and distills the organic phase under reduced pressure to remove the solvent to obtain brown yellow viscous liquid. 80mL of methylene chloride was added and the mixture was allowed to stand for 3 hours, whereby 9.9g of 2-chloro-6-methyl-N- (5-methyl-1H-pyrazol-3-yl) pyrimidin-4-amine (1 c) was precipitated, and the yield was 74%.
MS m/z(ESI):224.3[M+1]
And a second step of: synthesis of tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxylate
Figure BDA0001964875480000112
In a 50mL single vial was added 2-chloro-6-methyl-N- (5-methyl-1H-pyrazol-3-yl) pyrimidin-4-amine (1 c) (892 mg,4 mmol), 1-tert-butoxycarbonyl piperazine (1 d) (1.49 g,8 mmol), K 2 CO 3 (1.66 g,12 mmol) and 25mL DMF were reacted at 140℃for 6 hours, the reaction solution was cooled to room temperature, 150mL ethyl acetate was added for extraction, the organic phase was washed three times with water (15 mL. Times.3) and once with saturated brine, dried over anhydrous sodium sulfate, filtered and distilled off under reduced pressure to remove the solvent, and then column chromatography was carried out (eluent: methanol=20:1) to give tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxylate (1 f) 1.01g in 67% yield.
MS m/z(ESI):374.2[M+1]
And a third step of: synthesis of 6-methyl-N- (5-methyl-1H-pyrazol-3-yl) -2- (1-piperazinyl) pyrimidin-4-amine hydrochloride
Figure BDA0001964875480000121
To a 100mL single port flask was added tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxylate (1 f) (1.24 g,3.3 mmol) and 25mL of 1, 4-dioxane, 25. 25mL of a 2.6mol/L hydrogen chloride/1, 4-dioxane solution was added dropwise, the reaction was carried out at room temperature of 4H, filtration was carried out, the filter cake was washed with diethyl ether, and vacuum drying was carried out to obtain 6-methyl-N- (5-methyl-1H-pyrazol-3-yl) -2- (1-piperazinyl) pyrimidin-4-amine hydrochloride (1 g) 1.1 g, yield >99%.
MS m/z(ESI):274.2[M+1]
Fourth step: synthesis of (S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- (5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide
Figure BDA0001964875480000122
In a 50mL single port flask, (S) -1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl-1-amine (A) (279 mg,1 mmol) and methylene chloride 10mL were added, cooled to 0℃and triethylamine (1.02 g,10 mmol) and triphosgene (446 mg,1.5 mmol) were sequentially added, and the reaction was maintained at 0℃for 1H. The reaction was then cooled to-50 ℃, 6-methyl-N- (5-methyl-1H-pyrazol-3-yl) -2- (1-piperazinyl) pyrimidin-4-amine hydrochloride (1 g) (530 mg,1.7 mmol), reacted at-50 ℃ for 20 minutes, quenched with 2mL methanol and 10mL water, added 50mL dichloromethane, extracted, the organic phase washed twice with water (10 mL ×2), saturated brine once, dried over anhydrous sodium sulfate, filtered, and the solvent removed by distillation under reduced pressure, and the column chromatography was separated (eluent and volume ratio: dichloromethane: methanol=10:1) to give (S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- (5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide (I-1) mg in 57% yield.
1 H NMR(400MHz,DMSO)δ11.89(s,1H),9.35(s,1H),8.66(d,J＝4.5Hz,1H),8.40(d,J＝1.9Hz,1H),7.95(dd,J＝8.5,2.2Hz,1H),7.90(d,J＝4.2Hz,1H),7.86(d,J＝8.5Hz,1H),7.00(d,J＝7.6Hz,1H),6.33-5.98(m,2H),4.92(t,J＝7.2Hz,1H),3.67(s,4H),3.40(s,4H),2.19(s,3H),2.13(s,3H),1.43(d,J＝7.1Hz,3H)ppm.
MS m/z(ESI):506.3[M+1].
Referring to example 1, the following compounds (I-2), compound (I-3), compound (I-4), compound (I-5), compound (I-6) and compound (I-7) may be prepared. The structure and characterization data are as follows:
compound (I-2):
Figure BDA0001964875480000131
1 H NMR(400MHz,DMSO)δ12.01(s,1H),9.39(s,1H),8.66(d,J＝4.4Hz,1H),8.40(d,J＝1.7Hz,1H),7.94(dd,J＝8.6,2.0Hz,1H),7.90(d,J＝4.2Hz,1H),7.85(d,J＝8.4Hz,1H),6.97(s,1H),6.18(s,2H),4.94(t,J＝7.2Hz,1H),4.74(s,1H),4.32(d,J＝12.6Hz,1H),4.04(d,J＝11.4Hz,1H),3.93(d,J＝13.2Hz,1H),3.05(d,J＝11.0Hz,2H),2.94-2.79(m,1H),2.20(s,3H),2.18(s,3H),1.44(d,J＝7.1Hz,3H),1.10(d,J＝5.8Hz,3H)ppm.
MS m/z(ESI):520.6[M+1].
compound (I-3):
Figure BDA0001964875480000132
1 H NMR(400MHz,DMSO)δ11.83(s,1H),9.25(s,1H),8.66(d,J＝4.5Hz,1H),8.38(s,1H),7.93(dd,J＝8.5,1.9Hz,1H),7.89(d,J＝4.2Hz,1H),7.85(d,J＝8.5Hz,1H),6.76(d,J＝7.4Hz,1H),6.17(s,1H),6.14(s,1H),4.86(t,J＝7.2Hz,1H),3.78-3.63(m,4H),3.63-3.49(m,2H),2.18(s,3H),2.11(s,3H),1.40(d,J＝7.0Hz,3H),1.34(s,3H),1.26(s,3H)ppm.
MS m/z(ESI):534.3[M+1].
compound (I-4):
Figure BDA0001964875480000141
1 H NMR(400MHz,DMSO)δ11.85(s,1H),9.26(s,1H),8.66(d,J＝4.3Hz,1H),8.40(d,J＝1.6Hz,1H),7.94(dd,J＝8.5,2.0Hz,1H),7.89(d,J＝4.1Hz,1H),7.86(d,J＝8.5Hz,1H),7.09(d,J＝7.5Hz,1H),6.14(s,2H),4.94(t,J＝7.2Hz,1H),4.33-4.18(m,2H),3.38-3.36(m,2H),2.95(d,J＝9.4Hz,2H),2.19(s,3H),2.10(s,3H),1.73(s,2H),1.57(d,J＝7.4Hz,2H),1.43(d,J＝7.0Hz,3H)ppm.
MS m/z(ESI):532.7[M+1].
compound (I-5):
Figure BDA0001964875480000142
1 H NMR(400MHz,DMSO)δ11.83(s,1H),9.28(s,1H),8.67(d,J＝4.0Hz,1H),8.40(s,1H),7.95(d,J＝7.4Hz,1H),7.90(d,J＝4.1Hz,1H),7.86(d,J＝8.3Hz,1H),6.80(d,J＝7.1Hz,1H),6.16(s,2H),4.99(t,J＝7.2Hz,1H),4.53(d,J＝13.3Hz,2H),4.35-4.15(m,2H),2.95(d,J＝10.8Hz,2H),2.19(s,3H),2.11(s,3H),1.44(d,J＝6.8Hz,3H),1.11(s,6H)ppm.
MS m/z(ESI):534.3[M+1].
compound (I-6):
Figure BDA0001964875480000143
1 H NMR(400MHz,CDCl 3 )δ8.37(d,J＝1.9Hz,1H),8.35(d,J＝4.4Hz,1H),7.87(d,J＝8.5Hz,1H),7.76(dd,J＝8.5,2.1Hz,1H),7.56(d,J＝4.2Hz,1H),6.04(s,2H),5.69(d,J＝6.5Hz,1H),5.11-5.06(m,1H),3.83-3.63(m,6H),2.26(s,3H),2.22(s,3H),1.56(d,J＝7.0Hz,3H),1.12-0.94(m,4H)ppm.
MS m/z(ESI):532.7[M+1].
compound (I-7):
Figure BDA0001964875480000151
1 H NMR(400MHz,DMSO)δ11.85(s,1H),9.26(s,1H),8.66(d,J＝4.3Hz,1H),8.40(d,J＝1.6Hz,1H),7.94(dd,J＝8.5,2.0Hz,1H),7.89(d,J＝4.1Hz,1H),7.86(d,J＝8.5Hz,1H),7.09(d,J＝7.5Hz,1H),6.14(brs,2H),5.00-4.80(m,1H),4.33-4.15(m,2H),3.44-3.35(m,2H),2.95(d,J＝9.4Hz,2H),2.19(s,3H),2.10(s,3H),1.73(s,2H),1.57(d,J＝7.4Hz,2H),1.43(d,J＝7.0Hz,3H)ppm.
MS m/z(ESI):532.3[M+1].
example 2
(S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperazine-1-carboxamide
Figure BDA0001964875480000152
The first step: synthesis of tert-butyl 4- (6-bromo-4-methylpyridin-2-yl) piperazine-1-carboxylate
Figure BDA0001964875480000153
In a 100mL single flask were placed 2-bromo-6-fluoro-4-methylpyridine (2 a) (950 mg,5 mmol), 1-tert-butoxycarbonylpiperazine (1 d) (1.4 g,7.5 mmol), K 2 CO 3 (2.09g,15mmol) and 35ml dmf, at 140 c for 6h. The reaction mixture was cooled to room temperature, washed with 200mL of ethyl acetate, once with water (20 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to remove the solvent, and then separated by column chromatography (eluent: methanol=30:1 by volume) to give 1.45g of t-butyl 4- (6-bromo-4-methylpyridin-2-yl) piperazine-1-carboxylate (2 b) in 81% yield.
MS m/z(ESI):[M+1]357.1
And a second step of: synthesis of tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperidine-1-carboxylate
Figure BDA0001964875480000161
In a 100mL single vial was added tert-butyl 4- (6-bromo-4-methylpyridin-2-yl) piperazine-1-carboxylate (2 b) (1.07 g,3 mmol), 5-methyl-1H-3-aminopyrazole (a) (552 mg,7.5 mmol), pd 2 (dba) 3 (550 mg,0.6 mmol), t-BuXPhos (510 mg,1.2 mmol), KOAc (882 mg,9 mmol) and 15mLDMA, nitrogen was purged three times and reacted at 140℃for 2h. After the reaction solution was cooled to room temperature, 200mL of ethyl acetate was added, washed three times with water (20 ml×3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure, and separated by column chromatography (eluent: methanol=20:1 by volume) to give tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperidine-1-carboxylate (2 c) 421mg in 38% yield.
MS m/z(ESI):[M+1]373.2
And a third step of: synthesis of 4-methyl-N- (5-methyl-1H-pyrazol-3-yl) -6- (1-piperazinyl) pyridin-2-amine hydrochloride
Figure BDA0001964875480000162
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To a 50mL single vial was added tert-butyl 4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperidine-1-carboxylate (2 c) (426 mg,1.1 mmol) and 8mL dioxane, 2.6mol/L hydrogen chloride/1, 4-dioxane solution (20 mL) was added, reacted at room temperature for 4H, concentrated, and dried under vacuum to give 440mg of 4-methyl-N- (5-methyl-1H-pyrazol-3-yl) -6- (1-piperazinyl) pyridin-2-amine hydrochloride (2 d) in >99% yield.
MS m/z(ESI):[M+1]273.2
Fourth step: synthesis of (S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperazine-1-carboxamide
Figure BDA0001964875480000171
In a 50mL single-necked flask, (S) -1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl-1-amine (A) (140 mg,0.5 mmol) and 5mL of methylene chloride were added, cooled to 0℃and triethylamine (510 mg,5 mmol) and triphosgene (223 mg,0.75 mmol) were sequentially added, and the mixture was reacted at 0℃for 1 hour. The reaction was then cooled to-50 ℃, compound (2 d) (232 mg,0.75 mmol), reacted at-50 ℃ for 1 hour, quenched with 2mL of methanol and 10mL of water, and the organic phase was washed twice with 50mL of dichloromethane (10 ml×2), dried with saturated brine once, filtered, the solvent was removed under reduced pressure, column chromatography was separated (eluent and volume ratio: dichloromethane: methanol=30:1), the mixture was collected again and the plate was prepared with silica gel (developing reagent and volume ratio: dichloromethane: methanol=12:1) to give (S) -N- (1- (6- (4-fluoro-1H-pyrazol-1-yl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyridin-2-yl) piperazine-1-carboxamide (I-8) 11mg, yield 5%.
1 H NMR(400MHz,DMSO)δ11.60(s,1H),8.63(d,J＝4.3Hz,1H),8.58(s,1H),8.37(s,1H),7.91(d,J＝6.9Hz,1H),7.86(d,J＝4.2Hz,1H),7.83(d,J＝8.4Hz,1H),6.96(d,J＝7.5Hz,1H),6.30(s,1H),5.97(s,1H),5.93(s,1H),4.89(t,J＝7.2Hz,1H),3.38(s,8H),2.14(s,3H),2.08(s,3H),1.40(d,J＝6.9Hz,3H)ppm.
MS m/z(ESI):[M+1]505.3
Example 3
(S) -N- (1- (6- (4-fluoro-phenyl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide
Figure BDA0001964875480000172
The first step: synthesis of 1- (6- (4-fluorobenzene) pyridin-3-yl) ethyl-1-one
Figure BDA0001964875480000181
In a 250mL single vial was added 1- (6-bromo-pyridin-3-yl) ethyl-1-one (3 a) (4.0 g,20 mmol), 4-fluorobenzeneboronic acid (3 b) (4.2 g,30 mmol), pd 2 (dba) 3 (920mg,1mmol)、PCy 3 (560 mg,2 mmol), sodium carbonate (8.5 g,80 mmol) and 1, 4-dioxane/water (70 mL/7 mL) were mixed, nitrogen was purged three times, and reacted at 100℃overnight. Cooling to room temperature after the reaction, filtering by diatomite, washing by 200mL of ethyl acetate, washing by water for three times (40 mL multiplied by 3), washing by saturated saline water once, drying by anhydrous sodium sulfate, filtering, distilling under reduced pressure to remove the solvent, and separating by column chromatography (eluent: methanol=20:1) to obtain 4.5g of 1- (6- (4-fluorophenyl) pyridin-3-yl) -ethanone (3 c), and obtaining the yield>99％。
MS m/z(ESI):[M+1]216.1
And a second step of: synthesis of (S) -N- ((S) -1- (6- (4-fluorophenyl) pyridin-3-yl) ethyl) -2-methylpropyl-2-sulfonylimine
Figure BDA0001964875480000182
1- (6- (4-fluorobenzene) pyridin-3-yl) ethyl-1-one (3 c) (4.5 g,21 mmol), (R) - (+) -tert-butylsulfinamide (2.56 g,21 mmol) and 50mL tetrahydrofuran were added in a 250mL three-port flask, nitrogen was purged three times, ethyl tetratitanate (9.62 g,42 mmol) was added and reacted at 75℃for 17 hours, TLC was monitored until the starting material disappeared, the temperature was lowered to-78 ℃, lithium tri-sec-butylborohydride (11.97 g,63 mmol) was slowly added dropwise, after maintaining the reaction at-78℃for 2 hours, room temperature was recovered, 15mL of methanol was added dropwise for quenching, 100mL of ice water was added, the precipitated solid was filtered through celite, ethyl acetate was washed, the solvent was distilled off under reduced pressure, 300mL of ethyl acetate was added three times (40 mL. Times 3) and saturated brine was washed once, dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, column chromatography (eluent and volume ratio: petroleum ether: ethyl acetate=7:3) was separated to give (S) -N- (6-phenyl) -2-3- ((2-methyl) pyridine-2-yl) 2-sulfonyl imide yield (31 g).
MS m/z(ESI):321.1[M+1]
And a third step of: synthesis of (S) -1- (6- (4-fluorophenyl) pyridin-3-yl) ethyl-1-amine hydrochloride
Figure BDA0001964875480000191
To a 100mL single flask were added (S) -N- ((S) -1- (6- (4-fluorophenyl) pyridin-3-yl) ethyl) -2-methylpropyl-2-sulfonylimide (3 d) (2 g,6.2 mmol) and 20mL of 1, 4-dioxane, 25mL of a hydrogen chloride/1, 4-dioxane solution having a concentration of 2.6mol/L was added dropwise, the mixture was reacted at room temperature for 2 hours, filtered, the filter cake was washed with ethyl acetate, and dried under vacuum to give (S) -1- (6- (4-fluorophenyl) pyridin-3-yl) ethyl-1-amine hydrochloride (3 e) 1.5g, yield 84%.
MS m/z(ESI):217.1[M+1]
Figure BDA0001964875480000192
Fourth step: synthesis of (S) -N- (1- (6- (4-fluoro-phenyl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide
In a 50mL single-necked flask, (S) -1- (6- (4-fluorophenyl) pyridin-3-yl) ethyl-1-amine hydrochloride (3 e) (126 mg,0.5 mmol), triphosgene (225 mg,0.75 mmol) and 5mL methylene chloride were added, cooled to-20℃and triethylamine (758 mg,7.5 mmol) was added and the reaction was maintained at-20℃for 1 hour. 6-methyl-N- (5-methyl-1H-pyrazol-3-yl) -2- (1-piperazinyl) pyrimidin-4-amine hydrochloride (1 g) (186 mg,0.6 mmol) was added and reacted at-20℃for 30 minutes, 1mL of methanol and 5mL of water were added, 50mL of methylene chloride was added, the organic phase was washed twice with water (10 mL. Times.2), saturated brine was washed once with anhydrous sodium sulfate and dried, and after removal of the solvent by distillation under reduced pressure, column chromatography was carried out (eluent and volume ratio: methylene chloride: methanol=10:1), and the crude product obtained was again isolated by a silica gel preparation plate (developer and volume ratio: methylene chloride: methanol=10:1) to give (S) -N- (1- (6- (4-fluoro-phenyl) pyridin-3-yl) ethyl) -4- (4-methyl-6- ((5-methyl-1H-pyrazol-3-yl) amino) pyrimidin-2-yl) piperazine-1-carboxamide (I-9) 30mg, 12% yield.
1 H NMR(400MHz,DMSO-d 6 )δ11.85(s,1H),9.28(s,1H),8.61(s,1H),8.14-8.07(m,2H),7.90(d,J＝8.2Hz,1H),7.81(d,J＝8.3Hz,1H),7.30(t,J＝8.8Hz,2H),6.98(d,J＝7.5Hz,1H),6.19(s,1H),6.13(s,1H),4.99-4.82(m,1H),3.67(s,4H),3.40(s,4H),2.20(s,3H),2.12(s,3H),1.44(d,J＝7.1Hz,3H)ppm.
MS m/z(ESI):516.3[M+1].
Biological evaluation
Test example 1 measurement of RET kinase Activity by the Compounds of the invention
The method uses Cisbio company
Figure BDA0001964875480000201
The KinEASE-TK tyrosine kinase kit (cat. No. 62TK0 PEB) was assayed by time resolved fluorescence energy resonance transfer (TR-FRET) by determining the degree of phosphorylation of the biotinylated polypeptide substrate. Human RET protein (RET kinase) was purchased from Carna bioscience (Japan, cat. No. 08-159-5. Mu.g).
The experimental procedure was as follows:
the test compound (compound of the invention and compound 164 in WO2018017983A1 as a control) was dissolved in 100% DMSO to a final concentration of 10mM.
The solution of the compound to be tested prepared in the step (1) was dissolved in 46uL of 100% DMSO, and the solution obtained in the step was designated as No. 2.
Third, solution number 2 was subjected to subsequent gradient dilutions at 5 fold (i.e., 20 μl of 100% dmso plus 5 μl of compound) for a total of 9 gradients, numbered 3 to 11.
Note that: no. 2 is not used for the dilution in step (4).
(unless otherwise specified, the following steps are all required to be performed on ice)
The solution numbered 3 to 11 was further diluted in a gradient with the buffer provided in the kit (Cisbio, cat. No. 62TK0 PEB) at a dilution factor of 20 (i.e. 19. Mu.L buffer was added to the solution numbered 3 to 11). At this time, the final concentration of the test compound in the system No. 3 to 11 ranged from 3200nM to 0.008nM (9 gradients), and the final concentration of DMSO was 2%.
And fifthly, adding the 9 compound solutions to be tested with gradient concentration in the step (4) into 384-hole plates according to concentration, wherein each hole is 4 mu L, and two compound holes are arranged.
Add 2 μl of human RET protein per well and incubate on ice for 10 min.
mu.L of ATP (Sigma #A7699) and 2. Mu.L of biotinylated polypeptide substrate (Cisbio, cat. No. 62TK0 PEB) were added to each well to initiate phosphorylation. Incubate at 37℃for half an hour.
mu.L of an anti-phosphotyrosine antibody conjugated with europium-based element compound (supplied in the kit under the trade name 62TK0 PEB) and 5. Mu.L of streptavidin conjugated with modified allophycocyanin XL665 (Cisbio under the trade name 62TK0 PEB) were added to each well.
Incubation was continued for 1 hour at room temperature. After the incubation, the TF-FRET pattern of the microplate reader (BMG Labtech, model: FLUOStar Omega) was used to measure the fluorescence intensity of each well at excitation wavelength of 304nM, and the ratio was automatically calculated by reading the fluorescence intensity of each well at emission wavelengths of 615nM and 665 nM.
By comparing the fluorescence intensity ratio with that of a control group, calculating the inhibition rate of the compound at each concentration, and further performing curve fitting by using GraphPad Prism5 with logarithmic concentration-inhibition rate, and calculating the IC of the compound 50 The values are given in Table 2 below.
The control kinase selected is another receptor tyrosine kinase KDR which is similar to RET kinase in structure. Purchased from Carna bioscience (Japan, cat. No. 08-191-5. Mu.g). The gradient dilution step is the same as RET kinase, so that the final concentration of the compound to be tested in the reaction system is 16000 nM-0.04 nM (No. 2-10The solution was subjected to step 4 gradient dilution) and the other reaction conditions were as above, with a final DMSO concentration of 2%. IC for inhibiting KDR kinase by test compound 50 Value calculation method and RET kinase inhibition IC 50 The value calculation method is the same.
TABLE 2 IC of the inhibition of RET kinase and KDR kinase by the compounds of the invention 50 Value of
Numbering of compounds RET(IC 50 ) KDR(IC 50 ) KDR/RET ratio
I-1 0.7nM 295nM 433
I-8 2.5nM 157nM 63
I-9 5.3nM 264nM 50
164 73nM 2300nM 32
From the above table, it can be seen that the compounds of the present invention have a remarkable inhibitory effect on RET kinase activity, and the inhibitory effect is superior to that of compound 164 in WO2018017983A1. The inhibiting activity of the compound of the invention on RET kinase is better than that on KDR kinase. The compounds of the invention are therefore useful as a class of potent and selective RET kinase inhibitors.
Remarks: the structural formula of compound 164 is shown below, and the preparation method is shown in WO2018017983A1.
Figure BDA0001964875480000221
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Claims (10)

1. A compound of formula (I) or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof:
Figure FDA0004058595710000011
wherein:
x is selected from N;
R 1 ，R 1’ ，R 2 ，R 2’ ，R 3 ，R 3’ ，R 4 ，R 4’ independently selected from hydrogen;
the D ring is selected from phenyl, said phenyl optionally being further substituted with one or more R 5 Substituted, the R 5 Each independently selected from halogen.
2. A compound according to claim 1-or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, wherein the D-ring is selected from phenyl optionally further substituted with one fluoro.
3. The compound of claim 1, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, wherein the compound has the specific structure of:
Figure FDA0004058595710000012
4. a process for the preparation of a compound of formula (I) as defined in claim 1, or a stereoisomer, tautomer or pharmaceutically acceptable salt thereof, which comprises: in an organic solvent, under alkaline conditions, the compound of the formula (I-B) or salt thereof, the compound of the formula (I-A) or salt thereof and triphosgene react to prepare the compound of the formula (I),
Figure FDA0004058595710000021
wherein D ring, X, R 1 ，R 1’ ，R 2 ，R 2’ ，R 3 ，R 3’ ，R 4 And R is 4’ Is defined as in claim 1.
5. The method according to claim 4, wherein the organic solvent is methylene chloride, acetonitrile, tetrahydrofuran or N, N-dimethylformamide; the alkali used in the alkaline condition is diisopropylethylamine or triethylamine; the reaction temperature of the reaction is-60-0 ℃.
6. The method according to claim 5, wherein the organic solvent is methylene chloride; the alkali used in the alkaline condition is diisopropylethylamine.
7. A pharmaceutical composition comprising an effective amount of a compound according to any one of claims 1 to 3, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, or combination thereof.
8. Use of a compound according to any one of claims 1 to 3, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, for the preparation of a rearrangement kinase inhibitor during transfection.
9. Use of a compound according to any one of claims 1 to 3, or a stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the treatment of a disease driven by rearrangement of genes during transfection, wherein the disease is cancer.
10. The use according to claim 9, wherein the cancer is selected from lung cancer, thyroid cancer, colon cancer, breast cancer or pancreatic cancer.
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