WO2020177668A1 - 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途 - Google Patents

吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途 Download PDF

Info

Publication number
WO2020177668A1
WO2020177668A1 PCT/CN2020/077449 CN2020077449W WO2020177668A1 WO 2020177668 A1 WO2020177668 A1 WO 2020177668A1 CN 2020077449 W CN2020077449 W CN 2020077449W WO 2020177668 A1 WO2020177668 A1 WO 2020177668A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
cycloalkyl
alkyl
heteroaryl
alkylene
Prior art date
Application number
PCT/CN2020/077449
Other languages
English (en)
French (fr)
Inventor
郑苏欣
王中利
张建平
刘俊
安泉林
赵雯雯
Original Assignee
察略盛医药科技(上海)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 察略盛医药科技(上海)有限公司 filed Critical 察略盛医药科技(上海)有限公司
Priority to US17/435,703 priority Critical patent/US20220135560A1/en
Priority to EP20765744.6A priority patent/EP3936504A4/en
Priority to CN202080017586.6A priority patent/CN113508116A/zh
Publication of WO2020177668A1 publication Critical patent/WO2020177668A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention belongs to the field of medicine, and relates to a pyrazolo [1,5-a] pyridine derivative, a preparation method thereof, a pharmaceutical composition containing the derivative, and its use as a therapeutic agent, especially as a RET protein kinase inhibitor use.
  • RET protein (Rerranged during Transfection, rearrangement during transfection) belongs to the family of receptor tyrosine kinases. As a receptor for nerve growth factor, its function involves the normal growth and development of the nervous system, cell proliferation and survival. Glial cell line-derived neurotrophic factor (glial cell line-derived neurotrophic factor, GDNF) family ligands (GFL) regulate downstream signals by binding to RET. The GFL family also includes neuroturin (NTRN), artemin (ARTN) and persephin ( PSPN) (Arighi et al., Cytokine Growth Factor rev., 2005, 16, 441-67). Unlike other receptor tyrosine kinases, RET activation requires ligands.
  • NTRN neuroturin
  • ARTN artemin
  • PSPN persephin
  • the GDNF family receptor alpha protein and RET protein form a multimeric complex.
  • the GFR ⁇ receptor is connected to the co-receptor on the cell surface through glycosylphosphatidylinositol bonds. It has been found that this family includes 4 subtypes (GFR ⁇ 1-4) that can bind to different GFLs.
  • GFL and GFR ⁇ family members form a binary complex, and the complex binds to RET and recruits it to the cholesterol-rich secondary domain called lipid raft, which induces dimerization of RET. And the formation of GFL(2)-GFRa(2)-RET(2) heterohexamer complex, which activates RET kinase activity and downstream signals.
  • RET dimerizes and induces autophosphorylation of intracellular tyrosine residues of the protein, and then further recruits other connexins and signal transduction proteins to activate multiple downstream signal transduction pathways , Including Ras-MAPK pathway and PI3K-Akt/mTOR signaling pathway.
  • Abnormal RET is closely related to the occurrence and development of tumor diseases, mainly manifested as gain-of-function gene mutation or RET fusion protein due to gene rearrangement, both of which will cause the continuous abnormal activation of RET signal.
  • the proportion of RET point mutations is as high as 95%; in patients with papillary thyroid cancer, the incidence of RET gene rearrangement is about 20%-40%; in addition, in breast cancer, colon cancer, The overexpression of RET protein can also be observed in pancreatic cancer and acute leukemia. (Lois M. Mulligan; RETrevisited expanding theoncogenic portfolio, NatureReviewsCancer 14,173-186(2014)).
  • Ring A is selected from the group:
  • X 1 and X 2 are each independently selected from CH, CCH 3 , CF, CCl or N;
  • X 3 and X 4 are each independently selected from CH, CF or N;
  • L 1 is selected from a bond, -(R a R b C)-, -(R a R b N)- or -O-;
  • L 2 is selected from a bond, -(alkylene)-, -C(O)-, -S(O)-, -SO 2 -, -(alkylene)-O-, -(alkylene) -S-, -(alkylene)-NR 1 -, -C(O)-(alkylene)-, -SO 2 -(alkylene)-; where the right button in the L 2 group is connected to E; Wherein said alkylene group is optionally further selected by one or more selected from hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR 2 R 3 ,- C(O)NR 2 R 3 , -C(O)R 4 , -OC(O)R 4 , -NR 2 C(O)R 3 or -SO 2 NR 2 R 3 substituents;
  • R a is selected from a hydrogen atom, an alkyl group or a halogen
  • R b is selected from hydrogen atom, alkyl group or halogen
  • the cycloalkyl or heterocyclic group is optionally further selected by one or more selected from hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR 2 R 3 , -C(O)NR 2 R 3 , -C(O)R 4 , -OC(O)R 4 , -NR 2 C(O)R 3 or -SO 2 NR 2 R 3 replace;
  • B is selected from hydrogen atom, alkyl group, alkenyl group, alkynyl group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group, wherein said alkyl group, alkenyl group, alkynyl group, cycloalkyl group, heterocyclic group , Aryl or heteroaryl is optionally further substituted with one or more substituents selected from R c ;
  • E is selected from a hydrogen atom, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group; wherein the cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group is optionally further substituted by one or more One is selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR 2 R 3 , -C(O)NR 2 R 3 , -C(O)R 4 , -OC(O)R 4 , -NR 2 C(O)R 3 or -SO 2 NR 2 R 3 substituents; preferably phenyl or pyridyl;
  • R c is selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR 2 R 3 , -C(O)NR 2 R 3 , -C(O)R 4 , -OC(O)R 4 , -NR 2 C(O)R 3 or -S(O) 2 NR 2 R 3 , wherein the alkyl group, alkoxy group, Cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally further substituted by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl Group, heteroaryl, -NR 5 R 6 , -C(O)NR 5 R 6 , -C(O)R 7 , -OC(O)R 7 , -NR 5 C(O)R 6 or -
  • R 1 is the same or different, each independently selected from a hydrogen atom, a hydroxyl group, an alkyl group, a halogen or an alkoxy group, wherein the alkyl group or alkoxy group is optionally further substituted by one or more selected from the group consisting of hydroxyl, halogen or alkane. Substituted by substituents of oxy;
  • R 2 , R 3 and R 4 are each independently selected from a hydrogen atom, a hydroxyl group, a halogen, a nitro group, a cyano group, an alkyl group, an alkoxy group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein The alkyl group, alkoxy group, cycloalkyl group, heterocyclic group, aryl group or heteroaryl group is optionally further selected by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, Cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR 5 R 6 , -C(O)NR 5 R 6 , -C(O)R 7 , -OC(O)R 7 , -NR 5 C(O)R 6 or -SO 2 NR 5 R 6 substituents;
  • R 2 and R 3 and the connected N atom together form a 4- to 8-membered heterocyclic group, wherein the 4- to 8-membered heterocyclic ring contains one or more N, O, S(O) n atoms, and 4
  • R 5 , R 6 and R 7 are each independently selected from a hydrogen atom, an alkyl group, a cycloalkyl group, a heterocyclic group, an aryl group or a heteroaryl group, wherein the alkyl group, cycloalkyl group, heterocyclic group, aryl group Group or heteroaryl group is optionally further substituted by one or more selected from hydroxyl, halogen, nitro, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyl or carboxyl Substitution of ester group;
  • n is selected from 0, 1, 2, 3, 4 or 5;
  • n is selected from 0, 1 or 2;
  • L 1 is selected from -(R a R b C)-, -(R a R b N)- or -S-;
  • B is selected from alkenyl or alkynyl, wherein the alkenyl or alkynyl is optionally further substituted with one or more substituents selected from R c .
  • a compound represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof is a compound represented by general formula (II) or Stereoisomers, tautomers or their pharmaceutically acceptable salts:
  • L 1 is selected from a bond, -(R a R b C)-, -(R a R b N)- or -O-; preferably -O-;
  • L 2, B, E, X 1, X 2, X 3, X 4, R 1, R a, R b and m are as defined in formula (I) said.
  • a compound represented by general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof is a compound represented by general formula (III) or Stereoisomers, tautomers or their pharmaceutically acceptable salts:
  • L 1 is selected from -(R a R b C)-, -(R a N)- or -S-;
  • R a and R b are selected from a hydrogen atom or a methyl group
  • B is selected from alkenyl or alkynyl, wherein the alkenyl or alkynyl is optionally further substituted with one or more substituents selected from R c ;
  • X 1 , X 2 , X 3 and X 4 are selected from CH or N;
  • X 1 , X 2 , X 3 and X 4 are N;
  • X 1 is N
  • X 2 , X 3 , and X 4 are CH.
  • L 2 is selected from a bond, -(alkylene)-, -C(O)-, -SO 2 -, -(alkylene)-O-, -(alkylene)-S-, -(alkylene) Alkyl) -NR 1 -or -C(O)-(alkylene)-, where the right button of the L 2 group is connected to E;
  • it is -(alkylene)-, -C(O)-, -(alkylene)-S- or -C(O)-(alkylene)-;
  • the -(alkylene)- mentioned therein is preferably -(methylene)- or -CH(CH 3 )-.
  • B is selected from pyrazolyl, wherein said pyrazolyl is optionally further substituted by methyl;
  • alkyl group wherein the alkyl group is optionally further substituted with one or more hydroxy groups; preferably,
  • B is selected from the group:
  • B is selected from the group:
  • Typical compounds of the present invention include but are not limited to:
  • the present invention provides a method for preparing a compound represented by the general formula (I) or a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, the method comprising:
  • the general formula (IA) is reacted with E-(alkylene)-OMs, EC(O)X, E-(alkylene)-C(O)X or EC(O)H to obtain the general formula (I) ;
  • X is selected from hydroxyl or halogen, preferably hydroxyl, Cl or Br;
  • L 1 , L 2 , B, E, X 1 , X 2 , X 3 , X 4 , R 1 and m are as defined in the general formula (I).
  • the present invention provides a compound of general formula (IA) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof,
  • L 1 , B, X 1 , X 2 , X 3 , X 4 , R 1 and m are defined as described in the general formula (I).
  • Typical compounds of general formula (IA) include but are not limited to:
  • a pharmaceutical composition of the present invention contains an effective dose of the compound represented by the general formula (I), (II) or (III) or its stereoisomers, tautomers or Its pharmaceutically acceptable salts, and pharmaceutically acceptable carriers, excipients or combinations thereof.
  • the present invention provides a compound represented by the general formula (I), (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and their pharmaceutical compositions are in preparation Use of RET or mutant RET inhibitors in medicine.
  • the present invention provides a compound represented by the general formula (I), (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and their pharmaceutical compositions are in preparation For use in medicines for the treatment of diseases mediated by abnormal RET, wherein the diseases are preferably cancer, fibrodysplasia and genetic disorders, and the cancers are preferably non-small cell lung cancer, thyroid cancer, solid tumors, Colon tumor or pancreatic cancer, more preferably non-small cell lung cancer, thyroid cancer, solid tumor.
  • the present invention provides a compound represented by the general formula (I), (II) or (III) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and their pharmaceutical compositions are in preparation Use in medicine for treating subjects who have developed antibodies to cancer.
  • Alkyl refers to a saturated aliphatic hydrocarbon group, including 1-20 carbon atoms, or 1-10 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, or 1-3 carbon atoms , Or a saturated linear or branched monovalent hydrocarbon group with 1-2 carbon atoms, wherein the alkyl group can be independently optionally substituted by one or more substituents described in the present invention.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1 ,1-Dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, N-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2 -Dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-di Methyl butyl and so on.
  • Alkyl groups can be optionally substituted or unsubstituted.
  • Alkenyl refers to a linear or branched monovalent hydrocarbon group of 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, in which at least one CC is sp 2 double bond, where the alkenyl group can be independently optionally substituted by one or more substituents described in the present invention, and specific examples include, but are not limited to, vinyl, allyl and alkene Butyl and so on. Alkenyl groups can be optionally substituted or unsubstituted.
  • Alkynyl refers to a linear or branched monovalent hydrocarbon group of 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, in which at least one CC is sp triple bond, wherein the alkynyl group can be independently optionally substituted by one or more substituents described in the present invention. Specific examples include, but are not limited to, ethynyl, 1-propynyl, 2-propenyl Alkynyl, 1-, 2- or 3-butynyl, etc. Alkynyl groups can be optionally substituted or unsubstituted.
  • Cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring includes 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 Carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl , Cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls. Cycloalkyl groups can be optionally substituted or unsubstituted.
  • “Spirocycloalkyl” refers to a polycyclic group with 5 to 18 members, two or more cyclic structures, and the single rings share one carbon atom (called a spiro atom) with each other, and the ring contains one or more A double bond, but none of the rings have a fully conjugated ⁇ -electron aromatic system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spirocycloalkyl group is divided into mono-spiro, di-spiro or poly-spiro-cycloalkyl, preferably mono-spiro and bi-spiro-cycloalkyl, preferably 4-membered/5-membered, 4-membered Yuan/6 Yuan, 5 Yuan/5 Yuan or 5 Yuan/6 Yuan.
  • spirocycloalkyl include, but are not limited to:
  • “Fused cycloalkyl” refers to a 5- to 18-membered, all-carbon polycyclic group containing two or more cyclic structures sharing a pair of carbon atoms with each other.
  • One or more rings may contain one or more double bonds, However, none of the rings has a fully conjugated ⁇ -electron aromatic system, preferably 6 to 12 members, more preferably 7 to 10 members.
  • it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic condensed cycloalkyls, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic alkyl.
  • Non-limiting examples of "fused cycloalkyl” include, but are not limited to:
  • “Bridged cycloalkyl” refers to a 5- to 18-membered all-carbon polycyclic group containing two or more cyclic structures that share two carbon atoms that are not directly connected to each other.
  • One or more rings may contain one or An aromatic system with multiple double bonds, but none of the rings has a fully conjugated ⁇ electron, preferably 6 to 12 members, more preferably 7 to 10 members.
  • it can be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyls, preferably bicyclic, tricyclic or tetracyclic, and more preferably bicyclic or tricyclic.
  • Non-limiting examples of "bridged cycloalkyl” include, but are not limited to:
  • the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocyclyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, non-limiting examples include indanyl, tetrahydronaphthalene Group, benzocycloheptyl group, etc.
  • Alkylene refers to a divalent alkyl group or a divalent cycloalkyl group (cycloalkylene), wherein the divalent alkyl group is preferably a divalent alkyl group with 1-10 carbon atoms, more preferably 1- A divalent alkyl group of 6 carbon atoms, particularly preferably a divalent alkyl group of 1 to 4 carbon atoms; wherein the cycloalkylene group is preferably a cycloalkylene group of 3 to 12 carbon atoms, more preferably A cycloalkylene group of 3-8 carbon atoms is most preferably a cycloalkylene group of 3-6 carbon atoms.
  • alkylene groups include, but are not limited to, methylene, ethylene, -CH(CH 3 )-, -CH(CH 3 ) 2 -, n-propylene, cyclopropylene, cyclobutylene Group, cyclopentylidene, etc.
  • the alkylene group may be substituted or unsubstituted.
  • Heterocyclyl “heterocyclic” or “heterocyclic” are used interchangeably in this application. They are used interchangeably in this application and refer to a saturated or partially unsaturated monocyclic ring containing 3-12 ring atoms.
  • a bicyclic or tricyclic non-aromatic heterocyclic group in which at least one of the ring atoms is a heteroatom, such as oxygen, nitrogen, and sulfur atoms. It preferably has a 5- to 7-membered monocyclic ring or a 7- to 10-membered bi- or tricyclic ring, which may contain 1, 2 or 3 atoms selected from nitrogen, oxygen and/or sulfur.
  • heterocyclyl examples include, but are not limited to, morpholinyl, oxetanyl, thiomorpholinyl, tetrahydropyranyl, 1,1-dioxo-thiomorpholinyl, piperidine Group, 2-oxo-piperidinyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, piperazin-2-one, 8-oxa-3-aza-bicyclo[3.2.1]octyl and Piperazinyl.
  • the heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group.
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • “Spiroheterocyclic group” refers to a polycyclic group with 5 to 18 members, two or more cyclic structures, and the single rings share one atom with each other.
  • the ring contains one or more double bonds, but no A ring has a fully conjugated ⁇ -electron aromatic system, in which one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) m heteroatoms, and the remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiro heterocyclic group is classified into a single spiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a single spiro heterocyclic group and a dispiro heterocyclic group. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiro heterocyclic group.
  • spiroheterocyclyl include but are not limited to:
  • “Fused heterocyclic group” refers to an all-carbon polycyclic group containing two or more ring structures that share a pair of atoms with each other. One or more rings may contain one or more double bonds, but no ring has a complete A conjugated ⁇ -electron aromatic system, in which one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) m heteroatoms, and the remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups preferably bicyclic or tricyclic, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclyl include, but are not limited to:
  • “Bridge heterocyclic group” refers to a 5- to 18-membered polycyclic group containing two or more cyclic structures that share two atoms that are not directly connected to each other.
  • One or more rings may contain one or more Double bonds, but none of the rings has a fully conjugated ⁇ -electron aromatic system, where one or more ring atoms are selected from nitrogen, oxygen, sulfur or S(O) m heteroatoms, and the remaining ring atoms are carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bridged heterocyclyl include, but are not limited to:
  • Aryl refers to a carbocyclic aromatic system containing one or two rings, wherein the rings can be joined together in a fused manner.
  • aryl includes aromatic groups such as phenyl, naphthyl, and tetrahydronaphthyl.
  • the aryl group is a C 6 -C 10 aryl group, more preferably the aryl group is a phenyl group and a naphthyl group, and most preferably a phenyl group.
  • Aryl groups can be substituted or unsubstituted.
  • the "aryl group” can be fused with a heteroaryl group, a heterocyclic group or a cycloalkyl group, wherein the aryl ring is connected to the parent structure. Non-limiting examples include but are not limited to:
  • Heteroaryl refers to an aromatic 5- to 6-membered monocyclic ring or 9 to 10-membered bicyclic ring, which may contain 1 to 4 atoms selected from nitrogen, oxygen and/or sulfur.
  • 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, benzobis Oxolol, benzimidazolyl, indolyl, isoindolyl, 1,3-dioxo-isoindolyl, quinolin
  • Heteroaryl groups can be optionally substituted or unsubstituted.
  • the heteroaryl ring may be fused on an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring.
  • Non-limiting examples include but are not limited to:
  • Alkoxy refers to (alkyl-O-) groups. Among them, the alkyl group is defined in this article. C 1 -C 6 alkoxy groups are preferred. Examples thereof include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy and the like.
  • Haloalkyl refers to an alkyl group having one or more halogen substituents, wherein the alkyl group has the meaning as described in the present invention.
  • haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, perfluoroethyl, 1,1-dichloroethyl, 1,2-dichloropropyl, and the like.
  • Halogen refers to fluorine, chlorine, bromine and iodine.
  • Amino refers to -NH 2 .
  • Cyano refers to -CN.
  • Niro refers to -NO 2 .
  • Benzyl refers to -CH 2 -phenyl.
  • Carboxy refers to -C(O)OH.
  • Alcohol refers to -C(O)CH 3 or Ac.
  • Tf means trifluoromethanesulfonyl
  • Borate group refers to -B[O(alkyl) or (cycloalkyl)][O(alkyl) or (cycloalkyl)], alternatively, two alkyl groups and the attached O atom can be further A 5- to 6-membered heterocyclic group is formed, and the 5- to 6-membered heterocyclic group is optionally further substituted;
  • Carboxylic acid ester group refers to -C(O)O(alkyl) or (cycloalkyl), wherein the definitions of alkyl and cycloalkyl are as described above.
  • Substituted refers to one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of each other, substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (eg, olefinic) bond.
  • stereochemistry and the usage of conventions in the present invention usually refer to the following documents: SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S. , "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.
  • the compounds of the present invention may contain asymmetric centers or chiral centers, so there are different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to, diastereomers, enantiomers, atropisomers, and their mixtures, such as racemic mixtures, constitute the present invention Part.
  • Diastereomers can be separated into individual diastereomers by methods such as chromatography, crystallization, distillation or sublimation based on their physical and chemical differences.
  • Enantiomers can be separated to convert a chiral isomeric mixture into a diastereomeric mixture by reaction with a suitable optically active compound (for example, a chiral auxiliary, such as a chiral alcohol or Mosher's acid chloride) , Separate diastereomers, and convert individual diastereomers into corresponding pure enantiomers.
  • a suitable optically active compound for example, a chiral auxiliary, such as a chiral alcohol or Mosher's acid chloride
  • the intermediates and compounds of the present invention may also exist in different tautomeric forms, and all such forms are included in the scope of the present invention.
  • optically active compounds that is, they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D, L or R, S are used to indicate the absolute configuration of the molecular chiral center.
  • the prefixes d, l or (+), (-) are used to name the symbols of the plane-polarized light rotation of the compound, (-) or l means that the compound is levorotatory, and the prefix (+) or d means that the compound is dextrorotatory.
  • the atoms or atomic groups of these stereoisomers are connected to each other in the same order, but their stereo structures are different.
  • a specific stereoisomer may be an enantiomer, and a mixture of isomers is usually called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which may result in no stereoselectivity or stereospecificity during chemical reactions.
  • the terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomers, lacking optical activity.
  • Tautomers or “tautomeric forms” mean that isomers of different energy structures can be converted into each other through a low energy barrier.
  • proton tautomers that is, tautomers of proton transfer
  • interconversions by proton migration such as keto-enol and imine-enamine isomerization
  • Valence (valence) tautomers include the interconversion of recombined bond electrons.
  • the structural formula described in the present invention includes all isomeric forms (such as enantiomers, diastereomers, and geometric isomers): for example, R and S configurations containing asymmetric centers, The (Z) and (E) isomers of the double bond, and the (Z) and (E) conformational isomers. Therefore, a single stereochemical isomer of the compound of the present invention or a mixture of its enantiomers, diastereomers, or geometric isomers all belong to the scope of the present invention.
  • the present invention provides a method for preparing a compound of general formula (ID) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, which comprises the following steps:
  • R d is selected from a borate group or a borate ester group; the borate group is preferably:
  • G is a leaving group, preferably selected from halogen or trifluoromethanesulfonyloxy; halogen is more preferably Cl or Br;
  • PG is a protecting group for N, preferably tert-butoxycarbonyl
  • the palladium catalyst is [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride or tetrakis(triphenylphosphorus)palladium;
  • L 1 , L 2 , B, E, X 1 , X 2 , X 3 , X 4 , R 1 and m are as defined in the general formula (I).
  • the present invention provides a method for preparing a compound of general formula (I) or its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, which comprises the following steps:
  • the protective group PG is further removed from the compound of general formula (ID) to obtain compound of general formula (IA); general formula (IA) and E-(alkylene)-OMs, EC(O)X, E-(alkylene) -C(O)X or EC(O)H reaction to obtain general formula (I);
  • PG is a protecting group for N, preferably tert-butoxycarbonyl
  • X is selected from hydroxyl or halogen, preferably hydroxyl, Cl or Br;
  • L 1 , L 2 , B, E, X 1 , X 2 , X 3 , X 4 , R 1 and m are as defined in the general formula (I).
  • the mass spectrum is measured by an LC/MS instrument, and the ionization method can be ESI or APCI.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the size of thin layer chromatography separation and purification products is 0.4mm ⁇ 0.5mm.
  • the argon atmosphere means that the reaction flask is connected to an argon balloon with a volume of about 1L.
  • the compound was purified by silica gel column chromatography and thin layer chromatography, where the eluent or developing solvent system was selected from: A: petroleum ether and ethyl acetate system; B: dichloromethane and methanol system; C: dichloro Methane: ethyl acetate; the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents, such as acetic acid or triethylamine, can also be added for adjustment.
  • A petroleum ether and ethyl acetate system
  • B dichloromethane and methanol system
  • C dichloro Methane: ethyl acetate
  • the volume ratio of the solvent varies according to the polarity of the compound, and a small amount of acidic or alkaline reagents, such as acetic acid or triethylamine, can also be added for adjustment.
  • reaction solution was cooled to room temperature, spin-dried dioxane, 150mL of water was added, extracted with ethyl acetate (50mL ⁇ 3), washed with saturated sodium chloride solution (50mL), dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure , The residue was purified by silica gel column chromatography (eluent: system A) to obtain tert-butyl 5-bromo-3',6'-dihydro-[2,4'-dipyridine]-1'(2 'H)-formate 1c (2.82 g, white solid), yield 54%.
  • reaction solution was cooled to room temperature, spin-dried dioxane, 150mL water was added, extracted with ethyl acetate (40mL ⁇ 3), washed with saturated sodium chloride solution (30mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure , The residue was purified by silica gel column chromatography (eluent: system C) to obtain (1'-(tert-butoxycarbonyl)-1',2',3',6'-tetrahydro-[2, 4'-Dipyridyl]-5-yl)boronic acid 1d (936 mg, light brown solid), the yield was 37%.
  • reaction solution was cooled to room temperature, spin-dried dioxane, added 30 mL of water, extracted with ethyl acetate (20 mL ⁇ 3), washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure
  • the residue was purified by silica gel column chromatography (eluent: system A) to obtain tert-butyl 5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazole And [1,5-a]pyridin-4-yl)-3',6'-dihydro-[2,4'-dipyridine]-1'(2'H)-formate 1f (97mg, shallow Yellow solid), the yield is 61%.
  • tert-butyl 5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)-3',6 '-Dihydro-[2,4'-Dipyridine]-1'(2'H)-formate 1f (650mg, 1.35mmol) was added to a 50mL single-necked flask, and 4N hydrochloric acid/dioxane solution was added (10 mL), the reaction solution was heated to 30°C for 4 hours.
  • reaction solution was concentrated under reduced pressure to obtain 6-(2-hydroxy-2-methylpropoxy)-4-(1',2',3',6 '-Tetrahydro-[2,4'-dipyridine]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile hydrochloride 2f (441mg, light yellow solid), used directly Next step reaction.
  • reaction solution was cooled to room temperature, 20mL of water was added, stirred and filtered, and the filter cake was purified by high performance liquid chromatography to obtain 4-(1'-(3,5-difluorobenzyl)-1',2',3',6 '-Tetrahydro-[2,4'-dipyridin]-5-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-methan Nitrile 7 (50 mg, white solid), yield 50%.
  • 6-Bromo-4-methoxypyrazolo[1,5-a]pyridine-3-carbonitrile 10a (2.52g, 10mmol, prepared by the method disclosed in the patent application "WO2017011776"
  • methyl acrylate (3.2mL, 35mmol)
  • triethylamine (3.75mL, 27mmol)
  • tris(o-methylphenyl)phosphorus (0.51g, 1.67mmol)
  • palladium acetate (0.25g, 1.13mmol
  • nitrogen protection Lower the temperature to reflux temperature and react for 16 hours.
  • reaction solution was diluted with ethyl acetate (100 mL), quenched by adding saturated ammonium chloride solution (30 mL), washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • reaction solution was concentrated under reduced pressure to obtain 4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(3-hydroxy-3- Methylbutyl)pyrazolo[1,5-a]pyridine-3-carbonitrile 10i (40mg, yellow oil), proceed directly to the next step.
  • reaction solution was diluted with ethyl acetate (100 mL), washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • the residue was subjected to silica gel column chromatography (eluent: System B) ) Purification to obtain 6-(3-hydroxy-3-methylbutyl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazepine Heterobicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile 10 (8mg, pale yellow solid), yield 15.4% .
  • Test Example 1 Determination of the compound of the present invention on the proliferation inhibitory activity of RET abnormal tumor cells (TT cells)
  • the following method is used to determine the proliferation inhibitory activity of the preferred compounds of the present invention on TT cells under in vitro conditions.
  • the TT cell is a human medullary thyroid carcinoma cell line, which contains the RET C634W mutation.
  • the cell line was purchased from ATCC (Cat. No. CRL-1803).
  • the experimental method is briefly described as follows: the day before the formal experiment, the cells in the logarithmic growth phase were digested with trypsin (purchased from Thermofisher, Gibco, catalog number 25300054), and then an appropriate volume of medium was added to terminate the trypsin reaction. After centrifugation at 1000 rpm, aspirate the upper layer of medium, and add new medium to prepare a cell suspension. Add 50uL cell suspension to a 96-well cell culture plate (purchased from Corning, catalog number 3904), with a cell density of 6000 cells/well per well. Subsequently, the culture plate was cultured overnight at 37°C and 5% CO 2 .
  • test compound was first dissolved in DMSO (Sigma-Aldrich, Product No. v900090) to prepare a stock solution, and then serially diluted with DMSO at a ratio of 1:3, a total of 9 concentration points, and a DMSO control was set. Then another 96-well culture plate was taken, 250uL serum-free F12 medium was added to each well, and 0.5uL compound solutions of various concentrations were added to each well, shaken and mixed, and prepared into test samples. Then, 50 uL of the test sample solution from each well was added to the cell plate, and the final test concentration of the compound was in the range of 10000 nM to 1.52 nM, and each concentration contained a duplicate well control.
  • DMSO Sigma-Aldrich, Product No. v900090
  • the cell plate was cultured continuously for 8 days at 37°C and 5% CO 2 . After the incubation, add 50uL CellTiter-Glo reagent to each well, shake and mix, and incubate at room temperature for 10 minutes in the dark, and then read the signal of each well on the Envision plate reader (Perkin Elmer). Subsequently, a nonlinear regression analysis was performed in GraphPad Prism software based on the compound concentration logarithm-signal intensity, and the IC 50 value of the test compound for inhibiting cell proliferation was obtained.
  • the inhibitory activity of the compound of the present invention on the proliferation of RET abnormal tumor cells (TT cells) was measured by the above experiment, and the measured IC 50 values are shown in Table 1.
  • Table 1 The compounds of the present invention inhibit the proliferation activity of TT cells with IC 50
  • the compound of the present invention has a significant inhibitory effect on the proliferation of RET abnormal tumor cells (TT cells)
  • Test Example 2 Determination of the inhibitory activity of the compound of the present invention on RET enzyme
  • Steptavidin-XL665 1X enzyme reaction buffer diluted Steptavidin-XL665 to 250uM
  • the compound of the present invention has obvious inhibitory effect on RET enzyme activity.

Abstract

本发明涉及吡唑并[1,5-a]吡啶类衍生物、其制备方法及其在医药上的应用。具体而言，本发明涉及一种通式(I)所示的吡唑并[1,5-a]吡啶类衍生物、其制备方法及其可药用的盐，以及它们作为治疗剂，特别是RET抑制剂的用途，其中通式(I)中的各取代基的定义与说明书中的定义相同。（I）

Description

吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途
本申请要求申请日为2019年3月2日的中国专利申请CN201910157739.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于医药领域，涉及一种吡唑并[1,5-a]吡啶类衍生物、其制备方法、含有该衍生物的药物组合物以及其作为治疗剂特别是作为RET蛋白激酶抑制剂的用途。
背景技术
RET蛋白(Rerranged during Transfection，转染期间重排)属于受体型酪氨酸激酶家族，作为神经生长因子的受体，其功能涉及神经系统的正常生长发育，细胞增殖和存活。胶质细胞衍生的神经营养因子(glial cell line-derived neurotrophic factor,GDNF)家族配体(GFL)通过与RET结合来调节下游信号，GFL家族还包括neurturin(NTRN)、artemin(ARTN)和persephin(PSPN)(Arighi et al.,Cytokine Growth Factor rev.,2005,16,441-67)。与其他受体酪氨酸激酶不同，RET激活需要配体，GDNF家族受体α蛋白以及RET蛋白形成多聚体复合物。GFRα受体通过糖基磷脂酰肌醇键连接到细胞表面的共受体(co-receptor)，现已发现该家族包括4种亚型(GFRα1-4)，能够与不同GFL结合。首先GFL和GFRα家族成员形成二元复合物，复合物再与RET结合并将其募集到细胞膜上被称为脂筏(lipid raft)的胆固醇富集的次级结构域，诱导RET发生二聚化并形成GFL(2)-GFRa(2)-RET(2)异六聚体复合物，激活RET激酶活性以及下游信号。
在配体-共受体复合物结合后，RET发生二聚化并诱使蛋白胞内酪氨酸残基自磷酸化，之后进一步招募其他连接蛋白和信号传导蛋白从而激活多中下游信号传导途径，包括Ras-MAPK通路和PI3K-Akt/mTOR信号通路。
RET异常与肿瘤疾病的发生发展密切相关，主要表现为功能获得性基因突变或因基因重排产生的RET融合蛋白，两者均会引起RET信号的持续异常活化。例如在家族性甲状腺髓样癌患者中，RET点突变比例高达95％；在乳头状甲状腺癌患者中，RET基因重排发生率约为20％～40％；此外，在乳腺癌，结肠癌、胰腺癌、急性白血病中还可观察到RET蛋白过量表达的情况。(Lois M.Mulligan；RET revisited expanding the oncogenic portfolio,Nature Reviews Cancer 14,173-186(2014))。上述存在RET融合蛋白及RET点突变的肿瘤都依赖于RET激酶活化来维持肿瘤细胞的增殖和存活，这种依赖性通常被称为“致癌基因成瘾”(oncogene addiction)，使得这类肿瘤对靶向RET的小分子激酶抑制剂高度敏感。目前已有部分靶向多个激酶的小分子激酶抑制剂具有RET激酶的抑制活性，如凡德替尼(Vandetinib)，卡博替尼(Cabozantinib)已被FDA批准用甲状腺癌的治疗，其他如Ponatinib，Nintedanib，Lenvatinib等也在针对存在RET信号异常活化的肿瘤开展临床研究，但由于缺乏针对RET蛋白的选择 性，上述多重激酶抑制剂在药效和安全性上均存在不同的问题，从而限制了其在RET异常肿瘤中的进一步研究和应用。因此，临床上迫切需要高选择性靶向RET蛋白的小分子激酶抑制化合物。
目前已经公开了一系列的选择性RET激酶抑制剂专利，其中包括WO2017011776、WO2018071447、WO2018136661等，处于临床II期研究的药物主要为LOXO-292。但这些现有技术中公开的化合物以及试验药物在有效性、安全性或选择性等方面仍然存在问题，加之目前对于RET在肿瘤中所扮演的角色的认知也在不断深入，仍有必要研究和开发新的小分子选择性RET激酶抑制剂，以填补和满足临床针对RET异常疾病的治疗需要。
发明内容
本发明人通过实验研究出乎意料地发现，下式(I)的化合物可以有效抑制RET激酶，
Figure PCTCN2020077449-appb-000001
其中：
环A选自基团：
Figure PCTCN2020077449-appb-000002
X 1、X 2各自独立地选自CH、CCH 3、CF、CCl或N；
X 3、X 4各自独立地选自CH、CF或N；
其中X 1、X 2、X 3和X 4中的0、1或2个是N；
L 1选自一个键、-(R aR bC)-、-(R aR bN)-或-O-；
L 2选自一个键、-(亚烷基)-、-C(O)-、-S(O)-、-SO 2-、-(亚烷基)-O-、-(亚烷基)-S-、-(亚烷基)-NR 1-、-C(O)-(亚烷基)-、-SO 2-(亚烷基)-；其中L 2基团中右键与E相连；其中所述的亚烷基任选进一步被一个或多个选自羟基、卤素、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；
R a选自氢原子、烷基或卤素；
R b选自氢原子、烷基或卤素；
或者，R a和R b与其相连接的C或N原子一起形成3～6元环烷基或杂环基，其中杂环基内含有一个或多个N、O、S(O) n原子，其中所述的环烷基或杂环基任选进一步被一个或多个选自羟基、卤素、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；
B选自氢原子、烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自R c的取代基所取代；
E选自氢原子、烷氧基、环烷基、杂环基、芳基或杂芳基；其中所述的环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；优选为苯基或吡啶基；
R c选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-S(O) 2NR 2R 3，其中所述的烷基、烷氧基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
R 1相同或不同，各自独立地选自氢原子、羟基、烷基、卤素或烷氧基，其中所述的烷基或烷氧基任选进一步被一个或多个选自羟基、卤素或烷氧基的取代基所取代；
R 2、R 3和R 4各自独立地选自氢原子、羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、烷氧基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
或者，R 2和R 3与相连接的N原子一起形成一个4～8元杂环基，其中4～8元杂环内含有一个或多个N、O、S(O) n原子，并且4～8元杂环上任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、＝O、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
R 5、R 6和R 7各自独立地选自氢原子、烷基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基或羧酸酯基的取代基所取代；
m选自0，1，2，3，4或5；
n选自0，1或2；
条件是，当环A选自基团：
Figure PCTCN2020077449-appb-000003
时，
(1)L 1选自-(R aR bC)-，-(R aR bN)-或-S-；
(2)L 1选自一个健时，B选自烯基或炔基，其中烯基或炔基任选进一步被一个或多个选自R c的取代基所取代。
本发明的优选方案，一种通式(I)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(II)所示的化合物或其立体异构体、互变异构体或其可药用的盐：
Figure PCTCN2020077449-appb-000004
其中：
L 1选自一个键、-(R aR bC)-、-(R aR bN)-或-O-；优选为-O-；
L 2、B、E、X 1、X 2、X 3、X 4、R 1、R a、R b和m的定义如通式(I)所述。
本发明的优选方案，一种通式(I)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐：
Figure PCTCN2020077449-appb-000005
其中：
(1)L 1选自-(R aR bC)-、-(R aN)-或-S-；
优选地，R a和R b选自氢原子或甲基；
(2)当L 1选自一个键时，B选自烯基或炔基，其中烯基或炔基任选进一步被一个或多个选自R c的取代基所取代；
L 2、B、E、X 1、X 2、X 3、X 4、R 1、R a、R b、R c和m的定义如通式(I)中所述。
本发明的优选方案，一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中：
X 1，X 2，X 3，X 4选自CH或N；
X 1，X 2，X 3和X 4中的0、1或2个是N；
优选为，X 1为N，X 2，X 3，X 4为CH。
本发明的优选方案，一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中：
L 2选自一个键、-(亚烷基)-、-C(O)-、-SO 2-、-(亚烷基)-O-、-(亚烷基)-S-、-(亚烷基)-NR 1-或-C(O)-(亚烷基)-，其中L 2基团中右键与E相连；
优选为-(亚烷基)-、-C(O)-、-(亚烷基)-S-或-C(O)-(亚烷基)-；
其中所述的-(亚烷基)-优选为-(亚甲基)-或-CH(CH 3)-。
本发明的优选方案，一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，其中B选自：
(i)5～6元杂芳基，其中所述的杂芳基任选进一步被一个或多个选自烷基或卤素的取代基所取代；
优选地，B选自吡唑基，其中所述的吡唑基任选进一步被甲基取代；
(ii)烷基，其中所述的烷基任选进一步被一个或多个羟基所取代；优选地，
B选自基团：
Figure PCTCN2020077449-appb-000006
(iii)烯基或炔基，其中所述的烯基或炔基任选进一步被一个或多个羟基所取代；
优选地，当L 1为一个键时，B选自基团：
Figure PCTCN2020077449-appb-000007
本发明的典型化合物包括但不限于：
Figure PCTCN2020077449-appb-000008
Figure PCTCN2020077449-appb-000009
Figure PCTCN2020077449-appb-000010
上述典型化合物包括其立体异构体、互变异构体或其可药用的盐。
进一步，本发明提供一种通式(I)所示的化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，所述方法包括：
Figure PCTCN2020077449-appb-000011
将通式(IA)与E-(亚烷基)-OMs、E-C(O)X、E-(亚烷基)-C(O)X或E-C(O)H反应，得到通式(I)；
X选自羟基或卤素，优选为羟基、Cl或Br；
其中：L 1、L 2、B、E、X 1、X 2、X 3、X 4、R 1和m的定义如通式(I)中所述。
本发明提供一种通式(IA)所述的化合物或其立体异构体、互变异构体或其可药用的盐，
Figure PCTCN2020077449-appb-000012
其中：L 1、B、X 1、X 2、X 3、X 4、R 1和m的定义如通式(I)中所述。
通式(IA)的典型化合物包括但不限于：
Figure PCTCN2020077449-appb-000013
Figure PCTCN2020077449-appb-000014
上述典型化合物包括其立体异构体、互变异构体、游离态或其他可药用的盐。
进一步，本发明一种药物组合物，所述的药物组合物含有有效剂量的通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，及可药用的载体、赋形剂或它们的组合。
本发明提供一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，以及它们的药物组合物在制备RET或突变的RET抑制剂的药物中的用途。
本发明提供一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，以及它们的药物组合物在制备用于治疗与RET异常介导的疾病的药物中的用途，其中所述的疾病优选为癌症、纤维发育不良和遗传障碍，其中所述的癌症优选为非小细胞肺癌，甲状腺癌，实体瘤，结肠肿瘤或胰腺癌，更优选为非小细胞肺癌，甲状腺癌，实体瘤。
本发明提供一种通式(I)、(II)或(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐，以及它们的药物组合物在制备用于治疗对癌症产生抗体的受试者的药物中的用途。
发明的详细说明
除非有相反陈述，否则本发明在说明书和权利要求书中所使用的部分术语定义如下：
“烷基”指饱和脂肪族烃基团，包括1-20个碳原子，或1-10个碳原子，或1-6个碳原子，或1-4个碳原子，或1-3个碳原子，或1-2个碳原子饱和直链或支链的单价烃基，其中烷基可以独立任选地被一个或多个本发明所描述地取代基所取代。烷基基团更近一步地实例包括，包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是任选取代或未取代的。
“烯基”指2-12个碳原子，或2-8个碳原子，或2-6个碳原子，或2-4个碳原子直链或支链的一价烃基，其中至少一个C-C为sp 2双键，其中烯基的基团可以独立任选地被1个或多个本发明所描述的取代基所取代，其中具体的实例包括，但并不限于乙烯基、烯丙基和烯丁基等等。烯基可以是任选取代或未取代的。
“炔基”指2-12个碳原子，或2-8个碳原子，或2-6个碳原子，或2-4个碳原子直链或支链的一价烃基，其中至少一个C-C为sp三键，其中炔基基团可以独立任选地被一个或多个本发明所描述的取代基所取代，具体的实例包括，但并不限于乙炔基、1-丙炔基、2-丙炔基、1-、2-或3-丁炔基等。炔基可以是任选取代或未取代的。
“环烷基”是指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包括3至20个碳原子，优选包括3至12个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性实施例包括，但不限于环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。环烷基可以是任选取代的或未取代的。
“螺环烷基”指5至18元，两个或两个以上环状结构，且单环之间彼此共用一个碳原子(称螺原子)的多环基团，环内含有1个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺、双螺或多螺环烷基，优选为单螺和双螺环烷基，优选为4元/5元、4元/6元、5元/5元或5元/6元。“螺环烷基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000015
“稠环烷基”指5至18元，含有两个或两个以上环状结构彼此公用一对碳原子的全碳多环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统，优选为6至12元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。“稠环烷基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000016
“桥环烷基”指5至18元，含有两个或两个以上环状结构，彼此共用两个不直接相连接碳原子的全碳多环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统，优选为6至12元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更有选为双环或三环。“桥环烷基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000017
所述环烷基环可以稠合于芳基、杂芳基或杂环基环上，其中与母体结构连接在一起的环为环烷基，非限制性实施例包括茚满基、四氢萘基、苯并环庚烷基等。
“亚烷基”是指二价烷基或二价环烷基(亚环烷基)，其中所述的二价烷基优选为1-10碳原子的二价烷基,更优选为1-6个碳原子的二价烷基，特别优选为1-4个碳原子的二价烷基；其中所述的亚环烷基优选为3-12个碳原子的亚环烷基，更优选为3-8个碳原子亚环烷基，最优选为3-6个碳原子的亚环烷基。亚烷基基团的实施例包括但不限于亚甲基、亚乙基、-CH(CH 3)-、-CH(CH 3) 2-、亚正丙基、亚环丙基、亚环丁基、亚环戊基等。亚烷基可以是取代或未取代的。
“杂环基”、“杂环”或“杂环的”在本申请中可交换使用，本申请中可交换使用，都是指包含3-12个环原子的饱和或部分不饱和的单环、双环或三环的非芳香性杂环基，其中至少一个环原子原子是杂原子，如氧、氮、硫原子等。优选具有5至7元单环或7至10元双-或三环，其可以包含1，2或3个选自氮、氧和/或硫中的原子。“杂环基”的实例包括但不限于吗啉基，氧杂环丁烷基，硫代吗啉基，四氢吡喃基，1，1-二氧代-硫代吗啉基，哌啶基，2-氧代-哌啶基，吡咯烷基，2-氧代-吡咯烷基，哌嗪-2-酮，8-氧杂-3-氮杂-双环[3.2.1]辛基和哌嗪基。所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基。杂环基可以是任选取代的或未取代的。
“螺杂环基”指5至18元，两个或两个以上环状结构，且单环之间彼此共用一个原子的多环基团，环内含有1个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统，其中一个或多个环原子选自氮、氧、硫或S(O) m的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。“螺杂环基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000018
“稠杂环基”指含有两个或两个以上环状结构彼此公用一对原子的全碳多环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统，，其中一个或多个环原子选自氮、氧、硫或S(O) m的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。“稠杂环基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000019
“桥杂环基”指5至18元，含有两个或两个以上环状结构，彼此共用两个不直接相连接的原子的多环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子的芳香系统，其中一个或多个环原子选自氮、氧、硫或S(O) m的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更有选为双环或三环。“桥杂环基”的非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000020
“芳基”是指含有一个或者两个环的碳环芳香系统，其中所述环可以以稠合的方式连接在一起。术语“芳基”包括比如苯基、萘基、四氢萘基的芳香基团。优选芳基为C 6-C 10芳基，更优选芳基为苯基和萘基，最优选为苯基。芳基可以是取代或未取代的。所述“芳基”可与杂芳基、杂环基或环烷基稠合，其中与母体结构连接在一起的为芳基环，非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000021
“杂芳基”是指芳香族5至6元单环或9至10元双环，其可以包含1至4个选自氮、氧和/或硫中的原子。“杂芳基”的实施例包括但不限于呋喃基，吡啶基，2-氧代-1，2-二氢吡啶基、哒嗪基、嘧啶基、吡嗪基、噻吩基、异噁唑基、噁唑基、噁二唑基、咪唑基、吡咯基、吡唑基、三唑基、四唑基、噻唑基、异噻唑基、1，2，3-噻二唑基、苯并间二氧杂环戊烯基、苯并咪唑基、吲哚基、异吲哚基、1，3-二氧代-异吲哚基、喹啉基、吲唑基、苯并异噻唑基、苯并噁唑基和苯并异噁唑基。杂芳基可以是任选取代或未取代的。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，非限制性实施例包括但不限于：
Figure PCTCN2020077449-appb-000022
“烷氧基”是指(烷基-O-)的基团。其中，烷基见本文有关定义。C 1-C  6的烷氧基为优先选择。其实例包括，但不限于：甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基等。
“卤代烷基”指具有一个或者多个卤素取代基的烷基，其中烷基基团具有如本发明所述的含义。卤代烷基的实例包括，但并不限于氟甲基、二氟甲基、三氟甲基、全氟乙基、1,1-二氯乙基、1,2-二氯丙基等。
“羟基”指-OH基团。
“卤素”是指氟、氯、溴和碘。
“氨基”指-NH 2。
“氰基”指-CN。
“硝基”指-NO 2。
“苄基”指-CH 2-苯基。
“羧基”指-C(O)OH。
“乙酰基”指-C(O)CH 3或Ac。
“Tf”指三氟甲磺酰基；
“Ms”指甲磺酰基；
“硼酸基”指-B(OH) 2；
“硼酸酯基”指-B[O(烷基)或(环烷基)][O(烷基)或(环烷基)]，或者，两个烷基与相连接的O原子可以进一步形成5～6元杂环基，5～6元杂环基任选进一步被取代；
“羧酸酯基”指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基的定义如上所述。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
本说明书所述的“取代”或“取代的”，如无特别指出，均是指基团可被一个或多个选自以下的基团取代：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、疏基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氨基、卤代烷基、羟烷基、羧基、羧酸酯基、＝O、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3，其中，R 2、R 3和R 4的定义如通式(I)中所述。
本发明中立体化学的定义和惯例的使用通常参考以下文献：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.本发明的化合物可以包含不对称中心或手性中心，因此存在不同的立体异构体。本发明的化合物所有的立体异构形式，包括但绝不限于，非对映体，对映异构体，阻转异构体，和它们的混合物，如外消旋混合物，组成了本发明的一部分。非对映异构体可以以其物理化学差异为基础，通过层析、结晶、蒸馏或升华等方法被分离为个别非对映异构体。对映异构体可以通过分离，使手性异构混合物转化为非对映异构混合物，其方式是与适当光学活性化合物(例如手性辅助剂，譬如手性醇或Mosher氏酰氯)的反应，分离非对映异构体，且使个别非对映异构体转化为相应的纯对映异构体。本发明的中间体与化合物也可以不同互变异构形式存在，且所有此种形式被包含在本发明的范围内。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀d、l或(+)、(-)用来命名化合物平面偏振光旋转的符号，(-)或l是指化合物是左旋的，前缀(+)或d是指化合物是右旋的。这些立体异构体的原子或原子团互相连接次序相同，但是它们的立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50：50的对映体混合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。
“互变异构体”或“互变异构的形式”是指不同能量的结构的同分异构体可以通过低能垒互相转化。例如质子互变异构体(即质子移变的互变异构体)包括通过质子迁移的互变，如酮式-烯醇式和亚胺-烯胺的同分异构化作用。原子价(化合价)互变异构体包括重组成键电 子的互变。除非其他方面表明，本发明所描述的结构式包括所有的同分异构形式(如对映异构，非对映异构，和几何异构)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体，和(Z)、(E)的构象异构体。因此，本发明的化合物的单个立体化学异构体或其对映异构体，非对映异构体，或几何异构体的混合物都属于本发明的范围。
本发明化合物的合成方法
为了完成本发明的目的，本发明采用如下技术方案：
本发明提供一种通式(ID)化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2020077449-appb-000023
通式(IB)化合物和通式(IC)化合物，在钯催化剂存在的条件下，发生偶联反应，得到通式(ID)化合物；
其中：
R d选自硼酸基或硼酸酯基；所述的硼酸酯基优选为：
Figure PCTCN2020077449-appb-000024
G为离去基团，优选选自卤素或三氟甲磺酰氧基；卤素更优选为Cl或Br；
PG为N的保护基，优选为叔丁氧基羰基；
所述钯催化剂为[1,1'-双(二苯基膦基)二茂铁]二氯化钯或四(三苯基磷)钯；
L 1、L 2、B、E、X 1、X 2、X 3、X 4、R 1和m如权通式(I)中所定义。
本发明提供一种通式(I)化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，包括以下步骤：
Figure PCTCN2020077449-appb-000025
通式(ID)化合物进一步脱去保护基PG，得到通式(IA)化合物；通式(IA)与E-(亚烷基)-OMs、E-C(O)X、E-(亚烷基)-C(O)X或E-C(O)H反应，得到通式(I)；
其中：
PG为N的保护基，优选为叔丁氧基羰基；
X选自羟基或卤素，优选为羟基、Cl或Br；
L 1、L 2、B、E、X 1、X 2、X 3、X 4、R 1和m如权通式(I)中所定义。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。
实施例
实施例给出了式(I)所表示的代表性化合物的制备及相关结构鉴定数据。必须说明，下述实施例是用于说明本发明而不是对本发明的限制。 1H NMR图谱是用Bruker仪器(400MHz)测定而得，化学位移用ppm表示。使用四甲基硅烷内标准(0.00ppm)。1H NMR的表示方法：s＝单峰，d＝双重峰，t＝三重峰，q＝四重峰，m＝多重峰，br＝变宽的，dd＝双重峰的双重峰，dt＝三重峰的双重峰。若提供偶合常数时，其单位为Hz。
质谱是用LC/MS仪测定得到，离子化方式可为ESI或APCI。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶200～300目硅胶为载体。
在下列实例中，除非另有指明，所有温度为摄氏温度，除非另有指明，各种起始原料和试剂来自市售或者是根据已知的方法合成，市售原料和试剂均不经进一步纯化直接使用，除非另有指明，市售厂家包括但不限于Aldrich Chemical Company，ABCR GmbH & Co.KG，Acros Organics，广赞化工科技有限公司和景颜化工科技有限公司等。
CD 3OD：氘代甲醇
CDCl 3：氘代氯仿
DMSO-d 6：氘代二甲基亚砜
氩气氛是指反应瓶连接一个约1L容积的氩气气球。
实施例中无特殊说明，反应中的溶液是指水溶液。
对化合物进行纯化，采用硅胶柱层析和薄层色谱法，其中洗脱剂或展开剂体系选自：A：石油醚和乙酸乙酯体系；B：二氯甲烷和甲醇体系；C：二氯甲烷：乙酸乙酯；其中溶剂的体积比根据化合物的极性不同而不同，也可以加入少量的酸性或碱性试剂进行调节，如醋酸或三乙胺等。
实施例1
4-(1'-((6-methoxypyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-3-甲腈1
Figure PCTCN2020077449-appb-000026
第一步
tert-butyl 5-bromo-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-carboxylate叔丁基5-溴-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1c
将叔丁基4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-3,6-二氢吡啶-1(2H)-甲酸酯1a(5g,16.18mmol，采用专利申请“WO2003092686”公开的方法制备而得)加入250mL的三口瓶中，依次加入5-溴-2-碘吡啶1b(4.58g,16.18mmol)，二氧六环(162mL)，水(20mL)，碳酸钠(5.15g,48.54mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(1.18g,1.62mmol)，反应液升温至80℃反应4小时。反应液冷却至室温，旋干二氧六环，加入150mL水，用乙酸乙酯(50mL×3)萃取，用饱和氯化钠溶液(50mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶柱层析法(洗脱剂：A体系)纯化，得到叔丁基5-溴-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1c(2.82g，白色固体)，产率54％。
MS m/z(ESI):339.1[M+1]
第二步
(1'-(tert-butoxycarbonyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)boronic acid(1'-(叔丁氧基羰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)硼酸1d
将叔丁基5-溴-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1c(2.82g,8.3mmol)加入250mL的三口瓶中，依次加入联硼酸频那醇酯(2.54g,10mmol)，100mL二氧六环，15mL水，醋酸钾(2.44g,24.9mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(607mg,0.83mmol)，反应液升温至100℃反应7小时。反应液冷却至室温，旋干二氧六环，加入150mL水，用乙酸乙酯(40mL×3)萃取，用饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶柱层析法(洗脱剂：C体系)纯化，得到(1'-(叔丁氧基羰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)硼酸1d(936mg，浅褐色色固体)，产率37％。
MS m/z(ESI)：305.1[M+1]
第三步
tert-butyl 5-(3-cyano-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-4-yl)-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-carboxylate
叔丁基5-(3-氰基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1f
将(1'-(叔丁氧基羰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)硼酸1d(100mg,0.33mmol)加入50mL的三口瓶中，依次加入3-氰基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-4-基三氟甲磺酸酯1e(146mg,0.39mmol,采用专利申请“WO2017011776”公开的方法制备而得)，5mL二氧六环，15mL水，碳酸钠(70mg,0.66mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(24mg,0.03mmol)，反应液升温至100℃反应6小时。反应液冷却至室温，旋干二氧六环，加入30mL水，用乙酸乙酯(20mL×3)萃取，用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶柱层析法(洗脱剂：A体系)纯化，得到叔丁基5-(3-氰基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1f(97mg，浅黄色固体)，产率61％。
MS m/z(ESI)：481.6[M+1]
第四步
6-(1-methyl-1H-pyrazol-4-yl)-4-(1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(1-甲基-1H-吡唑-4-基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈1g
将叔丁基5-(3-氰基-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯1f(650mg,1.35mmol)加入50mL的单口瓶中，加入4N的盐酸/二氧六环溶液(10mL)，反应液升温至30℃反应4小时。反应液减压浓缩，得到6-(1-甲基-1H-吡唑-4-基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈1g(514.8mg，黄色固体)，直接用于下步反应。
MS m/z(ESI):381.4[M+1]
第五步
4-(1'-((6-methoxypyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-3-甲腈1
将6-(1-甲基-1H-吡唑-4-基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈1g(100mg,0.26mmol)加入25mL的单口瓶中，依次加入5mL N,N-二甲基甲酰胺，N,N-二异丙基乙胺(339mg,2.62mmol)，(6-甲氧基吡啶-3-基)甲基甲磺酸酯1h(171mg,0.79mmol,采用专利申请“WO2014066659”公开的方法制备而得)，反应液升温至70℃反应6小时。反应液冷却至室温，加入50mL乙酸乙酯，依次用水(20mL×3)，饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，残留物用薄层色谱法(洗脱剂：B体系)纯化，得到4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(1-甲基-1H-吡唑-4-基)吡唑并[1,5-a]吡啶-3-甲腈1(34mg，浅黄色固体)，产率25％。
MS m/z(ESI):503.3[M+1]
1H NMR(400MHz,DMSO):δ9.32(d,J＝1.4Hz,1H),8.79(d,J＝1.9Hz,1H),8.68(s,1H),8.41(s,1H),8.14(s,1H),8.11(s,1H),8.07-8.03(m,1H),7.92(d,J＝1.4Hz,1H),7.70(d,J＝8.2Hz,2H),6.85-6.79(m,2H),3.88(s,3H),3.85(s,3H),3.58(s,2H),3.17(d,J＝5.3Hz,2H),2.70-2.61(m,4H).
实施例2
6-(2-hydroxy-2-methylpropoxy)-4-(1'-((6-methoxypyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈2
Figure PCTCN2020077449-appb-000027
第一步
4-(6-chloropyridin-3-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile4-(6-氯吡啶-3-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈2c
将4-溴-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈2a(1.6g,5.12mmol，采用专利申请“WO2018071447”公开的方法制备而得)加入100mL的三口瓶中，依次加入35mL N,N-二甲基甲酰胺，2-氯吡啶-5-硼酸频哪醇酯2b(1.6g,6.7mmol)，磷酸钾(5.7g,27.1mmol)和四(三苯基磷)钯(0.295g,0.26mmol)，反应液升温至95℃反应16小时。反应液冷却至室温，加入70mL水，用二氯甲烷和甲醇的混合液(V:V＝10:1,100mL×3)萃取，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶柱层析法(洗脱剂：A体系)纯化，得到4-(6-氯吡啶-3-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈2c(1.26g，黄色固体)，产率72％。
MS m/z(ESI)：343.0[M+1]
第二步
tert-butyl 5-(3-cyano-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridin-4-yl)-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-carboxylate
叔丁基5-(3-氰基-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯2e
25mL微波管中，依次加入12mL 1,4-二氧六环，3mL水，4-(6-氯吡啶-3-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈2c(0.5g,1.46mmol)，叔丁基4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-3,6-二氢吡啶-1(2H)-甲酸甲酯2d(0.74g,2.39mmol)，碳酸钠(0.419g,3.9mmol)和四(三苯基磷)钯(0.2mg,0.13mmol)，反应液升温至110℃，微波反应1.5小时。反应液冷却至室温，加入30mL水，用乙酸乙酯(30mL×3)萃取，无水硫酸钠干燥，过滤，减压浓缩，残留物用硅胶柱层析法(洗脱剂：A体系)纯化，得到叔丁基5-(3-氰基-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯2e(460mg，黄色固体)，产率62.6％。
MS m/z(ESI)：490.0[M+1]
第三步
6-(2-hydroxy-2-methylpropoxy)-4-(1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile hydrogen chloride
6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f
将(叔丁基5-(3-氰基-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-4-基)-3',6'-二氢-[2,4'-二吡啶]-1'(2'H)-甲酸酯2e(460mg,0.94mmol)加入100mL的三口瓶中，依次加入30mL二氯甲烷和10mL 4N的盐酸/二氧六环溶液，反应液室温反应4小时。反应液减压浓缩，得到6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(441mg，浅黄色固体)，直接用于下步反应。
MS m/z(ESI)：390.0[M+1]
第四步
6-(2-hydroxy-2-methylpropoxy)-4-(1'-((6-methoxypyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈2
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(120mg,0.31mmol)加入10mL的三口瓶中，依次加入3mL乙腈，碳酸铯(200mg,0.61mmol)，5-(氯甲基)-2-甲氧基吡啶2g(148mg,0.86mmol,采用专利申请“WO2016033445”公开的方法制备而得)，反应液升温至75℃反应1.5小时。反应液冷却至室温，加入100mL水，搅拌过滤，滤饼用乙酸乙酯(30mL)溶解，无水硫酸钠干燥，过滤，减压浓缩，残留物用乙腈重结晶，得到6-(2-羟基-2-甲基丙氧基)-4-(1'-((6-甲氧基吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈2(50mg，灰色固体)，产率33.3％。
MS m/z(ESI)：511.2[M+1]
1H NMR(400MHz,DMSO):δ12.24-12.19(m,1H),8.75(dd,J＝8.2,2.1Hz,2H),8.60(s,1H),8.11(d,J＝2.0Hz,1H),7.99(dt,J＝15.9,7.9Hz,1H),7.73-7.63(m,2H),7.44(d,J＝2.0Hz,1H),6.81(t,J＝5.7Hz,2H),4.72(s,1H),3.89(s,2H),3.85(s,3H),3.57(s,2H),3.15(d,J＝2.4Hz,2H),2.67(d,J＝4.9Hz,2H),2.62(s,2H),1.23(s,6H).
实施例3
6-(2-hydroxy-2-methylpropoxy)-4-(1'-((5-methoxypyridin-2-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-((5-甲氧基吡啶-2-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈
Figure PCTCN2020077449-appb-000028
第一步
6-(2-hydroxy-2-methylpropoxy)-4-(1'-((5-methoxypyridin-2-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-((5-甲氧基吡啶-2-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈3
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(78mg,0.175mmol)加入10mL的三口瓶中，依次加入3mL乙腈，三乙胺(202mg,2mmol)，(5-甲氧基吡啶-2-基)甲基甲磺酸酯3a(176mg,0.8mmol,采用专利申请“WO2018130838”公开的方法制备而得)，反应液升温至70℃反应2小时。反应液冷却至室温，加入100mL水，搅拌过滤，收集滤饼，用高效液相色谱法纯化得到6-(2-羟基-2-甲基丙氧基)-4-(1'-((5-甲氧基吡啶-2-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈3(36mg，黄色固体)，产率36％。
MS m/z(ESI)：511.3[M+1]
1H NMR(400MHz,DMSO):δ8.80(dd,J＝9.5,2.2Hz,2H),8.61(s,1H),8.42(d,J＝2.3Hz,1H),8.09(dt,J＝8.8,4.4Hz,1H),7.80(d,J＝8.2Hz,1H),7.57(dt,J＝8.6,5.5Hz,2H),7.50-7.43(m,1H),6.85(s,1H),4.54(s,2H),4.00(s,2H),3.90(d,J＝2.3Hz,2H),3.89(s,3H),3.54(s,2H), 2.95(s,2H),1.23(s,6H).
实施例4
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(4-methoxybenzyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(4-甲氧基苄基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈
Figure PCTCN2020077449-appb-000029
第一步
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(4-methoxybenzyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(4-甲氧基苄基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈4
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(78mg,0.175mmol)加入10mL的三口瓶中，依次加入3mL乙腈，三乙胺(121mg,1.2mmol)，4-甲氧基苄基甲磺酸酯4a(250mg,1mmol,采用专利申请“US6194406”公开的方法制备而得)，反应液升温至70℃反应3小时。反应液冷却至室温，加入20mL水，用二氯甲烷和甲醇的混合液(V:V＝10:1，15mL×3)萃取，减压浓缩，残余物用高效液相色谱法纯化得到6-(2-羟基-2-甲基丙氧基)-4-(1'-(4-甲氧基苯基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈4(10mg，黄色固体)，产率10％。
MS m/z(ESI)：510.3[M+1]
1H NMR(400MHz,DMSO):δ12.24-12.18(m,1H),8.77(dd,J＝8.2,2.1Hz,2H),8.65(s,1H),8.08(d,J＝2.0Hz,1H),8.0(dt,J＝15.9,7.9Hz,1H),7.78-7.62(m,4H),7.44-7.21(m,3H),6.83(t,J＝5.7Hz,2H),4.71(s,1H),3.89(s,2H),3.84(s,3H),3.58(s,2H),3.14(d,J＝2.4Hz,2H),2.77(d,J＝4.9Hz,2H),2.69(s,2H),1.25(s,6H).
实施例5
4-(1'-((5-fluoropyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-((5-氟吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈5
Figure PCTCN2020077449-appb-000030
第一步
4-(1'-((5-fluoropyridin-3-yl)methyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-((5-氟吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈5
将(5-氟吡啶-3-基)甲基甲磺酸酯5a(150mg，0.73mmol，采用专利申请“US20080153883”公开的方法制备而得)溶于5mL乙腈中，加入6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(95mg，0.24mmol)，三乙胺(73mg，0.72mmol)，升温至70℃反应2小时。TLC检测反应完全，加入5mL水淬灭，用乙酸乙酯(5mL×3)萃取，用饱和氯化钠溶液(5mL)洗涤，无水硫酸钠干燥，过滤，减压浓缩，用高效液相色谱 法纯化得到4-(1'-((5-氟吡啶-3-基)甲基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈5(10mg，红色固体)，产率2.7％。
MS m/z(ESI):498.8[M+1]
1H NMR(400MHz,DMSO):δ8.81(d,J＝2.0Hz,1H),8.78(d,J＝2.0Hz,1H),8.73(d,J＝2.8Hz,1H),8.66(s,1H),8.61(s,1H),8.10(dd,J＝2.4,8.0Hz,1H),7.99(ddd,J＝2.4,1.6,9.6Hz,1H),7.81(d,J＝8.4Hz,1H),7.46(d,J＝2.4Hz,1H),6.85(s,1H),4.58(s,2H),3.98(s,2H),3.90(s,2H),3.73(s,1H),3.30(s,1H),3.00(t,2H),1.23(s,6H).
实施例6
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(2-(phenylthio)ethyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-(苯硫基)乙基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈6
Figure PCTCN2020077449-appb-000031
第一步
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(2-(phenylthio)ethyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-(苯硫基)乙基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈6
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(78mg,0.175mmol)加入10mL的三口瓶中，依次加入3mL乙腈，三乙胺(121mg,1.2mmol)，2-(苯硫基)乙基甲磺酸酯6a(93mg,0.4mmol,采用专利申请“JP2017137276”公开 的方法制备而得)，反应液升温至70℃反应5小时。反应液冷却至室温，加入20mL水，搅拌过滤，滤饼用高效液相色谱法纯化得到6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-(苯硫基)乙基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈6(30mg，白色固体)，产率30％。MS m/z(ESI)：526.3[M+1]
1H NMR(400MHz,DMSO):δ8.75(dd,J＝6.4,2.0Hz,2H),8.60(s,1H),8.01(dd,J＝8.2,2.3Hz,1H),7.70(d,J＝8.3Hz,1H),7.45(d,J＝2.0Hz,1H),7.34(dt,J＝15.3,7.6Hz,4H),7.19(t,J＝7.0Hz,1H),6.83(s,1H),4.71(s,1H),3.90(s,2H),3.25(s,2H),3.22-3.15(m,2H),2.70(d,J＝6.5Hz,4H),2.62(s,2H),1.23(s,6H).
实施例7
4-(1'-(3,5-difluorobenzyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-(3,5-二氟苄基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈7
Figure PCTCN2020077449-appb-000032
第一步
4-(1'-(3,5-difluorobenzyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)-6-(2-hydroxy-2-methylpropoxy)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(1'-(3,5-二氟苄基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈7
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(78mg,0.175mmol)加入10mL的三口瓶中，依次加入3mL乙腈，三乙胺(121mg, 1.2mmol)，3,5-二氟苄基甲磺酸酯7a(88mg,0.4mmol,采用专利申请“WO2012092442”公开的方法制备而得)，反应液升温至70℃反应5小时。反应液冷却至室温，加入20mL水，搅拌过滤，滤饼用高效液相色谱法纯化得到4-(1'-(3,5-二氟苄基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)-6-(2-羟基-2-甲基丙氧基)吡唑并[1,5-a]吡啶-3-甲腈7(50mg，白色固体)，产率50％。
MS m/z(ESI)：516.3[M+1]
1H NMR(400MHz,DMSO):δ8.80(dd,J＝10.8,2.1Hz,2H),8.61(s,1H),8.10(dd,J＝8.3,2.3Hz,1H),7.81(d,J＝8.2Hz,1H),7.47(d,J＝2.1Hz,1H),7.46-7.39(m,1H),7.37(d,J＝6.1Hz,2H),6.84(s,1H),4.50(s,2H),3.95(s,2H),3.90(s,2H),3.57(dd,J＝122.6,40.4Hz,2H),2.95(d,J＝49.9Hz,2H),1.23(s,6H)
实施例8
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(6-methoxynicotinoyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(6-甲氧基烟酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈8
Figure PCTCN2020077449-appb-000033
第一步
6-(2-hydroxy-2-methylpropoxy)-4-(1'-(6-methoxynicotinoyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(2-羟基-2-甲基丙氧基)-4-(1'-(6-甲氧基烟酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈8
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(39mg,0.087mmol)加入10mL的三口瓶中，依次加入2mL N,N-二甲基甲酰胺，6-甲氧基烟酸8a(30mg,0.196mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(38mg，0.1mmol)，N,N-二异丙基乙胺(64.5mg，0.5mmol)，室温搅拌16小时，加入30mL水，搅拌过滤，滤饼用高效液相色谱法纯化得到6-(2-羟基-2-甲基丙氧基)-4-(1'-(6-甲氧基烟酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈8(21mg，白色固体)，产率40％。MS m/z(ESI)：522.2[M+1]
1H NMR(400MHz,DMSO):δ8.80-8.75(m,2H),8.61(s,1H),8.35(d,J＝2.0Hz,1H),8.06(dd,J＝8.3,2.3Hz,1H),7.85(dd,J＝8.5,2.4Hz,1H),7.74(d,J＝8.1Hz,1H),7.46(d,J＝2.0Hz,1H),6.92(s,1H),6.90(s,1H),4.32(s,2H),3.91(s,3H),3.90(s,2H),3.69(d,J＝28.5Hz,2H),2.74(s,2H),1.23(s,6H).
实施例9
(R)-6-(2-hydroxy-2-methylpropoxy)-4-(1'-(2-phenylpropanoyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin]-5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
(R)-6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-苯基丙酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈9
Figure PCTCN2020077449-appb-000034
第一步
(R)-6-(2-hydroxy-2-methylpropoxy)-4-(1'-(2-phenylpropanoyl)-1',2',3',6'-tetrahydro-[2,4'-bipyridin] -5-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
(R)-6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-苯基丙酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈9
将6-(2-羟基-2-甲基丙氧基)-4-(1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈盐酸盐2f(39mg,0.087mmol)加入10mL的三口瓶中，依次加入2mL N,N-二甲基甲酰胺，(R)-2-苯基丙酸9a(30mg,0.2mmol)，2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(38mg，0.1mmol)，N,N-二异丙基乙胺(64.5mg，0.5mmol)，室温搅拌16小时，加入30mL水，搅拌过滤，滤饼用高效液相色谱法纯化得到6(R)-6-(2-羟基-2-甲基丙氧基)-4-(1'-(2-苯基丙酰基)-1',2',3',6'-四氢-[2,4'-二吡啶]-5-基)吡唑并[1,5-a]吡啶-3-甲腈9(21mg，白色固体)，产率40％。
MS m/z(ESI)：522.2[M+1]
1H NMR(400MHz,DMSO):δ8.81-8.69(m,2H),8.61(d,J＝13.6Hz,1H),8.03-7.98(m,1H),7.66(t,J＝9.4Hz,1H),7.43(d,J＝1.8Hz,1H),7.32(t,J＝7.0Hz,4H),7.23(t,J＝6.6Hz,1H),6.77(d,J＝43.6Hz,1H),4.37(dd,J＝43.8,17.1Hz,3H),4.25-4.11(m,2H),3.89(s,2H),3.70-3.57(m,2H),2.00(dd,J＝15.0,7.3Hz,1H),1.33(d,J＝6.7Hz,3H),1.23(s,6H).
实施例10
6-(3-hydroxy-3-methylbutyl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(3-羟基-3-甲基丁基)-4-(6-(6-((6-甲氧基吡啶-3-基)甲基)-3,6-二氮杂二环[3.1.1]庚烷-3-基)吡啶-3-基)吡唑并[1,5-a]吡啶-3-甲腈10
Figure PCTCN2020077449-appb-000035
Figure PCTCN2020077449-appb-000036
第一步
methyl(E)-3-(3-cyano-4-methoxypyrazolo[1,5-a]pyridin-6-yl)acrylate(E)-3-(3-氰基-4-甲氧基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10b
将6-溴-4-甲氧基吡唑并[1,5-a]吡啶-3-甲腈10a(2.52g,10mmol，采用专利申请“WO2017011776”公开的方法制备而得)，丙烯酸甲酯(3.2mL,35mmol)，三乙胺(3.75mL,27mmol)，三(邻甲基苯基)磷(0.51g,1.67mmol)，醋酸钯(0.25g，1.13mmol)加入15mL乙腈中，氮气保护下升温至回流温度，反应16小时。反应液减压浓缩，残留物用硅胶柱层析法(洗脱剂：C体系)纯化，得到(E)-3-(3-氰基-4-甲氧基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10b(1.8g，淡黄色固体)，产率70％。
MS m/z(ESI)：258.1[M+1]
1H NMR(400MHz,DMSO)：δ8.99(s,1H),8.65(s,1H),7.75(dd,J＝16.0,9.9Hz,1H),7.45(s,1H),6.96(d,J＝16.0Hz,1H),4.07(s,3H),3.76(s,3H).
第二步
methyl(E)-3-(3-cyano-4-hydroxypyrazolo[1,5-a]pyridin-6-yl)acrylate(E)-3-(3-氰基-4-羟基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10c
将(E)-3-(3-氰基-4-甲氧基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10b(1.54g,6.0mmol)加入80mL 1,2-二氯乙烷中，加入三氯化铝(2.4g,18mmol)，反应液升至80℃反应4小时。反应液冷却至室温，加入四氢呋喃(100mL)和十水硫酸钠(10g)淬灭，室温搅拌过夜，过滤，滤液减压浓缩，得到(E)-3-(3-氰基-4-羟基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10c(1.22g，黑棕色固体)，直接进行下步反应。
MS m/z(ESI)：244[M+1]
第三步
methyl(E)-3-(3-cyano-4-(((trifluoromethyl)sulfonyl)oxy)pyrazolo[1,5-a]pyridin-6-yl)acrylate(E)-3-(3-氰基-4-(((三氟甲基)磺酰基)氧基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10d
将(E)-3-(3-氰基-4-羟基吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10c(1.22g,5.0mmol)加入10mL N,N-二甲基乙酰胺中，加入N-苯基双(三氟甲烷磺酰)亚胺(3.57g,10.0mmol)，N,N-二异丙基乙胺(1.7mL,5.0mmol)，室温搅拌反应2小时。反应液加入乙酸乙酯(200mL)，依次用水(50mL×3)，饱和氯化钠溶液(50mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用硅胶柱层析法(洗脱剂：C体系)纯化，得到(E)-3-(3-氰基-4-(((三氟甲基)磺酰基)氧基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10d(0.76g，淡黄色固体)，产率40.4％。
MS m/z(ESI)：376[M+1]
1H NMR(400MHz,DMSO):δ9.56(s,1H),8.88(s,1H),8.43(s,1H),7.81(d,J＝16.1Hz,1H),7.01(d,J＝16.0Hz,1H),3.77(s,3H).
第四步
methyl(E)-3-(3-cyano-4-(6-fluoropyridin-3-yl)pyrazolo[1,5-a]pyridin-6-yl)acrylate(E)-3-(3-氰基-4-(6-氟吡啶-3-基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10e
将(E)-3-(3-氰基-4-(((三氟甲基)磺酰基)氧基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10d(751mg,2.0mmol)，2-氟吡啶-5-硼酸频哪醇酯(670mg,3.0mmol)，四(三苯基磷)钯(116mg,0.1mmol)，3mL 2N的碳酸钠溶液和9mL 1,2-二氯乙烷加入25mL微波管中，氮气保护，升温至100℃下微波反应1.5小时。过滤，滤饼用水(2mL×5)洗涤，干燥，得到(E)-3-(3-氰基-4-(6-氟吡啶-3-基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10e(560mg，淡黄色固体)，直接进行下步反应。
MS m/z(ESI)：323[M+1]
1H NMR(400MHz,DMSO):δ9.46(s,1H),8.78(s,1H),8.29(d,J＝5.2Hz,1H),8.15(s,1H),7.83(d,J＝16.2Hz,1H),7.49-7.37(m,2H),6.98(d,J＝16.0Hz,1H),3.75(s,3H).
第五步
tert-butyl(E)-3-(5-(3-cyano-6-(3-methoxy-3-oxoprop-1-en-1-yl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate
叔丁基(E)-3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙-1-烯-1-基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10f
将(E)-3-(3-氰基-4-(6-氟吡啶-3-基)吡唑并[1,5-a]吡啶-6-基)丙烯酸甲酯10e(500mg,1.55mmol)加入3mL二甲亚砜中，加入叔丁基3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯(309mg,1.55mmol)，碳酸钾(1.1g,7.75mmol)，升温至90℃下反应20小时。反应液加入乙酸乙酯(100mL)，依次用水(30mL×3)，饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用硅胶柱层析法(洗脱剂：C体系)纯化，得到叔丁基(E)-3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙-1-烯-1-基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10f (250mg，淡黄色固体)，产率32.3％。
MS m/z(ESI)：501[M+1]
第六步
tert-butyl 3-(5-(3-cyano-6-(3-methoxy-3-oxopropyl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate
叔丁基3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10g
将(E)-3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙-1-烯-1-基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10f(180mg,0.36mmol)加入5mL甲醇中，氢气氛下室温反应12小时。反应液过滤，滤液减压浓缩，得到叔丁基3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10g(171mg，浅黄色固体)，直接进行下步反应。
MS m/z(ESI)：503[M+1]
第七步
tert-butyl 3-(5-(3-cyano-6-(3-hydroxy-3-methylbutyl)pyrazolo[1,5-a]pyridin-4-yl)pyridin-2-yl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate
叔丁基3-(5-(3-氰基-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10h
将叔丁基3-(5-(3-氰基-6-(3-甲氧基-3-氧代丙基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10g(150mg,0.3mmol)加入10mL无水乙醚中，0℃下滴加3N的甲基溴化镁(1.0mL)，滴加完毕后升至室温反应2小时。反应液加入乙酸乙酯(100mL)稀释，加入饱和氯化铵溶液(30mL)淬灭，用饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用硅胶柱层析法(洗脱剂：C体系)纯化，得到叔丁基3-(5-(3-氰基-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10h(50mg，淡黄色固体)，产率33.％。
MS m/z(ESI):503[M+1]
第八步
4-(6-(3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)-6-(3-hydroxy-3-methylbutyl)pyrazolo[1,5-a]pyridine-3-carbonitrile
4-(6-(3,6-二氮杂二环[3.1.1]庚烷-3-基)吡啶-3-基)-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-3-甲腈10i
将叔丁基3-(5-(3-氰基-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-4-基)吡啶-2-基)-3,6-二氮杂二环[3.1.1]庚烷-6-甲酸酯10h(50mg,0.1mmol)加入1mL二氯甲烷中，加入4N的盐酸/二氧六环溶液(1mL)，室温反应12小时。反应液减压浓缩，得到4-(6-(3,6-二氮杂二环[3.1.1]庚 烷-3-基)吡啶-3-基)-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-3-甲腈10i(40mg，黄色油状物)，直接进行下步反应。
MS m/z(ESI):403[M+1]
第九步
6-(3-hydroxy-3-methylbutyl)-4-(6-(6-((6-methoxypyridin-3-yl)methyl)-3,6-diazabicyclo[3.1.1]heptan-3-yl)pyridin-3-yl)pyrazolo[1,5-a]pyridine-3-carbonitrile
6-(3-羟基-3-甲基丁基)-4-(6-(6-((6-甲氧基吡啶-3-基)甲基)-3,6-二氮杂二环[3.1.1]庚烷-3-基)吡啶-3-基)吡唑并[1,5-a]吡啶-3-甲腈10
将4-(6-(3,6-二氮杂二环[3.1.1]庚烷-3-基)吡啶-3-基)-6-(3-羟基-3-甲基丁基)吡唑并[1,5-a]吡啶-3-甲腈10i(40mg,0.1mmol)加入2mL 1,2-二氯乙烷中，加入三乙酰氧基硼氢化钠(27.4mg,0.2mmol)，6-甲氧基-3-吡啶甲醛(63.6mg,0.3mmol)，室温反应12小时。反应液加入乙酸乙酯(100mL)稀释，用饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，残留物用硅胶柱层析法(洗脱剂：B体系)纯化，得到6-(3-羟基-3-甲基丁基)-4-(6-(6-((6-甲氧基吡啶-3-基)甲基)-3,6-二氮杂二环[3.1.1]庚烷-3-基)吡啶-3-基)吡唑并[1,5-a]吡啶-3-甲腈10(8mg，淡黄色固体)，产率15.4％。
MS m/z(ESI):522[M+1]
1H NMR(400MHz,DMSO):δ12.24-12.19(m,1H),8.75(dd,J＝8.2,2.1Hz,2H),8.60(s,1H),8.11(d,J＝2.0Hz,1H),7.99(dt,J＝15.9,7.9Hz,1H),7.73-7.63(m,2H),7.44(d,J＝2.0Hz,1H),6.81(t,J＝5.7Hz,2H),4.72(s,1H),3.89(s,2H),3.85(s,3H),3.15(d,J＝2.4Hz,2H),2.67(d,J＝4.9Hz,2H),2.31-2.23(m,2H),1.32(s,6H),1.25-1.23(m,2H).
生物学评价
测试例1、本发明化合物对RET异常肿瘤细胞(TT细胞)增殖抑制活性的测定
以下方法用于测定本发明优选化合物在体外条件下对TT细胞的增殖抑制活性。TT细胞为人甲状腺髓样癌细胞株，该细胞株含RET C634W突变，细胞株购于美国ATCC(货号
Figure PCTCN2020077449-appb-000037
CRL-1803)。该细胞使用Ham's F-12K(Kaighn's)培养基(购于Thermofisher，Gibco，货号21127022)附加10％胎牛血清(FBS,购于Thermofisher，Gibco，货号12483-020)，1x Glutamax(购于Thermofisher，Gibco，货号35050061),1x non-essential amino acids(购于Thermofisher，Gibco，货号11140050),1x Pen/Strep antibiotics(购于Thermofisher，Gibco，货号15140122)在37℃，5％浓度CO 2条件下进行培养。细胞活性通过采用Promega公司的
Figure PCTCN2020077449-appb-000038
Luminescent Cell Viability Assay(货号G7573)方法进行检测。
实验方法简述如下：正式实验前日，将处于对数生长期的细胞用胰酶(trypsin，购于Thermofisher，Gibco，货号25300054)消化处理，随后加入适宜体积的培养基终止胰酶反应。以1000转/分速度离心后，吸去上层培养基，并加入新的培养基配成细胞悬液。向96孔细胞培养板(购于Corning，货号3904)中加入50uL细胞悬液，每孔细胞密度为6000个/孔。随后将培养板在37℃，5％浓度CO 2条件下培养过夜。实验当日，首先将测试化合物溶解于DMSO(Sigma-Aldrich，货号v900090)中配制为贮存液，随后用DMSO按1：3比例进行连 续稀释，共计9个浓度点，同时设DMSO对照。随后另取一块96孔培养板，向各孔中加入250uL不含血清的F12培养基，再向各孔中加入0.5uL各浓度化合物溶液，振荡混匀，配制成测试样品。之后从各孔中取50uL测试化样品溶液加入至细胞板中，最终化合物的测试浓度范围为10000nM至1.52nM，每个浓度含复孔对照。将细胞板在37℃，5％浓度CO 2条件下连续培养8天。培养结束后，向各孔中加入50uL CellTiter-Glo试剂，振荡混匀后在室温下避光孵育10分钟，随后在Envision酶标仪(Perkin Elmer)上读取各孔信号。随后在GraphPad Prism软件中以化合物浓度对数-信号强度进行非线性回归分析，得到测试化合物抑制细胞增殖的IC 50值。
本发明中化合物对RET异常肿瘤细胞(TT细胞)增殖抑制活性通过以上的实验进行测定，测得的IC 50值见表1
表1 本发明中化合物对TT细胞增值活性抑制IC 50
化合物编号 IC 50(nM)
1 28.9
8 87.8
10 68.6
结论：本发明化合物对RET异常肿瘤细胞(TT细胞)增殖具有明显的抑制作用
测试例2、本发明化合物对RET酶抑制活性的测定
1、实验材料
试剂名称 供货商 货号
GST-hRET激酶(658-1114) Carna 08-159
HTRF KinEASE-TK kit Cisbio 62TK0PEC
5X酶反应缓冲液 Cisbio 62EZBFDC
TK substrate-biotin Cisbio 61TK0BLE
SEB Cisbio 61SEBALF
Ultra Pure ATP Promega V915A
TK Antibody-Cryptate Cisbio —
streptavidin-XL665 Cisbio 610SAXLA
Detection buffer Cisbio 62SDBRDD
384孔板 PerkinELmer 6007290
2、试剂准备
(1)1X酶反应缓冲液:超纯水4：1稀释5x酶反应缓冲液，加入25nM SEB。
(2)5X RET激酶：1X酶反应缓冲液稀释RET激酶到2.5nM
(3)5X ATP：1X酶反应缓冲液稀释ATP到50uM
(4)5X底物：1X酶反应缓冲液稀释TK substrate-biotin到2500nM
(5)2.5X化合物：终浓度最高浓度为2500nm，3倍梯度稀释，10个稀释点，DMSO终浓度为0.4％
(6)4X Steptavidin-XL665：1X酶反应缓冲液稀释Steptavidin-XL665到250uM
3、实验过程
(1)384多孔板中加入2ul/孔5X RET激酶(终浓度0.5nM)，设定空白组(酶反应缓冲液)作为阴性对照
(2)加入4ul/孔2.5X化合物梯度，室温孵育30分钟，设定DMSO组作为阳性对照
(3)加入2ul/孔5X底物(终浓度500nM)和2ul/孔5X ATP(终浓度10uM)起始反应，室温反应30分钟
(4)加入5ul/孔TK Antibody-Cryptate和5ul/孔4X Steptavidin-XL665(终浓度62.5uM)，室温孵育60分钟
(5)Envision读取HTRF信号
4、数据分析
用Graph Pad prism软件对化合物的剂量效果(IC 50)进行分析
表1 本发明中化合物对RET酶活性抑制IC 50
化合物编号 IC 50(nM)
1 23.1
2 25.4
10 5.6
结论：本发明化合物对RET酶活性具有明显的抑制作用。

Claims (14)

  1. 一种通式(I)所示的化合物或其立体异构体、互变异构体或其可药用的盐：
    Figure PCTCN2020077449-appb-100001
    其中：
    环A选自基团：
    Figure PCTCN2020077449-appb-100002
    X 1、X 2各自独立地选自CH、CCH 3、CF、CCl或N；
    X 3、X 4各自独立地选自CH、CF或N；
    其中X 1、X 2、X 3和X 4中的0、1或2个是N；
    L 1选自一个键、-(R aR bC)-、-(R aR bN)-或-O-；
    L 2选自一个键、-(亚烷基)-、-C(O)-、-S(O)-、-SO 2-、-(亚烷基)-O-、-(亚烷基)-S-、-(亚烷基)-NR 1-、-C(O)-(亚烷基)-、-SO 2-(亚烷基)-；其中L 2基团中右键与E相连；其中所述的亚烷基任选进一步被一个或多个选自羟基、卤素、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；
    R a选自氢原子、烷基或卤素；
    R b选自氢原子、烷基或卤素；
    或者，R a和R b与其相连接的C或N原子一起形成3～6元环烷基或杂环基，其中杂环基内含有一个或多个N、O、S(O) n原子，其中所述的环烷基或杂环基任选进一步被一个或多个选自羟基、卤素、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；
    B选自氢原子、烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、烯基、炔基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自R c的取代基所取代；
    E选自氢原子、烷氧基、环烷基、杂环基、芳基或杂芳基；其中所述的环烷基、杂环基、 芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-SO 2NR 2R 3的取代基所取代；优选为苯基或吡啶基；
    R c选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 2R 3、-C(O)NR 2R 3、-C(O)R 4、-OC(O)R 4、-NR 2C(O)R 3或-S(O) 2NR 2R 3，其中所述的烷基、烷氧基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
    R 1相同或不同，各自独立地选自氢原子、羟基、烷基、卤素或烷氧基，其中所述的烷基或烷氧基任选进一步被一个或多个选自羟基、卤素或烷氧基的取代基所取代；
    R 2、R 3和R 4各自独立地选自氢原子、羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、烷氧基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
    或者，R 2和R 3与相连接的N原子一起形成一个4～8元杂环基，其中4～8元杂环内含有一个或多个N、O、S(O) n原子，并且4～8元杂环上任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、＝O、-NR 5R 6、-C(O)NR 5R 6、-C(O)R 7、-OC(O)R 7、-NR 5C(O)R 6或-SO 2NR 5R 6的取代基所取代；
    R 5、R 6和R 7各自独立地选自氢原子、烷基、环烷基、杂环基、芳基或杂芳基，其中所述的烷基、环烷基、杂环基、芳基或杂芳基任选进一步被一个或多个选自羟基、卤素、硝基、氰基、烷基、烷氧基、环烷基、杂环基、芳基、杂芳基、羧基或羧酸酯基的取代基所取代；
    m选自0，1，2，3，4或5；
    n选自0，1或2；
    条件是，当环A选自基团：
    Figure PCTCN2020077449-appb-100003
    时，
    (1)L 1选自-(R aR bC)-，-(R aR bN)-或-S-；
    (2)L 1选自一个健时，B选自烯基或炔基，其中烯基或炔基任选进一步被一个或多个选自R c的取代基所取代。
  2. 根据权利要求1所述的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(II)所示的化合物或其立体异构体、互变异构体或其可药用的盐：
    Figure PCTCN2020077449-appb-100004
    其中：
    L 1选自一个键、-(R aR bC)-、-(R aR bN)-或-O-；优选为-O-；
    L 2、B、E、X 1、X 2、X 3、X 4、R 1、R a、R b和m的定义如权利要求1所述。
  3. 根据权利要求1所述的化合物或其立体异构体、互变异构体或其可药用的盐，其为通式(III)所示的化合物或其立体异构体、互变异构体或其可药用的盐：
    Figure PCTCN2020077449-appb-100005
    其中：
    (1)L 1选自-(R aR bC)-、-(R aN)-或-S-；
    优选地，R a和R b选自氢原子或甲基；
    (2)当L 1选自一个键时，B选自烯基或炔基，其中烯基或炔基任选进一步被一个或多个选自R c的取代基所取代；
    L 2、B、E、X 1、X 2、X 3、X 4、R 1、R a、R b、R c和m的定义如权利要求1所述。
  4. 根据权利要求1～3任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中：
    X 1，X 2，X 3，X 4选自CH或N；
    X 1，X 2，X 3和X 4中的0、1或2个是N；
    优选为，X 1为N，X 2，X 3，X 4为CH。
  5. 根据权利要求1～3任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中：
    L 2选自一个键、-(亚烷基)-、-C(O)-、-SO 2-、-(亚烷基)-O-、-(亚烷基)-S-、-(亚烷基)-NR 1-或-C(O)-(亚烷基)-，其中L 2基团中右键与E相连；
    优选为-(亚烷基)-、-C(O)-、-(亚烷基)-S-或-C(O)-(亚烷基)-；
    其中所述的-(亚烷基)-优选为-(亚甲基)-或-CH(CH 3)-。
  6. 根据权利要求1～3任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中B选自：
    (i)5～6元杂芳基，其中所述的杂芳基任选进一步被一个或多个选自烷基或卤素的取代基所取代；
    优选地，B选自吡唑基，其中所述的吡唑基任选进一步被甲基取代；
    (ii)烷基，其中所述的烷基任选进一步被一个或多个羟基所取代；优选地，
    B选自基团：
    Figure PCTCN2020077449-appb-100006
    (iii)烯基或炔基，其中所述的烯基或炔基任选进一步被一个或多个羟基所取代；
    优选地，当L 1为一个键时，B选自基团：
    Figure PCTCN2020077449-appb-100007
  7. 根据权利要求1～6任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中所述的化合物选自：
    Figure PCTCN2020077449-appb-100008
  8. 一种根据权利要求1所述的通式(I)化合物或其立体异构体、互变异构体或其可药用的盐的制备方法，所述方法包括：
    Figure PCTCN2020077449-appb-100009
    将通式(IA)与E-(亚烷基)-OMs、E-C(O)X、E-(亚烷基)-C(O)X或E-C(O)H反应，得到通式(I)；
    X选自羟基或卤素，优选为羟基、Cl或Br；
    其中：L 1、L 2、B、E、X 1、X 2、X 3、X 4、R 1和m的定义如权利要求1所述。
  9. 一种通式(IA)所述的化合物或其立体异构体、互变异构体或其可药用的盐，
    Figure PCTCN2020077449-appb-100010
    其中：L 1、B、X 1、X 2、X 3、X 4、R 1和m的定义如权利要求1所述。
  10. 根据权利要求9所述的化合物或其立体异构体、互变异构体或其可药用的盐，其中所述的化合物包括：
    Figure PCTCN2020077449-appb-100011
    Figure PCTCN2020077449-appb-100012
  11. 一种药物组合物，所述的药物组合物含有有效剂量的根据权利要求1～7中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，及可药用的载体、赋形剂或它们的组合。
  12. 根据权利要求1～7中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求11所述的药物组合物在制备RET或突变的RET抑制剂的药物中的用途。
  13. 根据权利要求1～7中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求11所述的药物组合物在制备用于治疗与RET异常介导的疾病的药物中的用途，其中所述的疾病优选为癌症、纤维发育不良和遗传障碍，其中所述的癌症优选为非小细胞肺癌，甲状腺癌，实体瘤，结肠肿瘤或胰腺癌，更优选为非小细胞肺癌，甲状腺癌，实体瘤。
  14. 根据权利要求1～7中任一项所述的化合物或其立体异构体、互变异构体或其可药用的盐，或根据权利要求11所述的药物组合物在制备用于治疗对癌症产生抗体的受试者的药物中的用途。
PCT/CN2020/077449 2019-03-02 2020-03-02 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途 WO2020177668A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US17/435,703 US20220135560A1 (en) 2019-03-02 2020-03-02 Pyrazolo[1,5-a]pyridine derivatives, preparation method therefor and use thereof
EP20765744.6A EP3936504A4 (en) 2019-03-02 2020-03-02 PYRAZOLO[1,5-A]PYRIDINE DERIVATIVES, PROCESS FOR THEIR PRODUCTION AND THEIR USE
CN202080017586.6A CN113508116A (zh) 2019-03-02 2020-03-02 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910157739.6A CN111635400A (zh) 2019-03-02 2019-03-02 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途
CN201910157739.6 2019-03-02

Publications (1)

Publication Number Publication Date
WO2020177668A1 true WO2020177668A1 (zh) 2020-09-10

Family

ID=72328994

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/077449 WO2020177668A1 (zh) 2019-03-02 2020-03-02 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途

Country Status (4)

Country Link
US (1) US20220135560A1 (zh)
EP (1) EP3936504A4 (zh)
CN (2) CN111635400A (zh)
WO (1) WO2020177668A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021093720A1 (zh) * 2019-11-12 2021-05-20 浙江海正药业股份有限公司 吡唑[1,5-a]吡啶-3-腈化合物及其在医药上的用途
CN114181205A (zh) * 2020-09-14 2022-03-15 上海辉启生物医药科技有限公司 吡唑并吡啶类化合物或其盐及其制备方法和用途

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111592538A (zh) * 2019-02-21 2020-08-28 南京明德新药研发有限公司 作为ret抑制剂的脂肪环衍生物
TW202108582A (zh) * 2019-05-14 2021-03-01 大陸商上海翰森生物醫藥科技有限公司 含二並環類衍生物抑制劑、其製備方法和應用
CN112939967A (zh) * 2019-12-11 2021-06-11 深圳晶泰科技有限公司 吡唑并[1,5-a]吡啶类化合物及其制备方法和应用
US20230095530A1 (en) * 2019-12-27 2023-03-30 TYK Medicines, Inc Compound used as ret kinase inhibitor and application thereof
WO2022083741A1 (zh) * 2020-10-23 2022-04-28 上海辉启生物医药科技有限公司 吡唑并吡啶类化合物或其盐及其制备方法和用途
JP2023550069A (ja) * 2020-11-13 2023-11-30 上▲海▼翰森生物医▲薬▼科技有限公司 二環系誘導体を含む阻害剤の遊離塩基の結晶及びその製造方法と使用

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6194406B1 (en) 1995-12-20 2001-02-27 Aventis Pharmaceuticals Inc. Substituted 4-(1H-benzimidazol-2-yl)[1,4]diazepanes useful for the treatment of allergic disease
WO2003092686A1 (en) 2002-04-27 2003-11-13 Astrazeneca Ab Inhibitors of histone deacetylase
US20080153883A1 (en) 2006-12-22 2008-06-26 Abbott Laboratories Novel compounds as cannabinoid receptor ligands and uses thereof
WO2012092442A1 (en) 2010-12-29 2012-07-05 Agios Pharmaceuticals, Inc. Therapeutic compounds and compositions
WO2014066659A1 (en) 2012-10-25 2014-05-01 Tetra Discovery Partners, Llc. Heteroaryl inhibitors of pde4
WO2016033445A1 (en) 2014-08-29 2016-03-03 Chdi Foundation, Inc. Probes for imaging huntingtin protein
WO2017011776A1 (en) 2015-07-16 2017-01-19 Array Biopharma, Inc. Substituted pyrazolo[1,5-a]pyridine compounds as ret kinase inhibitors
JP2017137276A (ja) 2016-02-05 2017-08-10 東洋合成工業株式会社 化合物、それを含む組成物及び該組成物を用いたデバイスの製造方法
WO2018071447A1 (en) 2016-10-10 2018-04-19 Andrews Steven W Substituted pyrazolo[1,5-a]pyridine compounds as ret kinase inhibitors
WO2018130838A1 (en) 2017-01-13 2018-07-19 Redag Crop Protection Ltd Thiobenzoimidazole as fungicides
WO2018136661A1 (en) 2017-01-18 2018-07-26 Andrews Steven W SUBSTITUTED PYRAZOLO[1,5-a]PYRAZINE COMPOUNDS AS RET KINASE INHIBITORS

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220017539A1 (en) * 2018-12-06 2022-01-20 Shenzhen Targetrx, Inc. Substituted pyrazolo[1,5-a]pyridine compound, composition containing the same and use thereof
CN111592538A (zh) * 2019-02-21 2020-08-28 南京明德新药研发有限公司 作为ret抑制剂的脂肪环衍生物
CN112778337B (zh) * 2019-11-08 2023-09-26 杭州邦顺制药有限公司 作为ret激酶抑制剂的3、6二氮杂双环[3.1.1]庚烷衍生物
CN112939967A (zh) * 2019-12-11 2021-06-11 深圳晶泰科技有限公司 吡唑并[1,5-a]吡啶类化合物及其制备方法和应用

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6194406B1 (en) 1995-12-20 2001-02-27 Aventis Pharmaceuticals Inc. Substituted 4-(1H-benzimidazol-2-yl)[1,4]diazepanes useful for the treatment of allergic disease
WO2003092686A1 (en) 2002-04-27 2003-11-13 Astrazeneca Ab Inhibitors of histone deacetylase
US20080153883A1 (en) 2006-12-22 2008-06-26 Abbott Laboratories Novel compounds as cannabinoid receptor ligands and uses thereof
WO2012092442A1 (en) 2010-12-29 2012-07-05 Agios Pharmaceuticals, Inc. Therapeutic compounds and compositions
WO2014066659A1 (en) 2012-10-25 2014-05-01 Tetra Discovery Partners, Llc. Heteroaryl inhibitors of pde4
WO2016033445A1 (en) 2014-08-29 2016-03-03 Chdi Foundation, Inc. Probes for imaging huntingtin protein
WO2017011776A1 (en) 2015-07-16 2017-01-19 Array Biopharma, Inc. Substituted pyrazolo[1,5-a]pyridine compounds as ret kinase inhibitors
CN108349969A (zh) * 2015-07-16 2018-07-31 阵列生物制药公司 作为RET激酶抑制剂的取代的吡唑并[1,5-a]吡啶化合物
JP2017137276A (ja) 2016-02-05 2017-08-10 東洋合成工業株式会社 化合物、それを含む組成物及び該組成物を用いたデバイスの製造方法
WO2018071447A1 (en) 2016-10-10 2018-04-19 Andrews Steven W Substituted pyrazolo[1,5-a]pyridine compounds as ret kinase inhibitors
US10555944B2 (en) * 2016-10-10 2020-02-11 Eli Lilly And Company Substituted pyrazolo[1,5-A]pyridine compounds as RET kinase inhibitors
WO2018130838A1 (en) 2017-01-13 2018-07-19 Redag Crop Protection Ltd Thiobenzoimidazole as fungicides
WO2018136661A1 (en) 2017-01-18 2018-07-26 Andrews Steven W SUBSTITUTED PYRAZOLO[1,5-a]PYRAZINE COMPOUNDS AS RET KINASE INHIBITORS

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"McGraw-Hill Dictionary of Chemical Terms", 1984, MCGRAW-HILLBOOK COMPANY
ARIGHI ET AL., CYTOKINE GROWTH FACTOR, vol. 16, 2005, pages 441 - 67
ELIEL,E.WILEN,S.: "Stereochemistry of Organic Compounds", 1994, JOHN WILEY&SONS, INC.
M. MULLIGAN: "RET revisited expanding the oncogenic portfolio", NATURE REVIEWS CANCER, vol. 14, 2014, pages 173 - 186, XP055336166, DOI: 10.1038/nrc3680
See also references of EP3936504A4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021093720A1 (zh) * 2019-11-12 2021-05-20 浙江海正药业股份有限公司 吡唑[1,5-a]吡啶-3-腈化合物及其在医药上的用途
CN114181205A (zh) * 2020-09-14 2022-03-15 上海辉启生物医药科技有限公司 吡唑并吡啶类化合物或其盐及其制备方法和用途

Also Published As

Publication number Publication date
EP3936504A1 (en) 2022-01-12
CN113508116A (zh) 2021-10-15
US20220135560A1 (en) 2022-05-05
CN111635400A (zh) 2020-09-08
EP3936504A4 (en) 2022-10-26

Similar Documents

Publication Publication Date Title
WO2020177668A1 (zh) 吡唑并[1,5-a]吡啶类衍生物、及其制备方法和用途
TWI828677B (zh) 甲基修飾酵素之調節劑、其組成物及用途
TWI757256B (zh) 轉染過程重排之抑制劑
JP6820836B2 (ja) 変異イソクエン酸デヒドロゲナーゼ阻害剤としてのフェニルキノリノン誘導体
WO2017161269A1 (en) Inhibitors of ret receptor tyrosine kinases
CN108473434A (zh) 取代喹诺酮类衍生物或其药学上可接受的盐或立体异构体及其药用组合物和应用
JP2014511892A (ja) ブロモドメイン阻害剤として有用なテトラヒドロキノリン誘導体
TW201512185A (zh) 雜芳族化合物以及其作為多巴胺ｄ１配體的用途
JP6612857B2 (ja) ベンゾイミダゾールアナログおよび関連方法
WO2022206723A1 (zh) 杂环类衍生物、其制备方法及其医药上的用途
WO2022169948A1 (en) Tricyclic-amido-bicyclic prmt5 inhibitors
WO2016050171A1 (zh) 多环类间变性淋巴瘤激酶抑制剂
WO2018214866A1 (zh) 一种氮杂芳基衍生物、其制备方法和在药学上的应用
JP6386585B2 (ja) プロテインキナーゼのインヒビターとしてのフロピリジン
WO2023134647A1 (zh) 含哌嗪并环类衍生物、其药学上可接受的盐及其制备方法和应用
WO2020215998A1 (zh) 嘧啶并五元杂环类化合物及其作为突变型idh2抑制剂的用途
WO2018233526A9 (zh) 一种csf1r抑制剂及其制备方法和应用
TW201605858A (zh) 縮合吡唑衍生物
TW202028195A (zh) 作為TGF-βR1抑制劑的化合物及其應用
WO2020156319A1 (zh) N-甲酰胺衍生物、其制备方法及其在医药上的用途
WO2023036252A1 (zh) 吡咯并嘧啶类或吡咯并吡啶类衍生物及其医药用途
CN108264511B (zh) 杂环类衍生物及其制备方法和其在医药上的用途
EP4140997A1 (en) Pyridine acetamide derivative serving as cdk inhibitor, and preparation method therefor and use thereof
TWI749518B (zh) 哌嗪醯胺衍生物，其製備方法及其在醫藥上的用途
WO2022033455A1 (zh) 具有egfr抑制活性的三嗪衍生物及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20765744

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2020765744

Country of ref document: EP

Effective date: 20211004