WO2021129621A1 - 吡唑类化合物 - Google Patents

吡唑类化合物 Download PDF

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WO2021129621A1
WO2021129621A1 PCT/CN2020/138367 CN2020138367W WO2021129621A1 WO 2021129621 A1 WO2021129621 A1 WO 2021129621A1 CN 2020138367 W CN2020138367 W CN 2020138367W WO 2021129621 A1 WO2021129621 A1 WO 2021129621A1
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group
membered
pharmaceutically acceptable
compound
acceptable salt
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PCT/CN2020/138367
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English (en)
French (fr)
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唐锋
金国恒
吴清萍
王峰
彭少平
任晋生
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江苏先声药业有限公司
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Priority to CN202080075713.8A priority Critical patent/CN114728980A/zh
Publication of WO2021129621A1 publication Critical patent/WO2021129621A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • the present invention relates to a novel pyrazole compound or pharmaceutically acceptable salt, a pharmaceutical composition containing them, and use as a TGF- ⁇ inhibitor.
  • Transforming growth factor- ⁇ (transforming growth factor ⁇ , TGF- ⁇ ) is a multifunctional cytokine that participates in regulating cell proliferation, differentiation, and cell proliferation through complex receptor signaling pathways on the cell surface through autocrine, paracrine, and endocrine methods. Apoptosis.
  • TGF- ⁇ mainly exists in three forms, TGF- ⁇ 1, TGF- ⁇ 2 and TGF- ⁇ 3, which are located on different chromosomes, of which TGF- ⁇ 1 accounts for the highest proportion in somatic cells.
  • TGF- ⁇ has three main cell receptors, namely type I (ALK5), type II and type III receptors.
  • the type I (ALK5) and type II receptors are transmembrane serine/threonine kinases.
  • TGF- ⁇ 1 transduce signals through two highly conserved single-span membrane serine/threonine kinases, namely type I (ALK5) and type II TGF- ⁇ receptors, the latter through phosphorylation of serine/threonine in the ALK5GS region Acid residues create a binding site for the Smad protein to cause the activation of ALK5, and ultimately exert biological functions through Smad and other signaling pathways.
  • type I ALK5
  • type II TGF- ⁇ receptors the latter through phosphorylation of serine/threonine in the ALK5GS region
  • Acid residues create a binding site for the Smad protein to cause the activation of ALK5, and ultimately exert biological functions through Smad and other signaling pathways.
  • TGF- ⁇ is a superfamily of multifunctional growth factors with a wide range of biological activities. Studies have shown that TGF- ⁇ participates in early embryonic development, cartilage and bone formation, synthesis of extra-encapsulated matrix, inflammation, fibrosis, tumor formation and development, etc. Therefore, abnormalities in TGF- ⁇ signaling are related to the occurrence of various diseases.
  • cancers such as colon cancer, melanoma, hepatocellular carcinoma, kidney cancer, glioblastoma, pancreatic cancer, lung cancer and gastric cancer, etc.
  • fibrotic diseases such as kidney fibrosis, liver fibrosis, lung fibrosis, etc.
  • inflammatory diseases such as arthritis, chronic nephritis, acute nephritis, etc.
  • TGF- ⁇ small molecule inhibitors have also been reported in the existing literature for the treatment of cancer and/or treatment of fibrotic diseases (such as patent document WO2016057278A1), but there are still a large number of patients who cannot obtain satisfactory clinical treatment effects. Therefore, there is still a need for better and more effective clinical treatments and solutions.
  • the present invention creatively designs a series of compounds on the basis of the existing technology to provide a series of TGF- ⁇ inhibitors with novel structure, excellent pharmacodynamics, high bioavailability and good druggability, and are used for effective treatment of TGF- ⁇ Related diseases and disorders, including but not limited to tumors, fibrotic diseases, inflammatory diseases, autoimmune diseases, etc.
  • the present invention provides a compound represented by general formula (A) or a pharmaceutically acceptable salt thereof:
  • ring Q is selected from phenyl or 5-6 membered heteroaryl
  • R 1, R 2 is independently selected from hydrogen, F, Cl, Br, I , CN, OH, NO 2 , or R a optionally substituted following groups: NH 2, SH, C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, 3-10 membered heterocyclyl, C 1 -C 10 alkoxy, C 3 -C 10 cycloalkyloxy, 3-10 membered heterocyclyloxy, C 1 -C 10 alkylsulfonyl, C 3 -C 10 cycloalkylsulfonyl, 3-10 membered heterocyclic sulfonyl, aminosulfonyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 6- C 10 aryl, 5-10 membered heteroaryl, C 6 -C 10 aryloxy, 5-10 membered heteroaryloxy, C 6 -C 10 arylsulfonyl, 5-10 membered
  • Ring Q is phenyl or 5-membered heteroaryl.
  • ring Q is phenyl, thienyl, or furyl.
  • ring Q is phenyl or thienyl.
  • R 1 and R 2 are independently selected from H, C 1 -C 6 alkoxy, 3-10 membered heterocyclyl, C 6 -C 10 aryl, or 5-10 membered heteroaryl, so said C 1 -C 6 alkoxy, 3-10 membered heterocyclyl, C 6 -C 10 aryl or 5-10 membered heteroaryl optionally substituted with R a.
  • ring Q is phenyl
  • ring Q is thienyl
  • Ring Q is furyl
  • R 1, R 2 is independently selected from H or R a is optionally substituted C 1 -C 6 alkoxy.
  • R 1, R 2 is independently selected from H or R a optionally substituted with the following groups: 3-10 member heterocyclyl, C 6 -C 10 aryl or 5-10 membered heteroaryl .
  • each Ra is independently selected from the following groups optionally substituted by R b : C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl , 3-8 membered heterocyclic group, C 1 -C 10 alkylsulfonyl group, phenyl group or 5-6 membered heteroaryl group.
  • each Ra is independently selected from the following groups optionally substituted by R b : C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 3 -C 6 cycloalkyl , 3-8 membered heterocyclic group, C 1 -C 10 alkylsulfonyl or phenyl.
  • each Ra is independently selected from the following groups optionally substituted with R b : C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl, 3-8 membered heterocyclyl, C 1 -C 10 alkylsulfonyl, phenyl or 5-6 membered heteroaryl.
  • the ring Q is a phenyl group
  • the R 1 and R 2 are independently selected from H or a C 1 -C 6 alkoxy group optionally substituted by Ra.
  • the ring Q is phenyl
  • said R 1, R 2 is independently selected from H or R a is optionally substituted C 1 -C 6 alkoxy, said R a is selected from 3 10-membered heterocyclic group.
  • the ring Q is a phenyl group
  • the R 1 and R 2 are independently selected from H or a C 1 -C 6 alkoxy group optionally substituted by Ra
  • the R a is selected from 6-membered Heterocyclic group.
  • the ring Q is phenyl, said R 1, R 2 independently selected from H or optionally substituted with R a C 1 -C 3 alkoxy, R a is selected from the morpholine base.
  • the ring Q is a thienyl group
  • the R 1, R 2 is independently selected from H or R a optionally substituted with the following groups: 3-10 member heterocyclyl, C 6 -C 10 Aryl or 5-10 membered heteroaryl.
  • the ring Q is a thienyl group
  • the R 1, R 2 is independently selected from H or an optionally substituted group of R a group of the following: phenyl, pyridyl, pyrazolyl, tetrahydropyridine base, Azetidinyl, benzopyrazolyl, benzimidazolyl.
  • each Ra is independently selected from the following groups optionally substituted by R b : C 1 -C 3 alkylsulfonyl, C 1 -C 6 alkyl, C 3 -C 6 cycloalkane Group, 3-6 membered heterocyclic group, C 1 -C 3 alkoxy group or phenyl group.
  • the ring Q is thienyl
  • R 1 and R 2 are independently selected from H, phenyl, pyridyl, pyrazolyl, or tetrahydropyridyl, and the phenyl, pyridyl, pyridyl thiazolyl, or optionally substituted tetrahydropyridinyl R a.
  • the ring Q is thienyl
  • R 1 and R 2 are independently selected from H, phenyl, pyridyl, pyrazolyl, or tetrahydropyridyl, and the phenyl, pyridyl, pyridyl thiazolyl, or optionally substituted tetrahydropyridinyl R a, R a the optionally substituted group consisting of R b: C 1 -C 6 alkylsulfonyl, C 1 -C 6 alkyl or 3-6 Membered heterocyclic group.
  • the ring Q is thienyl
  • the ring Q is a furyl group
  • the R 1 and R 2 are independently selected from H or a 5-6 membered heteroaryl group optionally substituted with a C 1 -C 6 alkyl group.
  • the ring Q is a furyl group
  • the R 1 and R 2 are independently selected from H or a pyrazolyl group optionally substituted with a C 1 -C 3 alkyl group.
  • the ring Q is furyl, and R 1 and R 2 are independently selected from H or
  • the R 1 and R 2 are independently selected from a hydrogen atom, a methoxy group,
  • the R 1 and R 2 are independently selected from a hydrogen atom, a methoxy group,
  • the compound or pharmaceutically acceptable salt represented by the general formula (A) is selected from the following compounds or pharmaceutically acceptable salts:
  • the present invention also provides a pharmaceutical composition, which comprises a compound represented by formula (A) or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.
  • the present invention relates to the use of the compound represented by the general formula (A) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a medicine for preventing or treating TGF- ⁇ -mediated diseases.
  • the present invention relates to the use of the compound represented by general formula (A) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the prevention or treatment of diseases mediated by TGF- ⁇ .
  • the present invention relates to a compound of general formula (A) or a pharmaceutically acceptable salt thereof for preventing or treating diseases mediated by TGF- ⁇ , or a pharmaceutical composition thereof.
  • the TGF- ⁇ -mediated disease is selected from cancer, fibrotic disease or inflammatory disease.
  • the present invention also relates to a method for preventing or treating TGF- ⁇ -mediated diseases, the method comprising administering to a patient a therapeutically effective dose of a medicament comprising the compound of general formula (A) of the present invention or a pharmaceutically acceptable salt thereof preparation.
  • the compound of the present invention has novel structure, excellent pharmacokinetic properties, and remarkable pharmacodynamics, can effectively treat related diseases and disorders mediated by TGF- ⁇ , and has strong drug-making properties.
  • pharmaceutically acceptable salts refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, and organic acids and bases.
  • stereoisomer refers to the isomers produced by the different arrangements of atoms in the molecule in space, including cis and trans isomers, enantiomers, diastereomers and conformational isomers.
  • the compound of the present invention may have an asymmetric carbon atom (optical center) or an asymmetric double bond. Racemates, enantiomers, diastereomers, geometric isomers and individual isomers are all included in the scope of the present invention.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms may be present in substituents such as alkyl groups.
  • the asymmetric atom-containing compound of the present application can be isolated in an optically pure form or a racemic form.
  • the optically active pure form can be resolved from the racemic mixture or synthesized by using chiral raw materials or chiral reagents.
  • tautomer refers to an isomer of a functional group resulting from the rapid movement of an atom in a molecule at two positions.
  • the compounds of the present invention may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more mutually convertible species.
  • Proton shift tautomers result from the migration of covalently bonded hydrogen atoms between two atoms.
  • Tautomers generally exist in an equilibrium form, and an attempt to separate a single tautomer usually produces a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties of the molecule.
  • the ketone type is dominant; in phenol, the enol type is dominant.
  • the present invention encompasses all tautomeric forms of the compound.
  • pharmaceutical composition means a mixture of one or more of the compounds described in the text or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable excipients .
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence of the specific atom is normal and the substituted compound is stable.
  • it means that two hydrogen atoms are replaced, and the oxo will not occur on the aromatic group.
  • the term “optional” or “optionally” means that the event or situation described later can occur or not occur, and the description includes occurrence of said event or situation and non-occurrence of said event or situation.
  • the ethyl group is "optionally" substituted by halogen, meaning that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (such as CH 2 CH 2 F), or polysubstituted (such as CHFCH 2 F, CH 2 CHF 2 etc.) or completely substituted (CF 2 CF 3 ).
  • CH 2 CH 3 unsubstituted
  • monosubstituted such as CH 2 CH 2 F
  • polysubstituted such as CHFCH 2 F, CH 2 CHF 2 etc.
  • CF 2 CF 3 completely substituted
  • any variable such as R
  • its definition in each case is independent. For example, if a group is replaced by 2 Rs, then each R has independent options.
  • the substituent can be bonded to any atom on the ring.
  • the structural unit It means that R 1 and R 2 can be substituted at any position on the ring Q.
  • halo or halogen refers to fluorine, chlorine, bromine and iodine.
  • C 1 -C 10 alkyl should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
  • the alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl Group, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-d
  • C 1 -C 6 alkyl should be understood to mean having 1, 2, 3, 4, 5, 6 A straight chain or branched chain saturated monovalent hydrocarbon group having three carbon atoms
  • C 1 -C 3 alkyl should be understood to mean a straight chain or branched chain saturated monovalent hydrocarbon group having 1, 2, 3 carbon atoms.
  • C 1 -C 10 alkoxy should be understood as “C 1 -C 10 alkyloxy” or “C 1 -C 10 alkyl-O", preferably, "C 1 -C 10 alkoxy”" May include “C 1 -C 6 alkoxy” and "C 1 -C 3 alkoxy”.
  • C 2 -C 10 alkenyl should be understood to preferably mean a linear or branched monovalent hydrocarbon group, which contains one or more double bonds and has 2, 3, 4, 5, 6, 7, 8, 9 Or 10 carbon atoms.
  • the alkenyl groups can be classified into “cis” and “trans” orientations (or “E” and “Z” orientations).
  • C 2 -C 6 alkenyl should be understood as a linear or branched monovalent hydrocarbon group containing one or more double bonds and having 2, 3, 4, 5 or 6 carbon atoms.
  • C 2 -C 10 alkynyl should be understood to preferably mean a linear or branched monovalent hydrocarbon group, which contains one or more triple bonds and has 2, 3, 4, 5, 6, 7, 8, 9 Or 10 carbon atoms.
  • Examples of "C 2 -C 10 alkynyl” include, but are not limited to, ethynyl (-C ⁇ CH), prop-1-ynyl (1-propynyl, -C ⁇ CCH 3 ), prop-2-ynyl (Propargyl), but-1-ynyl, but-2-ynyl or but-3-ynyl.
  • C 2 -C 3 alkynyl examples include ethynyl (-C ⁇ CH), prop-1-ynyl (1-propynyl, -C ⁇ CCH 3 ), prop-2-ynyl (propargyl ).
  • C 3 -C 10 cycloalkyl refers to a carbocyclic ring that is fully saturated and can exist as a monocyclic, bridged or spiro ring, and the ring atoms of the carbocyclic ring are 3-10 carbon atoms.
  • Non-limiting examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl Group), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decane, etc.
  • C 3 -C 10 cycloalkyl preferably includes “C 3 -C 8 cycloalkyl", “C 3 -C 6 cycloalkyl”, etc., and the non-limiting of “C 3 -C 8 cycloalkyl” Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl; non-limiting examples of "C 3 -C 6 cycloalkyl” include, but are not limited to, cyclopropyl Group, cyclobutyl, cyclopentyl, cyclohexyl.
  • cycloalkyloxy can be understood as “cycloalkyl-O-”.
  • 3-10 membered heterocyclic group means a saturated or partially saturated monovalent monocyclic, fused ring, spiro ring or bridged ring, which contains 1-5, preferably 1-3 selected from N, O and S heteroatom.
  • “3-10 membered heterocyclic group” includes “3-8 membered heterocyclic group”, “3-6 membered heterocyclic group” and the like.
  • the "3-10 membered heterocyclic group” may include but is not limited to: 4-membered ring, such as azetidinyl, oxetanyl; 5-membered ring, such as tetrahydrofuranyl, dioxolane Group, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or 6-membered ring, such as tetrahydropyranyl, piperidinyl, morpholinyl, dithiadiyl, thiomorpholinyl, Piperazinyl or trithiayl; or partially saturated 6-membered ring such as tetrahydropyridyl; or 7-membered ring such as diazeppanyl.
  • 4-membered ring such as azetidinyl, oxetanyl
  • 5-membered ring such as tetrahydrofuranyl, dioxolane
  • the heterocyclic group may be benzo-fused, in particular, the heterocyclic group may also be the above-mentioned 4-membered heterocyclic group, 5-membered heterocyclic group or 6-membered heterocyclic group of benzo-fused. ⁇ The ring group.
  • the heterocyclic group may be bicyclic, such as but not limited to a 5, 5-membered ring, such as hexahydrocyclopenta[c]pyrrole-2(1H)-yl ring, or a 5, 6-membered bicyclic ring, such as hexahydropyrrole And [1,2-a]pyrazine-2(1H)-yl ring.
  • the ring containing the nitrogen atom may be partially unsaturated, that is, it may contain one or more double bonds, such as but not limited to 2,5-dihydro-1H-pyrrolyl, 4H-[1,3,4]thiadi Azinyl, 4,5-dihydrooxazolyl or 4H-[1,4]thiazinyl, optionally, the heterocyclic group may be the above-mentioned partially unsaturated nitrogen-containing heterocyclic benzo Condensed ring groups, such as but not limited to According to the present invention, the heterocyclic group is non-aromatic (when the heterocyclic ring is a benzo-fused ring, the part of the heterocyclic group fused with the benzene ring is non-aromatic).
  • heterocyclyloxy can be understood as “heterocyclyl-O-”.
  • C 6 -C 10 aryl group means a monovalent aromatic or partially aromatic hydrocarbon ring having 6 to 10 carbon atoms.
  • rings with 6 carbon atoms such as phenyl;
  • C 6 -C 10 aryl also includes two fused aromatic rings with non-aromatic rings or partially saturated rings.
  • a ring group, and the atom on the aromatic ring is the group attachment point.
  • Non-limiting examples include, but are not limited to, phenyl, naphthyl, anthryl, indenyl, indanyl, 1,2-dihydronaphthalene And 1,2,3,4-tetrahydronaphthyl.
  • heteroaryl should be understood to include such a monovalent monocyclic, bicyclic or tricyclic aromatic ring system: unless otherwise indicated, the aromatic ring system usually has 5 to 20 ring atoms, and the ring atoms Contains 1-5 heteroatoms independently selected from N, O and S.
  • heteroaryl should be understood to include monovalent monocyclic, bicyclic or tricyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, especially 5 Or 6 or 9 or 10 ring atoms, and it contains 1-5, preferably 1-3 heteroatoms independently selected from N, O and S. And, in addition, it may be benzo-fused in each case.
  • the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiol Diazolyl, etc.
  • 5-6 membered heteroaryl refers to an aromatic ring system having 5 or 6 ring atoms, and it contains 1-3, preferably 1-2, heteroatoms each independently selected from N, O, and S.
  • "5-10 membered heteroaryl group” also includes the above-mentioned aromatic ring system and non-aromatic ring system fused
  • “5-10 membered heteroaryl group” includes the above-mentioned 5-6 membered heteroaryl group A polycyclic group formed by condensing with a saturated or partially saturated heterocyclic ring, and using the atoms of the above 5-6 membered heteroaryl group as the group connection point.
  • Non-limiting examples include, but are not limited to, 4, 5, 6, 7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl, 2,3-dihydro-1H-pyrrolo[3,2-b]pyridin-5-yl and the like.
  • treatment means administering the compound or formulation described in this application to prevent, ameliorate or eliminate a disease or one or more symptoms associated with the disease, and includes:
  • terapéuticaally effective amount means (i) treatment or prevention of a particular disease, condition or disorder, (ii) reduction, amelioration or elimination of one or more symptoms of a particular disease, condition or disorder, or (iii) prevention or delay
  • the amount of the compound of the present invention that constitutes a “therapeutically effective amount” varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but it can be routinely determined by those skilled in the art. Determined by its own knowledge and this disclosure.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious stimulating effect on the organism and will not damage the biological activity and performance of the active compound.
  • Suitable auxiliary materials are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the present application also includes compounds of the present application that are the same as those described herein, but have one or more atoms replaced by an isotope-labeled atom having an atomic weight or mass number different from the atomic weight or mass number commonly found in nature.
  • isotopes that can be bound to the compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • isotope-labeled compounds of the application can be used in compound and/or substrate tissue distribution analysis. Tritiated (ie 3 H) and carbon-14 (ie 14 C) isotopes are especially preferred due to their ease of preparation and detectability. Positron emission isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy.
  • PET positron emission tomography
  • the isotopically-labeled compounds of the present application can be prepared by the following procedures similar to those disclosed in the schemes and/or examples below, by replacing non-isotopically-labeled reagents with isotope-labeled reagents.
  • substitution with heavier isotopes can provide certain therapeutic advantages resulting from higher metabolic stability (for example, increased in vivo half-life or reduced dosage requirements), and therefore in certain situations
  • deuterium substitution can be partial or complete, and partial deuterium substitution refers to the substitution of at least one hydrogen by at least one deuterium.
  • the pharmaceutical composition of the present application can be prepared by combining the compound of the present application with suitable pharmaceutically acceptable excipients, for example, can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, and powders. , Granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.
  • Typical routes for administering the compound of the present application or a pharmaceutically acceptable salt or pharmaceutical composition thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, Intramuscular, subcutaneous, and intravenous administration.
  • the pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing method, dissolution method, granulation method, sugar-coated pill method, grinding method, emulsification method, freeze-drying method, etc.
  • the pharmaceutical composition is in an oral form.
  • the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These auxiliary materials enable the compound of the present application to be formulated into tablets, pills, lozenges, sugar-coated agents, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.
  • the solid oral composition can be prepared by conventional mixing, filling or tableting methods. For example, it can be obtained by the following method: mixing the active compound with solid excipients, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain tablets Or the core of the dragee.
  • suitable excipients include, but are not limited to: binders, diluents, disintegrants, lubricants, glidants, sweeteners or flavoring agents, and the like.
  • the pharmaceutical composition may also be suitable for parenteral administration, such as a sterile solution, suspension or lyophilized product in a suitable unit dosage form.
  • the daily dose is 0.01 to 100 mg/kg body weight, preferably 0.05 to 50 mg/kg body weight, more preferably 0.1 to 30 mg/kg body weight, in single or divided doses form.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the unit of NMR shift is 10 -6 (ppm).
  • the solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS);
  • TMS tetramethylsilane
  • IC 50 refers to the half inhibitory concentration, which refers to the half of the maximum inhibitory effect concentration.
  • Step 2 Preparation of 1-(dimethylamino)-3-oxo-3-(tetrahydro-2H-pyran-4-yl)prop-1-en-2-yl benzoate
  • Step 7 4-((1-Cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)-6-methoxyquinoline- Preparation of 7-alcohol
  • step 6 Put the compound prepared in step 6 into a single-necked flask, add 10 mL of methanol and 50 mg of 10% palladium on carbon, evacuate, and then stir under a hydrogen atmosphere at room temperature for 2 h, and TLC monitors until the reaction is complete. The palladium carbon was removed by suction filtration, and the filtrate was concentrated to obtain the title compound.
  • Step 8 4-(3-((4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)-6 -Methoxyquinolin-7-yl)oxy)propyl)morpholine
  • step 7 Take the compound prepared in step 7 and place it in a single-necked flask, add 4-(3-chloropropyl)morpholine (164mg, 1mmol), cesium carbonate (625mg, 2mmol) and a catalyst amount of NaI in the single-necked flask, and then add 10mL of acetonitrile, stirred and refluxed for 12h, TLC monitored until the reaction was complete. After the reaction solution was concentrated by suction filtration, the title compound was separated by column chromatography. LC-MS m/z: 509.3 [M+H] + .
  • the preparation method is the same as in Example 1, except that the commercially available raw material 7-benzyloxy-4-chloroquinoline (CAS: 178984-56-0) is used instead of 7-benzyloxy-4-chloro-6-methyl. Oxyquinoline to obtain the title compound.
  • Step 2 7-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy)-2-(4-(methyl Preparation of sulfonyl)phenyl)thieno[3,2-b]pyridine
  • the preparation method is the same as in Example 3, except that the commercially available raw material 1-acetyl-5,6-dihydro-2H-pyridine-4-boronic acid pinacol ester (CAS: 1227068-67-8) is used instead 4-Methanesulfonylphenylboronic acid, to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the commercially available raw material 4-pyrazoleboronic acid pinacol ester (CAS: 269410-08-4) is used instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the commercially available 1-(oxetan-3-yl)-4-(4,4,5,5-tetramethyl-1,3,2 -Dioxaborolan-2-yl)-1H-pyrazole (CAS: 1339890-99-1) instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as that in Example 3, except that the commercially available 1-(tetrahydropyran-4-yl)-1H-pyrazole-4-boronic acid pinacol ester (CAS: 1040377-03-4) is used. Instead of 4-methanesulfonylphenylboronic acid, the title compound was prepared.
  • the preparation method is the same as in Example 3, except that the commercially available 2-methyl-1-[4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H- Pyrazol-1-yl]propan-2-ol (CAS: 1082503-77-2) replaces 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the commercially available 1-methylpyrazole-4-boronic acid pinacol ester (CAS: 761446-44-0) is used instead of 4-methanesulfonylphenylboronic acid to prepare Title compound.
  • the preparation method is the same as in Example 12, except that the commercially available iodocyclobutane (CAS: 38557-29-8) is used instead of 3-fluoro-1-iodopropane (CAS: 462-40-8) to prepare Get the title compound.
  • the preparation method is the same as that in Example 12. The difference is that commercially available 1,1-difluoro-2-iodoethane (CAS: 598-39-0) is used instead of 3-fluoro-1-iodopropane (CAS :462-40-8) to obtain the title compound.
  • the preparation method is the same as that of Example 12. The difference is that commercially available 2,2,2-trifluoroethyl trifluoromethanesulfonate (CAS: 6226-25-1) is used instead of 3-fluoro-1-iodo Propane (CAS: 462-40-8) to obtain the title compound.
  • Step 1 tert-Butyl 3-(4-(7-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)oxy) Thieno[3,2-b]pyridin-2-yl)-1H-pyrazol-1-yl)azetidine-1-carboxylic acid
  • Step 2 2-(1-(azetidin-3-yl)-1H-pyrazol-4-yl)-7-(((1-cyclopropyl-3-(tetrahydro-2H-pyridine (Pyran-4-yl)-1H-pyrazole-4-(yl)oxy)thieno[3,2-b]pyridine
  • step 1 The product obtained in step 1 (284 mg, 0.50 mmol) was added to 5 mL of hydrogen chloride dioxane solution, and stirred at room temperature until the reaction was completed as monitored by TLC. Concentrate under reduced pressure to obtain the crude product, which was directly put into the next step.
  • the preparation method is the same as that in Example 12, except that commercially available 1,3,2-dioxane, 5,5-difluoro, 2,2-dioxide (CAS: 1215071-15-0) is used instead 3-Fluoro-1-iodopropane (CAS: 462-40-8) to obtain the title compound.
  • the preparation method is the same as in Example 12, except that the commercially available 2,2-difluoropropyl trifluoromethanesulfonate (CAS: 784193-15-3) is used instead of 3-fluoro-1-iodopropane ( CAS: 462-40-8) to obtain the title compound.
  • the preparation method is the same as in Example 3, except that commercially available 1-(difluoromethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboron) is used.
  • Alk-2-yl)-1H-pyrazole (CAS: 1206640-82-5) replaces 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the commercially available 1-methyl-1H-pyrazole-5-boronic acid pinacol ester (CAS: 847818-74-0) is used instead of 4-methanesulfonylphenylboronic acid. , The title compound was obtained.
  • the preparation method is the same as in Example 3, except that commercially available phenylboronic acid (CAS: 98-80-6) is used instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that commercially available 4-trifluoromethoxyphenylboronic acid (CAS: 139301-27-2) is used instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the commercially available 2-methylindazole-5-boronic acid pinacol ester (CAS:1189746-27-7) is used instead of 4-methanesulfonylphenylboronic acid to prepare Title compound.
  • the preparation method is the same as that in Example 3, except that the commercially available 1-methyl-1H-benzimidazole-5-boronic acid nacol ester (CAS: 1107627-02-0) is used instead of 4-methanesulfonylphenylboronic acid , The title compound was obtained.
  • the preparation method is the same as in Example 3, except that commercially available 6-(2-hydroxypropane-2-yl)pyridine-3-boronic acid (CAS: 1088496-42-7) is used instead of 4-methanesulfonylphenylboronic acid , The title compound was obtained.
  • the preparation method is the same as in Example 16, except that the 3-fluoro-1-iodopropane in step 3 is replaced with methanesulfonic anhydride (CAS: 7143-01-3) to obtain the title compound.
  • the preparation method is the same as that in Example 16, except that the 1-Boc-3-iodoazetidine in step 1 is replaced with N-Boc-4-iodopiperidine, and the 3-fluoro- in step 3
  • the 1-iodopropane was replaced with cyclopropanesulfonyl chloride (CAS: 139631-62-2) to prepare the title compound.
  • the preparation method is the same as in Example 16, except that the 3-fluoro-1-iodopropane in step 3 is replaced with 2,2,2-trifluoroethyl trifluoromethanesulfonate (CAS: 6226-25 -1)
  • the title compound was prepared.
  • the preparation method is the same as in Example 3, except that commercially available p-methoxyphenylboronic acid (CAS: 5720-07-0) is used instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that commercially available 4-trifluoromethylphenylboronic acid (CAS: 128796-39-4) is used instead of 4-methanesulfonylphenylboronic acid to obtain the title compound.
  • the preparation method is the same as in Example 3, except that the 2-bromo-7-chlorothieno[3,2-b]pyridine in step 1 is replaced with 7-chloro-2-iodofuro[3,2-B] Pyridine (CAS: 1071540-54-9), while replacing 4-methanesulfonylphenylboronic acid in step 2 with 1-methyl-4-(4,4,5,5-tetramethyl-1,3, 2-Dioxaborolan-2-yl)-1H-pyrazole (CAS: 761446-44-0), to obtain the title compound.
  • the preparation method is the same as in Example 3, the difference is that commercially available (1-(4-fluorophenyl)-1H-pyrazol-4-yl)boronic acid (CAS:1072945-89-1) is used instead of 4-methane Sulfonylphenylboronic acid, to obtain the title compound.
  • the preparation method is similar to that in Example 16, except that the 1-Boc-3-iodoazetidine in step 1 is replaced with N-Boc-4-iodopiperidine, and the 3-fluoro -1-iodopropane was replaced with 2,2,2-trifluoroethyl trifluoromethanesulfonate (CAS: 6226-25-1) to obtain the title compound.
  • the preparation method is similar to Example 12, except that 3-fluoro-1-iodopropane is replaced with 2-[1-(ethylsulfonyl)-3-azetidinylidene]acetonitrile (CAS:1187595-85 -2) to obtain the title compound.
  • the preparation method is the same as that in Example 3, except that 5-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-4 ,5,6,7-Tetrahydropyrazolo[1,5-a]pyrazine (CAS: 2057507-40-9) instead of 4-methanesulfonylphenylboronic acid to prepare the title compound.
  • Test Example 1 Determination of the inhibitory effect of the compound of the present invention on the activity of TGF ⁇ RI kinase
  • the inhibitory effect of the compound of the present invention on the activity of TGF ⁇ RI kinase ALK5 was tested by the following method.
  • Enzyme activity detection uses ADP-Glo kinase detection kit (V9102/3, Promega).
  • V9102/3 Promega
  • a 384-well plate Cat.No.4512, Corning
  • 50nl of the compound dissolved in 100% DMSO in 3-fold dilutions (the initial concentration of the test is 10 ⁇ M)
  • 2.5 ⁇ l of reaction buffer 40mM Tris, pH 7.5; 20mM MgCl2; 0.10% BSA; 1mM DTT
  • ALK5 Cat.09-141, Carna
  • enzyme solution final concentration 12.5nM
  • Table IC 50 of Compound 1 of the present invention TGF ⁇ RI ALK5 kinase inhibition activity
  • Test Example 2 Determination of the inhibitory effect of the compound of the present invention on the activity of p38 ⁇ kinase
  • Enzyme activity detection uses p38 ⁇ kinase detection kit (V9591, Promega), in a 384-well plate (Cat.No.4512, Corning), add 5 ⁇ l of 3-fold serially diluted compounds dissolved in 100% DMSO (the first concentration of the test is 10 ⁇ M). ), 10 ⁇ l of p38 ⁇ enzyme (Cat.04-152, Carna) solution (final concentration 6nM) prepared with reaction buffer (50mM HEPES, pH 7.5; 0.0015% Brij-35), placed at room temperature for 10 minutes.
  • reaction buffer 50mM HEPES, pH 7.5; 0.0015% Brij-35
  • Test Example 3 Determination of the inhibitory activity of the compound of the present invention on the Smad signaling pathway of TGF ⁇ RI
  • the TGF ⁇ /SMAD reporter gene cell line (BPS Bioscience, Cat. 60653) was used to detect the inhibitory effect of the molecule on the TGF- ⁇ 1 protein signaling pathway.
  • the TGF- ⁇ 1 protein binds to receptors on the cell surface and initiates a signal cascade, which leads to the phosphorylation and activation of SMAD2 and SMAD3, and forms a complex with SMAD4.
  • the SMAD complex transfers to the nucleus and binds to the SMAD binding element in the nucleus, resulting in the transcription and expression of TGF ⁇ /SMAD responsive genes.
  • the TGF ⁇ /SAMD reporter gene cell line contains the firefly luciferase gene, which is stably integrated into HEK293 cells and is controlled by SMAD response elements.
  • the cells When the cells are stimulated by human TGF- ⁇ 1, the cells express firefly luciferase.
  • the luciferase After adding the substrate, the luciferase catalyzes the substrate to emit light, and the light intensity value can be read on the microplate reader. After adding the molecule to be tested, if it can inhibit the signal pathway of TGF- ⁇ 1, a decrease in the reading can be detected.
  • the cells were added to a 96-well white impermeable plate (Corning, Cat. 3917) at a density of 0.35 ⁇ 10 6 cells/mL, and cultured in a 5% CO2 incubator at 37° C. for 24 hours.
  • the molecules to be detected were diluted 4-fold with a starting concentration of 5 ⁇ M, and there were 7 gradients in total. Subsequently, the diluted molecule was mixed with 2 ⁇ g/ml TGF- ⁇ 1 protein (Perpotech, Cat.100-21) at a ratio of 1:1, and 100 ⁇ l per well was added to the cells cultured overnight, and each gradient was 3 replicate wells.
  • luciferase substrate Promega, G7940
  • the biological activity of the compound of the present invention was determined by the above test, and the measured IC 50 value is shown in Table 3 below.
  • Test compound IC 50 (nM) 001 18.31 002 32.90 003 13.06 004 64.28 005 56.80 006 48.66 007 51.52 008 26.77 009 64.24 010 37.13
  • mice Using mice as test animals, the LC/MS/MS method was used to determine the drug concentration in plasma at different times after the mice were given the compound of the invention intragastrically and intravenously. Study the pharmacokinetic behavior of the compound of the present invention in rats and evaluate its pharmacokinetic characteristics.
  • Positive control compound compound 001, 004, 005, 007, 009, 010.
  • mice 42 healthy adult BALB/c mice, male, divided into 7 groups, 6 in each group, 3 gavage, 3 intravenous, the mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license Certificate number: SCXK (Zhejiang) 2019-0001.
  • Gavage group BALB/c mice were fasted overnight and then administered by gavage. The dose was 10 mg/kg and the volume was 10 mL/kg.
  • Intravenous group BALB/c mice were given intravenously after fasting overnight. The dose was 1 mg/kg and the volume was 5 mL/kg.
  • mice After gavage or intravenous administration of mice, 40 ⁇ L of blood was collected from the orbit at 5min, 15min, 30min, 1h, 2h, 6h, 24h after administration, 5 ⁇ L EDTA-K2 anticoagulation, 12000rpm, 4°C, 5 minutes centrifugal separation of plasma, Store at -20°C.
  • LC/MS/MS conditions mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: 0.1% formic acid acetonitrile, column: ACE C18 5 ⁇ m (3.0mm*50mm), column temperature: 35°C, flow rate 0.5ml/min.
  • the pharmacokinetic parameters of the compound of the present invention are as follows:
  • Test Example 5 Anti-tumor efficacy test of the compound of the present invention on CT26 tumor-bearing mice
  • Compounds 001, 004, 005, 007, 009, 010 were prepared with 20% PG + 5% ethanol + 10% solutol + 65% water to prepare a 3 mg/ml aqueous solution, the administration dose was 30 mg/kg, and the oral gavage volume was 10 ml /kg.
  • the positive control drug mPD-1-RMP1-14 (purchased from bioxcell, Catalog Number: BE0188) was injected intraperitoneally.
  • mice Female, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 16-18g when purchased. After the animals were purchased, the experiment was started after 3 days of adaptive breeding. Feeding conditions: SPF level. Animal feeding method: 12/12 hours light/dark cycle adjustment, temperature 20-26 °C, humidity 40-70%, animals are given standard sterilized rat feed, free to eat and drink.
  • mice The right ribs of the mice were prepared one day in advance, and CT26 cells (1*10 ⁇ 5/mouse, purchased from ATCC) were subcutaneously inoculated. When the tumor volume grew to about 60-80mm 3 , the mice were administered in groups.
  • V tumor volume
  • Relative tumor volume (RTV) V T /V 0
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volumes of the blank control group and the experimental group at the end of the experiment, respectively.
  • Table 7 shows the efficacy data of Compound 1 on CT26 tumor-bearing mice.

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Abstract

式(A)所示化合物或其药学上可接受的盐、药物组合物，以及用于有效治疗TGF-β介导相关的疾病或病症，包括但不限于癌症、纤维化疾病或炎性疾病等。

Description

吡唑类化合物 技术领域
本发明涉及一种新型的吡唑类化合物或药学可接受的盐，含有它们的药物组合物以及作为TGF-β抑制剂的用途。
背景技术
转化生长因子-β(transforming growth factorβ,TGF-β)是一种多功能细胞因子，以自分泌、旁分泌和内分泌等方式通过细胞表面复杂的受体信号传导途径参与调节细胞的增殖，分化和凋亡。在哺乳动物中TGF-β主要以TGF-β1、TGF-β2和TGF-β3三种形式存在，它们位于不同的染色体上，其中TGF-β1在体细胞中所占比例最高。TGF-β有3个主要的细胞受体，分别为I型(ALK5)、II型和III型受体。其中I型(ALK5)和II型受体是跨膜丝氨酸/苏氨酸激酶。
TGF-β1通过两种高保守性单跨膜丝氨酸/苏氨酸激酶转导信号，即I型(ALK5)和II型TGF-β受体，后者通过磷酸化ALK5GS区中的丝氨酸/苏氨酸残基，创建Smad蛋白的结合位点引起ALK5的活化，并通过Smad等信号通路最终发挥生物学功能。
TGF-β是一个多功能生长因子超家族，具有广泛的生物学活性。研究表明，TGF-β参与早期胚胎发育、软骨和骨的形成、包外基质的合成、炎症、纤维化、肿瘤的形成和发展等，因此TGF-β信号的异常和多种疾病的发生相关，包括癌症(如结肠癌、黑素瘤、肝细胞癌、肾癌、成胶质细胞瘤、胰腺癌、肺癌和胃癌等)、纤维化疾病(如肾纤维化、肝纤维化、肺纤维化等)、炎性疾病(如关节炎、慢性肾炎、急性肾炎等)。
虽然恶性肿瘤、纤维化疾病和炎性疾病等领域相关疾病的治疗取得了一定的进展，现有文献中也报道了一些TGF-β小分子抑制剂用于治疗癌症和/或治疗纤维化疾病(如专利文献WO2016057278A1)，但仍有大量的患者无法得到满意的临床治疗效果，因此目前仍然需要更优、更有效的临床治疗和解决方案。鉴于此，本发明在现有技术基础上创造性地设计了系列化合物，提供一系列结构新颖、药效优良、生物利用度高、成药性好的TGF-β抑制剂，并用于有效治疗TGF-β相关的疾病、病症，包括但不限于肿瘤、纤维化疾病、炎性疾病、自身免疫性疾病等。
发明内容
本发明提供一种通式(A)所示化合物或其药学上可接受的盐：
Figure PCTCN2020138367-appb-000001
其中环Q选自苯基或5-6元杂芳基；
R 1、R 2独立选自氢、F、Cl、Br、I、CN、OH、NO 2或任选被R a取代的以下基团：NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、3-10元杂环基磺酰基、氨基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基、5-10元杂芳基氧基、C 6-C 10芳基磺酰基、5-10元杂芳基磺酰基；
每一个R a独立地选自F、Cl、Br、I、OH、CN、＝O、NO 2或任选被R b取代的下列基团：NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、3-10元杂环基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 2-C 10烯氧基、C 2-C 10炔氧基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基、5-10元杂芳基氧基、C 6-C 10芳基磺酰基、5-10元杂芳基磺酰基；
每一个R b独立选自F、Cl、Br、I、OH、CN、＝O、NO 2、NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基或5-10元杂芳基氧基，所述C 1-C 10烷基任选被F、Cl、Br、I、CN取代。
在一些实施方案中，每一个R b独立选自F、Cl、Br、I、OH、CN、＝O、NO 2、NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 2-C 10烯基、C 2-C 10炔基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基或5-10元杂芳基氧基。
在一些实施方案中，环Q为苯基或5元杂芳基。
在一些实施方案中，环Q为苯基、噻吩基或呋喃基。
在一些实施方案中，环Q为苯基或噻吩基。
在一些实施方案中，R 1、R 2独立选自H、C 1-C 6烷氧基、3-10元杂环基、C 6-C 10芳基或5-10元杂芳基，所述C 1-C 6烷氧基、3-10元杂环基、C 6-C 10芳基或5-10元杂芳基任选被R a取代。
在一些实施方案中，环Q为苯基。
在一些实施方案中，环Q为噻吩基。
在一些实施方案中，环Q为呋喃基。
在一些实施方案中，R 1、R 2独立选自H或任选被R a取代的C 1-C 6烷氧基。
在一些实施方案中，R 1、R 2独立选自H或任选被R a取代的下列基团：3-10元杂环基、C 6-C 10芳基或5-10元杂芳基。
在一些实施方案中，每一个R a独立地选自任选被R b取代的下列基团：C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、3-8元杂环基、C 1-C 10烷基磺酰基、苯基或5-6元杂芳基。
在一些实施方案中，每一个R a独立地选自任选被R b取代的下列基团：C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、3-8元杂环基、C 1-C 10烷基磺酰基或苯基。
在一些实施方案中，每一个R a独立地选自任选被R b取代的下列基团：C 1-C 6烷基、C 3-C 6环烷基、3-8元杂环基、C 1-C 10烷基磺酰基、苯基或5-6元杂芳基。
在一些实施方案中，每一个R b独立选自F、Cl、Br、I、OH、＝O、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 8环烷基、3-8元杂环基、C 1-C 6烷基磺酰基、C 3-C 8环烷基磺酰基、苯基或5-6元杂芳基，所述C 1-C 6烷基任选被F、Cl、Br、I、CN取代。
在一些实施方案中，每一个R b独立选自F、Cl、Br、I、OH、＝O、C 1-C 6烷基、C 1-C 6烷基磺酰基或C 3-C 8环烷基磺酰基，所述C 1-C 6烷基任选被F、Cl、Br、I、CN取代。
在一些实施方案中，每一个R b独立选自F、Cl、Br、I、OH、＝O、NH 2、C 1-C 6烷基、C 3-C 8环烷基、3-8元杂环基、苯基或5-6元杂芳基。
在一些实施方案中，所述环Q为苯基，所述R 1、R 2独立选自H或任选被R a取代的C 1-C 6烷氧基。
在一些实施方案中，所述环Q为苯基，所述R 1、R 2独立选自H或任选被R a取代的C 1-C 6烷氧基，所述R a选自3-10元杂环基。
在一些实施方案中，所述环Q为苯基，所述R 1、R 2独立选自H或任选被R a取代的C 1-C 6烷氧基，所述R a选自6元杂环基。
在一些实施方案中，所述环Q为苯基，所述R 1、R 2独立选自H或任选被R a取代的C 1-C 3烷氧基，所述R a选自吗啉基。
在一些实施方案中，所述环Q为噻吩基，所述R 1、R 2独立选自H或任选被R a取代的下 列基团：3-10元杂环基、C 6-C 10芳基或5-10元杂芳基。
在一些实施方案中，所述环Q为噻吩基，所述R 1、R 2独立选自H或任选被R a取代的下列基团：苯基、吡啶基、吡唑基、四氢吡啶基、
Figure PCTCN2020138367-appb-000002
氮杂环丁烷基、苯并吡唑基、苯并咪唑基。
在一些实施方案中，每一个R a独立地选自任选被R b取代的下列基团：C 1-C 3烷基磺酰基、C 1-C 6烷基、C 3-C 6环烷基、3-6元杂环基、C 1-C 3烷氧基或苯基。
在一些实施方案中，每一个R b独立地选自F、Cl、Br、I、OH、＝O、C 1-C 6烷基、C 1-C 3烷基磺酰基或C 3-C 6环烷基磺酰基，所述C 1-C 6烷基任选被F、Cl、Br、I、CN取代。
在一些实施方案中，所述环Q为噻吩基，所述R 1、R 2独立选自H、苯基、吡啶基、吡唑基或四氢吡啶基，所述苯基、吡啶基、吡唑基或四氢吡啶基任选被R a取代。
在一些实施方案中，所述环Q为噻吩基，所述R 1、R 2独立选自H、苯基、吡啶基、吡唑基或四氢吡啶基，所述苯基、吡啶基、吡唑基或四氢吡啶基任选被R a取代，所述R a任选被R b取代的下列基团：C 1-C 6烷基磺酰基、C 1-C 6烷基或3-6元杂环基。
在一些实施方案中，所述环Q为噻吩基，所述R 1、R 2独立选自H、苯基、吡啶基、吡唑基或四氢吡啶基，所述苯基、吡啶基、吡唑基或四氢吡啶基任选被R a取代，所述R a任选被R b取代的下列基团：C 1-C 6烷基磺酰基、C 1-C 6烷基或3-6元杂环基，所述R b选自羟基、C 1-C 6烷基或＝O。
在一些实施方案中，所述环Q为呋喃基，所述R 1、R 2独立选自H或任选被C 1-C 6烷基取代的5-6元杂芳基。
在一些实施方案中，所述环Q为呋喃基，所述R 1、R 2独立选自H或任选被C 1-C 3烷基取代的吡唑基。
在一些实施方案中，所述环Q为呋喃基，所述R 1、R 2独立选自H或
Figure PCTCN2020138367-appb-000003
在一些实施方案中，所述R 1、R 2独立选自氢原子、甲氧基、
Figure PCTCN2020138367-appb-000004
Figure PCTCN2020138367-appb-000005
Figure PCTCN2020138367-appb-000006
在一些实施方案中，所述R 1、R 2独立选自氢原子、甲氧基、
Figure PCTCN2020138367-appb-000007
Figure PCTCN2020138367-appb-000008
在一些实施方案中，所述通式(A)所示的化合物或药学可接受的盐，选自以下化合物或药学可接受的盐：
Figure PCTCN2020138367-appb-000009
Figure PCTCN2020138367-appb-000010
Figure PCTCN2020138367-appb-000011
本发明还提供药物组合物，其包含式(A)所示化合物或其药学可接受的盐和药学上可接受的辅料。
进一步，本发明涉及通式(A)所示的化合物或其药学上可接受的盐，或其药物组合物在制备预防或者治疗TGF-β介导的疾病的药物中的用途。
进一步，本发明涉及通式(A)所示的化合物或其药学上可接受的盐，或其药物组合物在预防或者治疗TGF-β介导的疾病中的用途。
进一步，本发明涉及预防或者治疗TGF-β介导的疾病的通式(A)化合物或其药学上可接受的盐，或其药物组合物。
本发明的优选方案，其中所述的TGF-β介导的疾病选自癌症、纤维化疾病或炎性疾病。
本发明还涉及预防或者治疗TGF-β介导的疾病的方法，该方法包括给以患者治疗上有效剂量的包含本发明所述的通式(A)化合物或其药学上可接受的盐的药物制剂。
本发明化合物结构新颖、药代动力学性质优良、药效显著，能够有效治疗TGF-β介导的相关的疾病、病症，具有很强的成药性。
术语定义和说明
除非另有说明，本申请说明书和权利要求书中记载的基团和术语定义，包括其作为实例 的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。这样的组合和结合后的基团定义及化合物结构，应当属于本申请说明书记载的范围内。
术语“药学上可接受的盐”是指药学上可接受的无毒酸或碱的盐，包括无机酸和碱、有机酸和碱的盐。
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体、非对应异构体和构象异构体。
本发明的化合物可以具有不对称碳原子(光学中心)或不对称双键。外消旋体、对映异构体、非对映异构体、几何异构体和单个的异构体都包括在本发明的范围之内。
本文中消旋体或者对映体纯的化合物的图示法来自Maehr，J.Chem.Ed.1985，62:114-120。除非另有说明，用楔形键和虚线键
Figure PCTCN2020138367-appb-000012
表示一个立体中心的绝对构型。当本文所述化合物含有烯属双键或其它几何不对称中心，除非另有规定，它们包括E、Z几何异构体。同样地，所有的互变异构形式均包括在本发明的范围之内。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。本申请的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来。光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本发明化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。质子移变互变异构体来自两个原子之间共价键合的氢原子的迁移。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本发明包含化合物的所有互变异构形式。
术语“药物组合物”表示一种或多种文本所述化合物或其生理学/药学上可接受的盐或前体药物与其它化学组分的混合物，其它组分例如生理学/药学上可接受的辅料。药物组合物的目的是促进化合物对生物体的给药。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，指乙基可以是未被取代的(CH 2CH 3)、单取代的(如CH 2CH 2F)、多取代的(如CHFCH 2F、CH 2CHF 2等)或完全被取代的(CF 2CF 3)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个R所取代，则每个R都有独立的选项。
当一个取代基的键交叉连接到一个环上时，这种取代基可以与这个环上的任意原子相键合。例如，结构单元
Figure PCTCN2020138367-appb-000013
表示R 1、R 2可在环Q上的任意一个位置发生取代。
术语“卤”或“卤素”是指氟、氯、溴和碘。
术语“C 1-C 10烷基”应理解为表示具有1、2、3、4、5、6、7、8、9或10个碳原子的直链或支链饱和一价烃基。所述烷基是例如甲基、乙基、丙基、丁基、戊基、己基、异丙基、异丁基、仲丁基、叔丁基、异戊基、2-甲基丁基、1-甲基丁基、1-乙基丙基、1，2-二甲基丙基、新戊基、1，1-二甲基丙基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、2-乙基丁基、1-乙基丁基、3，3-二甲基丁基、2，2-二甲基丁基、1，1-二甲基丁基、2，3-二甲基丁基、1，3-二甲基丁基或1，2-二甲基丁基等；优选地，“C 1-C 10烷基”可以包含“C 1-C 6烷基”和“C 1-C 3烷基”，“C 1-C 6烷基”应理解为表示具有1、2、3、4、5、6个碳原子的直链或支链饱和一价烃基，“C 1-C 3烷基”应理解为表示具有1、2、3个碳原子的直链或支链饱和一价烃基。
术语“C 1-C 10烷氧基”应理解为“C 1-C 10烷基氧基”或“C 1-C 10烷基-O”，优选地，“C 1-C 10烷氧基”可以包含“C 1-C 6烷氧基”和“C 1-C 3烷氧基”。
术语“C 2-C 10烯基”应理解为优选表示直链或支链的一价烃基，其包含一个或多个双键并且具有2、3、4、5、6、7、8、9或10个碳原子。所述烯基可分为“顺式”和“反式”取向(或者″E″和″Z″取向)。“C 2-C 6烯基”应理解为直链或支链的一价烃基，其包含一个或多个双键并且具有2、3、4、5或6个碳原子。“C 2-C 10烯基”的实例包括但不限于乙烯基(-CH＝CH 2)、丙-1-烯基(-CH＝CHCH 3)、丙-2-烯基(-CH 2CH＝CH 2)、2-甲基丙-1-烯基、丁-1-烯基、丁-2-烯基、丁-3-烯基、丁-1，3-二烯基、2-甲基-1，3-丁二烯基、己-1-烯基、己-2-烯基、己-3-烯基或己-4-烯基。
术语“C 2-C 10炔基”应理解为优选表示直链或支链的一价烃基，其包含一个或多个三键并且具有2、3、4、5、6、7、8、9或10个碳原子。“C 2-C 10炔基”的实例包括但不限于乙炔基(-C≡CH)、丙-1-炔基(1-丙炔基、-C≡CCH 3)、丙-2-炔基(炔丙基)、丁-1-炔基、丁-2-炔基或丁-3-炔基。“C 2-C 3炔基”实例包括乙炔基(-C≡CH)、丙-1-炔基(1-丙炔基、-C≡CCH 3)、丙-2-炔基(炔丙基)。
术语“C 3-C 10环烷基”指完全饱和的并且可以以呈单环、桥环或螺环存在的碳环,该碳环的环原子为3～10个碳原子。非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、环癸基、降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基、螺[4.5]癸烷等。“C 3-C 10环烷基”优选地包含了“C 3-C 8环烷基”、“C 3-C 6环烷基”等，“C 3-C 8环烷基”的非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基；“C 3-C 6环烷基”的非限制性实例包括但不限于环丙基、环丁基、环戊基、环己基。
术语“环烷基氧基”可理解为“环烷基-O-”。
术语“3-10元杂环基”意指饱和的或部分饱和的一价单环、并环、螺环或桥环，其包含1-5个，优选1-3个选自N、O和S的杂原子。特别地，“3-10元杂环基”包括“3-8元杂环基”、“3-6元杂环基”等。所述“3-10元杂环基”可以包括但不限于：4元环，如氮杂环丁烷基、氧杂环丁烷基；5元环，如四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基；或6元环，如四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基或三噻烷基；或部分饱和的6元环如四氢吡啶基；或7元环，如二氮杂环庚烷基。任选地，所述杂环基可以是苯并稠合的，特别地，所述杂环基还可以是上述4元杂环基、5元杂环基或6元杂环基的苯并稠合环基。所述杂环基可以是双环的，例如但不限于5,5元环，如六氢环戊并[c]吡咯-2(1H)-基环，或者5,6元双环，如六氢吡咯并[1,2-a]吡嗪-2(1H)-基环。含氮原子的环可以是部分不饱和的，即它可以包含一个或多个双键，例如但不限于2,5-二氢-1H-吡咯基、4H-[1,3,4]噻二嗪基、4,5-二氢噁唑基或4H-[1,4]噻嗪基，任选地，所述杂环基可以是上述部分不饱和的含氮原子的杂环基的苯并稠合环基，例如但不限于
Figure PCTCN2020138367-appb-000014
根据本发明，所述杂环基是无芳香性的(当所述杂环为苯并稠合环时，与苯环稠合的杂环基部分是无芳香性的)。
术语“杂环基氧基”可理解为“杂环基-O-”。
术语“C 6-C 10芳基”表示具有6～10个碳原子的一价芳香性或部分芳香性的烃环。特别是具有6个碳原子的环(“C 6芳基”)，例如苯基；“C 6-C 10芳基”还包括芳族环与非芳族环或部分饱和的环稠合的二环基团，且以芳香环上的原子为基团连接位点，非限制性实例包括但不限于，苯基、萘基、蒽基、茚基、茚满基、1，2-二氢萘基和1，2，3，4-四氢萘基。
术语“杂芳基”应理解为包括这样的一价单环、双环或三环芳族环系：除非另有指示，该芳族环系通常具有5～20个环原子，且所述环原子包含1-5个独立选自N、O和S的杂原子。
术语“5-10元杂芳基”应理解为包括这样的一价单环、双环或三环芳族环系：其具有5、6、7、8、9或10个环原子，特别是5或6或9或10个环原子，且其包含1-5个，优选1-3个独立选自N、O和S的杂原子。并且，另外在每一种情况下可为苯并稠合的。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、噻二唑基等以及它们的苯并衍生物，例如苯并呋喃基、苯并噻吩基、苯并噁唑基、苯并异噁唑基、苯并咪唑基、苯并三唑基、吲唑基、吲哚基、异吲哚基等；或吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基等，以及它们的苯并衍生物，例如喹啉基、喹唑啉基、异喹啉基等；或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物；或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基、吩噁嗪基等。术语“5-6元杂芳基”指具有5或6个环原子的芳族环系，且其包含1-3个，优选1-2各独立选自N、O和S的杂原子。任选地，“5-10元杂芳基”还包括上述芳族环系与非芳香性环系稠合，特别地，“5-10元杂芳基”包括上述5-6元杂芳基与饱和或部分饱和的杂环稠合形成的多环基团，并以上述5-6元杂芳基的原子为基团连接位点，非限制性实例包括但不限于4,5,6,7-四氢吡唑并[1,5-a]吡嗪-2-基、2,3-二氢-1H-吡咯并[3,2-b]吡啶-5-基等。
术语“治疗”意为将本申请所述化合物或制剂进行给药以预防、改善或消除疾病或与所述疾病相关的一个或多个症状，且包括：
(i)预防疾病或疾病状态在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病状态，但尚未被诊断为已患有该疾病状态时；
(ii)抑制疾病或疾病状态，即遏制其发展；
(iii)缓解疾病或疾病状态，即使该疾病或疾病状态消退。
术语“治疗有效量”意指(i)治疗或预防特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本发明化合物的用量。构成“治疗有效量”的本发明化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”、“含有(comprise)”或“包含(comprise)”及其英文变体例如 comprises或comprising应理解为开放的、非排他性的意义，即“包括但不限于”。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本申请还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本申请化合物。可结合到本申请化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为 2H、 3H、 11C、 13C、 14C、 13N、 15N、 15O、 17O、 18O、 31P、 32P、 35S、 18F、 123I、 125I和 36Cl等。
某些同位素标记的本申请化合物(例如用 3H及 14C标记的那些)可用于化合物和/或底物组织分布分析中。氚化(即 3H)和碳-14(即 14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如 15O、 13N、 11C和 18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本申请化合物。
此外，用较重同位素(诸如氘(即 2H))取代可以提供某些由更高的代谢稳定性产生的治疗优点(例如增加的体内半衰期或降低的剂量需求)，并且因此在某些情形下可能是优选的，其中氘取代可以是部分或完全的，部分氘取代是指至少一个氢被至少一个氘取代。
本申请的药物组合物可通过将本申请的化合物与适宜的药学上可接受的辅料组合而制备，例如可配制成固态、半固态、液态或气态制剂，如片剂、丸剂、胶囊剂、粉剂、颗粒剂、膏剂、乳剂、悬浮剂、栓剂、注射剂、吸入剂、凝胶剂、微球及气溶胶等。
给予本申请化合物或其药学上可接受的盐或其药物组合物的典型途径包括但不限于口服、直肠、局部、吸入、肠胃外、舌下、阴道内、鼻内、眼内、腹膜内、肌内、皮下、静脉内给药。
本申请的药物组合物可以采用本领域众所周知的方法制造，如常规的混合法、溶解法、制粒法、制糖衣药丸法、磨细法、乳化法、冷冻干燥法等。
在一些实施方案中，药物组合物是口服形式。对于口服给药，可以通过将活性化合物与本领域熟知的药学上可接受的辅料混合，来配制该药物组合物。这些辅料能使本申请的化合物被配制成片剂、丸剂、锭剂、糖衣剂、胶囊剂、液体、凝胶剂、浆剂、悬浮剂等，用于对患者的口服给药。
可以通过常规的混合、填充或压片方法来制备固体口服组合物。例如，可通过下述方法获得：将所述的活性化合物与固体辅料混合，任选地碾磨所得的混合物，如果需要则加入其它合适的辅料，然后将该混合物加工成颗粒，得到了片剂或糖衣剂的核心。适合的辅料包括 但不限于：粘合剂、稀释剂、崩解剂、润滑剂、助流剂、甜味剂或矫味剂等。
药物组合物还可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。
本文所述的通式Ⅰ化合物的所有施用方法中，每天给药的剂量为0.01到100mg/kg体重，优选为0.05到50mg/kg体重，更优选0.1到30mg/kg体重，以单独或分开剂量的形式。
本发明具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本发明的化学变化及其所需的试剂和物料。为了获得本发明的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
具体实施方式
以下实施例详细说明发明的技术方案，但本发明的保护范围包括但不限于此。
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移的单位为10 -6(ppm)。NMR测定的溶剂为氘代二甲基亚砜、氘代氯仿、氘代甲醇等，内标为四甲基硅烷(TMS)；“IC 50”指半数抑制浓度，指达到最大抑制效果一半时的浓度。
实施例1 化合物001的制备
4-(3-((4-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-6-甲氧基喹啉-7-基)氧基)丙基)吗啉的合成(化合物001)
Figure PCTCN2020138367-appb-000015
化合物001的合成路线如下所示：
Figure PCTCN2020138367-appb-000016
步骤1:2-氧代-2-(四氢-2H-吡喃-4-基)乙基苯甲酸酯的制备
Figure PCTCN2020138367-appb-000017
取苯甲酸(185g,1.52mol)溶于2升DMF中，加入碳酸钾(383g,2.75mol)搅拌10分钟，再向体系中加入化合物2-溴-1-(四氢-2H-吡喃-4-基)乙酮(285g,1.38mol)，室温搅拌反应3h，TLC监测反应完全。加入饱和食盐水，乙酸乙酯萃取,合并有机相，饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得标题化合物。LC-MS m/z:249.2[M+H]+。
步骤2:1-(二甲基氨基)-3-氧代-3-(四氢-2H-吡喃-4-基)丙-1-烯-2-基苯甲酸酯的制备
Figure PCTCN2020138367-appb-000018
取2-氧代-2-(四氢-2H-吡喃-4-基)乙基苯甲酸酯(90.9g,0.3mol)和DMF-DMA(160mL,1.2mol)置于反应瓶中，100℃搅拌反应3h，TLC监控至反应完全。反应结束后，减压浓缩得棕色油状标题化合物。LC-MS m/z:304.3[M+H] +。
步骤3:3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基苯甲酸甲酯的制备
Figure PCTCN2020138367-appb-000019
将步骤2合成的1-(二甲基氨基)-3-氧代-3-(四氢-2H-吡喃-4-基)丙-1-烯-2-基苯甲酸酯溶解到装有1L乙酸的反应瓶中，冰浴条件下缓慢滴加80％水合肼150mL，滴加完毕后，升温至室温，TLC监控至反应完全。反应结束后加入乙酸乙酯和饱和碳酸氢钠溶液，无水硫酸钠干燥有机相，减压浓缩得标题化合物。LC-MS m/z:273.2[M+H] +。
步骤4:1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基苯甲酸酯的制备
Figure PCTCN2020138367-appb-000020
将2,2'-联吡啶(51.5g,0.33mol)、乙酸铜(59.9g,0.33mol)和1L 1,2-二氯乙烷置于反应瓶中，于75℃条件下搅拌反应25分钟后冷却至室温。取环丙基硼酸(51.5g,0.6mol)、碳酸钠(63.6g,0.6mol)和步骤3合成的化合物3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基苯甲酸甲酯加至上述反应体系中，在氧气氛围下于75℃搅拌反应2h，TLC监控至反应完全。冷却至室温，加入水并用二氯甲烷萃取，有机层分别用0.5M的稀盐酸和饱和氯化钠水溶液洗涤，无水硫酸钠干燥有机相，减压浓缩，柱层析纯化得标题化合物。LC-MS m/z:313.0[M+H] +。
步骤5:1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-醇的制备
Figure PCTCN2020138367-appb-000021
取步骤4合成的化合物1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基苯甲酸酯和750mL的甲醇置于反应瓶，加入1M NaOH溶液900mL后室温反应2h，TLC监控至反应完全。反应完浓缩后用1M HCl调PH值为5-6，二氯甲烷萃取，有机层用饱和食盐水洗涤，无水硫酸钠干燥后减压浓缩得标题化合物。LC-MS m/z:209.1[M+H] +。
步骤6:7-(苄氧基)-4-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-6-甲氧基喹啉的制备
Figure PCTCN2020138367-appb-000022
取1-环丙基-3(-四氢-2H-吡喃-4-基)-1H-吡唑-4-醇(229mg,1.1mmol)、7-苄氧基-4-氯-6-甲氧基喹啉(299mg,1mmol)和碳酸铯(625mg,2mmol)于单口瓶中，加入10mL乙腈，搅拌回流反应12h，TLC监控至反应完全。将反应液浓缩后，加入冰水混合物，有大量固体析出，抽滤烘干后得标题化合物。LC-MS m/z:472.2[M+H] +。
步骤7:4-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-6-甲氧基喹啉-7-醇的制备
Figure PCTCN2020138367-appb-000023
取步骤6制备的化合物置于单口瓶中，加入10mL甲醇和50mg 10％钯碳，抽真空，然后氢气氛围下室温搅拌2h，TLC监控至反应完全。抽滤除去钯碳，滤液浓缩后得标题化合物。LC-MS m/z:382.2[M+H] +。
步骤8:4-(3-((4-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-6-甲氧基喹啉-7-基)氧基)丙基)吗啉的制备
Figure PCTCN2020138367-appb-000024
取步骤7制备的化合物置于单口瓶中，依次加入4-(3-氯丙基)吗啉(164mg,1mmol)、碳酸铯(625mg,2mmol)以及催化剂量的NaI于单口瓶中，再加入10mL乙腈，搅拌回流反应12h，TLC监控至反应完全。将反应液抽滤浓缩后，柱层析分离得标题化合物。LC-MS m/z:509.3[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.50-8.48(d,J＝5.2Hz,1H),7.95(s,1H),7.48(s,1H),7.38(s,1H),6.63-6.61(d,J＝5.2Hz,1H),4.21-4.18(m,2H),3.93(s,3H),3.80-3.77(m,2H),3.72-3.68(m,1H),3.60-3.58(m,4H),3.29-3.23(m,2H),2.78-2.71(m,1H),2.49-2.45(m,2H), 2.39(s,4H),2.00-1.94(m,2H),1.70-1.66(m,4H),1.08-0.93(m,4H)
实施例2 化合物002的制备
4-(3-((4-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)喹啉-7-基)氧基)丙基)吗啉的合成(化合物002)
Figure PCTCN2020138367-appb-000025
制备方法同实施例1，不同之处为用商业可得的原料7-苄氧基-4-氯喹啉(CAS：178984-56-0)代替7-苄氧基-4-氯-6-甲氧基喹啉，制得标题化合物。LC-MS m/z:479.3[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.62-8.61(d,J＝5.2Hz,1H),8.18-8.16(d,J＝9.1Hz,1H),7.95(s,1H),7.39-7.38(m,1H),7.30-7.27(m,1H),6.62-6.61(m,1H),4.21-4.18(m,2H),3.79-3.76(m,2H),3.72-3.66(m,1H),3.60-3.58(m,4H),3.28-3.22(m,2H),2.78-2.70(m,1H),2.49-2.45(m,2H),2.39(s,4H),1.99-1.93(m,2H),1.67-1.63(m,4H),1.09-0.93(m,4H)
实施例3 化合物003的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(4-(甲基磺酰基)苯基)噻吩并[3,2-b]吡啶的合成(化合物003)
Figure PCTCN2020138367-appb-000026
合成路线：
Figure PCTCN2020138367-appb-000027
步骤1：2-溴-7-(((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩并[3,2-b]吡啶的制备
Figure PCTCN2020138367-appb-000028
称取实施例1中步骤5制备的1-环丙基-3(-四氢-2H-吡喃-4-基)-1H-吡唑-4-醇(2.29g,11mmol)与商业可购得的2-溴-7-氯噻吩并[3,2-b]吡啶(2.49g,10mmol)、碳酸铯(6.25g,20mmol)于单口瓶中，加入50mL乙腈，搅拌回流反应12h，TLC监控至反应完全。将反应液抽滤，滤液浓缩后柱层析分离后得标题化合物。LC-MS m/z:420.2,422.2[M+H] +。
步骤2：7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(4-(甲基磺酰基)苯基)噻吩并[3,2-b]吡啶的制备
Figure PCTCN2020138367-appb-000029
称取步骤1合成的化合物2-溴-7-(((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩并[3,2-b]吡啶(420mg,1mmol)、4-甲烷磺酰苯硼酸(240mg,1.2mmol)、碳酸钾(417mg,3mmol)和1,1'-双二苯基膦二茂铁二氯化钯(37mg,0.05mmol)置于反应瓶，加入二氧六环(20mL)和水(5mL)，体系抽真空并用氩气置换,氩气氛围下100℃搅拌反应1.5h，TLC监控至反应完全。加入水和乙酸乙酯萃取，有机相用水和饱和氯化钠水溶液洗涤，无水硫酸钠干燥，减压浓缩，柱层析纯化得标题化合物。LC-MS m/z:496.1[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.58-8.57(d,J＝5.4Hz,1H),8.28(s,1H),8.19-8.17(d,2H),8.06-8.02(m,3H),6.83-6.82(d,J＝5.4Hz,1H),3.82-3.79(m,2H),3.74-3.68(m,1H),3.29-3.26(m,5H),2.82-2.74(m,1H),1.70-1.64(m,4H),1.09-0.94(m,4H)。
实施例4 化合物004的制备
2-(4-(7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩[3,2-b]吡啶-2-基)吡啶-2-基)丙醇的合成(化合物004)
Figure PCTCN2020138367-appb-000030
合成路线(采用一锅法)：
Figure PCTCN2020138367-appb-000031
将2-(4-溴吡啶-2-基)-2-丙醇(260mg,1.2mmol)、联硼酸频那醇酯(380mg,1.5mmol)、1,1'-双二苯基膦二茂铁二氯化钯(80mg,0.12mmol)和醋酸钾(537mg,3.6mmol)置于三口反应瓶中，加入10mL二氧六环，氩气氛围下100℃搅拌反应12h，TLC监控至反应完全。降温至室温，分别用注射器加入5mL 2-溴-7-(((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩并[3,2-b]吡啶(420mg,1mmol)的二氧六环溶液和5mL碳酸钾(417mg,3mmol)的水溶液，氩气氛围下100℃搅拌反应2h,TLC监控至反应完全。反应结束后，加入水和乙酸乙酯萃取，有机相依次用水和饱和氯化钠水溶液洗涤，无水硫酸钠干燥，减压浓缩，柱层析纯化得标题化合物。LC-MS m/z:477.2[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.63-8.58(dd,J＝5.1Hz,J＝5.4Hz,2H),8.30(s,1H),8.03(s,2H),7.79-7.77(m,1H),6.85-6.84(d,J＝5.4Hz,1H),5.40(s,1H),3.81-3.79(m,2H),3.74-3.68(m,1H),3.30-3.26(m,2H),2.82-2.74(m,1H),1.69–1.64(m,4H),1.50(s,6H),1.10-0.94(m,4H)
实施例5 化合物005的制备
1-(4-(7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩[3,2-b]吡啶-2-基)-3,6-二氢吡啶-1(2H)-基)乙烷-1-酮的合成
Figure PCTCN2020138367-appb-000032
制备方法同实施例3,不同之处为用商业可得的原料1-乙酰基-5,6-二氢-2H-吡啶-4-硼酸频哪醇酯(CAS：1227068-67-8)代替4-甲烷磺酰苯硼酸，制得标题化合物。LC-MS m/z:465.2[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.48-8.47(d,J＝5.4Hz,1H),7.99(s,1H),7.55-7.53(d,1H),6.74-6.72(m,1H),6.42-6.40(m,1H),4.21-4.15(d,2H),3.80-3.78(m,2H),3.71-3.65(m,3H),3.31-3.24(m,2H),2.78-2.69(m,2H),2.58(s,1H),2.09-2.05(m,3H),1.67-1.62(m,4H),1.08-0.93(m,4H)。
实施例6 化合物006的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(1H-吡唑-4-基)噻吩[3,2-b]吡啶的合成
Figure PCTCN2020138367-appb-000033
制备方法同实施例3，不同之处为用商业可得的原料4-吡唑硼酸频哪醇酯(CAS：269410-08-4)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:408.1[M+H] +。
1H NMR(400MHz,DMSO-d6)δ13.30(bs,1H),8.50-8.49(d,J＝5.4Hz,1H),8.42(s,1H),8.07(s,1H),8.03(s,1H),7.74(s,1H),6.72-6.71(d,J＝5.4Hz,1H),3.85-3.82(m,2H),3.76-3.71(m,1H),3.34-3.29(m,2H),2.83-2.76(m,1H),1.72-1.67(m,4H),1.12-0.97(m,4H)
实施例7 化合物007的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(1-(氧杂环丁-3-基)-1H-吡唑-4-基)噻吩并[3,2-b]吡啶的合成
Figure PCTCN2020138367-appb-000034
制备方法同实施例3，不同之处为用商业可得的1-(氧杂环丁烷-3-基)-4-(4,4,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)-1H-吡唑(CAS:1339890-99-1)代替4-甲烷磺酰苯硼酸，制得标题化合物。LC-MS m/z:464.2[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.53(s,1H),8.47-8.46(d,J＝5.4Hz,1H),8.16(s,1H),7.99(s,1H),7.73(s,1H),6.71-6.70(d,J＝5.4Hz,1H),5.65-5.61(m,1H),4.96-4.93(m,4H),3.81-3.78(m,2H),3.72-3.66(m,1H),3.31-3.25(m,2H),2.79-2.72(m,1H),1.68-1.63(m,4H),1.08-0.93(m,4H)
实施例8 化合物008的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(1-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)噻吩并[3,2-b]吡啶的合成
Figure PCTCN2020138367-appb-000035
制备方法同实施例3，不同之处为用商业可得的1-(四氢吡喃-4-基)-1H-吡唑-4-硼酸频哪醇酯(CAS:1040377-03-4)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:492.2[M+H] +。
1H NMR(400MHz,DMSO-d6)δ8.52-8.50(m,2H),8.08-8.05(d,2H),7.75(s,1H),6.76-6.75(d,J＝5.4Hz,1H),4.55-4.49(m,1H),4.06-4.03(m,2H),3.87-3.84(m,2H),3.78-3.73(m,1H),3.57-3.51(m,2H),3.37-3.31(m,2H),2.85-2.78(m,1H),2.08-1.97(m,4H),1.74-1.69(m,4H),1.14-0.99(m,4H)
实施例9 化合物009的制备
1-(4-(7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基]噻吩并[3,2-b]吡啶-2-基)-1H-吡唑-1-基)-2-甲基丙烷-2-醇的合成
Figure PCTCN2020138367-appb-000036
制备方法同实施例3,不同之处为用商业可得的2-甲基-1-[4-(四甲基-1,3,2-二噁硼戊环-2-基)-1H-吡唑-1-基]丙烷-2-醇(CAS:1082503-77-2)代替4-甲烷磺酰苯硼酸，制得标题化合物。LC-MS m/z:480.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.46-8.45(d,J＝5.4Hz,1H),8.23(s,1H),8.00(s,2H),7.70(s,1H),6.70-6.69(d,J＝5.4Hz,1H),4.79(s,1H),4.07(s,2H),3.81-3.78(m,2H),3.72-3.67(m,1H),3.31-3.25(m,2H),2.79-2.72(m,1H),1.68-1.63(m,4H),1.11-0.93(m,10H)
实施例10 化合物010的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(1-甲基-1H-吡唑-4-基)噻吩并[3,2-b]吡啶的合成
Figure PCTCN2020138367-appb-000037
制备方法同实施例3，不同之处为用商业可得的1-甲基吡唑-4-硼酸频哪醇酯(CAS:761446-44-0)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:422.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.46-8.45(d,J＝5.4Hz,1H),8.31(s,1H),7.99(s,2H),7.67(s,1H),6.70-6.69(d,J＝5.4Hz,1H),3.90(s,3H),3.81-3.78(m,2H),3.72-3.68(m,1H),3.29-3.25(m,2H),2.77-2.74(m,1H),1.68-1.66(m,4H),1.06-0.95(m,4H)。
实施例12 化合物012的制备
7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)-2-(1-(3-氟丙基)-1H-吡唑-4-基)噻吩并[3,2-b]吡啶的合成
Figure PCTCN2020138367-appb-000038
将化合物006(204mg，0.50mmol)加入到5mL的N,N-二甲基甲酰胺中，加入碳酸铯(325mg,1.00mmol)和3-氟-1-碘代丙烷(95.0mg,0.50mml)，加热至80℃搅拌直至TLC监控反应完全。向反应液中加入50mL水，乙酸乙酯萃取，有机层用无水硫酸钠干燥后浓缩，柱层析分离得标题化合物。
1H NMR(400MHz,DMSO-d 6)δ8.48-8.46(m,1H),8.41(s，1H),8.04(s，1H),8.00(s，1H),7.70(s,1H),6.72-6.70(m,1H),4.57-4.55(m,1H),4.46-4.43(m,1H),4.31-4.28(m,2H),3.82-3.79(m,2H),3.72-3.70(m,1H),3.29-3.26(m,2H),2.78-2.74(m,1H),2.28-2.18(m,2H),1.68-1.67(m,4H),1.07-0.96(m,4H).
LC-MS m/z:468.2[M+H] +。
实施例13 化合物013的制备
Figure PCTCN2020138367-appb-000039
制备方法同实施例12，不同之处为用商业可得的碘环丁烷(CAS:38557-29-8)代替3-氟-1-碘代丙烷(CAS:462-40-8)，制得标题化合物。
LC-MS m/z:462.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.48-8.47(m,2H),8.05-8.01(m,2H),7.71(s,1H),6.72-6.71(m,1H),4.93-4.89(m,1H),3.83-3.80(m,2H),3.72-3.70(m,1H),3.28-3.24(m,4H),2.78-2.76(m,1H),2.45-2.43(m,2H),1.86-1.84(m,2H),1.70-1.66(m,4H),1.08-0.97(m,4H).
实施例14 化合物014的制备
Figure PCTCN2020138367-appb-000040
制备方法同实施例12，不同之处为用商业可得的1,1-二氟-2-碘代乙烷(CAS:598-39-0)代替3-氟-1-碘代丙烷(CAS:462-40-8)，制得标题化合物。
LC-MS m/z:472.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.49-8.47(m,1H),8.41(s,1H),8.13(s,1H),8.00(s,1H),7.75(s,1H),6.73-6.72(m,1H),6.57-6.30(m,1H),4.75-4.66(m,2H),3.82-3.70(m,3H),3.30-3.28(m,2H),2.76-2.74(m,1H),1.67-1.66(m,4H),1.07-0.96(m,4H).
实施例15 化合物015的制备
Figure PCTCN2020138367-appb-000041
制备方法同实施例12，不同之处为用商业可得的2,2,2-三氟乙基三氟甲烷磺酸酯(CAS:6226-25-1)代替3-氟-1-碘代丙烷(CAS:462-40-8)，制得标题化合物。
LC-MS m/z:490.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.50-8.47(m,2H),8.19(s,1H),8.00(s,1H),7.79(s,1H),6.74-6.73(m,1H),5.24-5.21(m,2H),3.82-3.69(m,3H),3.29-3.27(m,2H),2.78-2.74(m,1H),1.69-1.66(m,4H),1.07-0.96(m,4H).
实施例16 化合物016的制备
Figure PCTCN2020138367-appb-000042
步骤1：叔丁基3-(4-(7-((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-基)氧基)噻吩并[3,2-b]吡啶-2-基)-1H-吡唑-1-基)氮杂环丁烷-1-甲酸
Figure PCTCN2020138367-appb-000043
将化合物006(408mg，1.00mmol)加入到5mL的N,N-二甲基甲酰胺中，加入碳酸铯(325mg,1.00mmol)和1-Boc-3-碘氮杂环丁烷(285mg,1.00mml)，加热至80℃搅拌直至TLC监控反应完全。向反应液中加入50mL水，乙酸乙酯萃取，有机层用无水硫酸钠干燥后浓缩，柱层析分离得标题化合物。
LC-MS m/z:563.2[M+H] +。
步骤2：2-(1-(氮杂环丁烷-3-基)-1H-吡唑-4-基)-7-(((1-环丙基-3-(四氢-2H-吡喃-4-基)-1H-吡唑-4-(基)氧基)噻吩并[3,2-b]吡啶
Figure PCTCN2020138367-appb-000044
将步骤1所得产物(284mg，0.50mmol)加入到5mL的氯化氢二氧六环溶液中，室温搅拌直至TLC监控反应完全。减压浓缩得到粗品，直接投入下一步。
LC-MS m/z:463.2[M+H] +。
步骤3：化合物016的制备
Figure PCTCN2020138367-appb-000045
将上一步所得粗品(232mg，0.50mmol)加入到5mL的N,N-二甲基甲酰胺中，加入碳酸铯(325mg,1.00mmol)和3-氟-1-碘代丙烷(95.0mg,0.50mml)，室温搅拌，TLC监控至反应完全。向反应液中加入50mL水，乙酸乙酯萃取，有机层用无水硫酸钠干燥后浓缩，柱层析分离得标题化合物。
LC-MS m/z:523.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.51(s,1H),8.47-8.45(m,1H),8.08(s,1H),7.98(s,1H),7.71(s,1H),6.71-6.69(m,1H),5.05-5.01(m,1H),4.57-4.54(m,1H),4.45-4.42(m,1H),3.78-3.71(m,5H),3.36-3.28(m,4H),2.77-2.73(m,1H),2.61-2.58(m,2H),1.75-1.65(m,6H),1.06-0.97(m,4H).
实施例17 化合物017的制备
Figure PCTCN2020138367-appb-000046
制备方法同实施例12，不同之处为用商业可得的1，3，2-二氧硫烷，5，5-二氟，2，2-二氧化物CAS:1215071-15-0)代替3-氟-1-碘代丙烷(CAS:462-40-8)，制得标题化合物。
LC-MS m/z:502.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.48-8.46(m,1H),8.38(s,1H),8.10(s,1H),7.99(s,1H),7.75(s,1H),6.72-6.71(m,1H),5.80(br,1H),4.78-4.70(m,2H),3.81-3.67(m,5H),3.29-3.28(m,2H),2.77-2.73(m,1H),1.68-1.65(m,4H),1.06-0.95(m,4H).
实施例18 化合物018的制备
Figure PCTCN2020138367-appb-000047
制备方法同实施例12，不同之处为用商业可得的2,2-二氟丙基三氟甲烷磺酸酯(CAS:784193-15-3)代替3-氟-1-碘代丙烷(CAS:462-40-8)，制得标题化合物。
LC-MS m/z:486.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.48-8.46(m,1H),8.38(s,1H),8.11(s,1H),7.99(s,1H),7.75(s,1H),6.72-6.71(m,1H),4.71-4.68(m,2H),3.81-3.69(m,3H),3.28-3.26(m,2H),2.76-2.74(m,1H),1.71-1.62(m,7H),1.06-0.95(m,4H).
实施例19 化合物019的制备
Figure PCTCN2020138367-appb-000048
制备方法同实施例3，不同之处为用商业可得的1-(二氟甲基)-4-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-1H-吡唑(CAS：1206640-82-5)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:458.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.90(s,1H),8.52-8.50(m,1H),8.41(s,1H),7.99(s,1H),7.90(s,1H),8.02-7.73(m,1H),6.76-6.74(m,1H),3.81-3.69(m,3H),3.32-3.25(m,2H),2.76-2.74(m,1H),1.69-1.66(m,4H),1.06-0.96(m,4H).
实施例20 化合物020的制备
Figure PCTCN2020138367-appb-000049
制备方法同实施例3，不同之处为用商业可得的1-甲基-1H-吡唑-5-硼酸频哪醇酯(CAS：847818-74-0)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:422.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.47-8.45(m,1H),7.54(s,1H),7.47(s,1H),7.33(s,1H),6.61-6.60(m,1H),6.49(s,1H),4.04(s,3H),3.89-3.86(m,2H),3.54-3.50(m,1H),3.35-3.29(m,2H),2.77-2.74(m,1H),1.84-1.68(m,4H),1.07-0.95(m,4H).
实施例21 化合物021的制备
Figure PCTCN2020138367-appb-000050
制备方法同实施例3，不同之处为用商业可得的苯硼酸(CAS:98-80-6)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:418.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.50-8.48(m,1H),7.77-7.75(m,3H),7.49-7.41(m,4H),6.63-6.62(m,1H),3.97-3.93(m,2H),3.60-3.58(m,1H),3.43-3.36(m,2H),2.86-2.83(m,1H),1.92-1.76(m,4H),1.13-1.04(m,4H).
实施例22 化合物022的制备
Figure PCTCN2020138367-appb-000051
制备方法同实施例3，不同之处为用商业可得的4-三氟甲氧基苯硼酸(CAS:139301-27-2)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:502.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.51-8.50(m,1H),7.78-7.73(m,3H),7.41(s,1H),7.33-7.31(m,2H),6.65-6.64(m,1H),3.97-3.94(m,2H),3.60-3.59(m,1H),3.43-3.36(m,2H),2.86-2.82(m,1H),1.92-1.76(m,4H),1.14-1.04(m,4H).
实施例23 化合物023的制备
Figure PCTCN2020138367-appb-000052
制备方法同实施例3，不同之处为用商业可得的2-甲基吲唑-5-硼酸频哪醇酯(CAS:1189746-27-7)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:472.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.41-8.40(m,1H),7.97(s,1H),7.92(s,1H),7.72-7.61(m,3H),7.34(s,1H),6.55-6.54(m,1H),4.18(s,3H),3.90-3.87(m,2H),3.52-3.51(m,1H),3.36-3.30(m,2H),2.80-2.76(m,1H),1.86-1.70(m,4H),1.08-0.96(m,4H).
实施例24 化合物024的制备
Figure PCTCN2020138367-appb-000053
制备方法同实施例3，不同之处为用商业可得的1-甲基-1H-苯并咪唑-5-硼酸那醇酯(CAS:1107627-02-0)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:472.2[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.51-8.49(m,1H),7.94(s,1H),7.89-7.86(m,1H),7.78(s,1H),7.76(s,1H),7.73-7.71(m,1H),7.42(s,1H),6.64-6.62(m,1H),4.0-3.97(m,1H),3.94(s,3H),3.63-3.58(m,1H),3.44-3.38(m,2H),2.89-2.82(m,2H),1.97-1.87(m,2H),1.80-1.77(m,2H),1.17-1.12(m,2H),1.08-1.03(m,2H).
实施例25 化合物025的制备
Figure PCTCN2020138367-appb-000054
制备方法同实施例3，不同之处为用商业可得的6-(2-羟基丙烷-2-基)吡啶-3-硼酸(CAS:1088496-42-7)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:477.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ9.04-9.04(m,1H),8.57-8.56(m,1H),8.29-8.26(m,1H),8.16(s,1H),8.03(s,1H),7.82-7.80(m,1H),6.81-6.79(m,1H),5.39(s,1H),3.83-3.80(m,2H),3.75-3.70(m,1H),3.32-3.30(m,2H),2.83-2.75(m,1H),1.72-1.67(m,4H),1.51-1.49(m,6H),1.11-1.08(m,2H),1.01-0.96(m,2H).
实施例26 化合物026的制备
Figure PCTCN2020138367-appb-000055
制备方法同实施例16，不同之处为将步骤3中的3-氟-1-碘代丙烷替换为甲基磺酸酐(CAS：7143-01-3)制得标题化合物。
LC-MS m/z:541.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.53(s,1H),8.50-8.48(m,1H),8.21(s,1H),8.00(s,1H),7.76(s,1H),6.73-6.72(m,1H),5.39-5.32(m,1H),4.35-4.33(m,4H),3.82-3.80(m,2H),3.72-3.70(m,1H),3.32-3.30(m,2H),3.16(s,3H),2.81-2.73(m,1H),1.68-1.67(m,4H),1.07-1.06(m,2H),0.98-0.96(m,2H).
实施例27 化合物027的制备
Figure PCTCN2020138367-appb-000056
制备方法同实施例16，不同之处为将步骤1中的1-Boc-3-碘氮杂环丁烷替换为N-Boc-4-碘哌啶，同时将步骤3中的3-氟-1-碘代丙烷替换为将环丙磺酰氯(CAS：139631-62-2)制得标题化合物。
LC-MS m/z:595.2[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.40-8.38(s,1H),7.77(s,1H),7.71(s,1H),7.41(s,1H),7.33(s,1H),6.54-6.53(m,1H),4.30-4.22(m,1H),3.93-3.86(m,4H),3.55-3.50(m,1H),3.35-3.29(m,2H),3.03-2.97(m,2H),2.79-2.72(m,1H),2.29-2.22(m,3H),2.17-2.07(m,2H),1.87-1.77(m,2H),1.72-1.70(m,2H),1.17-1.13(m,2H),1.09-1.04(m,2H),1.00-0.95(m,4H).
实施例28 化合物028的制备
Figure PCTCN2020138367-appb-000057
制备方法同实施例16，不同之处为将步骤3中的3-氟-1-碘代丙烷替换为的2,2,2-三氟乙基三氟甲烷磺酸酯(CAS:6226-25-1)制得标题化合物。
LC-MS m/z:545.2[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.40-8.38(s,1H),7.81-7.80(m,2H),7.42(s,1H),7.33(s,1H),6.54-6.53(m,1H),5.00-4.97(m,1H),3.99-3.95(m,2H),3.89-3.86(m,2H),3.78-3.74(m,2H),3.55-3.49(m,1H),3.35-3.29(m,2H),3.16-3.09(m,2H),2.80-2.72(m,1H),1.87-1.80(m,2H),1.70-1.68(m,2H),1.08-1.03(m,2H),1.00-0.95(m,2H).
实施例29 化合物029的制备
Figure PCTCN2020138367-appb-000058
制备方法同实施例3，不同之处为用商业可得的对甲氧基苯硼酸(CAS:5720-07-0)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:545.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.51-8.50(m,1H),8.00(s,1H),7.91(s,1H),7.85-7.83(m,2H),7.10-7.08(m,2H),6.75-6.73(m,1H),3.85(s,3H),3.84-3.79(m,2H),3.74-3.69(m,1H),3.34-3.28(m,2H),2.82-2.74(m,1H),1.71-1.66(m,4H),1.10-1.06(m,2H),1.00-0.95(m,2H).
实施例30 化合物030的制备
Figure PCTCN2020138367-appb-000059
制备方法同实施例3，不同之处为用商业可得的4-三氟甲基苯硼酸(CAS:128796-39-4)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:486.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.57-8.56(m,1H),8.24(s,1H),8.14-8.12(m,2H),8.02(s,1H),7.89-7.87(m,2H),6.82-6.81(m,1H),3.82-3.78(m,2H),3.73-3.68(m,1H),3.29-3.26(m,2H),2.81-2.74(m,1H),1.70-1.64(m,4H),1.09-1.05(m,2H),0.99-0.94(m,2H).
实施例31 化合物031的制备
Figure PCTCN2020138367-appb-000060
制备方法同实施例3，不同之处将步骤1中的2-溴-7-氯噻吩并[3,2-b]吡啶替换为7-氯-2-碘呋喃并[3,2-B]吡啶(CAS：1071540-54-9)，同时将步骤2中的4-甲烷磺酰苯硼酸替换为1-甲基-4-(4,4,5,5-四甲基-1,3,2-二氧杂戊硼烷-2-基)-1H-吡唑(CAS：761446-44-0)，制得标题化合物。
LC-MS m/z:406.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.35(s,1H),8.28-8.27(m,1H),7.99(s,1H),7.94(s,1H),7.18(s,1H),6.66-6.61(m,1H),3.92(s,3H),3.82-3.78(m,2H),3.72-3.66(m,1H),3.34-3.26(m, 2H),2.82-2.74(m,1H),1.71-1.66(m,4H),1.07-1.03(m,2H),0.97-0.92(m,2H).
实施例32 化合物032的制备
Figure PCTCN2020138367-appb-000061
制备方法同实施例3，不同之处为用商业可得的(1-(4-氟苯基)-1H-吡唑-4-基)硼酸(CAS:1072945-89-1)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:502.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ9.13(s,1H),8.50-8.49(m,1H),8.35(s,1H),8.00(s,1H),7.98-7.94(m,2H),7.84(s,1H),7.44-7.39(m,2H),6.74-6.72(m,1H),3.82-3.78(m,2H),3.73-3.67(m,1H),3.30-3.26(m,2H),2.81-2.73(m,1H),1.70-1.64(m,4H),1.09-1.05(m,2H),0.98-0.93(m,2H).
实施例33 化合物033的制备
Figure PCTCN2020138367-appb-000062
制备方法类似于实施例16，不同之处为将步骤1中的1-Boc-3-碘氮杂环丁烷替换为N-Boc-4-碘哌啶，同时将步骤3中的3-氟-1-碘代丙烷替换为将2,2,2-三氟乙基三氟甲烷磺酸酯(CAS:6226-25-1)制得标题化合物。
LC-MS m/z:573.2[M+H] +。
1H NMR(400MHz,CDCl3)δ8.46-8.44(m,1H),7.83(s,1H),7.78(s,1H),7.46(s,1H),7.40(s,1H),6.60-6.59(m,1H),4.23-4.16(m,1H),3.97-3.93(m,2H),3.62-3.57(m,1H),3.43-3.36(m,2H),3.15-3.12(m,2H),3.10-3.03(m,2H),2.87-2.79(m,1H),2.64-2.58(m,2H),2.21-2.11(m, 4H),1.95-1.84(m,2H),1.79-1.78(m,2H),1.16-1.11(m,2H),1.08-1.02(m,2H).
实施例34 化合物034的制备
Figure PCTCN2020138367-appb-000063
制备方法类似于实施例12，不同之处为将3-氟-1-碘代丙烷替换为2-[1-(乙基磺酰基)-3-氮杂环丁亚基]乙腈(CAS:1187595-85-2)，制得标题化合物。
LC-MS m/z:594.2[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.49-8.48(m,1H),7.96(s,1H),7.94(s,1H),7.54(s,1H),7.41(s,1H),6.64-6.62(m,1H),4.61-4.59(m,2H),4.24-4.22(m,2H),3.97-3.94(m,2H),3.61-3.57(m,1H),3.43-3.37(m,4H),3.12-3.07(m,2H),2.87-2.80(m,1H),1.95-1.84(m,2H),1.79-1.76(m,2H),1.44-1.41(m,3H),1.16-1.11(m,2H),1.07-1.02(m,2H).
实施例35 化合物035的制备
Figure PCTCN2020138367-appb-000064
制备方法同实施例3，不同之处为用5-甲基-3-(4,4,5,5-四甲基-1,3,2-二氧杂硼烷-2-基)-4,5,6,7-四氢吡唑并[1,5-a]吡嗪(CAS:2057507-40-9)代替4-甲烷磺酰苯硼酸，制得标题化合物。
LC-MS m/z:477.2[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ8.50-8.48(m,1H),8.00(m,2H),7.53(s,1H),6.72-6.70(m,1H),4.20-4.18(m,2H),3.89(s,2H),3.84-3.79(m,2H),3.74-3.68(m,1H),3.50-3.27(m,5H),2.94-2.92(m,2H),2.81-2.73(m,1H),1.70-1.65(m,4H),1.10-1.06(m,2H),1.00-0.95(m,2H).生 物学活性及相关性质测试例
测试例1、本发明化合物对TGFβRI激酶活性的抑制作用的测定
本发明化合物对TGFβRI激酶ALK5活性的抑制作用采用如下的方法进行测试。
酶活性检测使用ADP-Glo激酶检测试剂盒(V9102/3，Promega)。在384孔板(Cat.No.4512，Corning)中依次加入50nl溶于100％DMSO的3倍梯度稀释的化合物(测试首浓度为10μM)，2.5μl用反应缓冲液(40mM Tris,pH 7.5；20mM MgCl2；0.10％BSA；1mM DTT)配制的ALK5(Cat.09-141，Carna)酶溶液(终浓度12.5nM)，2.5μl反应缓冲液后室温孵育10分钟。加入2.5μl ATP和TGFβRI底物多肽的混合溶液(多肽：Cat.T36-58，SignalChem，多肽终浓度0.1mg/ml，ATP在Km浓度下)，28℃反应2小时后，每孔加入5μl试剂盒中的ADP-Glo溶液，28℃放置30分钟。使用Envision多功能酶标仪检测化学发光信号值。根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC 50值。
本发明化合物的生物活性通过以上的试验进行测定，测得的IC 50值见下表1.
表1本发明化合物对TGFβRI激酶ALK5活性抑制的IC 50
测试化合物 IC 50(nM)
001 19
002 15
003 17
004 104
005 47
006 163
007 40
008 17
009 63
010 49
012 33
013 63
014 30
015 56
016 30
017 82
018 89
019 103
020 75
021 100
022 546
023 50
024 71
025 34
026 21
027 19
028 44
029 151
030 279
031 733
032 571
033 63
034 28
035 199
测试例2、本发明化合物对p38α激酶活性的抑制作用的测定
以下所述实验方法用来测定本发明化合物对p38α激酶活性的抑制作用：
酶活性检测使用p38α激酶检测试剂盒(V9591，Promega)，在384孔板(Cat.No.4512，Corning)中依次加入5μl溶于100％DMSO的3倍梯度稀释的化合物(测试首浓度为10μM)，10μl用反应缓冲液(50mM HEPES,pH 7.5；0.0015％Brij-35)配制的p38α酶(Cat.04-152，Carna)溶液(终浓度6nM)，室温放置10分钟。加入10μl ATP和p38底物多肽5-FAM-IPTSPITTTYFFFKKK-COOH(Cat.112396，GL)的混合溶液(多肽终浓度3μM，ATP在Km浓度下)，28℃反应60min，每孔加入25μl终止液(100mM HEPES,pH 7.5；0.015％Brij-35；0.2％Coating Reagent#3；50mM EDTA)终止反应。使用Envision多功能酶标仪检测化学发生信号值。根据化合物各浓度与相应的信号值计算化合物对酶抑制作用的IC 50值。
本发明化合物的生物活性通过以上的试验进行测定，测得IC 50值见下表2.
表2本发明化合物对p38α激酶活性的抑制作用的IC 50
测试化合物 IC 50(nM)
004 >10000
005 >10000
007 >10000
009 >10000
010 >10000
测试例3、本发明化合物对TGFβRI的Smad信号通路的抑制活性的测定
以下所述实验方法用来测定本发明化合物对TGFβRI的Smad信号通路的抑制活性：
本实施例采用TGFβ/SMAD报告基因细胞系(BPS Bioscience，Cat.60653)检测分子对于TGF-β1蛋白信号通路的抑制作用。TGF-β1蛋白与细胞表面的受体结合，启动信号级联反应，导致SMAD2和SMAD3磷酸化和活化，并与SMAD4形成复合物。然后，SMAD复合物转移至细胞核并与细胞核中的SMAD结合元件结合，从而导致TGFβ/SMAD响应基因的转录和表达。TGFβ/SAMD报告基因细胞系包含萤火虫荧光素酶基因，该基因稳定整合到HEK293细胞中，并受SMAD响应元件的控制。当细胞受到人TGF-β1的刺激时，细胞表达萤火虫荧光素酶，加入底物后，荧光素酶催化底物发光，可以于酶标仪上读取光强度数值。加入待测 分子后，如果其可以抑制TGF-β1的信号通路，则能够检测到读值的降低。
将细胞以0.35×10 6个/mL的密度，每孔100μl加入96孔白不透板(Corning，Cat.3917)中，于37℃，5％CO2培养箱中培养24小时。将待检测的分子以5μM的起始浓度进行4倍梯度稀释，共7个梯度。随后将稀释后的分子与2μg/ml的TGF-β1蛋白(Perpotech，Cat.100-21)以1：1的比例混合，每孔100μl加入培养过夜的细胞，每个梯度3复孔。继续于37℃，5％CO2培养箱中培养18个小时后，每孔100μl加入荧光素酶底物(Promega，G7940)，于酶标仪上检测。将读值以4参数方程进行拟合，得到IC 50值。
本发明化合物的生物活性通过以上的试验进行测定，测得IC 50值见下表3。
表3本发明化合物对Smad信号通路的抑制作用的IC 50
测试化合物 IC 50(nM)
001 18.31
002 32.90
003 13.06
004 64.28
005 56.80
006 48.66
007 51.52
008 26.77
009 64.24
010 37.13
测试例4、本发明化合物的药代动力学测试
以小鼠为受试动物，应用LC/MS/MS法测定了小鼠灌胃和静脉给予本发明化合物后，不同时刻血浆中的药物浓度。研究本发明化合物在大鼠体内的药代动力学行为，评价其药动学特征。
试验方案
2.1试验药品
阳性对照组化合物、化合物001、004、005、007、009、010。
2.2试验动物
健康成年BALB/c小鼠42只，雄性，平均分为7组，每组6只，3只灌胃，3只静脉，小鼠购自北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(浙)2019-0001。
2.3药物配制
称取一定量药物，溶于20％PG 5％乙醇75％水，配制成1mg/ml，用于灌胃。称取一定量药物，溶于4％PG 1％乙醇95％水，配制成0.2mg/ml，用于静脉注射。
2.4给药
灌胃组：BALB/c小鼠禁食过夜后灌胃给药，给药剂量均为10mg/kg，给药体积均为 10mL/kg。
静脉组：BALB/c小鼠禁食过夜后静脉给药，给药剂量均为1mg/kg，给药体积均为5mL/kg。
操作
小鼠灌胃或静脉给药后，于给药后5min，15min，30min，1h，2h，6h，24h由眼眶采血40μL，5μL EDTA-K2抗凝，12000rpm，4℃，5分钟离心分离血浆，于-20℃保存。
测定不同浓度的药物灌胃或静脉给药后小鼠血浆中待测化合物含量：取样品室温融解，涡旋1min；定量转移15μL至2ml 96孔板中，加入150μL内标沉淀剂，振荡(1200rpm*3min)；离心(4000rpm*15min)，转移上清100μL至1ml 96孔板中；氮气吹干，加入100μL复溶液(乙腈水1：9)，振荡摇匀(900rpm*3min),20μL进样分析。LC/MS/MS条件：流动相A:0.1％甲酸水溶液，流动相B：0.1％甲酸乙腈，色谱柱：ACE C18 5μm(3.0mm*50mm)，柱温：35℃，流速0.5ml/min。
本发明化合物的药代动力学参数如下：
表5小鼠单次静脉注射化合物后药代动力学参数
Figure PCTCN2020138367-appb-000065
表6小鼠单次灌胃给予化合物后药代动力学参数
Figure PCTCN2020138367-appb-000066
测试例5、本发明化合物对CT26荷瘤鼠抗肿瘤药效测试
1、实验目的
评价化合物001、化合物002、化合物005、化合物007、化合物009、化合物010抑制鼠结肠癌细胞CT26移殖瘤在BALB/c小鼠上生长的作用。
2、实验药品
化合物001、004、005、007、009、010用20％PG+5％乙醇+10％solutol+65％水配制成3mg/ml的水溶液，给药剂量为30mg/kg，口服灌胃体积为10ml/kg。
阳性对照药物mPD-1-RMP1-14(购自bioxcell，Catalog Number：BE0188)通过腹腔注射。
3、实验方法和实验材料
3.1实验动物和饲养条件
实验用BALB/c小鼠，雌性，购自北京维通利华实验动物科技有限公司，购入时16-18g。动物购入后，进行3天适应性饲养后开始实验。饲养条件：SPF级。动物饲养方式：给予12/12小时光/暗周期调节，温度20-26℃，湿度40-70％，动物均给予标准灭菌鼠饲料，自由进食饮水。
3.2模型购建
小鼠右肋部提前一天备皮，皮下接种CT26细胞(1*10^5/只，购自ATCC)，待肿瘤体积长至60～80mm 3左右时进行分组给药。
3.3动物分组
肿瘤体积达到分组条件后，按照肿瘤体积进行随机分组，每组6只。
受试物 剂量(mg/kg) 给药方式 给药频率
空白对照组 NA IG Bid
mPD-1-RMP1-14 10 IP Biw
001 30 IG Bid
004 30 IG Bid
005 30 IG Bid
007 30 IG Bid
009 30 IG Bid
010 30 IG Bid
注：Bid为一天给药2次；Biw为一周给药2次；IG为灌胃给药，IP为腹腔注射给药
3.4实验方法
动物分组后当天，开始给药。每只小鼠按照上表的给药频率进行给药，空白组灌胃20％PG+5％乙醇+10％solutol+65％水配制成3mg/ml的水溶液。每周测量肿瘤体积3次，称量动物体重3次。共给药2周。
3.5数据统计
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对肿瘤体积(RTV)＝V T/V 0
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。
相对肿瘤增殖率T/C(％)＝T RTV/C RTV
C RTV、T RTV分别为实验结束时的空白对照组及实验组的相对肿瘤体积。
4、结果
化合物1对CT26荷瘤鼠肿瘤疗效数据见表7。
表7本发明化合物对CT26荷瘤鼠移植瘤的疗效
实施例编号 RTV T/C(％)
空白对照 4.01 -
mPD-1-RMP1-14 2.16 53.80
001 0.86 21.49
004 0.92 22.93
005 1.42 35.42
007 1.84 46.13
009 0.98 24.53
010 1.52 37.98

Claims (13)

  1. 一种式(A)所示化合物或其药学上可接受的盐：
    Figure PCTCN2020138367-appb-100001
    其中环Q选自苯基或5-6元杂芳基；
    R 1、R 2独立选自氢、F、Cl、Br、I、CN、OH、NO 2或任选被R a取代的以下基团：NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、3-10元杂环基磺酰基、氨基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基、5-10元杂芳基氧基、C 6-C 10芳基磺酰基、5-10元杂芳基磺酰基；
    每一个R a独立地选自F、Cl、Br、I、OH、CN、＝O、NO 2或任选被R b取代的下列基团：NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、3-10元杂环基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 2-C 10烯氧基、C 2-C 10炔氧基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基、5-10元杂芳基氧基、C 6-C 10芳基磺酰基、5-10元杂芳基磺酰基；
    每一个R b独立选自F、Cl、Br、I、OH、CN、＝O、NO 2、NH 2、SH、C 1-C 10烷基、C 3-C 10环烷基、3-10元杂环基、C 1-C 10烷氧基、C 3-C 10环烷基氧基、3-10元杂环基氧基、C 1-C 10烷基磺酰基、C 3-C 10环烷基磺酰基、C 2-C 10烯基、C 2-C 10炔基、C 6-C 10芳基、5-10元杂芳基、C 6-C 10芳基氧基或5-10元杂芳基氧基，所述C 1-C 10烷基任选被F、Cl、Br、I、CN取代。
  2. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述环Q为苯基、噻吩基或呋喃基。
  3. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述R 1、R 2独立选自H、C 1-C 6烷氧基、3-10元杂环基、C 6-C 10芳基或5-10元杂芳基，所述C 1-C 6烷氧基、3-10元杂环基、C 6-C 10芳基或5-10元杂芳基任选被R a取代。
  4. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述环Q为噻吩基，所述R 1、R 2独立选自H或任选被R a取代的下列基团：3-10元杂环基、C 6-C 10芳基或5-10元杂芳基。
  5. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述环Q为噻吩基，所述R 1、R 2独立选自H或任选被R a取代的下列基团：苯基、吡啶基、吡唑基、四氢吡啶基、
    Figure PCTCN2020138367-appb-100002
    氮杂环丁烷基、苯并吡唑基、苯并咪唑基。
  6. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述环Q为苯基，所述R 1、R 2独立选自H或任选被R a取代的C 1-C 6烷氧基。
  7. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述环Q为呋喃基，所述R 1、R 2独立选自H或任选被C 1-C 6烷基取代的5-6元杂芳基。
  8. 根据权利要求1-6任一项所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，R a选自任选被R b取代的下列基团：C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 6环烷基、3-8元杂环基、C 1-C 10烷基磺酰基或苯基。
  9. 根据权利要求8所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，R b选自F、Cl、Br、I、OH、＝O、NH 2、C 1-C 6烷基、C 1-C 6烷氧基、C 3-C 8环烷基、3-8元杂环基、C 1-C 6烷基磺酰基、C 3-C 8环烷基磺酰基、苯基或5-6元杂芳基，所述C 1-C 6烷基任选被F、Cl、Br、I、CN取代。
  10. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述R 1、R 2独立选自氢原子、甲氧基、
    Figure PCTCN2020138367-appb-100003
    Figure PCTCN2020138367-appb-100004
    Figure PCTCN2020138367-appb-100005
  11. 根据权利要求1所述的式(A)所示化合物或其药学上可接受的盐，其特征在于，所述式(A)所示化合物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：
    Figure PCTCN2020138367-appb-100006
    Figure PCTCN2020138367-appb-100007
  12. 一种药物组合物，所述组合物包含权利要求1至11任一项的化合物或其药学上可接 受的盐，以及药学上可接受的辅料。
  13. 权利要求1至11任一项的化合物或其药学上可接受的盐、或权利要求12所述的药物组合物在制备预防或者治疗TGF-β介导的疾病的药物中的用途。
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