WO2020083377A1 - 吡咯烷基脲衍生物及其应用 - Google Patents

吡咯烷基脲衍生物及其应用 Download PDF

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WO2020083377A1
WO2020083377A1 PCT/CN2019/113278 CN2019113278W WO2020083377A1 WO 2020083377 A1 WO2020083377 A1 WO 2020083377A1 CN 2019113278 W CN2019113278 W CN 2019113278W WO 2020083377 A1 WO2020083377 A1 WO 2020083377A1
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compound
isomer
pharmaceutically acceptable
solution
acceptable salt
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French (fr)
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张杨
伍文韬
滕明星
李志祥
李婕
龚珍
黎健
陈曙辉
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Medshine Discovery Inc
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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
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/38—Nitrogen atoms
    • C07D231/40—Acylated on said nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • 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
    • 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
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems

Definitions

  • the present invention relates to a class of TrkA inhibitors and their application in the preparation of drugs for treating TrkA-related diseases. Specifically, it relates to the compounds represented by formula (I) and (II), their isomers and pharmaceutically acceptable salts thereof.
  • Promyosin-related kinase is a high-affinity receptor tyramine activated by a group of soluble growth factors called nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic factor (NT) Acid kinase, whose family consists of three members (TrkA, TrkB, TrkC).
  • NGF, BDNF, NT-4 / 5 play an important role in many physiological adjustment processes such as signal maintenance of neuronal cells, signal transmission of neuronal cells, cell proliferation, cell differentiation, and cell survival through the receptor Trk.
  • TrkA inhibitors described in the present invention can solve the treatment needs of pain, cancer, inflammation, neurodegenerative diseases and certain infectious diseases.
  • Patent WO2015175788 reports compounds having inhibitory activity against TrkA and pharmaceutically acceptable salts thereof (Reference Example 8: Reference compound D1).
  • the present invention provides compounds represented by formula (I), (II), isomers thereof or pharmaceutically acceptable salts thereof,
  • T 1 , T 2 and T 3 are independently selected from N and C (R 3 );
  • X 1 , X 2 and X 3 are independently selected from O, N (R 4 ) and C (R 5 ) (R 6 );
  • Z 1 , Z 2 and Z 3 are independently selected from N and CH;
  • R 1 is selected C 1-6 alkyl and C 1-6 alkoxy, said C 1-6 alkyl and C 1-6 alkoxy optionally substituted with 1, 2 or 3 R a;
  • R 2 is selected from C 1-3 alkyl optionally substituted with 1, 2 or 3 R b ;
  • R 3 is independently selected from H, F, Cl, Br, I, OH, and NH 2 ;
  • R 4 is independently selected from H and C 1-3 alkyl optionally substituted with 1, 2 or 3 R c ;
  • R 5 and R 6 are independently selected from H, F, Cl, Br, I, OH, and NH 2 ;
  • n is selected from 1, 2, and 3;
  • R a is selected from H, F, Cl, Br, I, OH, NH 2 , CN, C 1-3 alkyl and C 1-3 alkoxy, the C 1-3 alkyl and C 1-3 alkyl
  • the oxy group is optionally substituted with 1, 2 or 3 R;
  • R b and R c are independently selected from H, F, Cl, Br, I, OH and NH 2 ;
  • R is selected from F, Cl, Br, I, OH and NH 2 ;
  • the carbon atom with "*" is a chiral carbon atom and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer;
  • the carbon atom with "#" is a chiral carbon atom and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
  • R a is selected from H, F, Cl, Br,, OH, NH 2, CN, CH 3 , and CH 3 O, CH 3 a CH 3 O, and optionally substituted with 1,2 Or 3 R substitutions, other variables are as defined in the present invention.
  • R a is selected from H, F, Cl, Br, I, OH, NH 2, CN, CH 3, CH 2 F, CHF 2, CF 3 , and CH 3 O, other variables such as the present Definition of invention.
  • R 1 is selected from CH 3, CH 3 CH 2 and CH 3 O, the CH 3, CH 3 CH 2 and CH 3 O optionally substituted with 1, 2 or 3 R a, Other variables are as defined in the present invention.
  • R 1 is selected from Other variables are as defined in the present invention.
  • R 2 is selected from CH 3 and CH 3 CH 2 , and other variables are as defined in the invention.
  • R 4 is selected from H, CH 3 and CH 3 CH 2 , wherein the CH 3 and CH 3 CH 2 are optionally substituted with 1, 2 or 3 R c , and other variables are as described in the present invention definition.
  • R 4 is selected from H, CH 3 , CH 2 F, CHF 2 , CF 3 , CH 3 CH 2 and Other variables are as defined in the present invention.
  • T 1 , T 2 and T 3 are independently selected from N, CH and CF, and other variables are as defined in the present invention.
  • X 1 , X 2 and X 3 are independently selected from O, N (CH 3 ), NH, CH 2 and Other variables are as defined in the present invention.
  • the above compound, its isomer or pharmaceutically acceptable salt thereof is selected from
  • T 1 , T 2 and T 3 are independently selected from N and C (R 3 );
  • D is selected from N (R 4 ) and O;
  • R 1 , R 2 , R 3 and R 4 are as defined in the present invention.
  • the above compound, its isomer or pharmaceutically acceptable salt thereof is selected from
  • D is selected from N (R 4 ) and O;
  • R 1 , R 2 , R 3 and R 4 are as defined in the present invention.
  • the present invention also provides the following compounds, isomers or pharmaceutically acceptable salts thereof, which are selected from
  • the above compound, its isomer or pharmaceutically acceptable salt thereof is selected from
  • the present invention also provides the use of the above-mentioned compound, its isomer or a pharmaceutically acceptable salt thereof in the preparation of drugs related to the treatment of TrkA inhibitors.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and / or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit / risk ratio.
  • pharmaceutically acceptable salt refers to a salt of a compound of the present invention, prepared from a compound having a specific substituent and a relatively non-toxic acid or base found in the present invention.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Bisulfate, hydroiodic acid, phosphorous acid, etc .; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; also includes salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain basic and acidic functional groups and can be converted to any base or
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of appropriate base or acid in water or an organic solvent or a mixture of both.
  • the term “isomer” is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers isomer.
  • 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 enantiomerically or diastereomerically enriched mixtures, all of which belong to this Within the scope of the invention. Additional asymmetric carbon atoms may be present in the substituents such as alkyl. All these isomers and their mixtures are included in the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers in a mirror image relationship with each other.
  • cis-trans isomer or “geometric isomer” is caused by the fact that double bonds or single bonds of ring-forming carbon atoms cannot rotate freely.
  • diastereomer refers to a stereoisomer in which a molecule has two or more chiral centers and there is a non-mirror relationship between the molecules.
  • wedge-shaped solid line key And wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center
  • using straight solid line keys And straight dotted keys Represents the relative configuration of the three-dimensional center
  • wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dotted key Or with wavy lines Represents a straight solid line key And straight dotted keys
  • the following formula (A) indicates that the compound exists as a single isomer of formula (A-1) or (A-2) or as two isomers of formula (A-1) and formula (A-2) Exists in the form of a mixture;
  • the following formula (B) indicates that the compound exists as a single isomer of formula (B-1) or formula (B-2) or in both formula (B-1) and formula (B-2) There is a mixture of isomers.
  • the following formula (C) indicates that the compound exists as a single isomer of formula (C-1) or (C-2) or as two isomers of formula (C-1) and formula (C-2) In the form of a mixture.
  • tautomer or “tautomeric form” means that at room temperature, isomers of different functional groups are in dynamic equilibrium and can quickly convert to each other. If tautomers are possible (as in solution), the chemical equilibrium of tautomers can be achieved.
  • proton tautomers also called prototropic tautomers
  • proton migration such as ketone-enol isomerization and imine-ene Amine isomerization.
  • Valence tautomers include some recombination of bond-forming electrons for mutual conversion.
  • keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “rich in one isomer”, “isomer enriched”, “rich in one enantiomer” or “enantiomerically enriched” refer to one of the isomers or pairs
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater or equal 99.9%.
  • the terms “isomer excess” or “enantiomeric excess” refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the excess of isomer or enantiomer (ee value) is 80% .
  • optically active (R)-and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If an enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, in which the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then by conventional methods known in the art The diastereomers are resolved and the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography, which uses a chiral stationary phase, and is optionally combined with chemical derivatization methods (for example, the formation of amino groups from amines) Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes in one or more atoms constituting the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • the hydrogen can be replaced by heavy hydrogen to form a deuterated drug.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have lower toxicity and increase drug stability. , Strengthen the efficacy, extend the biological half-life of drugs and other advantages.
  • the conversion of all isotopic compositions of the compounds of the present invention, whether radioactive or not, is included within the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include heavy hydrogen and hydrogen variants, as long as the valence state of the specific atom is normal and the substituted compound is stable of.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents may be arbitrary on the basis of chemical realization.
  • any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent.
  • R when any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent.
  • the group can optionally be substituted with up to two Rs, and R in each case has independent options.
  • combinations of substituents and / or variants thereof are only allowed if such combinations will produce stable compounds.
  • linking group When the number of a linking group is 0, such as-(CRR) 0- , it means that the linking group is a single bond.
  • one of the variables When one of the variables is selected from a single bond, it means that the two groups to which it is connected are directly connected. For example, when L represents a single bond in A-L-Z, it means that the structure is actually A-Z.
  • substituents listed do not indicate through which atom they are connected to the substituted group, such substituents can be bonded through any of their atoms.
  • substituents can be bonded through any of their atoms.
  • pyridyl can be used as a substituent through any of the pyridine rings. The carbon atom is attached to the substituted group.
  • connection direction is arbitrary, for example,
  • the linking group L in the middle is -MW-, then -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right It can also be formed by connecting ring A and ring B in the opposite direction to the reading order from left to right
  • Combinations of the linking group, substituents, and / or variants thereof are only allowed if such a combination will produce a stable compound.
  • C 1-6 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 6 carbon atoms.
  • the C 1-6 alkyl group includes C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 and C 5 alkyl groups; etc .; Is monovalent (such as methyl), divalent (such as methylene) or multivalent (such as methine).
  • C 1-6 alkyl examples include but are not limited to methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl , S-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl and so on.
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc .; it may be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methine) .
  • Example C 1- 3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n- propyl and isopropyl) and the like.
  • the term “leaving group” refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, an affinity substitution reaction).
  • representative leaving groups include triflate; chlorine, bromine, and iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, and p-toluenesulfonate Ester, etc .; acyloxy, such as acetoxy, trifluoroacetoxy, etc.
  • C 1-6 alkoxy refers to those alkyl groups containing 1 to 6 carbon atoms connected to the rest of the molecule through one oxygen atom.
  • the C 1-6 alkoxy group includes C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-4 , C 6 , C 5 , C 4 and C 3 alkoxy groups, etc. .
  • C 1-6 alkoxy groups include but are not limited to methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy Oxy, s-butoxy and t-butoxy), pentyloxy (including n-pentyloxy, isopentyloxy and neopentyloxy), hexyloxy, etc.
  • C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms connected to the rest of the molecule by one oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy groups and the like.
  • Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C n-n + m or C n -C n + m includes any specific case of n to n + m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4, C 5, C 6, C 7, C 8, C 9, C 10, C 11, and C 12, also including any one of n + m to n ranges, for example C 1- 3 comprises a C 1-12 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc .; similarly, n yuan to n + m member means that the number of atoms in the ring is n to n + m, for example, 3-12 member ring includes 3 member ring, 4 member ring, 5 member ring, 6 member ring, 7 member ring, 8 member ring, 9 member ring , 10-membered ring, 11-membered ring, and 12-membered
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (eg, an affinity substitution reaction).
  • representative leaving groups include triflate; chlorine, bromine, and iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, and p-toluenesulfonate Ester, etc .; acyloxy, such as acetoxy, trifluoroacetoxy, etc.
  • protecting group includes but is not limited to "amino protecting group", “hydroxy protecting group” or “mercapto protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-methoxyphenyl) methyl; silyl, such as trimethylsilyl (TMS) and tert-butyld
  • hydroxyl protecting group refers to a protecting group suitable for preventing side reactions of hydroxyl groups.
  • Representative hydroxy protecting groups include but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl groups (such as acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl, such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl groups such as methyl, ethyl and tert-butyl
  • acyl groups such as alkanoyl groups (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-flu
  • 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 the combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalently, preferred embodiments include but are not limited to the embodiments of the present invention.
  • aq stands for water
  • HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurea hexafluorophosphate Eq stands for equivalent and equivalent
  • DCM stands for dichloromethane
  • PE stands for petroleum ether
  • DMF stands for N, N-dimethylformamide
  • DMSO stands for dimethyl sulfoxide
  • EtOAc stands for ethyl acetate
  • EtOH stands for ethanol
  • MeOH stands for Methanol
  • CBz stands for benzyloxycarbonyl, an amine protecting group
  • BOC stands for tert-butoxycarbonyl, an amine protecting group
  • HOAc stands for acetic acid
  • rt stands for room temperature
  • O / N stands for overnight
  • THF stands for tetrahydrofuran
  • Boc 2 O stands for di-tert-butyl dicarbonate
  • the compound of the present invention has significant TrkA enzyme inhibitory activity; the compound of the present invention has a higher Caco-2 cell membrane penetration efflux ratio, which can better avoid the risk of entering the brain and reduce the movement caused by the inhibition of Trk target in the central nervous system Side effects such as disorders and sleep disturbances; the compound of the present invention has a higher free binding rate of human plasma proteins, lower risk of drug-drug interactions, and better metabolic stability of liver microsomes; a single drug administration in rats
  • the compound of the present invention had higher plasma exposure and higher oral bioavailability than the reference compound D1; in the rat model of CFA-induced mechanical pain, the same oral
  • the compound of the present invention has a longer-lasting analgesic effect than the reference compound D1; the compound of the present invention has a longer half-life and a shorter peak time, which indicates that the effect may be faster and have a longer time in vivo It has good pharmacokinetic properties and oral bioavail
  • the reaction solution was diluted with 1L of water, and the diluted reaction solution was extracted with ethyl acetate (1L ⁇ 3).
  • the organic phases were combined, and the organic phase was washed successively with water (800mL ⁇ 3), saturated brine (1L), organic
  • the phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness.
  • reaction solution was diluted with 100 mL of ethyl acetate and washed once with saturated brine (80.0 mL).
  • the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness.
  • the obtained crude product was subjected to column chromatography (eluent: 20 -40% acetone / petroleum ether) was isolated and purified to obtain compound A1-5.
  • Compound A3-1 (38.50 g, 231.7 mmol) was dispersed in a mixed solvent of ethyl acetate (200.0 mL) and n-heptane (200.0 mL), followed by addition of trifluoroacetic acid (2.64 g, 23.2 mmol, 1.7 mL), and cooled to At 0 ° C, N- (methoxymethyl) -N- (trimethylsilylmethyl) benzylamine (137.53g, 579.3mmol) was slowly added dropwise. After the addition was completed, the temperature was naturally raised to 25 ° C and reacted for 20 hours.
  • reaction solution Concentrate the reaction solution to about 300.0mL, then add 300mL n-heptane, continue to concentrate to about 300.0mL, repeat the above operation 6 times, until the last time after adding 300mL n-heptane, filter, filter cake with n-heptane (100.0mL ⁇ 2) Wash and filter with suction twice, the filter cake is suction dried, and the filter cake is collected to obtain compound A3-2.
  • A3-2 (53.00 g, 177.06 mmol) was dispersed in toluene (400.0 mL), N, N-diisopropylethylamine (25.17 g, 194.77 mmol, 34.0 mL) was added, and protected with nitrogen, Subsequently, diphenyl azide phosphate (53.60g, 194.77mmol, 42.2mL) was slowly added dropwise. After the addition was completed, the reaction was performed at 25 ° C for 0.5 hours, followed by heating to 90 ° C for 3 hours, and then tert-butanol (80.0 mL), the reaction was continued for 16 hours at 90 ° C.
  • reaction solution was cooled to room temperature, 500.0 mL of saturated sodium bicarbonate solution was added, followed by extraction with ethyl acetate (600.0 mL ⁇ 2), the organic phases were combined, the organic phase was washed with saturated brine (800.0 mL), and the organic phase was anhydrous It was dried over sodium sulfate, filtered, and the filtrate was concentrated to dryness.
  • the obtained crude product was separated and purified by column chromatography (eluent: 0-10% ethyl acetate / petroleum ether) to obtain compound A3-3.
  • A3-3 (29.40g, 79.36mmol) was dissolved in a mixed solvent of methanol (300.0mL) and tetrahydrofuran (75.0mL), followed by the addition of dry palladium carbon (3.00g, purity 10%), at 50psi Under hydrogen pressure, the reaction was carried out at 25 ° C for 18 hours. The reaction solution was filtered, and the filtrate was concentrated to dryness to obtain A3-4.
  • A3-5 (16.50 g, 48.76 mmol) was suspended in ethyl acetate (50.0 mL), hydrochloric acid / ethyl acetate (4.0 M, 50.0 mL) was added, and the reaction was performed at 25 ° C. for 0.5 hour.
  • the reaction solution was concentrated to dryness, 15% sodium hydroxide solution (50.0 mL) was added to the obtained crude product, the aqueous phase was extracted with dichloromethane (60.0 mL ⁇ 3), the organic phases were combined, and the organic phase was dried over anhydrous sodium sulfate. Filter and concentrate the filtrate to dryness to give crude compound A3-6.
  • MS m / z 239.1 [M + 1] + .
  • the filter cake was washed with ethyl acetate (30.0mL ⁇ 2), the filter cake was drained, the filter cake was collected and suspended in 15% sodium hydroxide solution (100.0mL), and extracted with ethyl acetate (60.0mL ⁇ 3) Extract the aqueous phase, combine the organic phases, wash the organic phase once with saturated sodium chloride solution (150.0 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate to dryness to obtain compound A3.
  • n-butylamine (16.6g, 227.81mmol) was dropped into acetic acid (147.8g, 2.46mol), the internal temperature was controlled to be less than 15 ° C, and compound A4-1 (28.2g, 198.10mmol) and Nitromethane (36.3g, 594.30mmol), the reaction solution was heated to 85 °C and continued to stir for 4 hours. After cooling, the reaction solution was poured into ice water (150 mL), a solid was precipitated, dissolved with ethyl acetate (50 mL), the aqueous layer was separated, and the aqueous layer was extracted with ethyl acetate (20 mL).
  • Compound A4-4 (48.2g, 151.26mmol) was dissolved in a mixed solution of ethanol (700mL) and water (140mL), and amine chloride (80.9g, 1.51mol) and iron powder (84.5g, 1.51mol) were added. The reaction liquid was heated to 78 ° C and stirred for 4 hours.
  • compound C1-4 (1.50g, crude) and compound B1-3 (292mg, 910.56 ⁇ mol) were dissolved in a mixed solution of dioxane (15.0mL) and water (3.0mL), and sodium carbonate was added (193 mg, 1.82 mmol) and 1,1′-bis (diphenylphosphino) ferrocene palladium dichloride (74 mg, 91.06 ⁇ mol), the reaction solution was heated to 100 ° C. and stirring was continued for 4 hours.
  • reaction solution was diluted with 100.0 mL of ethyl acetate, filtered, and the filtrate was washed with 80.0 mL of saturated brine, dried over anhydrous sodium sulfate, and passed through column chromatography (eluent: 10-50% ethyl acetate / petroleum ether) After separation and purification, crude C1 was obtained.
  • aqueous phase was extracted with ethyl acetate (120.0 mL ⁇ 3), the organic phases were combined, the organic phase was washed with saturated brine (100.0 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness, and the resulting crude product was dissolved After cooling to 0 ° C in toluene (30.0mL), trifluoroacetic anhydride (27.4g, 130.29mmol, 18.1mL) was added dropwise, followed by heating to 25 ° C for 23.5 hours.
  • compound C4-4 (2.50 g, 10.00 mmol) was dissolved in tetrahydrofuran (25.0 mL), followed by addition of isopropanol pinacol borate (2.80 g, 15.00 mmol), the system was replaced with nitrogen for three times and then dropped A solution of isopropylmagnesium chloride-lithium chloride in tetrahydrofuran (1.3M, 7.7mL) was added, and the reaction was continued at 0 ° C for 4 hours. A saturated ammonium chloride solution (20.0 mL) was added to the reaction system to quench, and the aqueous phase was extracted with ethyl acetate (50.0 mL ⁇ 3).
  • reaction solution was slowly poured into ice water (50 mL), extracted with ethyl acetate (100 mL ⁇ 2), the combined organic phase was washed with water (100 mL), saturated aqueous sodium chloride solution (50 mL), anhydrous sodium sulfate It was dried, filtered, and the organic solvent was removed under reduced pressure.
  • the obtained crude product was separated and purified by silica gel column chromatography (eluent: 16.7-50% ethyl acetate / petroleum ether) to obtain compound C6-4.
  • the hydrochloride of compound 6 can be adjusted to pH 7-8 by saturated aqueous sodium bicarbonate solution, extracted with methylene chloride, and the organic solvent is removed under reduced pressure to obtain compound 6.
  • This experiment uses homogeneous time-resolved fluorescence conjugated energy transfer from Cisbio ( Method) Perform activity test.
  • enzyme, biotin-labeled peptide substrate, ATP and detection compound are mixed, and the reaction is incubated.
  • EDTA was added to terminate the reaction, and Eu-labeled antibody was added at the same time, and XL665 labeled with streptavidin was reacted and detected.
  • the data are represented by the readings of the fluorescent signals at 665nm and 620nm, respectively, where a high ratio of 665nm / 620nm indicates higher activity, and a low ratio of 665nm / 620nm indicates that activity is inhibited.
  • Compound dilution The compound to be tested is diluted 3 times, a total of 11 concentrations, the final system concentration is from 10 ⁇ M to 0.17 nM
  • Caco-2 cells are human colon cancer cells.
  • the monolayer Caco-2 cell model is widely used to evaluate the passive diffusion and active transport of test compounds in the small intestine.
  • the efflux transporters in the small intestine include P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). This experiment was used to determine the bidirectional permeability of the test compound through the Caco-2 cell model, and to investigate its efflux transport.
  • MEM Minimum Essential Media
  • FBS fetal bovine serum
  • penicillin-G 100 U / mL penicillin-G
  • 100 ⁇ g / mL streptomycin 100 ⁇ g / mL streptomycin was used.
  • the cell culture conditions are 37 ⁇ 1 °C, 5% CO 2 and saturation humidity.
  • trypsin 0.05%, w / v) / EDTA (0.02%, w / v) digestion solution to digest the cells and plant the plates.
  • the 38th generation Caco-2 cells were used.
  • the cells were seeded in BD Falcon's Transwell-96 well plate (Cat. No. 359274) with a seeding density of 1 ⁇ 105 cells / cm 2 .
  • the cells were placed in a carbon dioxide incubator for 22 days and used for transport experiments, during which the medium was changed every four to five days.
  • Hank's balanced salt buffer pH 7.40 ⁇ 0.05
  • HEPES Hank's balanced salt buffer
  • the concentration of DMSO in the incubation system is controlled below 1%.
  • liquid chromatography tandem mass spectrometry (LC / MS / MS) method was used to semi-quantitatively analyze the test compound and the control products fenoterol, propranolol, digoxin and digoxin Peak area ratio of internal standard.
  • dC r / d t is the cumulative concentration of the compound at the receiving end in unit time ( ⁇ M / s); V r is the volume of the receiving end solution (the volume of the solution at the top and basal end are 0.075mL and 0.250mL, respectively); A is the cell monolayer Relative surface area (0.0804cm 2 ); C 0 is the initial concentration (nM) of the test article at the administration end or the peak area ratio of the control article.
  • the efflux ratio is calculated using the following formula:
  • the recovery rate is calculated using the following formula:
  • C 0 is the initial concentration (nM) of the test article at the administration end or the peak area ratio of the reference substance
  • V d is the volume at the administration end (0.075 mL on the top side and 0.250 mL on the basal side)
  • C d and C r It is the final concentration (nM) of the test article at the administration end and the receiving end, respectively, or the peak area ratio of the control article.
  • the compound of the present invention has a higher Caco-2 cell membrane efflux efflux ratio, can better avoid the risk of entering the brain, and reduce side effects such as movement disorders and sleep disturbances caused by Trk target inhibition in the central nervous system.
  • test compounds in human, SD rat and Beagle dog plasma was measured.
  • test compound working solution 400 ⁇ M
  • warfarin working solution 400 ⁇ M
  • the final forest concentration was 2 ⁇ M. Mix the sample thoroughly.
  • the final concentration of the organic phase DMSO is 0.5%; pipette 50 ⁇ L of the test compound and warfarin plasma sample to the sample receiving plate, and immediately add the corresponding volume of corresponding blank plasma or buffer to make the final volume of each sample well 100 ⁇ L ,
  • the volume ratio of plasma: dialysis buffer is 1: 1, and then 400 ⁇ L of stop solution is added to these samples.
  • This sample will be used as a T 0 sample for recovery and stability determination.
  • the dialysis plate was sealed with a gas-permeable membrane, placed in a humidified 5% CO 2 incubator, and incubated at 37 ° C with shaking at 100 rpm for 4 hours. After dialysis, 50 ⁇ L of the dialyzed buffer sample and the dialyzed plasma sample were transferred to a new sample receiving plate.
  • F C is the concentration of the compound at the buffer end of the dialysis plate
  • T C is the concentration of the compound at the plasma end of the dialysis plate
  • T 0 is the concentration of the compound in the plasma sample at time zero.
  • the inhibitory activity of the test compounds on different isoforms of human cytochrome P450 isoenzyme was determined.
  • test compound Prepare the test compound, standard inhibitor (100 ⁇ final concentration) and mixed substrate working solution; remove the microsomes frozen in the refrigerator at -80 ° C and thaw.
  • NADPH coenzyme factor
  • Test article (10mM), testosterone (Testosterone, control substance, 10mM), diclofenac (Diclofenac, control substance, 10mM), propafenone (Propafenone, control substance, 10mM).
  • Working solution 450 ⁇ L of 100mM potassium phosphate buffer is used to dilute the intermediate solution.
  • liver microsome solution final concentration: 0.5 mg protein / mL
  • Dispense 680 ⁇ L / well liver microsome solution on a 96-well plate then add 80 ⁇ L / well to each plate, and place the above incubation plate at 37 ° C for pre-incubation for approximately 10 minutes.
  • Incubate for an appropriate time eg 5, 10, 20, 30 and 60 minutes.
  • liver weights of mice, rats, dogs, monkeys and humans are 88g / kg, 40g / kg, 32g / kg, 30g / kg and 20g / kg, respectively.
  • C t is the concentration at time t
  • t is the incubation time
  • C 0 is the concentration at 0
  • Ke is the elimination rate constant
  • Cl int (mic) is the intrinsic clearance of liver microparticles
  • Cl int (liver) is the intrinsic clearance of liver rate.
  • test compound was given by intragastric administration at 10 mg / kg.
  • the vehicle for intravenous administration is 10% ethanol + 20% castor oil polyoxyethylene ether + 70% physiological saline, and the vehicle for intragastric administration is 0.5% sodium carboxymethyl cellulose + 0.2% Tween 80.
  • Plasma samples were collected from animals in the intravenous group at 0.0833, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, and 24 hours after administration, while animals in the gavage group were at 0.25, 0.5, 1.0, 2.0, 4.0, 6.0 after administration. Plasma samples were collected at 8.0, 24 hours.
  • the plasma drug concentration was determined by LC-MS / MS method, and the relevant pharmacokinetic parameters were calculated by the non-compartment model linear log trapezoid method using WinNonlin TM Version 6.3 (Pharsight, Mountain View, CA) pharmacokinetic software.
  • C 0 is the initial concentration
  • T 1/2 is the elimination half-life
  • Vd ss is the steady-state apparent volume of distribution
  • Cl is the total clearance
  • AUC 0-last is the plasma from time 0 to the last quantifiable time point Area under the concentration-time curve
  • AUC 0-inf is the area under the plasma concentration-time curve from time 0 to extrapolation to infinity
  • C max is the peak concentration
  • T max is the peak time.
  • CD-1 mice male, 20-40g, 6-9 weeks old, Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.
  • the test compound was formulated as a clear solution or a homogeneous suspension, and given to mice for single intravenous injection and oral administration.
  • the solvent is a certain proportion of ethanol, Cremophor EL and physiological saline solution, vortexed to prepare a 1 mg / mL clear solution, and the microporous filter is filtered for use;
  • the oral solvent is a certain ratio of methyl cellulose solution or a certain ratio Methyl cellulose and Tween 80 aqueous solution.
  • test compound After the test compound is mixed with the solvent, vortex to prepare a 10 mg / mL clear or homogeneous suspension for use. After 2 mg / kg intravenous administration or 100 mg / kg oral administration of mice, a certain amount of whole blood samples were collected, centrifuged at 3200 g for 10 minutes, and the supernatant plasma samples were separated, and the samples were diluted with blank plasma by a certain multiple according to actual needs.
  • Plasma samples were added to 20-fold volume of acetonitrile solution containing internal standard to precipitate protein, centrifuged to take supernatant, added 2 volumes of water and then centrifuged to take supernatant for injection, quantitative analysis of plasma drug concentration by LC-MS / MS analysis method, and Phoenix WinNonlin software (Pharsight, USA) calculates pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the curve of drug time, bioavailability, etc.
  • C 0 is the initial concentration
  • T 1/2 is the elimination half-life
  • Vd ss is the steady-state apparent volume of distribution
  • Cl is the total clearance
  • AUC 0-inf is the plasma concentration from time 0 to extrapolation to infinity -The area under the time curve
  • C max is the peak concentration
  • T max is the peak time.
  • Compound 12 of the present invention has a longer half-life and a shorter time to peak, which indicates that the body may have a faster effect and a longer period of drug efficacy, reducing the frequency of administration; it has good mouse pharmacokinetics Nature and oral bioavailability.
  • the blood concentration of the compound was measured after a single administration and the pharmacokinetic behavior was evaluated.
  • test The purpose of the test is to test the pharmacokinetic characteristics of the test compound after intravenous injection and oral administration.
  • the test compound is formulated into a clear solution or a uniform suspension, and the beagle dog is given a single intravenous injection or oral administration .
  • the solvent is a certain proportion of HP- ⁇ -cyclodextrin solution of dimethyl sulfoxide or a certain proportion of ethanol, polyethylene glycol 400 and physiological saline solution, vortex and ultrasound to prepare 2mg / mL or 1mg / kg Clarified solution, ready for use after filtration through microporous membrane; oral solvent is a certain proportion of HP- ⁇ of dimethyl sulfoxide Cyclodextrin solution or a certain proportion of sodium carboxymethylcellulose solution.
  • the test compound is mixed with the solvent, vortex and sonicate to prepare a 2 mg / mL clear solution or 1 mg / mL homogeneous suspension for use.
  • C 0 is the initial concentration
  • T 1/2 is the elimination half-life
  • Vd ss is the steady-state apparent volume of distribution
  • Cl is the total clearance
  • AUC 0-inf is the plasma concentration from time 0 to extrapolation to infinity -The area under the time curve
  • C max is the peak concentration
  • T max is the peak time.
  • the compound 12 of the present invention has a longer half-life and shorter peak time, which indicates that the body may have a faster effect and a longer period of drug effect, reducing the frequency of administration; it has good Beagle dog pharmacokinetics Academic properties and oral bioavailability.

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Abstract

本发明公开了一类吡咯烷基脲衍生物，及其在制备治疗与TrkA相关疾病的药物中的应用。具体公开了式(Ⅰ)、(Ⅱ)所示化合物、其异构体及其药学上可接受的盐。

Description

吡咯烷基脲衍生物及其应用
本申请主张如下优先权：
CN201811255982.3，申请日2018-10-26；
技术领域
本发明涉及一类TrkA抑制剂，及其在制备治疗与TrkA相关疾病的药物中的应用。具体涉及式(Ⅰ)、(Ⅱ)所示化合物、其异构体及其药学上可接受的盐。
背景技术
原肌球蛋白相关激酶(Trk)是由称为神经增长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养因子(NT)的一群可溶性增长因子所激活的高亲和力受体酪氨酸激酶，其家族由三个成员(TrkA、TrkB、TrkC)组成。NGF、BDNF、NT-4/5通过受体Trk在神经元细胞的信号维持、神经元细胞的信号传递、细胞增殖、细胞分化、细胞存活等许多生理学调节过程中发挥重要作用。有许多证据显示NGF/Trk信号通路的抑制剂在疼痛的许多临床前模型中有效；还显示NGF/Trk信号通路的抑制剂在炎症性疾病的许多临床前模型中有效。此外，Trk激酶的过度表达、激活、扩增和/或突变与许多肿瘤或癌症相关。因此，Trk成为了一类重要治疗靶点，吸引了广泛的研发兴趣。本发明所述的TrkA抑制剂可以解决疼痛、癌症、炎症、神经变性疾病以及某些感染性疾病的治疗需求。
专利WO2015175788中报道了对TrkA有抑制活性的化合物及其药学上可接受的盐(参考例8:参考化合物D1)。
发明内容
本发明提供了式(Ⅰ)、(Ⅱ)所示化合物、其异构体或其药学上可接受的盐，
Figure PCTCN2019113278-appb-000001
其中，
T 1、T 2和T 3分别独立地选自N和C(R 3)；
X 1、X 2和X 3分别独立地选自O、N(R 4)和C(R 5)(R 6)；
Z 1、Z 2和Z 3分别独立地选自N和CH；
R 1选自C 1-6烷基和C 1-6烷氧基，所述C 1-6烷基和C 1-6烷氧基任选被1、2或3个R a取代；
R 2选自任选被1、2或3个R b取代的C 1-3烷基；
R 3分别独立地选自H、F、Cl、Br、I、OH和NH 2；
R 4分别独立地选自H和任选被1、2或3个R c取代的C 1-3烷基；
R 5和R 6分别独立地选自H、F、Cl、Br、I、OH和NH 2；
n选自1、2和3；
R a选自H、F、Cl、Br、I、OH、NH 2、CN、C 1-3烷基和C 1-3烷氧基，所述C 1-3烷基和C 1-3烷氧基任选被1、2或3个R取代；
R b和R c分别独立地选自H、F、Cl、Br、I、OH和NH 2；
R选自F、Cl、Br、I、OH和NH 2；
带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；
带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。
本发明的一些方案中，上述R a选自H、F、Cl、Br、I、OH、NH 2、CN、CH 3和CH 3O，所述CH 3和CH 3O任选被1、2或3个R取代，其它变量如本发明所定义。
本发明的一些方案中，上述R a选自H、F、Cl、Br、I、OH、NH 2、CN、CH 3、CH 2F、CHF 2、CF 3和CH 3O，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自CH 3、CH 3CH 2和CH 3O，所述CH 3、CH 3CH 2和CH 3O任选被1、2或3个R a取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 1选自
Figure PCTCN2019113278-appb-000002
其它变量如本发明所定义。
本发明的一些方案中，上述R 2选自CH 3和CH 3CH 2，其它变量如本发明所定义。
本发明的一些方案中，上述R 4选自H、CH 3和CH 3CH 2，所述CH 3和CH 3CH 2任选被1、2或3个R c取代，其它变量如本发明所定义。
本发明的一些方案中，上述R 4选自H、CH 3、CH 2F、CHF 2、CF 3、CH 3CH 2和
Figure PCTCN2019113278-appb-000003
其它变量如本发明所定义。
本发明的一些方案中，上述T 1、T 2和T 3分别独立地选自N、CH和CF，其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000004
选自
Figure PCTCN2019113278-appb-000005
Figure PCTCN2019113278-appb-000006
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000007
选自
Figure PCTCN2019113278-appb-000008
Figure PCTCN2019113278-appb-000009
其它变量如本发明所定义。
本发明的一些方案中，上述X 1、X 2和X 3分别独立地选自O、N(CH 3)、NH、CH 2和
Figure PCTCN2019113278-appb-000010
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000011
选自
Figure PCTCN2019113278-appb-000012
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000013
选自
Figure PCTCN2019113278-appb-000014
Figure PCTCN2019113278-appb-000015
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000016
选自
Figure PCTCN2019113278-appb-000017
其它变量如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2019113278-appb-000018
选自：
Figure PCTCN2019113278-appb-000019
其它变量如本发明所定义。
本发明还有一些方案由上述变量任意组合而来。
本发明的一些方案中，上述化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2019113278-appb-000020
其中，
T 1、T 2和T 3分别独立地选自N和C(R 3)；
D选自N(R 4)和O；
R 1、R 2、R 3和R 4如本发明所定义。
本发明的一些方案中，上述化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2019113278-appb-000021
其中，
D选自N(R 4)和O；
R 1、R 2、R 3和R 4如本发明所定义。
本发明还提供了下列化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2019113278-appb-000022
Figure PCTCN2019113278-appb-000023
本发明的一些方案中，上述化合物、其异构体或其药学上可接受的盐，其选自
Figure PCTCN2019113278-appb-000024
Figure PCTCN2019113278-appb-000025
本发明还提供了上述的化合物、其异构体或其药学上可接受的盐在制备治疗TrkA抑制剂相关药物上的应用。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一 氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
除非另有说明，术语“异构体”意在包括几何异构体、顺反异构体、立体异构体、对映异构体、旋光异构体、非对映异构体和互变异构体。
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。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。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(D)”或者“(+)”表示右旋，“(L)”或者“(-)”表示左旋，“(DL)”或者“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2019113278-appb-000026
和楔形虚线键
Figure PCTCN2019113278-appb-000027
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2019113278-appb-000028
和直形虚线键
Figure PCTCN2019113278-appb-000029
表示立体中心的相对构型，用波浪线
Figure PCTCN2019113278-appb-000030
表示楔形实线键
Figure PCTCN2019113278-appb-000031
或楔形虚线键
Figure PCTCN2019113278-appb-000032
或用波浪线
Figure PCTCN2019113278-appb-000033
表示直形实线键
Figure PCTCN2019113278-appb-000034
和直形虚线键
Figure PCTCN2019113278-appb-000035
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线
Figure PCTCN2019113278-appb-000036
连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示 该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
Figure PCTCN2019113278-appb-000037
本发明的化合物可以存在特定的。除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用 放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，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(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
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。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。
当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2019113278-appb-000038
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2019113278-appb-000039
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2019113278-appb-000040
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，术语“C 1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。所述C 1-6烷基包括C 1-5、C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6和C 5烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-6烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异 丙基)、丁基(包括n-丁基，异丁基，s-丁基和t-丁基)、戊基(包括n-戊基，异戊基和新戊基)、己基等。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1- 3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
除非另有规定，术语“C 1-6烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至6个碳原子的烷基基团。所述C 1-6烷氧基包括C 1-4、C 1-3、C 1-2、C 2-6、C 2-4、C 6、C 5、C 4和C 3烷氧基等。C 1-6烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)、丁氧基(包括n-丁氧基、异丁氧基、s-丁氧基和t-丁氧基)、戊氧基(包括n-戊氧基、异戊氧基和新戊氧基)、己氧基等。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1-12包括C 1- 3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲和取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等 等。
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。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：aq代表水；HATU代表O-(7-氮杂苯并三唑-1-基)-N，N，N'，N'-四甲基脲六氟磷酸盐；eq代表当量、等量；DCM代表二氯甲烷；PE代表石油醚；DMF代表N，N-二甲基甲酰胺；DMSO代表二甲亚砜；EtOAc代表乙酸乙酯；EtOH代表乙醇；MeOH代表甲醇；CBz代表苄氧羰基，是一种胺保护基团；BOC代表叔丁氧羰基是一种胺保护基团；HOAc代表乙酸；r.t.代表室温；O/N代表过夜；THF代表四氢呋喃；Boc 2O代表二叔丁基二碳酸酯；TFA代表三氟乙酸；DIPEA代表二异丙基乙基胺；SOCl 2代表氯化亚砜；mp代表熔点；DEA代表二乙基胺。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2019113278-appb-000041
软件命名，市售化合物采用供应商目录名称。
技术效果
本发明化合物具有显著的TrkA酶抑制活性；本发明化合物具有较高的Caco-2细胞膜渗透外排比，可以更好地规避进脑风险，减少因为中枢神经系统中的Trk靶点抑制所引起的运动失调、睡眠紊乱等副作用；本发明化合物具有较高的人血浆蛋白游离结合率，更低的药物-药物相互作用风险，更好的肝微粒体代谢稳定性；在大鼠单次给药药代研究中，相同的口服灌胃给药剂量下，本发明化合物具有比参考化合物D1更高的血浆暴露量和更高的口服生物利用度；在大鼠CFA诱导的机械痛模型中，相同的口服灌胃给药剂量下，本发明化合物具有比参考化合物D1更持久的镇痛效果；本发明化合物具有较长的半衰期和较短的达峰时间，预示体内可能起效较快且有较长时间的药效，降低给药频率，具有良好的小鼠和比格犬药代动力学性质和口服生物利用度。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
参考例1:合成中间体A1
Figure PCTCN2019113278-appb-000042
步骤1:化合物A1-2的制备
向化合物A1-1(50.00g,284.11mmol)和丙烯酸甲酯(24.46g,284.11mmol,25.6mL)的DMF(500.0mL)溶液中加入三乙胺(86.25g,852.34mmol,118.6mL)，用氮气置换体系三次，接着加入醋酸钯(6.38g,28.41mmol)和三(邻甲基苯基)膦(8.65g,28.41mmol)，再次用氮气置换体系三次，100℃下反应18小时。往反应液中加入1L水稀释，稀释后的反应液用乙酸乙酯(1L×3)萃取，合并有机相，有机相再依次用水(800mL×3)洗涤、饱和食盐水(1L)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品分散在500mL石油醚和乙酸乙酯的混合溶剂(石油醚:乙酸乙酯＝5:1)中打浆，过滤，抽干，收集滤饼得到化合物A1-2。 1H NMR(400MHz,CDCl 3)δ:8.28(d,J＝5.2Hz,1H),7.62(d,J＝16.0Hz,1H),7.31-7.28(m,1H),7.02(s,1H),6.62(d,J＝16.0Hz,1H),3.88(s,3H).
以下化合物使用与化合物A1-2类似的方法合成得到：
Figure PCTCN2019113278-appb-000043
步骤2:化合物A1-3的制备
将化合物A1-2(1.00g,5.52mmol)和2-甲氧基-N-(甲氧甲基)-N-((三甲基硅基)甲基)乙基胺(2.27g,11.04mmol)溶于正庚烷(10.0mL)和乙酸乙酯(10.0mL)的混合溶剂中，然后加入三氟乙酸(63mg,551.99μmol)，20℃下反应16小时。将反应液浓缩至干。所得粗品经柱层析(洗脱剂:10-50％乙酸乙酯/石油醚)分离纯化得到化合物A1-3。 1H NMR(400MHz,CDCl 3)δ:8.08(d,J＝5.2Hz,1H),7.14-7.10(m,1H),6.87(s,1H),3.66(s,3H),3.65-3.61(m,1H),3.52-3.47(m,2H),3.33(s,3H),3.17-3.10(m,1H),3.05-2.92(m,2H),2.86-2.79(m,2H),2.78-2.70(m,1H),2.67-2.60(m,1H).MS m/z:282.9[M+1] +.
以下化合物使用与化合物A1-3类似的方法合成得到：
Figure PCTCN2019113278-appb-000044
步骤3:化合物A1-4的制备
向化合物A1-3(1.40g,4.96mmol)的甲醇(15.0mL)和水(5.0mL)的混合溶液中加入氢氧化钠(595mg,14.88mmol)，20℃下反应2小时。浓缩除去反应液中的有机溶剂，用1.0M的盐酸溶液调节体系pH至4-5，接着用二氯甲烷(20.0mL)萃取，收集水相，将水相浓缩至干。所得粗品分散在50.0mL丙酮中，过滤，将滤液浓缩至干，得到化合物A1-4粗品。 1H NMR(400MHz,MeOD)δ:8.20(d,J＝5.2Hz,1H),7.41(d,J＝5.2Hz,1H),7.20(s,1H),4.89(s,1H),4.03-3.79(m,4H),3.76-3.70(m,2H),3.55-3.48(m,3H),3.43(s,3H),3.37-3.29(m,1H).
以下化合物使用与化合物A1-4类似的方法合成得到：
Figure PCTCN2019113278-appb-000045
步骤4:化合物A1-5的制备
向化合物A1-4(1.10g,4.10mmol)的甲苯(25.0mL)和N,N-二甲基甲酰胺(2.5mL)的混合溶液中加入N,N-二异丙基乙胺(1.59g,12.30mmol)，接着加入叠氮磷酸二苯酯(1.35g,4.92mmol)，20℃下反应0.5小时后升温至110℃下反应1小时，接着加入苄醇(4.43g,41.00mmol)，继续在110℃下反应15小时。反应液用100mL乙酸乙酯稀释后再用饱和食盐水(80.0mL)洗涤一次，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:20-40％丙酮/石油醚)分离纯化得到化合物A1-5。 1H NMR(400MHz,CDCl 3)δ:8.05(d,J＝5.2Hz,1H),7.33-7.23(m,5H),7.07(d,J＝4.0Hz,1H),6.82(s,1H),5.25(d,J＝8.0Hz,1H),5.06-4.97(m,2H),4.25-4.15(m,1H),3.44(t,J＝5.2Hz,2H),3.29(s,3H),3.24-3.10(m,2H),2.74-2.59(m,3H),2.53-2.43(m,1H).
以下化合物使用与化合物A1-5类似的方法合成得到：
Figure PCTCN2019113278-appb-000046
步骤5:化合物(+)-A1-5和(-)-A1-5的制备
将化合物A1-5(9.20g,24.64mmol)经过制备SFC(手性柱:ChiralPak AD 250mm×50mm I.D.,10μm；流动相:[0.1％NH 3·H 2O-EtOH])分离得到化合物(+)-A1-5和(-)-A1-5。
(+)-A1-5: 1H NMR(400MHz,CDCl 3)δ:8.14(d,J＝5.2Hz,1H),7.40-7.29(m,5H),7.19-7.11(m,1H),6.90(s,1H),5.27-5.17(m,1H),5.13-5.03(m,2H),4.33-4.22(s,1H),3.55-3.48(m,2H),3.38(s,3H),3.33-3.25(m,1H),3.24-3.15(m,1H),3.00-2.91(m,1H),2.84-2.63(m,3H),2.54(t,J＝8.0Hz,1H).MS m/z:374.1[M+1] +.SFC:ChiralPak AD-3；Rt＝3.716min；99.3％ee.
(-)-A1-5: 1H NMR(400MHz,CDCl 3)δ:8.14(d,J＝5.2Hz,1H),7.40-7.29(m,5H),7.19-7.12(m,1H),6.90(s,1H),5.27-5.19(m,1H),5.13-5.02(m,2H),4.32-4.23(m,1H),3.54-3.49(m,2H),3.38(s,3H),3.33-3.25(m,1H),3.24-3.16(m,1H),2.94(t,J＝8.4Hz,1H),2.83-2.64(m,3H),2.54(t,J＝8.4Hz,1H).MS m/z:374.1[M+1] +.SFC:ChiralPak AD-3；Rt＝3.913min；99.2％ee.
以下化合物使用与化合物(+)-A1-5和(-)-A1-5类似的方法制备分离得到:
SFC分析条件：
柱子：Chiralpak AD-3 150×4.6mm I.D.,3μm
流动相：A:CO 2B:异丙醇(0.05％DEA)
梯度：B在5.5分钟内从5％到40％并在40％保持3分钟,然后B在5％保持1.5min,
Figure PCTCN2019113278-appb-000047
步骤6:化合物A1的制备
将化合物(-)-A1-5(3.10g,8.57mmol)溶于三氟乙酸(15.0mL),50℃下反应19小时。将反应液浓缩至干得到化合物A1粗品。 1H NMR(400MHz,MeOD)δ:8.26(d,J＝5.2Hz,1H),7.45-7.36(m,5H),7.24(s,1H),4.43-4.35(m,1H),4.21-4.14(m,1H),4.11-4.03(m,1H),3.98-3.88(m,2H),3.78-3.69(m,3H),3.61-3.54(m,3H),3.40(s,3H).MS m/z:240.0[M+1] +.
以下化合物使用与化合物A1类似的方法合成得到：
Figure PCTCN2019113278-appb-000048
参考例2:合成中间体A3
Figure PCTCN2019113278-appb-000049
步骤1:化合物A3-2的制备
将化合物A3-1(38.50g,231.7mmol)分散在乙酸乙酯(200.0mL)和正庚烷(200.0mL)混合溶剂中，随后加入三氟乙酸(2.64g,23.2mmol,1.7mL)，冷却到0℃，慢慢滴加N-(甲氧甲基)-N-(三甲基硅甲基)苄胺(137.53g,579.3mmol)，滴加完毕后自然升温至25℃反应20小时。将反应液浓缩至约300.0mL，再加入300mL正庚烷，继续浓缩至浓缩至约300.0mL，重复上述操作6次，直至最后一次加完300mL正庚烷后，过滤，滤饼用正庚烷(100.0mL×2)洗涤抽滤两次，将滤饼抽干，收集滤饼得到化合物A3-2。 1H NMR(400MHz,CDCl 3)δ:7.45-4.43(m,2H),7.37-7.21(m,4H),7.13-6.98(m,2H),6.95-6.85(m,1H),4.51(d,J＝12.4Hz,1H),4.16-3.99(m,2H),3.78(d,J＝12.4Hz,1H),3.49(t,J＝10.0Hz,1H),3.20-3.10(m,2H),2.75(t,J＝10.0Hz,1H).
步骤2:化合物A3-3的制备
氮气氛围下，将A3-2(53.00g,177.06mmol)分散在甲苯(400.0mL)中，加入N,N-二异丙基乙胺(25.17g,194.77mmol,34.0mL)，用氮气保护，随后缓慢滴加叠氮磷酸二苯酯(53.60g,194.77mmol,42.2mL)，滴加完毕后，25℃下反应0.5小时，接着升温至90℃反应3小时，之后再加入叔丁醇(80.0mL)，90℃下继续反应16小时。将反应液冷却至室温，加入500.0mL饱和碳酸氢钠溶液，接着用乙酸乙酯(600.0mL×2)萃取，合并有机相，有机相用饱和食盐水(800.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-10％乙酸乙酯/石油醚)分离纯化得到化合物A3-3。 1H NMR(400MHz,CDCl 3)δ:7.27-7.25(m,4H),7.21-7.15(m,2H),7.01-6.89(m,2H),6.86-6.80(m,1H),4.84(br s,1H),4.11(br s,1H),3.57(s,2H),3.15-2.94(m,2H),2.90-2.82(m,1H),2.68-2.58(m,1H),2.44-2.34(m,1H),1.39(s,9H).
步骤3:化合物A3-4的制备
氮气氛围下，将A3-3(29.40g,79.36mmol)溶在甲醇(300.0mL)和四氢呋喃(75.0mL)的混合溶剂中，随后加入干钯碳(3.00g,纯度10％)，在50psi的氢气压力下，25℃下反应18小时。将反应液过滤，将滤液浓缩至干，得到A3-4。 1H NMR(400MHz,CDCl 3)δ:7.33-7.26(m,1H),7.05(d,J＝7.6Hz,1H),7.01-6.96(m,1H),6.96-6.90(m,1H),4.92(br s,1H),4.18-4.02(m,1H),3.47-3.36(m,2H),3.17-2.84(m,3H),1.41(s,9H).MS m/z:281.1[M+1] +.
步骤4:化合物A3-5的制备
将A3-4(14.50g,51.72mmol)溶于N,N-二甲基甲酰胺(100.0mL)，分别加入N,N-二异丙基乙胺(20.05g,155.16mmol,27.1mL)和2-溴乙基甲基醚(8.63g,62.06mmol,5.8mL)，25℃下反应16小时。向体系中加入400.0mL水稀释，用乙酸乙酯(400.0mL×3)萃取水相，合并有机相，有机相再用饱和食盐水(800.0mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干。所得粗品中加入200.0mL石油醚，过滤，将滤饼抽干，收集滤饼得到A3-5。 1H NMR(400MHz,CDCl 3)δ:7.30-7.25(m,1H),7.08(d,J＝7.6Hz,1H),7.01(d,J＝10.0Hz,1H),6.95-6.90(m,1H),4.98(br s,1H),4.21(br s,1H),3.53(t,J＝5.6Hz,2H),3.39(s,3H),3.35-3.31(m,1H),3.15-3.11(m,1H),2.90-2.80(m,2H),2.81-2.65(m,2H),2.51-2.39(m,1H),1.43(s,9H).MS m/z:339.2[M+1] +.
以下化合物使用与化合物A3-5类似的方法合成得到：
Figure PCTCN2019113278-appb-000050
步骤5:化合物A3-6的制备
将A3-5(16.50g,48.76mmol)悬浮在乙酸乙酯(50.0mL)中，加入盐酸/乙酸乙酯(4.0M，50.0mL)，25℃下反应0.5小时。将反应液浓缩至干，向所得粗品中加入15％氢氧化钠溶液(50.0mL)，用二氯甲烷(60.0mL×3)萃取水相，合并有机相，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干得到化合物A3-6粗品。MS m/z＝239.1[M+1] +.
以下化合物使用与化合物A3-6类似的方法合成得到：
Figure PCTCN2019113278-appb-000051
步骤6:化合物A3的制备
将化合物A3-6(11.40g,47.84mmol)溶在甲醇(99.0mL)和水(11.0mL)的混合溶剂中，然后加入D-(+)-二对甲基苯甲酰酒石酸(20.33g,52.62mmol)，50℃下反应1小时。随后再在25℃下静置16小时。过滤，滤饼用乙酸乙酯(30.0mL×2)洗涤，将滤饼抽干，收集滤饼将其悬浮在15％的氢氧化钠溶液(100.0mL)中，用乙酸乙酯萃取(60.0mL×3)萃取水相，合并有机相，有机相用饱和氯化钠溶液(150.0mL)洗涤一次，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干得到化合物A3。 1H NMR(400MHz,CDCl 3)δ:7.31-7.24(m,1H),7.06(d,J＝7.6Hz,1H),7.03-7.00(m,1H),6.95-6.88(m,1H),3.52(t,J＝5.6Hz,2H),3.48-3.42(m,1H),3.38(s,3H),3.22-3.15(m,1H),3.05-2.90(m,2H),2.83-2.73(m,1H),2.72-2.61(m,3H).MS m/z:239.1[M+1] +.SFC:Lux Cellulose-2；Rt＝4.889min；97.7％ee.
SFC分析条件：
柱子：Lux Cellulose-2 150×4.6mm I.D.,3μm
流动相：A:CO 2B:甲醇(0.1％乙醇胺)
梯度：B在5.5分钟内从5％到40％并在40％保持3分钟,然后B在5％保持1.5min,Rt＝4.889min
以下化合物使用与化合物A3类似的方法合成得到：
Figure PCTCN2019113278-appb-000052
参考例3:合成中间体A4-7
Figure PCTCN2019113278-appb-000053
步骤1：化合物A4-2的制备
在冷水浴条件下，将正丁胺(16.6g,227.81mmol)滴入乙酸(147.8g,2.46mol)中，控制内温小于15℃，依次加入化合物A4-1(28.2g,198.10mmol)和硝基甲烷(36.3g,594.30mmol)，反应液升温至85℃继续搅拌4小时。冷却，将反应液倒入冰水(150mL)中，析出固体，用乙酸乙酯(50mL)溶解，静置分出水层，水层用乙酸乙酯(20mL)萃取，合并后的有机相用饱和氯化钠溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂得到化合物A4-2。 1H NMR(400MHz,CDCl 3)δ:7.85(d,J＝14.0Hz,1H),7.43(d,J＝13.6Hz,1H),7.35-7.13(m,3H).
以下化合物使用与化合物A4-2类似的方法合成得到：
Figure PCTCN2019113278-appb-000054
步骤2：化合物A4-4的制备
将化合物A4-2(28.0g,151.25mmol)和化合物A4-3(89.8g,378.13mmol)溶于正庚烷(100mL)和乙酸乙酯(100mL)的混合溶液中，10℃条件下滴加三氟乙酸(1.7g,15.13mmol)，反应液升温至25℃继续搅拌4小时。将反应液滴入饱和碳酸钠溶液(50mL)中，静置分层，用乙酸乙酯(20mL)萃取水层一次，合并后的有机相用饱和氯化钠溶液(50mL)洗涤一次，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到化合物A4-4粗品。MS m/z＝319.0[M+H] +.
以下化合物使用与化合物A4-4类似的方法合成得到：
Figure PCTCN2019113278-appb-000055
Figure PCTCN2019113278-appb-000056
步骤3:化合物A4-5的制备
将化合物A4-4(48.2g,151.26mmol)溶解在乙醇(700mL)和水(140mL)的混合溶液中，加入氯化胺(80.9g,1.51mol)和铁粉(84.5g,1.51mol)，反应液升温至78℃搅拌4小时。过滤，用100mL乙酸乙酯洗涤滤饼，减压除去有机溶剂，所得粗品分散于乙酸乙酯(200mL)和水(100mL)中，分层，水相用乙酸乙酯(200mL×2)萃取，合并后的有机相用饱和氯化钠溶液(200mL)洗涤，最后用无水硫酸钠干燥，过滤，减压除去有机溶剂，得到化合物A4-5粗品。MS m/z＝289.1[M+H] +.
以下化合物使用与化合物A4-5类似的方法合成得到:
Figure PCTCN2019113278-appb-000057
步骤4：化合物A4-6的制备
将化合物A4-5(46.2g,160.27mmol)溶于四氢呋喃(1.0L)中，加入三乙胺(32.4g,320.54mmol)，0℃下滴加Boc酸酐(59.5g,272.46mmol)，反应液升温至25℃继续搅拌19小时。减压除去有机溶剂，将粗产品分散于乙酸乙酯(300mL)和水(200mL)的混合溶剂中，分液，乙酸乙酯(200mL×2)萃取，合并后的有机相用饱和氯化钠溶液(200mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱剂：0-10％乙酸乙酯/石油醚)分离纯化，得到化合物A4-6。 1H NMR(400MHz,CDCl 3)δ:7.30-7.28(m,3H),7.25-7.19(m,2H),7.10-6.90(m,3H),4.96(s,1H),4.10(s,1H),3.59(s,2H),3.20-2.85(m,3H),2.70-2.55(m,1H),2.50-2.35(m,1H),1.36(m,9H).
以下化合物使用与化合物A4-6类似的方法合成得到：
Figure PCTCN2019113278-appb-000058
Figure PCTCN2019113278-appb-000059
步骤5：化合物A4-7的制备
将化合物A4-6(26.5g,68.32mmol)溶于乙醇(300mL)中，加入湿钯碳(2.6g,10％纯度)，氩气置换三次，氢气置换三次，反应液升温至60℃在压力50psi下继续搅拌40小时。冷却，用硅藻土助滤，减压除去有机溶剂得到化合物A4-7粗品，该化合物不经进一步纯化直接用于下一步反应。 1H NMR(400MHz,CDCl 3)δ:7.15-6.90(m,3H),4.78(s,1H),4.07(s,1H),3.49-3.35(m,2H),3.10-2.80(m,3H),1.48-1.33(m,9H).
以下化合物使用与化合物A4-7类似的方法合成得到：
Figure PCTCN2019113278-appb-000060
参考例4:合成中间体A5
Figure PCTCN2019113278-appb-000061
步骤1：化合物A5-8的制备
将化合物A5-7(4.4g,粗品)溶于N,N-二甲基甲酰胺(15mL)中，加入2-溴乙基甲基醚(6.15g,44.25mmol)和二异丙基乙胺(5.72g,44.25mmol)，反应液在30℃下继续搅拌18小时。向反应液中加入水(40mL)，乙酸乙酯(200mL)萃取，有机相用水(20mL×3)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，所得粗品经硅胶柱层析(洗脱液：30-100％乙酸乙酯/石油醚)分离纯化，得到的化合物经SFC(column: AD(250mm*30mm,5μm)；流动相:[0.1％NH 3H 2O EtOH]；B％:5％-5％,min)分离纯化，得到化合物A5-8，取0.9mg样品溶于甲醇(1mL)中，测得比旋光度-33.602。 1H NMR(400MHz,CDCl 3)δ:6.86-6.80(m,2H),6.71-6.63(m,1H),5.07-4.95(m,1H),4.22-4.11(m,1H),3.53(t,J＝5.5Hz,2H),3.39-3.30(m,4H),3.20-3.07(m,1H),2.97-2.65(m,4H),2.60-2.42(m,1H),1.48-1.31(m,9H).MS m/z:356.9[M+H] +.[α]20D＝-33.602(C＝0.9mg/mL,CH 3OH).SFC:柱子：Lux Cellulose-2(150mm*4.6mm,3μm)；流动相：A:CO 2B:甲醇(0.1％DEA)；B％:5％5min；Rt＝2.546min；99.77％ee.
步骤2：化合物A5的制备
将化合物A5-8(170mg,476.98μmol)溶于二氯甲烷(2mL)中，加入三氟乙酸(1mL)，反应液在25℃继续搅拌1.5小时。向反应液中加入碳酸氢钠固体(4.0g)淬灭反应，过滤，减压除去有机溶剂，得到化合物A5粗品，该化合物不经进一步纯化直接用于下一步反应。MS m/z:257.1[M+H] +.
参考例5:合成中间体B1
Figure PCTCN2019113278-appb-000062
步骤1:化合物B1-2的制备
将苯肼(20.00g,184.95mmol,18.2mL)和2-氰基丙酸乙酯(23.51g,184.95mmol)溶于1,4-二氧六环(40.0mL)，110℃下反应72小时。将反应液浓缩至约20.0mL，析出固体，过滤，滤饼用乙酸乙酯(30.0mL)洗涤抽滤至干，收集滤饼得到化合物B1-2. 1H NMR(400MHz,MeOD)δ:7.53-7.46(m,2H),7.42-7.35(m,3H),1.77(s,3H).
步骤2:化合物B1-3的制备
将化合物B1-2(10.00g,52.85mmol)溶于N,N-二甲基甲酰胺(150mL)中，随后依次加入N,N-二异丙基乙胺(20.49g,158.55mmol,27.7mL)和N-苯基双(三氟甲烷磺酰)亚胺(19.82g,55.49mmol)，25℃下反应16小时。将反应液倒入500.0mL水中，随后用乙酸乙酯(150.0mL×3)萃取，合并有机相，有机相用饱和食盐水(300.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，得到化合物B1-3粗品。 1H NMR(400MHz,CDCl 3)δ:7.54-7.44(m,4H),7.40-7.34(m,1H),3.76(br s,2H),1.95(s,3H).
步骤3:化合物B1-4的制备
将化合物B1-3(1.00g,3.11mmol)溶于二氯甲烷(5.0mL)中，随后分别加入二碳酸二叔丁酯(2.04g,9.34mmol)、三乙胺(945mg,9.34mmol,1.3mL)、4-二甲氨基吡啶(38mg,311.26μmol)，15℃搅拌反应16.5小时。将反应液浓缩至干，所得粗品经柱层析分离纯化(洗脱液:0-11％乙酸乙酯/石油醚)得到化合物B1-4。 1H NMR(400MHz,CDCl 3)δ:7.37-7.26(m,5H),1.89(s,3H),1.20(s,18H).MS m/z:522.0[M+1] +.
步骤4:化合物B1的制备
将化合物B1-4(1.00g,1.92mmol)溶于1,4-二氧六环(10.0mL)中，随后分别加入联硼酸频哪醇酯(584mg,2.30mmol)、乙酸钾(565mg,5.57mmol)、1,1-双(二苯基膦)二茂铁二氯化钯二氯甲烷络合物(157mg,191.75μmol)，95℃下反应16小时。将反应液用硅藻土过滤，将滤液浓缩至干，所得粗品经柱层析分离纯化(洗脱液:0-50％乙酸乙酯/石油醚)得到化合物B1粗品。 1H NMR(400MHz,CDCl 3)δ:7.49-7.45(m,2H),7.42-7.32(m,3H),2.13(s,3H),1.38(s,12H),1.31(s,18H).
参考例6:合成中间体C1
Figure PCTCN2019113278-appb-000063
步骤1:化合物C1-2的制备
0℃下，向丙炔醇(2.00g,35.68mmol)的四氢呋喃(30.0mL)溶液中缓慢加入氢化钠(1.54g,38.50mmol,纯度60％)，加完后在0℃下反应0.5小时，接着再加入化合物C1-1(4.50g,35.00mmol)，随后升温至80℃下反应3小时。往反应液中加入100.0mL水淬灭，用乙酸乙酯(100.0mL×3)萃取水相，合并有机相，有机相用饱和食盐水(100.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-50％乙酸乙酯/石油醚)分离纯化得到化合物C1-2。 1H NMR(400MHz,CDCl 3)δ:8.75(d,J＝4.8Hz,2H),7.23(t,J＝4.8Hz,1H),4.87(s,2H),4.41(d,J＝2.4Hz,2H),2.49(t,J＝2.4Hz,1H).
步骤2:化合物C1-3的制备
氮气氛围下，将化合物C1-2(3.10g,20.92mmol)溶于硝基苯(10.0mL)，140℃下反应18小时。将反应液浓缩至干，所得粗品经柱层析(洗脱剂:0-50％乙酸乙酯/石油醚)分离纯化得到化合物C1-3。 1H NMR(400MHz,CDCl 3)δ:8.49(d,J＝4.8Hz,1H),7.57(d,J＝8.0Hz,1H),7.18(dd,J＝8.0,4.8Hz,1H),5.18(s,2H),5.09(s,2H).
步骤3:化合物C1-4的制备
将化合物C1-3(1.87g,15.44mmol)和联硼酸频哪醇酯(3.92g,15.44mmol)溶于四氢呋喃(30.0mL)，随后分别加入4,4’-二叔丁基-2,2’-联吡啶(249mg,926μmol)和二(1,5-环辛二烯)二-μ-甲氧基二铱(I)(307mg,463μmol)，用氮气置换体系三次，75℃下反应18小时。将反应液浓缩至干，所得粗品经柱层析(洗脱剂:0-50％乙酸乙酯/石油醚)分离纯化，得到化合物C1-4粗品。 1H NMR(400MHz,CDCl 3)δ:8.83(s,1H),7.95(s,1H),5.16(s,2H),5.08(s,2H),1.36(s,12H).
步骤4:化合物C1的制备
氮气保护下，将化合物C1-4(1.50g,粗品)和化合物B1-3(292mg,910.56μmol)溶于二氧六环(15.0mL)和水(3.0mL)的混合溶液中，加入碳酸钠(193mg,1.82mmol)和1,1'-双(二苯基磷)二茂铁二氯化钯(74mg,91.06μmol)，反应液升温至100℃继续搅拌4小时。将反应液用100.0mL乙酸乙酯稀释，过滤，将滤液用80.0mL饱和食盐水洗涤，用无水硫酸钠干燥，通过柱层析(洗脱剂：10-50％乙酸乙酯/石油醚)分离纯化，得到C1粗品。 1H NMR(400MHz,CDCl 3)δ:8.73(s,1H),7.86(s,1H),7.56-7.54(m,2H),7.46-7.42(m,2H),7.34-7.32(m,1H),5.13(s,2H),5.04(s,2H),3.63(s,2H),2.07(s,3H).MS m/z:293.1[M+1] +.
以下化合物使用与化合物C1类似的方法合成得到：
Figure PCTCN2019113278-appb-000064
Figure PCTCN2019113278-appb-000065
参考例7:合成中间体C2-3
Figure PCTCN2019113278-appb-000066
步骤1:化合物C2-2的制备
化合物C2-1(10.0g,86.86mmol)溶于浓盐酸(9.9mL)和水(54.0mL)的混合溶剂中，冷却至0℃后加入亚硝酸钠(8.4g,121.60mmol)的水(18.0mL)溶液，随后升温至25℃下反应23.5小时。用乙酸乙酯(120.0mL×3)萃取水相，合并有机相，有机相用饱和食盐水(100.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品溶于甲苯(30.0mL)，冷却至0℃后滴加三氟乙酸酐(27.4g,130.29mmol,18.1mL)，随后升温至25℃下反应23.5小时。将反应液浓缩至干，所得粗品经柱层析(洗脱剂:0-100％乙酸乙酯/石油醚)分离纯化得到化合物C2-2。 1H NMR(400MHz,CDCl 3)δ:4.42(t,J＝8.0Hz,2H),2.93-2.87(m,2H),2.82-2.73(m,2H).
步骤2:化合物C2-3的制备
将化合物C2-2(0.50g,3.96mmol)和乙炔基硼酸频那醇酯(1.2g,7.93mmol)溶于均三甲苯(4.0mL)，160℃下反应20小时。将反应液浓缩至干，所得粗品经柱层析(洗脱剂:0-5％甲醇/二氯甲烷)分离纯化得到化合物C2-3粗品。 1H NMR(400MHz,CDCl 3)δ:6.29(s,1H),4.09(t,J＝7.6Hz,2H),2.81(t,J＝7.6Hz,2H),2.60-2.50(m,2H),1.28(s,12H).
以下化合物使用与化合物C2-3类似的方法合成得到：
Figure PCTCN2019113278-appb-000067
参考例8:合成中间体C4-5
Figure PCTCN2019113278-appb-000068
步骤1:化合物C4-2的制备
0℃下，将丙炔酸甲酯(19.20g,228.26mmol,19.0mL)溶于甲醇(210.0mL)，随后加入水合肼(11.4g,228.26mmol,11.1mL)，加完后升温至20℃下反应1小时。将反应液浓缩除去甲醇，然后加入水(100.0mL)稀释，用乙酸乙酯(200.0mL×3)萃取水相，合并有机相，有机相用饱和食盐水(100.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，得到化合物C4-2粗品。 1H NMR(400MHz,DMSO-d 6)δ:7.34(d,J＝2.4Hz,1H),5.43(d,J＝2.0Hz,1H).
步骤2:化合物C4-3的制备
将化合物C4-2(12.00g,142.72mmol)溶于N,N-二甲基甲酰胺(250.0mL)，随后分别加入碳酸钾(69.00g,499.52mmol)和1,3-二溴丙烷(34.6g,171.26mmol,17.5mL)，130℃下反应21小时。将反应液浓缩除去大部分溶剂，随后加入乙酸乙酯(500.0mL)稀释，有机相用水(50.0mL×3)洗涤，再用饱和氯化钠溶液(50.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，得到化合物C4-3粗品。 1H NMR(400MHz,CDCl 3)δ:7.30(d,J＝1.6Hz,1H),5.47(d,J＝2.0Hz,1H),4.27(d,J＝5.2Hz,2H),4.17(d,J＝6.0Hz,2H),2.28-2.20(m,2H).
步骤3:化合物C4-4的制备
将化合物C4-3(2.0g,16.11mmol)溶于乙腈(20.0mL)，随后加入碘代丁二酰亚胺(4.00g,17.72mmol)，20℃下反应4小时。往反应体系中加入饱和硫代硫酸钠溶液(20.0mL)淬灭，用乙酸乙酯(100.0mL×2)萃取水相，合并有机相，有机相用饱和氯化钠溶液(50.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓 缩至干，所得粗品经柱层析(洗脱剂:0-100％乙酸乙酯/石油醚)分离纯化得到化合物C4-4。 1H NMR(400MHz,DMSO-d 6)δ:7.28(s,1H),4.32(t,J＝5.2Hz,2H),4.08(t,J＝6.0Hz,2H),2.21-2.11(m,2H).
步骤4:化合物C4-5的制备
0℃下，将化合物C4-4(2.50g,10.00mmol)溶于四氢呋喃(25.0mL)，随后加入异丙醇频哪醇硼酸酯(2.80g,15.00mmol)，用氮气置换体系三次后滴加异丙基氯化镁-氯化锂的四氢呋喃溶液(1.3M,7.7mL)，继续在0℃下反应4小时。向反应体系中加入饱和氯化铵溶液(20.0mL)淬灭，用乙酸乙酯(50.0mL×3)萃取水相。合并有机相，有机相用饱和氯化钠溶液(50.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-100％乙酸乙酯/石油醚)分离得到化合物C4-5。 1H NMR(400MHz,CDCl 3)δ:7.58(s,1H),4.37(t,J＝5.2Hz,2H),4.16(t,J＝6.4Hz,2H),2.28-2.19(m,2H),1.31(s,12H).
参考例9:合成中间体C5
Figure PCTCN2019113278-appb-000069
步骤1：化合物C5-2的制备
将化合物C5-1(15.0g,89.7mmol)溶于甲醇(200mL)中，慢慢加入浓硫酸(7.4g,75.0mmol)，反应液升温至90℃继续搅拌48小时。冷却，减压浓缩除去反应液中的甲醇，用乙酸乙酯(50mL)将粗产品溶解，用氨水调pH＝7-8，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱液：33-100％乙酸乙酯/石油醚～11％甲醇/二氯甲烷)分离纯化，得到化合物C5-2。 1H NMR(400MHz,CDCl 3)δ9.08(s,1H),8.84(d,J＝5.2Hz,1H),7.52(d,J＝5.2Hz,1H),3.96(d,J＝1.6Hz,6H).MS m/z:195.9[M+1] +.
步骤2：化合物C5-3的制备
将化合物C5-2(7.0g,35.8mmol)溶于二氯甲烷(100mL)中，慢慢加入间氯过氧苯甲酸(11.6g,53.8mmol,80％纯度)，反应液在25℃继续搅拌16小时。反应液用饱和碳酸钠水溶液(100mL)洗涤，水(100mL)洗涤，饱和氯化钠水溶液(50mL)洗涤，有机相用无水硫酸钠干燥，过滤，减压除去有机溶剂，得到 化合物C5-3。 1H NMR(400MHz,CDCl 3)δ8.38(d,J＝1.2Hz,1H),8.29(dd,J＝1.2,6.8Hz,1H),7.71(d,J＝6.4Hz,1H),3.95(d,J＝12.4Hz,6H).MS m/z:211.9[M+1] +.
步骤3：化合物C5-4的制备
将化合物C5-3(7.0g,33.2mmol)溶于氧氯化磷(70.0mL)中，反应液升温至100℃继续搅拌16小时。冷却，直接将反应液浓缩，然后将粗品慢慢倒入水(100mL)中，用饱和碳酸钠的水溶液调pH＝8，二氯甲烷(100mL×2)萃取，最后用饱和氯化钠的水溶液(50mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱液：9-25％乙酸乙酯/石油醚)分离纯化，得到化合物C5-4和化合物C5-4’。化合物C5-4： 1H NMR(400MHz,CDCl 3)δ:8.88(s,1H),7.52(s,1H),3.97(s,3H),3.95(s,3H).MS m/z:229.9[M+1] +.化合物C5-4’： 1H NMR(400MHz,CDCl 3)δ8.59(d,J＝4.8Hz,1H),7.79(d,J＝4.8Hz,1H),4.01(s,3H),3.96(s,3H).MS m/z:230.0[M+1] +.
步骤4：化合物C5-5的制备
将化合物C5-4(1.6g,6.9mmol)溶于乙醇(40mL)中，加入硼氢化钠(1.3g,34.8mmol)，反应液在25℃继续搅拌1小时，升温至80℃继续搅拌15小时。冷却，向反应液中加入饱和氯化铵的水溶液(20mL)，减压除去有机溶剂，向得到的粗品中加入水(20mL)，用乙酸乙酯(50mL×2)萃取，合并后的有机相用饱和氯化钠水溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：33-100％乙酸乙酯/石油醚)分离纯化，得到化合物C5-5。 1H NMR(400MHz,MeOD)δ8.29(s,1H),7.60(s,1H),4.77(s,2H),4.65(s,2H).MS m/z:173.6[M+1] +.
步骤5：化合物C5-6的制备
将化合物C5-5(650.0mg,3.7mmol)溶于N,N-二甲基甲酰胺(20mL)中，在0℃下加入钠氢(449.3mg,11.2mmol,60％纯度)，反应液继续搅拌0.5小时，然后加入对甲基苯磺酰氯(713.8mg,3.7mmol)，反应液缓慢升温至25℃继续搅拌15.5小时。将反应液倒入水(30mL)中，用乙酸乙酯(75mL×2)萃取，合并后的有机相用水(30mL)洗涤，饱和氯化钠的水溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：9-25％乙酸乙酯/石油醚)分离纯化，得到化合物C5-6。 1H NMR(400MHz,CDCl 3)δ8.30(s,1H),7.25(s,1H),5.12(s,2H),5.08(s,2H).
步骤6：化合物C5-7的制备
氮气保护下，将化合物C5-6(170.0mg,1.1mmol)和化合物B1(545.7mg,1.1mmol)溶于二氧六环(10mL)和水(1mL)的混合溶剂中，加入Pd(dppf)Cl 2(80.0mg,109.3μmol)和碳酸钠(231.6mg,2.2mmol)， 反应液升温至100℃继续搅拌10小时。冷却，过滤，滤饼用乙酸乙酯(30mL)洗涤，减压除去有机溶剂，得到化合物C5-7粗品，该化合物不经进一步纯化直接用于下一步反应。MS m/z:493.0[M+1] +.
以下化合物使用与化合物C5-7类似的方法合成得到：
Figure PCTCN2019113278-appb-000070
Figure PCTCN2019113278-appb-000071
步骤7:化合物C5的制备
将化合物C5-7(538.0mg,1.1mmol)溶于二氯甲烷(5mL)中，加入三氟乙酸(1.5g,13.5mmol)，反应液在25℃继续搅拌2.5小时。将反应液浓缩，向得到的粗品中慢慢加入饱和碳酸氢钠水溶液调pH＝8，用二氯甲烷(50mL×2)萃取，合并后的有机相用饱和氯化钠水溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱液：16.7-50％乙酸乙酯/石油醚)分离纯化，得到化合物C5。 1H NMR(400MHz,CDCl 3)δ:8.58(s,1H),7.91(s,1H),7.68-7.63(m,2H),7.52(t,J＝8.0Hz,2H),7.42-7.37(m,1H),5.20(s,2H),5.13(s,2H),3.69(s,2H),2.35(s,3H).MS m/z:293.0[M+1] +.
以下化合物使用与化合物C5类似的方法合成得到：
Figure PCTCN2019113278-appb-000072
Figure PCTCN2019113278-appb-000073
参考例10:合成中间体C6-8
Figure PCTCN2019113278-appb-000074
步骤1：化合物C6-2的制备
将化合物C6-1(10.0g,65.7mmol)溶于丙酮(30mL)中，在0℃下逐滴加入碘甲烷(10.6g,74.7mmol)，反应液缓慢升温至25℃继续搅拌20小时。过滤，滤饼用丙酮(50mL)洗涤，滤饼经真空干燥得到化合物C6-2。 1H NMR(400MHz,DMSO-d 6)δ8.19-8.17(m,1H),7.41-7.35(m,2H),4.52(s,2H),3.11(s,9H).
步骤2：化合物C6-4的制备
将化合物C6-2(9.0g,40.8mmol)溶于二甲亚砜(60mL)中，慢慢加入氢化钠(3.3g,81.6mmol,60％纯度)，反应液在25℃继续搅拌1小时，缓慢滴加化合物C6-3(10.0g,34.0mmol)的二甲亚砜(40mL)溶液，反应液在25℃继续搅拌16小时。将反应液缓慢倒入到冰水(50mL)中，用乙酸乙酯(100mL×2)萃取，合并后的有机相用水(100mL)洗涤，饱和氯化钠水溶液(50mL)洗涤，无水硫酸钠干燥，过滤，减 压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：16.7-50％乙酸乙酯/石油醚)分离纯化，得到化合物C6-4。 1H NMR(400MHz,CDCl 3)δ:8.08-8.02(m,1H),7.02(d,J＝2.8Hz,2H),4.67(t,J＝9.2Hz,2H),3.34(t,J＝9.2Hz,2H).MS m/z:121.7[M+1] +.
步骤3：化合物C6-5的制备
在0℃下，将化合物C6-4(700.0mg,5.8mmol)溶于浓硫酸(4mL)中，慢慢加入浓硝酸(1.1g,11.5mmol,68％纯度)的浓硫酸(2mL)的溶液，反应液在0℃继续搅拌1小时。将反应液慢慢倒入到冰水(30mL)中，二氯甲烷(50mL×2)萃取，合并后的有机相用饱和氯化钠水溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到化合物C6-5粗品，该化合物不经进一步纯化直接用于下一步反应。 1H NMR(400MHz,CDCl 3)δ＝8.17(d,J＝8.4Hz,1H),7.20(d,J＝8.4Hz,1H),4.89(t,J＝9.2Hz,2H),3.47(t,J＝8.8Hz,2H)MS m/z:166.6[M+1] +.
步骤4：化合物C6-6的制备
氮气保护下，将化合物C6-5(800.0mg,4.8mmol)溶于乙醇(20mL)中，加入Pd/C(100.0mg,10％纯度)，反应液在25℃和15psi氢气氛围下继续反应16小时。过滤，滤饼用甲醇(30mL)洗涤，减压除去有机溶剂，得到化合物C6-6粗品，该化合物不经进一步纯化直接用于下一步反应。 1H NMR(400MHz,CDCl 3)δ6.91(d,J＝8.8Hz,1H),6.28(d,J＝8.8Hz,1H),4.59(t,J＝8.8Hz,2H),4.11(br s,2H),3.20(t,J＝8.8Hz,2H).MS m/z:136.7[M+1] +.
步骤5：化合物C6-7的制备
将化合物C6-6(650.0mg,4.8mmol)溶于醋酸(5mL)中，加入液溴(465.0mg,2.9mmol)，反应液在25℃继续搅拌1小时。将反应液倒入到冰水(20mL)中，用饱和的碳酸钠溶液调pH＝7-8，用乙酸乙酯(50mL×2)萃取，合并后的有机相用饱和氯化钠水溶液(30mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱液：20-33％乙酸乙酯/石油醚)分离纯化，得到化合物C6-7。 1H NMR(400MHz,CDCl 3)δ7.15(s,1H),4.62(t,J＝8.8Hz,2H),4.54(s,2H),3.17(t,J＝8.8Hz,2H).MS m/z:216.6[M+1] +.
步骤6：化合物C6-8的制备
将化合物C6-7(570.0mg,2.6mmol)溶于四氢呋喃(10mL)中，加入亚硝酸叔丁酯(494.2mg,4.8mmol)，反应液升温至80℃继续搅拌2小时。减压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：14-25％乙酸乙酯/石油醚)分离纯化，得到化合物C6-8。 1H NMR(400MHz,CDCl 3)δ:8.12(d,J＝2.0Hz,1H), 7.18(d,J＝1.6Hz,1H),4.73(t,J＝8.8Hz,2H),3.30(t,J＝8.8Hz,2H).MS m/z:201.5[M+1] +.
参考例11:合成中间体C7-2
Figure PCTCN2019113278-appb-000075
步骤1：化合物C7-1的制备
将化合物C6-4(2.2g,18.2mmol)溶于二氯甲烷(50mL)中，缓慢加入间氯过氧苯甲酸(5.9g,27.2mmol,80％纯度)，反应液在25℃继续搅拌16小时。反应液用饱和碳酸钠水溶液(100mL)洗涤，水洗涤(100mL)，饱和氯化钠水溶液(50mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到化合物C7-1。 1H NMR(400MHz,CDCl 3)δ7.83(d,J＝6.4Hz,1H),7.06(t,J＝7.2Hz,1H),6.74(d,J＝8.0Hz,1H),4.76(t,J＝8.8Hz,2H),3.51(t,J＝8.8Hz,2H)MS m/z:137.8[M+1] +.
步骤2：化合物C7-2的制备
将化合物C7-1(800.0mg,5.8mmol)溶于氧氯化磷(10mL)中，反应液升温至100℃继续搅拌20小时。减压除去有机溶剂，所得粗品用二氯甲烷(50mL)溶解，饱和碳酸钠的水溶液(30mL)洗涤，饱和氯化钠的水溶液(20mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱液：9-16％乙酸乙酯/石油醚)分离纯化，得到化合物C7-2。 1H NMR(400MHz,CDCl 3)δ7.09-6.95(m,2H),4.70(t,J＝8.8Hz,2H),3.33(t,J＝8.8Hz,2H)MS m/z:155.9[M+1] +.
参考例12:合成中间体C8-5
Figure PCTCN2019113278-appb-000076
步骤1：化合物C8-2的制备
将化合物C8-1(20.00g,119.68mmol)溶于甲醇(200mL)中，滴入浓硫酸(7.36g,75.04mmol)，反应液升温至70℃继续搅拌29小时。冷却至40℃，滴加液溴(47.81g,299.19mmol)，反应液升温至55℃继续搅拌48小时。冷却至室温，向反应液中缓慢加入硫代硫酸钠溶液(100mL)，将反应液体积浓缩至一半，乙酸乙酯(200mL×4)萃取，合并后的有机相经饱和食盐水(50mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗品经柱层析分离纯化(洗脱液：0％～20％乙酸乙酯/石油醚)，得到化合物C8-2。 1H NMR(400MHz,CDCl 3)δ:8.82(d,J＝2.28Hz,1H),8.30(d,J＝2.28Hz,1H),4.00(s,3H),3.96(s,3H).MS m/z＝273.8 [M+H] +.
步骤2：化合物C8-3的制备
氮气保护下，将化合物C8-2(12.00g,43.78mmol)溶于乙醇(150mL)中，加入硼氢化钠(8.28g,218.92mmol)，反应液在15℃搅拌1.5小时，升温至80℃继续搅拌15.5小时。热过滤，减压除去有机溶剂，得到的粗品经高效液相色谱法(column:Phenomenex luna C18 250*50mm*10μm；流动相:[H 2O(10mM NH 4HCO 3)-ACN]；B(乙腈)％:0％-30％,25min)分离纯化，得到化合物C8-3。 1H NMR(400MHz,MeOD)δ:8.51-8.48(m,1H),8.08-8.06(m,1H),4.74(s,2H),4.69(s,2H).
步骤3：化合物C8-4的制备
将化合物C8-3(1.4g,6.42mmol)溶于溴化氢(55.13g,258.92mmol,38％纯度)中，反应液升温至130℃搅拌24小时，加入硫酸(9.2g,91.92mmol)，反应液在该温度下继续搅拌24小时。冷却，向反应液中加入饱和碳酸氢钠溶液调节pH＝7-8，二氯甲烷(150mL×3)萃取，合并后的有机相经无水硫酸钠干燥，过滤，减压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：0-10％乙酸乙酯/石油醚)分离纯化，得到化合物C8-4。 1H NMR(400MHz,CDCl 3)δ:8.58(d,J＝2.0Hz,1H),7.85(d,J＝2.0Hz,1H),4.67(s,2H),4.56(s,2H).MS m/z:343.7[M+H] +.
步骤4：化合物C8-5的制备
冰水浴条件下，将化合物C8-4(800mg,2.33mmol)和甲胺盐酸盐(471.27mg,6.98mmol,HCl)溶于二氯甲烷(5mL)中，加入DIEA(1.2g,9.31mmol)，反应液在该温度下搅拌1小时，升温至25℃继续搅拌17小时。减压除去有机溶剂，得到的粗产物经硅胶柱层析(洗脱液：10-100％乙酸乙酯/石油醚)分离纯化，得到化合物C8-5。 1H NMR(400MHz,CDCl 3)δ:8.46(s,1H),7.64(s,1H),3.98-3.91(m,4H),2.62(s,3H).
参考例13:合成中间体C9-2
Figure PCTCN2019113278-appb-000077
步骤1：化合物C9-2的制备
将化合物C9-1(0.9g,4.71mmol,盐酸盐)分散在二氯甲烷(30mL)中，依次加入冰乙酸(565.75mg,9.42mmol)，甲醛(1.15g,14.13mmol)和醋酸硼氢化钠(1.40g,6.59mmol)，反应液在20℃下继续搅拌16小时。加入1mL氨水，减压除去有机溶剂，得到的粗品经硅胶柱层析(洗脱剂0～10％甲醇/二氯甲烷) 分离纯化，得到化合物C9-2。 1H NMR(400MHz,CDCl 3)δ:8.26(s,1H),7.22(s,1H),4.03(d,J＝2.4Hz,4H),2.65(s,3H).
参考例14:合成中间体C10-3
Figure PCTCN2019113278-appb-000078
步骤1：化合物C12-1的制备
冰水浴条件下，将化合物C12-1(1.8g,8.26mmol)加入到二氯亚砜(5.89g,49.53mmol)中，缓慢升温至20℃，氮气保护下继续搅拌16小时，加入甲基叔丁基醚(4.75mL)。加入10mL甲基叔丁基醚，减压除去有机溶剂，得到化合物C12-1粗品，该化合物不经进一步纯化直接用于下一步反应。MS m/z:256.0[M+H] +.
步骤2：化合物C12-3的制备
将化合物C12-1(1.0g,3.92mmol)溶于N,N-二甲基甲酰胺(40mL)中，加入化合物C12-2(2.06g,11.76mmol)和二异丙基乙胺(2.03g,15.69mmol)，反应液升温至80℃继续搅拌7小时。冷却，加入20mL水，用40mL乙酸乙酯萃取，无水硫酸钠干燥，过滤，减压除去有机溶剂，粗品经硅胶柱层析(洗脱液：0-20％乙酸乙酯/石油醚)分离纯化，得到化合物C12-3。 1H NMR(400MHz,CDCl 3)δ:8.46(s,1H),7.63(s,1H),4.10-3.95(m,4H),3.83(t,J＝6.0Hz,2H),2.91(t,J＝6.0Hz,2H),0.92(s,9H),0.09(s,6H)，MS m/z:359.1[M+H] +.
参考例15:合成参考化合物D1
Figure PCTCN2019113278-appb-000079
步骤1:化合物D1-2的制备
将化合物D1-1(5.00g,28.90mmol)溶于1,4-二氧六环(120.0mL)，随后分别加入联硼酸频哪醇酯(8.81g,34.68mmol)和乙酸钾(5.67g,57.80mmol)，用氮气置换体系三次，接着再加入1,1-双(二苯基膦)二茂铁二氯化钯(1.06g,1.45mmol)，再次用氮气置换体系三次，100℃下反应18小时。将反应液过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-50％乙酸乙酯/石油醚)分离纯化得到化合物D1-2粗品。 1H NMR(400MHz,CDCl 3)δ:8.92(s,2H),2.75(s,3H),1.35(s,12H).
步骤2:化合物D1-3的制备
将化合物D1-2(2.67g,12.14mmol)溶于乙醇(15.0mL)和甲苯(45.0mL)的混合溶剂中，接着分别加入化合物B1-3(3.00g,9.34mmol)和碳酸钠(1.98g,18.68mmol)，用氮气置换体系三次，接着再加入四(三苯基膦)钯(1.08g,933μmol)，继续用氮气置换体系三次，100℃下反应17小时。将反应液过滤，滤饼用乙酸乙酯(50.0mL)淋洗，收集滤液，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-100％乙酸乙酯/石油醚)分离纯化得到化合物D1-3。 1H NMR(400MHz,CDCl 3)δ:8.99(s,2H),7.65-7.60(m,2H),7.56-7.50(m 2H),7.43-7.37(m,1H),3.71(s,2H),2.79(s,3H),2.14(s,3H).MS m/z:266.0[M+1] +.
步骤3:化合物D1的制备
向化合物D1-3(500mg,1.88mmol)的二氯甲烷(6.0mL)溶液中分别加入二异丙基乙胺(972mg,7.52μmol,1.3mL)和三光气(390mg,1.32mmol)，25℃下反应20分钟后再分别加入化合物A3(448mg,1.88mmol)和二异丙基乙胺(972mg,7.52mmol,1.3mL)，继续在25℃下反应17小时。往反应液中加入二氯甲烷(30.0 mL)稀释，有机相用饱和食盐水(30.0mL)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经柱层析(洗脱剂:0-100％乙酸乙酯/石油醚)分离纯化得到化合物D1。 1H NMR(400MHz,MeOD)δ:8.91(s,2H),7.45-7.23(m,6H),7.11-6.91(m,3H),4.50-4.38(m,1H),3.86-3.75(m,1H),3.70-3.47(m,6H),3.36(s,3H),3.24-3.06(m,3H),2.63(s,3H),2.00(s,3H).MS m/z:530.1[M+1] +.
实施例1:化合物1的制备
Figure PCTCN2019113278-appb-000080
步骤1:化合物1的制备
向化合物C1(49mg,168μmol)的二氯甲烷(5.0mL)溶液中分别加入二异丙基乙胺(65mg,503μmol,88μL)和三光气(25mg,84μmol)，25℃下反应15分钟后再分别加入化合物A3(40mg,168μmol)和二异丙基乙胺(65mg,503μmol,88μL)，继续在25℃下反应14小时。往反应液中加入二氯甲烷(30.0mL)稀释，有机相用饱和食盐水(30.0mL)洗涤一次，有机相用无水硫酸钠干燥，过滤，将滤液浓缩至干，所得粗品经反相制备HPLC(中性)分离得到化合物1。 1H NMR(400MHz,MeOD)δ:8.80(s,1H),8.11(s,1H),7.58-7.53(m,2H),7.52-7.40(m,3H),7.37-7.30(m,1H),7.12-6.94(m,3H),5.24(s,2H),5.08(s,2H),4.40-4.28(m,1H),3.54(t,J＝5.6Hz,2H),3.37(s,3H),3.37-3.25(m,2H),3.21-3.12(m,1H),3.02-2.93(m,1H),2.83-2.69(m,3H),2.63-2.55(m,1H),2.16(s,3H).MS m/z:557.3[M+1] +.
以下化合物使用与化合物1类似的方法合成得到，其中化合物6在经过反相制备高效液相色谱法(柱子:
YMC-Actus Triart C 18 100*30mm*5μm；流动相:[水(0.05％盐酸)-乙腈]；B(乙腈)％:25％-48％,7min)分离纯化，得到化合物6的盐酸盐。化合物6的盐酸盐可以通过饱和碳酸氢钠水溶液调节pH至7-8，二氯甲烷萃取，减压除去有机溶剂后得到化合物6。
Figure PCTCN2019113278-appb-000081
Figure PCTCN2019113278-appb-000082
Figure PCTCN2019113278-appb-000083
Figure PCTCN2019113278-appb-000084
实施例14:化合物14的制备
Figure PCTCN2019113278-appb-000085
步骤1:化合物14-1的制备
氮气保护下，将化合物C10(0.04g,88.96μmol)溶于二氯甲烷(6mL)中，然后加入二异丙基乙胺(45.99mg,355.82μmol,61.98μL)和三光气(21.12mg,71.16μmol)，反应液在20℃下搅拌1小时，然后加入二异丙基乙胺(45.99mg,355.82μmol,61.98μL)和化合物A3(22.26mg,93.40μmol)，反应液在20℃继续搅拌3小时。将反应体系直接减压浓缩，所得粗产品通过薄层层析硅胶板(乙酸乙酯：甲醇＝10:1)分离纯化，得到化合物14-1。 1H NMR(400MHz,CDCl 3)δ:8.76(d,J＝1.6Hz,1H),7.92(d,J＝2.0Hz,1H),7.55(d,J＝7.2Hz,2H),7.42(t,J＝7.2Hz,2H),7.35(t,J＝7.2Hz,1H),7.01-6.87(m,4H),5.53(s,1H),4.11(s,4H),3.87(t,J＝6.0Hz,2H),3.65-3.62(m,1H),3.49(s,2H),3.40-3.10(m,4H),2.96(t,J＝6.0Hz,3H),2.83-2.71(m,4H),2.46-2.39(m,1H),2.18(s,3H),0.94(s,9H),0.11(s,6H).MS m/z:357.8[M/2+1] +.
步骤2:化合物14的制备
氮气保护下，将化合物14-1(11mg,15.41μmol)溶于THF(1.6mL)中，加入吡啶(8.04mg,101.69μmol)和氟化氢吡啶(14.40mg,101.69μmol,70％纯度)，反应液在20℃继续搅拌5小时。加入饱和碳酸氢钠溶液(8mL)，乙酸乙酯(8mL×3)萃取，合并后的有机相用饱和食盐水(10mL)洗涤，无水硫酸钠干燥，过滤，减压除去有机溶剂，所得粗产品通过薄层层析硅胶板(乙酸乙酯：甲醇＝10:1)分离纯化，得到化合物14。 1H NMR(400MHz,CDCl 3)δ:8.63(s,1H),7.77(s,1H),7.66(d,J＝7.6Hz,2H),7.48(t,J＝7.6Hz,2H),7.37(t,J＝7.2Hz,1H),7.07(d,J＝7.2Hz,1H),6.99-6.92(m,3H),4.01-3.97(m,1H),3.91-3.87(m,1H),3.78(s,2H),3.74-3.70(m,3H),3.54(s,2H),3.50(s,2H),3.39(s,3H),2.85-2.79(m,6H),2.36-2.19(m,2H),1.92(s,3H).MS m/z:300.7[M/2+1] +.
TrkA酶活性测试
实验材料
TrkA                     Invitrogen-PV4114
TK检测试剂盒             Cisbio-62TK0PEJ
检测板                   PerkinElmer-6007299
Envision                 PerkinElmer-2104
激酶反应缓冲液
50mM Hepes(pH 7.5)，5mM MgCl 2，0.01mM Orthovanadate，1％BSA，1mM DTT
实验方法
本次试验使用Cisbio公司的均相时间分辨的荧光共轭能量转移(
Figure PCTCN2019113278-appb-000086
方法)进行活性检测。在检测板中，将酶、生物素标记的多肽底物、ATP以及检测化合物混合，孵育反应。反应后，加入EDTA终止反应，并同时加入Eu标记的抗体，链合亲和素标记的XL665进行反应并检测。数据分别用荧光信号665nm和620nm的读数来表示，其中665nm/620nm的高比值表示活性较高，而665nm/620nm的低比值则表示活性受到抑制。
实验步骤
1.化合物稀释：待测化合物3倍进行稀释，共11个浓度，最终体系浓度从10μM至0.17nM；
2.在缓冲液为50mM Hepes(pH 7.5)，5mM MgCl 2，0.01mM Orthovanadate，1％BSA，1mM DTT的10μL反应体系中，包括0.5nM TrkA，0.3μM biotin-TK peptide，90μM ATP，在23℃孵育90分钟。加入10μL含有20mM EDTA，0.67nM TK抗体，50nM XL-665的终止溶液，在23℃孵育60分钟，Envision读数；
3.将仪器读取的数据计算出化合物的抑制率，然后运用IDBS的XLFIT5中mode 205计算出IC 50值。
实验结果
结果见表1。
表1化合物对TrkA酶抑制的IC 50值
化合物编号 TrkA IC 50(nM)
化合物1 0.20
化合物2 2.90
化合物3 0.49
化合物4 0.79
化合物5 0.64
化合物6盐酸盐 1.40
化合物7 0.17
化合物8 0.19
化合物9 0.60
化合物10 1.1
化合物11 1.2
化合物12 0.44
化合物13 1.6
化合物14 0.43
结果表明：本发明化合物具有显著的TrkA酶抑制活性。
Caco-2细胞双向渗透性研究
实验目的
Caco-2细胞是是一种人的结肠癌细胞。单层Caco-2细胞模型被广泛地应用于评估受试化合物在小肠中的被动扩散和主动转运的吸收情况。小肠中的外排转运体包含P糖蛋白(P-gp)和乳腺癌耐药蛋白(BCRP)等。本实验用于测定受试化合物穿过Caco-2细胞模型的双向渗透性，同时可考察其外排转运情况。
实验操作
1.1细胞培养
细胞培养使用添加了2mM的L-谷氨酰胺，10％胎牛血清(FBS)，100U/mL青霉素-G以及100μg/mL链霉素的MEM(Minimum Essential Media)培养基。细胞培养条件为37±1℃，5％CO 2及饱和湿度。待细胞长至80-90％密集度，加入胰酶(0.05％,w/v)/EDTA(0.02％,w/v)消化液消化细胞后种板。本实验采用的是第38代Caco-2细胞，细胞接种于BD Falcon的Transwell-96孔板里(货号359274)，接种密度为1×105细胞/cm 2。细胞置于二氧化碳培养箱中培养22天后用于转运实验，期间每隔四到五天更换一次培养基。
1.2转运实验
本项目采用含10mM HEPES的Hank’s平衡盐缓冲液(pH 7.40±0.05)为转运缓冲液。测试受试化合物和阳性药地高辛在2μM浓度下的双向转运，各两个平行样本，测试非诺特罗和普萘洛尔从顶端到基底端(A-B)的转运。孵育体系中DMSO的浓度控制在1％以下，加样后，将细胞板置于37±1℃，5％CO 2及饱和湿度条件下孵育120分钟，所有的样品漩涡震荡后于3220rpm，20℃离心20分钟，对照品和供试品用超纯水1:1(v:v)稀释后储存于4℃，采用液相色谱串联质谱(LC/MS/MS)进行分析测试。转运实验完成后，采用荧光黄外排实验(the Lucifer Yellow Rejection Assay)来测试Caco-2细胞层的完整性。
1.3样品分析
本研究采用液相色谱串联质谱(LC/MS/MS)方法半定量分析起始溶液、接收液和给药孔上清液中受试化合物和对照品非诺特罗、普萘诺尔以及地高辛与内标的峰面积比值。
1.4数据计算
采用如下公式计算表观渗透系数(P app,cm/s)，外排率以及回收率。
表观渗透系数(P app,cm/s)采用如下公式计算：
P app＝(dC r/d t)×V r/(A×C 0)
dC r/d t是化合物在单位时间内接收端的累积浓度(μM/s)；V r是接收端溶液的体积(顶端和基底端的溶液体积分别为0.075mL和0.250mL)；A是胞单层的相对表面积(0.0804cm 2)；C 0是给药端供试品的起始浓度(nM)或对照品的峰面积比值。
外排比采用如下公式计算：
外排比＝P app(BA)/P app(AB)
回收率采用如下公式计算：
％回收率＝100×[(V r×C r)+(V d×C d)]/(V d×C 0)
C 0是给药端供试品的起始浓度(nM)或对照品的峰面积比值；V d是给药端的体积(顶侧为0.075mL，基底侧为0.250mL)；C d和C r分别为给药端和接收端供试品的终浓度(nM)或对照品的峰面积比值。
实验结果
结果见表2。
表2化合物1和化合物12的Caco-2细胞膜渗透性数据
化合物编号 P app(AB)(10 -6cm/s) P app(BA)(10 -6cm/s) 外排比
参考化合物D1 0.06 34.81 580.17
化合物1 0.05 28.55 571.00
化合物12 0.01 19.54 1508.34
结果表明：本发明化合物具有较高的Caco-2细胞膜渗透外排比，可以更好地规避进脑风险，减少因为中枢神经系统中的Trk靶点抑制所引起的运动失调、睡眠紊乱等副作用。
血浆蛋白结合率(PPB)测试
实验目的
测定受试化合物在人、SD大鼠及Beagle犬血浆中的蛋白结合率。
实验操作
取人、SD大鼠及Beagle犬的空白血浆796μL(血浆购买自BioreclamationIVT)，加入4μL受试化合物工作溶液(400μM)或华法林工作溶液(400μM)，使血浆样品中受试化合物与华法林终浓度均为2μM。将样品充分混合。有机相DMSO的终浓度为0.5％；移取50μL受试化合物和华法林血浆样品到样品接收板中，立即加入相应体积的对应空白血浆或缓冲液，使得每个样品孔的终体积为100μL，血浆：透析缓冲液的体积比为1:1，然后向这些样品中加入400μL终止液，此样品将作为T 0样品用于回收率及稳定性测定。将T 0样品存储于2℃-8℃，等待与其它透析完的样品一起进行后续处理；将150μL受试化合物和华法林血浆样品加入到每个透析孔的给药端，在透析孔对应的接收端中加入150μL空白透析缓冲液。然后将透析板封上透气膜后置于湿润的5％CO 2的培养箱中，在37℃、100rpm振荡孵育4小时。透析结束后，移取50μL透析后的缓冲液样品和透析后的血浆样品到新的样品接收板。在样品中加入相应体积的对应空白血浆或缓冲液，使得每个样品孔的终体积为100μL，血浆：透析缓冲液的体积比为1:1。所有样品经过蛋白沉淀后进行LC/MS/MS分析，并通过以下公式计算化合物的游离率(Unbound)％、结合率
(Bound)％和回收率(Recovery)％：
％Unbound＝100*F C/T C,
％Bound＝100-％Unbound,
％Recovery＝100*(F C+T C)/T 0。
其中F C是透析板缓冲液端化合物的浓度；T C是透析板血浆端化合物的浓度；T 0是零时刻血浆样品中化合物的浓度。
实验结果
结果见表3。
表3化合物的人、大鼠、犬血浆蛋白游离结合率
化合物编号 血浆蛋白游离结合率
  人血浆 SD大鼠血浆 Beagle犬血浆
参考化合物D1 13.6％ 9.6％ 2.2％
化合物1 12.1％ 1.5％ 2.9％
化合物2 11.5％ 1.6％ 6.9％
化合物3 11.5％ 2.5％ 3.9％
化合物4 26.1％ 3.8％ 9.7％
化合物5 37.7％ 5.2％ 10.6％
化合物9 21.7％ 1.2％ --
化合物12 16.8％ 7.1％ --
“--”：未进行犬种属PPB测试。
结果表明：本发明化合物具有与参考化合物D1相当甚至更高的人血浆蛋白游离结合率。
细胞色素P450同工酶抑制活性测试
实验目的
测定受试化合物对人细胞色素P450同工酶不同亚型的抑制活性。
实验操作
准备受试化合物、标准抑制剂(100×最终浓度)和混合底物工作溶液；将冷冻于-80℃冰箱的微粒体取出解冻。将2μL的待测化合物和标准抑制剂溶液加至相应孔位，同时将2μL相应的溶剂加至无抑制剂对照孔位(NIC)和空白对照孔位(Blank)孔位；其次将20μL混合底物溶液加至相应孔位，Blank孔位除外(将20μL PB加至Blank孔位)；准备人肝微粒体溶液(使用后标记日期立刻放回冰箱)，随即将158μL人肝微粒体溶液加至所有孔位；将上述样品板放入37℃水浴预孵育，随即准备辅酶因子(NADPH)溶液；10分钟后，添加20μL NADPH溶液到所有孔位，样品板摇匀后，放入37℃水浴孵育10分钟；在相应时间点，加入400μL冷的乙腈溶液(内标为200ng/mL甲苯磺丁脲和拉贝洛尔)终止反应；样品板混合均匀后，4000rpm离心20分钟，沉淀蛋白质；取200μL上清加至100μL水中，摇匀后送LC/MS/MS检测。
实验结果
结果见表4。
表4化合物1、化合物7和化合物12对P450同工酶抑制的IC 50值
Figure PCTCN2019113278-appb-000087
Figure PCTCN2019113278-appb-000088
结果表明：本发明化合物具有比参考化合物D1更低的药物-药物相互作用风险。
肝微粒体中的代谢稳定性(MMS)研究
实验目的
测试供试品在人、大鼠和犬肝微粒体中的代谢稳定性。
实验材料
供试品(10mM),睾酮(Testosterone,对照品,10mM),双氯芬酸(Diclofenac,对照品,10mM),普罗帕酮(Propafenone,对照品,10mM)。
缓冲体系
1. 100mM磷酸钾缓冲剂(pH 7.4)。
2. 10mM MgCl 2。
化合物稀释
1.中间体溶液：采用45μL DMSO(带有450μL 1:1甲醇/水)来稀释5μL供试品或对照品。
2.工作液：采用450μL 100mM磷酸钾缓冲剂来稀释中间体溶液。
NADPH再生体系
1.β-磷酸酰胺腺嘌呤二核苷酸，来源于Sigma,Cat.No.N0505。
2.异柠檬酸，来源于Sigma,Cat.No.I1252。
3.异柠檬酸脱氢酶，来源于Sigma,Cat.No.I2002。
肝微粒体溶液制备(最终浓度：0.5mg蛋白/mL)
Figure PCTCN2019113278-appb-000089
终止液
含100ng/mL Tolbutamide和100ng/mL Labetalol的冷乙腈作为内标物。
实验方法
加10μL供试品或对照品工作液到所有板中(T 0,T 5,T 10,T 20,T 30,T 60,NCF 60)。
分配680μL/well肝微粒体溶液到96孔板上，然后添加80μL/well到每块板上，将上述孵育板放置于37℃预孵育大约10分钟。
在NCF 60板上每孔添加10μL 100mM磷酸钾缓冲液。
预孵育结束后，分配90μL/well NADPH再生体系工作液到96孔板上，然后添加10μL/well到每块板上以 启动反应。
孵化适当的时间(如5、10、20、30和60分钟)。
分别在每个样品孔中加入300μL/well终止液(于4℃冷藏，含100ng/mL Tolbutamide和100ng/mL Labetalol)。样品板摇匀约10分钟并在4℃下4000转离心20分钟。
离心时，加300μL HPLC水到每孔中，取100μL上清液用于LC-MS/MS分析。
数据分析
通过下面公式中计算T 1/2和C lint(mic)
Figure PCTCN2019113278-appb-000090
当
Figure PCTCN2019113278-appb-000091
Figure PCTCN2019113278-appb-000092
Figure PCTCN2019113278-appb-000093
Figure PCTCN2019113278-appb-000094
每克肝含45mg微粒体蛋白，小鼠、大鼠、犬、猴和人的肝重分别为88g/kg,40g/kg,32g/kg,30g/kg和20g/kg。
C t为时间t时的浓度，t为孵育时间，C 0为0时的浓度，K e为消除速率常数，Cl int(mic)为肝微粒固有清除率，Cl int(liver)为肝固有清除率。
实验结果
结果见表5。
表5化合物7和化合物12在人、大鼠、犬肝微粒中的清除率
Figure PCTCN2019113278-appb-000095
“--”：未进测试。
结果表明：本发明化合物7在人、大鼠、犬三个种属上，都具有比参考化合物D1更好的肝微粒体代谢稳定性。
大鼠单次给药药代研究
实验目的
以雄性SD大鼠为受试动物，单次给药后测定化合物血药浓度并评估药代动力学行为。
实验操作
选择健康成年雄性SD大鼠4只(7-9周龄，购自上海维通利华实验动物有限公司)，随机分成两组，每组2只，一组静脉注射给予受试化合物2mg/kg，另一组灌胃给予受试化合物10mg/kg。静脉给药的溶媒为10％乙醇+20％蓖麻油聚氧乙烯醚+70％生理盐水，灌胃给药的溶媒为0.5％羧甲基纤维素钠+0.2％吐温80。静脉组动物于给药后0.0833,0.25,0.5,1.0,2.0,4.0,6.0,8.0,24小时采集血浆样品，而灌胃组动物于给药后0.25,0.5,1.0,2.0,4.0,6.0,8.0,24小时采集血浆样品。采用LC-MS/MS法测定血药浓度，使用WinNonlin TM Version6.3(Pharsight,Mountain View,CA)药动学软件，以非房室模型线性对数梯形法计算相关药代动力学参数。实验结果
结果见表6。
表6化合物1和化合物12在大鼠上的药代动力学数据
Figure PCTCN2019113278-appb-000096
其中，C 0为起始浓度，T 1/2为消除半衰期，Vd ss为稳态表观分布容积，Cl为总清除率，AUC 0-last为从0时间到最后一个可定量时间点的血浆浓度-时间曲线下面积，AUC 0-inf为从0时间到外推至无穷大时的血浆浓度-时间曲线下面积，C max为达峰浓度，T max为达峰时间。
结果表明：在大鼠单次给药药代研究中，相同的口服灌胃给药剂量下，本发明化合物1具有比参考化合物D1更高的血浆暴露量(AUC 0-last)和更高的口服生物利用度，本发明化合物12具有比参考化合物D1更大的Vd和更长的T 1/2。
小鼠单次给药后体内药代动力学研究
实验目的
以雄性CD-1小鼠为受试动物，单次给药后测定化合物血药浓度并评估药代动力学行为。
实验材料：
CD-1小鼠(雄性，20-40g，6～9周龄，上海西普尔－必凯实验动物有限公司)
实验操作：
以标准方案测试待测化合物静脉注射及口服给药后的啮齿类动物药代特征，实验中待测化合物配成澄清溶液或均一混悬液，给予小鼠单次静脉注射及口服给药。静脉注射组溶媒为一定比例的乙醇，Cremophor EL和生理盐水溶液，涡旋，制备得到1mg/mL澄清溶液，微孔滤膜过滤后备用；口服溶媒为一定比例的甲基纤维素溶液或一定比例甲基纤维素和吐温80水溶液，待测化合物与溶媒混合后，涡旋，制备得到10mg/mL澄清或均一混悬液备用。小鼠2mg/kg静脉给药或100mg/kg口服给药后，收集一定量的全血样品，3200g离心10分钟，分离上清得血浆样品，根据实际需要将样品用空白血浆稀释一定倍数。将血浆样品加入20倍体积含内标的乙腈溶液沉淀蛋白，离心取上清液加入2倍体积的水再离心取上清进样，以LC-MS/MS分析方法定量分析血药浓度，并用Phoenix WinNonlin软件(美国Pharsight公司)计算药代参数，如达峰浓度，达峰时间，清除率，半衰期，药时曲线下面积，生物利用度等。
实验结果：
表7化合物12在小鼠体内的药代动力学性质
Figure PCTCN2019113278-appb-000097
其中，C 0为起始浓度，T 1/2为消除半衰期，Vd ss为稳态表观分布容积，Cl为总清除率，AUC 0-inf为从0时间到外推至无穷大时的血浆浓度-时间曲线下面积，C max为达峰浓度，T max为达峰时间。
结果表明：本发明化合物12具有较长的半衰期和较短的达峰时间，预示体内可能起效较快且有较长时间的药效，降低给药频率；具有良好的小鼠药代动力学性质和口服生物利用度。
比格犬单次给药后体内药代动力学研究
实验目的
以雄性比格犬为受试动物，单次给药后测定化合物血药浓度并评估药代动力学行为。
实验材料：
比格犬(雄性，6～12kg，大于6月龄，北京玛斯生物技术公司)
实验操作：
试验目的是测试待测化合物静脉注射及口服给药后的非啮齿类动物药代特征，实验中待测化合物配成澄清溶液或均一混悬液，给予比格犬单次静脉注射或口服给药。静脉注射组溶媒为一定比例二甲亚砜的HP-β-环糊精溶液或一定比例的乙醇，聚乙二醇400和生理盐水溶液，涡旋并超声，制备得到2mg/mL或1mg/kg澄清溶液，微孔滤膜过滤后备用；口服溶媒为一定比例二甲亚砜的HP-β
Figure PCTCN2019113278-appb-000098
环糊精溶液或一定比例的羧甲基纤维素钠溶液，待测化合物与溶媒混合后，涡旋并超声，制备得到2mg/mL澄清溶液或1mg/mL均一混悬液备用。比格犬2mg/kg或1mg/kg静脉给药,10mg/kg或5mg/kg口服给药后，收集一定量的全血样品，3000g离心10分钟，分离上清得血浆样品，加入10倍体积含内标的乙腈溶液沉淀蛋白，离心取上清液进样，以LC-MS/MS分析方法定量分析血药浓度，并用Phoenix WinNonlin软件(美国Pharsight公司)计算药代参数，如达峰浓度，达峰时间，清除率，半衰期，药时曲线下面积，生物利用度等。
实验结果：
表8化合物12在犬体内的药代动力学性质
Figure PCTCN2019113278-appb-000099
其中，C 0为起始浓度，T 1/2为消除半衰期，Vd ss为稳态表观分布容积，Cl为总清除率，AUC 0-inf为从0时间到外推至无穷大时的血浆浓度-时间曲线下面积，C max为达峰浓度，T max为达峰时间。
结果表明：本发明化合物12具有较长的半衰期和较短的达峰时间，预示体内可能起效较快且有较长时间的药效，降低给药频率；具有良好的比格犬药代动力学性质和口服生物利用度。

Claims (20)

  1. 式(Ⅰ)、(Ⅱ)所示化合物、其异构体或其药学上可接受的盐，
    Figure PCTCN2019113278-appb-100001
    其中，
    T 1、T 2和T 3分别独立地选自N和C(R 3)；
    X 1、X 2和X 3分别独立地选自O、N(R 4)和C(R 5)(R 6)；
    Z 1、Z 2和Z 3分别独立地选自N和CH；
    R 1选自C 1-6烷基和C 1-6烷氧基，所述C 1-6烷基和C 1-6烷氧基任选被1、2或3个R a取代；
    R 2选自任选被1、2或3个R b取代的C 1-3烷基；
    R 3分别独立地选自H、F、Cl、Br、I、OH和NH 2；
    R 4分别独立地选自H和任选被1、2或3个R c取代的C 1-3烷基；
    R 5和R 6分别独立地选自H、F、Cl、Br、I、OH和NH 2；
    n选自1、2和3；
    R a选自H、F、Cl、Br、I、OH、NH 2、CN、C 1-3烷基和C 1-3烷氧基，所述C 1-3烷基和C 1-3烷氧基任选被1、2或3个R取代；
    R b和R c分别独立地选自H、F、Cl、Br、I、OH和NH 2；
    R选自F、Cl、Br、I、OH和NH 2；
    带“*”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在；
    带“#”碳原子为手性碳原子，以(R)或(S)单一对映体形式或富含一种对映体形式存在。
  2. 根据权利要求1所述化合物、其异构体或其药学上可接受的盐，其中，R a选自H、F、Cl、Br、I、OH、NH 2、CN、CH 3和CH 3O，所述CH 3和CH 3O任选被1、2或3个R取代。
  3. 根据权利要求2所述化合物、其异构体或其药学上可接受的盐，其中，R a选自H、F、Cl、Br、I、OH、NH 2、CN、CH 3、CH 2F、CHF 2、CF 3和CH 3O。
  4. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，R 1选自CH 3、CH 3CH 2和CH 3O，所述CH 3、CH 3CH 2和CH 3O任选被1、2或3个R a取代。
  5. 根据权利要求4所述化合物、其异构体或其药学上可接受的盐，其中，R 1选自
    Figure PCTCN2019113278-appb-100002
  6. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，R 2选自CH 3和CH 3CH 2。
  7. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，R 4选自H、CH 3和CH 3CH 2，所述CH 3和CH 3CH 2任选被1、2或3个R c取代。
  8. 根据权利要求7所述化合物、其异构体或其药学上可接受的盐，其中，R 4选自H、CH 3、CH 2F、CHF 2、CF 3、CH 3CH 2和
    Figure PCTCN2019113278-appb-100003
  9. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，T 1、T 2和T 3分别独立地选自N、CH和CF。
  10. 根据权利要求9所述化合物、其异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019113278-appb-100004
    选自
    Figure PCTCN2019113278-appb-100005
    Figure PCTCN2019113278-appb-100006
  11. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，X 1、X 2和X 3分别独立地选自O、N(CH 3)、NH、CH 2和
    Figure PCTCN2019113278-appb-100007
  12. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019113278-appb-100008
    选自
    Figure PCTCN2019113278-appb-100009
  13. 根据权利要求12任意一项所述化合物、其异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019113278-appb-100010
    选自
    Figure PCTCN2019113278-appb-100011
  14. 根据权利要求1～3任意一项所述化合物、其异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019113278-appb-100012
    选自
    Figure PCTCN2019113278-appb-100013
  15. 根据权利要求14所述化合物、其异构体或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2019113278-appb-100014
    选自
    Figure PCTCN2019113278-appb-100015
  16. 根据权利要求1～9任意一项所述化合物、其异构体或其药学上可接受的盐，其选自
    Figure PCTCN2019113278-appb-100016
    其中，
    T 1、T 2和T 3分别独立地选自N和C(R 3)；
    D选自N(R 4)和O；
    R 1、R 2、R 3和R 4如权利要求1～9任意一项所定义。
  17. 根据权利要求16所述化合物、其异构体或其药学上可接受的盐，其选自
    Figure PCTCN2019113278-appb-100017
    其中，
    D选自N(R 4)和O；
    R 1、R 2、R 3和R 4如权利要求1～9任意一项所定义。
  18. 下列化合物、其异构体或其药学上可接受的盐
    Figure PCTCN2019113278-appb-100018
    Figure PCTCN2019113278-appb-100019
  19. 根据权利要求18所述的化合物、其异构体或其药学上可接受的盐，其选自
    Figure PCTCN2019113278-appb-100020
    Figure PCTCN2019113278-appb-100021
  20. 根据权利要求1～19任意一项所述的化合物、其异构体或其药学上可接受的盐在制备治疗TrkA抑制剂相关药物上的应用。
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CN106459013A (zh) * 2014-05-15 2017-02-22 阵列生物制药公司 作为trka激酶抑制剂的1‑((3s，4r)‑4‑(3‑氟苯基)‑1‑(2‑甲氧基乙基)吡咯烷‑3‑基)‑3‑(4‑甲基‑3‑(2‑甲基嘧啶‑5‑基)‑1‑苯基‑1h‑吡唑‑5‑基)脲
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