WO2022063190A1 - 吡嗪硫联苯基类化合物及其应用 - Google Patents

吡嗪硫联苯基类化合物及其应用 Download PDF

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WO2022063190A1
WO2022063190A1 PCT/CN2021/119985 CN2021119985W WO2022063190A1 WO 2022063190 A1 WO2022063190 A1 WO 2022063190A1 CN 2021119985 W CN2021119985 W CN 2021119985W WO 2022063190 A1 WO2022063190 A1 WO 2022063190A1
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compound
pharmaceutically acceptable
acceptable salt
added
reaction
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PCT/CN2021/119985
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English (en)
French (fr)
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付志飞
罗妙荣
帅斌
张杨
李秋
黎健
陈曙辉
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南京明德新药研发有限公司
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Priority to CN202180065019.2A priority Critical patent/CN116348466A/zh
Priority to TW111107823A priority patent/TW202246279A/zh
Publication of WO2022063190A1 publication Critical patent/WO2022063190A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having 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
    • C07D241/20—Nitrogen atoms
    • 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
    • C07D401/02—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 two hetero rings
    • C07D401/04—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 two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • 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
    • C07D498/10—Spiro-condensed systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Definitions

  • the present invention relates to pyrazinethiobiphenyl compounds and their application, in particular to the compound of formula (II) or a pharmaceutically acceptable salt thereof.
  • Shp2 SH2 domain-containing protein-tyrosine phosphatase-2
  • PTP protein tyrosine phosphatase
  • PTPN11 gene which can not only positively regulate the downstream signal transduction pathway through the catalytic activity of phosphatase, but also play a positive regulatory role as a phosphatase-independent adaptor protein, and can also play a positive role in specific conditions. It has a negative regulatory effect, and thus widely participates in the regulation of biological functions such as cell differentiation, migration, and related signal transduction processes.
  • PTPN11 mutation is considered to be a high-risk factor for juvenile myelomonocytic leukemia (JMML). At the same time, it is considered to be the proto-oncogene of leukemia because of the abnormal activation and mutation of Shp2 in different types of leukemia.
  • Shp2 In cancer, pancreatic cancer, gastric cancer and glioma, Shp2 has also been reported to be overactivated; in lung cancer, Shp2 acts as an oncogene to promote tumor occurrence and development by regulating various mechanisms. But in the process of hepatocarcinogenesis, Shp2 plays the role of tumor suppressor gene under the influence of specific environment. In conclusion, as an important node molecule, Shp2 plays an important regulatory role in the process of tumorigenesis and development, and is a potential therapeutic target.
  • the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof,
  • E 1 is O or CH 2 ;
  • T 1 is N or CH
  • R 11 , R 13 and R 14 are each independently C 1-3 alkyl
  • R 12 is H or C 1-3 alkyl
  • R 2 is F, Cl, Br or I
  • R 3 is C 1-3 alkyl optionally substituted with 1 , 2 or 3 R a ;
  • R 4 is H, F, Cl, Br, I or C 1-3 alkyl optionally substituted with 1 , 2 or 3 R b ;
  • Ra and Rb are each independently F, Cl, Br, I, OH or NH2 ;
  • n 0, 1, 2 or 3;
  • n 1, 2 or 3;
  • n 1, 2 or 3
  • the structural unit for the structural unit Structural units for
  • the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • R 11 , R 13 and R 14 are each independently C 1-3 alkyl
  • R 12 is H or C 1-3 alkyl
  • R 2 is F, Cl, Br or I
  • R 3 is C 1-3 alkyl optionally substituted with 1 , 2 or 3 R a ;
  • R 4 is H, F, Cl, Br, I or C 1-3 alkyl optionally substituted with 1 , 2 or 3 R b ;
  • Ra and Rb are each independently F, Cl, Br, I, OH or NH2 ;
  • n 1, 2 or 3.
  • R 11 , R 13 and R 14 are each independently CH 3 , and other variables are as defined in any technical solution of the present invention.
  • R 12 is H or CH 3 , and other variables are as defined in any technical solution of the present invention.
  • R 3 is CH 3 , and other variables are as defined in any technical solution of the present invention.
  • R 4 is F, Cl, Br or I, and other variables are as defined in any technical solution of the present invention.
  • the above compound has the structure of formula (II-1)
  • the above-mentioned compound has the structure of formula (I-1) or (I-2)
  • the above compound has the structure of formula (I-1A), (I-1B), (I-2A), (II-1A) or (II-1B)
  • n, m, E 1 , T 1 , R 11 , R 12 , R 13 , R 14 , R 2 , R 3 and R 4 are as defined in any technical solution of the present invention.
  • the above-mentioned compound has the structure of formula (I-1A-1), (I-2A-1), (II-1A-1) or (II-1B-1)
  • the present invention also provides the following compounds or pharmaceutically acceptable salts thereof,
  • the above-mentioned compound of the present invention is compound 004,
  • the compound of the present invention is a stereoisomer of compound 004 or a formate of the stereoisomer, and the stereoisomer or its formate is subjected to chiral supercritical fluid chromatography
  • the retention time after analysis is 3.1-3.5min, preferably 3.2-3.4min, more preferably about 3.3min; the conditions for the chiral supercritical fluid chromatography analysis are: chromatographic column: Chiralpak AS-3 (100mm*4.6mm, 3 ⁇ m) ; Mobile phase: carbon dioxide; [0.05% triethylamine, % ethanol]: 40% ⁇ 40%.
  • the above-mentioned compound of the present invention is another stereoisomer of compound 004, and the retention time of the stereoisomer after analysis by chiral supercritical fluid chromatography is 4.3-4.7min, preferably 4.4-4.6min , more preferably about 4.5min; the conditions for the chiral supercritical fluid chromatography analysis are: chromatographic column: Chiralpak AS-3 (100mm*4.6mm, 3 ⁇ m); mobile phase: carbon dioxide; [0.05% triethylamine, ethanol% ]: 40% ⁇ 40%.
  • the compound of the present invention is a stereoisomer of compound 005; the retention time of the stereoisomer after analysis by chiral supercritical fluid chromatography is 4.8-5.2min, preferably 4.9-5.1min, more Preferably about 5.0min; the conditions of the chiral supercritical fluid chromatography analysis are: chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 48%-78 %,7min.
  • the compound of the present invention is another stereoisomer of compound 005; the retention time of the stereoisomer after analysis by chiral supercritical fluid chromatography is 5.6-6.0min, preferably 5.7-5.9min, More preferably about 5.8min; the conditions for the chiral supercritical fluid chromatography analysis are: chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 48%- 78%, 7min.
  • the above compound is a stereoisomer of compound 006, and the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 4.9-5.3min, preferably 5.0-5.2min, more preferably about 5.1min;
  • the conditions for the chiral supercritical fluid chromatographic separation are: chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-methanol]; methanol %: 40%-40%.
  • the above compound is another stereoisomer of compound 006, and the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 6.6-7.0min, preferably 6.7-6.9min, more Preferably about 6.8min;
  • the conditions for the chiral supercritical fluid chromatographic separation are: chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-methanol]; methanol %: 40%-40 %.
  • the compound of the present invention is a stereoisomer of compound 007; the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 6.6-7.0 min, preferably 6.7-6.9 min, more Preferably about 6.8min; the conditions for the chiral supercritical fluid chromatographic separation are chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 48%-78% ,10min.
  • the compound of the present invention is another stereoisomer of compound 007; the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 7.1-7.5min, preferably 7.2-7.4min , more preferably about 7.4min; the conditions for the chiral supercritical fluid chromatographic separation are chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 48%- 78%, 10min.
  • the compound of the present invention is another stereoisomer of compound 008; the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 3.6-4.0min, preferably 3.7-3.9min , more preferably about 3.9min; the conditions for the chiral supercritical fluid chromatographic separation are: chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 45% -45%.
  • the compound of the present invention is a stereoisomer of compound 010; the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 2.3-2.7min, preferably 2.4-2.6min, more Preferably about 2.6min; the conditions for the chiral supercritical fluid chromatographic separation are: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 40%-40%.
  • the compound of the present invention is another stereoisomer of compound 010; the retention time of the stereoisomer after separation by chiral supercritical fluid chromatography is 3.2-3.6min, preferably 3.3-3.5min , more preferably about 3.4min; the conditions for the chiral supercritical fluid chromatographic separation are: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 40%-40% .
  • the second aspect of the present invention also provides a pharmaceutical composition, which comprises the compound defined in any of the above technical solutions or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for treating a disease associated with SHP2 in a subject in need thereof, comprising providing the subject with an effective dose of a compound as defined in any of the above technical solutions or a pharmaceutically acceptable salt or pharmaceutical combination thereof thing.
  • the present invention also provides the use of the above compound, its isomer or its pharmaceutically acceptable salt or pharmaceutical composition in preparing a medicine for treating SHP2-related diseases.
  • the compounds of the present invention exhibit good inhibitory activity on protein tyrosine phosphatase SHP2, and will have excellent therapeutic effect in patients with abnormal SHP2 tumors.
  • the term "pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base in neat solution or in 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 such compounds with a sufficient amount of acid in neat solution or in 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, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base
  • the pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
  • 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 racemic mixtures thereof 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 substituents such as alkyl. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.
  • diastereomer refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.
  • tautomer or “tautomeric form” refers to isomers of different functional groups that are in dynamic equilibrium at room temperature and can rapidly interconvert.
  • a chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution).
  • proton tautomers also called prototropic tautomers
  • Valence tautomers include interconversions by recombination of some bonding electrons.
  • keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • atoms with "*" or “#” are chiral atoms or chiral centers that exist as (R) or (S) as a single enantiomer or in enriched form; for example, expressed as
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in one enantiomer” refer to one of the isomers or pairs
  • the enantiomer content is less than 100%, and the isomer or enantiomer content 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 Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • isomeric excess or “enantiomeric excess” refer to the difference between two isomers or relative percentages of 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 isomer or enantiomeric excess (ee value) is 80% .
  • Optically active (R)- and (S)-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art
  • the diastereoisomers were resolved and the pure enantiomers recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
  • deuterated drugs can be formed by replacing hydrogen with deuterium, and the bonds formed by deuterium and carbon are stronger than those formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a specified atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence of the specified atom is normal and the substituted compound is stable.
  • oxygen it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups.
  • any variable eg, R
  • its definition in each case is independent.
  • the group may optionally be substituted with up to two Rs, with independent options for R in each case.
  • combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • substituents When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate through which atom it is attached to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be through any one of the pyridine ring The carbon atom is attached to the substituted group.
  • the direction of attachment is arbitrary, for example,
  • the linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right. It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.
  • Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
  • any one or more sites in the group can be linked to other groups by chemical bonds.
  • connection method of the chemical bond is not located, and there is an H atom at the linkable site, when the chemical bond is connected, the number of H atoms at the site will be correspondingly reduced with the number of chemical bonds connected to the corresponding valence. the group.
  • the chemical bond connecting the site to other groups can be represented by straight solid line bonds straight dotted key or wavy lines Express.
  • a straight solid bond in -OCH 3 indicates that it is connected to other groups through the oxygen atom in this group;
  • the straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;
  • the wavy line in the phenyl group indicates that it is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;
  • C 1-3 alkyl is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it can be monovalent (eg methyl), divalent (eg methylene) or multivalent (eg methine) .
  • Examples of C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • Cn-n+m or Cn - Cn+m includes any particular instance of n to n+ m carbons, eg C1-12 includes C1 , C2 , C3, C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also including any one range from n to n+m, eg C 1-12 includes C 1-3 , 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.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-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, a nucleophilic substitution reaction).
  • a substitution reaction eg, a nucleophilic substitution reaction
  • representative leaving groups include triflate; chlorine, bromine, iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy, such as acetoxy, trifluoroacetoxy, and the like.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol 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 groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, 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 groups such as trimethylsilyl (TMS) and tert-
  • hydroxy protecting group refers to a protecting group suitable for preventing hydroxyl side reactions.
  • Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.
  • alkyl groups such as methyl, ethyl and tert-butyl
  • acyl groups such as alkanoyl (eg acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenyl
  • supercritical fluid chromatography for example, supercritical fluid chromatography (chromatographic column: DAICEL CHIRALPAK AS (250mm*30mm, 10 ⁇ m); mobile phase: [0.1% ammonia water-ethanol]; ethanol%: 48%- 78%, 7min, where 7min represents the time required to increase the ethanol concentration from 48% to 78%.
  • chromatographic column: DAICEL CHIRALPAK AS 250mm*30mm, 10 ⁇ m
  • mobile phase [0.1% ammonia water-ethanol]
  • ethanol% 48%- 78%, 7min, where 7min represents the time required to increase the ethanol concentration from 48% to 78%.
  • 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, embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • SXRD single crystal X-ray diffractometry
  • the cultivated single crystal is collected by Bruker D8venture diffractometer
  • the light source is CuK ⁇ radiation
  • the scanning method is as follows: After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • the solvent used in the present invention is commercially available.
  • the present invention adopts the following abbreviations: aq represents water; eq represents equivalent, equivalent; NaCNBH 3 represents sodium cyanoborohydride; rt represents room temperature; mp represents melting point; DCM represents dichloromethane; MeOH represents methanol; Critical fluid chromatography; MMS indicates hepatic intrinsic clearance.
  • Step 1 Synthesis of Compound 001-2:
  • compound 001-2 (5 g, 15.76 mmol, 1 eq) and dimethylphosphine oxide (1.23 g, 15.76 mmol, 1 eq) were dissolved in dioxane (50 mL), and tris(dioxane) was added in one portion.
  • the concentrate was dissolved in 30 mL of water and 50 mL of ethyl acetate, and the layers were extracted while the aqueous phase was extracted three times with ethyl acetate (30 mL, 30 mL, 30 mL).
  • the organic phases were combined, washed once with saturated sodium chloride solution (30 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • compound 001-3 (2.3g, 8.60mmol, 1eq) and compound 001-3A (2.25g, 10.32mmol, 1.2eq) were dissolved in dioxane (25mL), and N,N was added at one time -Diisopropylethylamine (3.33g, 25.80mmol, 4.49mL, 3eq), Tris(dibenzylideneacetone)dipalladium (787.38mg, 859.86 ⁇ mol, 0.1eq), 4,5-bisdiphenylphosphine -9,9-Dimethylxanthene (497.53 mg, 859.86 ⁇ mol, 0.1 eq), then the temperature was raised to 110° C.
  • compound 001-5 (270 mg, 1.22 mmol, 1 eq) and compound 001-5A (382.59 mg, 1.84 mmol, 1.5 eq) were dissolved in dioxane (3 mL), and tris(dibenzylidene) was added at one time acetone) dipalladium (112.05mg, 122.36 ⁇ mol, 0.1eq), 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (70.80mg, 122.36 ⁇ mol, 0.1eq), N,N - Diisopropylethylamine (474.43 mg, 3.67 mmol, 639.40 ⁇ L, 3 eq), then warmed to 110° C.
  • Step 1 Synthesis of Compound 002-1:
  • compound 001-1 (200 mg, 968.68 ⁇ mol, 1 eq) was dissolved in acetonitrile (5 mL), and diiodomethane (73.00 mg, 774.94 ⁇ mol, 69.52 ⁇ L, 0.8 eq) was added at one time, and the temperature was raised to 60 °C, tert-Butyl nitrite (149.83 mg, 1.45 mmol, 172.82 ⁇ L, 1.5 eq) was slowly added, then the temperature was raised to 80° C. and stirred for 1 hour. The reaction solution was concentrated under reduced pressure at 43°C.
  • compound 002-2 (1 g, 3.72 mmol, 1 eq) and compound 001-3A (894.37 mg, 4.10 mmol, 1.1 eq) were dissolved in dioxane (10 mL), and diisopropyl group was added at one time Ethylamine (1.44g, 11.17mmol, 1.95mL, 3eq), tris(dibenzylideneacetone)dipalladium (340.98mg, 372.36 ⁇ mol, 0.1eq), 4,5-bisdiphenylphosphine-9,9- Dimethylxanthene (215.45 mg, 372.36 ⁇ mol, 0.1 eq) was then heated to 110° C. and stirred for 5 hours.
  • the reaction liquid was cooled to 20°C, and concentrated under reduced pressure at 43°C.
  • the concentrate was dissolved in 50 mL of water and 50 mL of ethyl acetate, and the layers were extracted while the aqueous phase was extracted three times with ethyl acetate (50 mL, 30 mL, 30 mL).
  • the organic phases were combined, washed once with saturated sodium chloride solution (30 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • Column chromatography The crude product was isolated by column chromatography (0-10% methanol in dichloromethane). Compound 002-3 was obtained.
  • Step 5 Synthesis of Compound 002-5:
  • compound 002-4 50 mg, 225.50 ⁇ mol, 1 eq
  • compound 001-5A 70.51 mg, 338.25 ⁇ mol, 1.5 eq
  • the concentrate was dissolved in 30 mL of water and 30 mL of ethyl acetate, and the layers were extracted, while the aqueous phase was extracted three times with ethyl acetate (30 mL ⁇ 3).
  • the organic phases were combined, washed once with saturated sodium chloride solution (30 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • the crude product was isolated by column chromatography (0-10% methanol in dichloromethane). Compound 002-5 was obtained. MS (ESI) m/z: 348.9 [M+H]+.
  • compound 002-5 50 mg, 143.16 ⁇ mol, 1 eq
  • compound 001-6A 39.29 mg, 143.16 ⁇ mol, 1 eq
  • dimethylacetamide 2 mL
  • water 2 mL
  • Potassium carbonate 59.36 mg, 429.48 ⁇ mol, 3 eq
  • the reaction solution was directly spin-dried to obtain compound 002-6.
  • Step 1 Synthesis of Compound 003-1:
  • compound 002-2 (1.9 g, 7.07 mmol, 1 eq) was dissolved in N,N-dimethylformamide (20 mL), and sodium hydride (424.45 mg, 10.61 mmol, 60%) was added in one portion at 0°C. Purity, 1.5eq), the mixture was stirred at 0°C for 10min, then iodomethane (2.01g, 14.15mmol, 880.86 ⁇ L, 2eq) was added and the temperature was raised to 25°C and stirred for hours. The reaction solution was added dropwise to 10 mL of ice water, diluted with 50 mL of ethyl acetate, and the solution was separated.
  • compound 003-1 (1.5 g, 5.31 mmol, 1 eq) and compound 001-3A (1.27 g, 5.84 mmol, 1.1 eq) were dissolved in dioxane (20 mL), and diiso was added at one time Propylethylamine (2.06g, 15.92mmol, 2.77mL, 3eq), tris(dibenzylideneacetone)dipalladium (486.08mg, 530.81 ⁇ mol, 0.1eq), 4,5-bisdiphenylphosphine-9, 9-Dimethylxanthene (307.14 mg, 530.81 ⁇ mol, 0.1 eq), then warmed to 110° C.
  • Step 3 Synthesis of Compound 003-3:
  • compound 003-3 (56 mg, 237.54 ⁇ mol, 1 eq) and compound 001-5A (74.27 mg, 356.30 ⁇ mol, 1.5 eq) were dissolved in dioxane (3 mL), and diisopropyl group was added in one portion.
  • Ethylamine (92.10mg, 712.61 ⁇ mol, 124.12uL, 3eq), Tris(dibenzylideneacetone)dipalladium (21.75mg, 23.75 ⁇ mol, 0.1eq), 4,5-bisdiphenylphosphine-9,9- Dimethylxanthene (13.74 mg, 23.75 ⁇ mol, 0.1 eq) was then heated to 110° C. and stirred for 5 hours. The reaction liquid was cooled to 20°C, and concentrated under reduced pressure at 43°C.
  • the concentrate was dissolved in 30 mL of water and 30 mL of ethyl acetate, and the layers were extracted, while the aqueous phase was extracted three times with ethyl acetate (30 mL ⁇ 3).
  • the organic phases were combined, washed once with saturated sodium chloride solution (30 mL), and finally the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure.
  • the crude product was isolated by column chromatography (0-10% methanol in dichloromethane) to give compound 003-4. MS (ESI) m/z: 362.9 [M+H] + .
  • Step 1 Synthesis of Compound 004-2:
  • compound 004-1 (5.0 g, 19.43 mmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (60 mL), replaced with nitrogen three times and then cooled to -78°C.
  • anhydrous tetrahydrofuran 60 mL
  • To the reaction mixture was slowly added dropwise a solution of lithium diisopropylamide (2.0 M, 10.69 mL, 1.1 eq) in tetrahydrofuran, the mixture was stirred at -78°C for 1 hour, and then compound 004-2A in tetrahydrofuran was slowly added dropwise to the system The solution was reacted at -78°C for 30 minutes, and then the reaction system was slowly warmed to -25°C for 15 hours.
  • compound 004-2 (6.91 g, 18.91 mmol, 1 eq) was dissolved in dioxane (80 mL) and methanol (32 mL), and then sodium hydroxide aqueous solution (6 M, 16 mL, 5.08 eq) was added. Then, the temperature was raised to 100°C and the reaction was refluxed for 15 hours. After the reaction was completed, it was cooled to room temperature, the organic solvent was removed under reduced pressure, and the pH was adjusted to 3-4 with dilute hydrochloric acid (1.0M), filtered, and the filter cake was washed with water.
  • dioxane 80 mL
  • methanol 32 mL
  • sodium hydroxide aqueous solution 6 M, 16 mL, 5.08 eq
  • compound 004-4 (2g, 6.26mmol, 1eq) and compound 004-5A (2.28g, 18.79mol, 3.0eq) were added to a single-neck flask, followed by tetraethyl titanate (5mL) 100
  • the reaction was refluxed at °C for 18 hours. After the reaction was completed, it was cooled to room temperature, the mixture was poured into ice water to quench, ethyl acetate (50 mL) was added, and the mixture was stirred for 1 hour, and the layers were separated. The aqueous phase was extracted with ethyl acetate (50 mL ⁇ 3).
  • Step 8 Synthesis of Compound 004:
  • Step 9 Synthesis of Compounds 004-8A and 004-8B:
  • Compound 004-8 was sent to chiral supercritical fluid chromatography for separation (chromatographic column: Chiralpak AS-3 (100mm*4.6mm, 3 ⁇ m); mobile phase: carbon dioxide; [0.05% triethylamine, ethanol%]: 40%- 40%), two isomers were separated, and isomer 1 was 004-8A (retention time 2.557 min). MS (ESI) m/z: 637.1 [M+H] + . Isomer 2 was 004-8B (retention time 3.028 min), MS (ESI) m/z: 637.1 [M+H] + .
  • Step 10 Synthesis of Compound 004A:
  • Step 1 Synthesis of Compound 005-2:
  • Step 1 Synthesis of Compound 006-3:
  • compound 006-3 (12.00 g, 30.21 mmol, 1 eq) was dissolved in a mixed solution of NN dimethylacetamide (100 mL) and water (10 mL), and dichlorobis[di-tert-butyl] was added.
  • the system was purged with nitrogen three times, The temperature was raised to 130°C for 5 hours.
  • reaction solution was cooled to room temperature, 150 ml of water was added, extracted with ethyl acetate (200 mL ⁇ 3), the organic phases were combined, concentrated under reduced pressure to a concentrated solution volume of about 150 mL, washed with water 4 times, washed with saturated brine 2 times, and anhydrous Dry over sodium sulfate and concentrate under reduced pressure to obtain crude product.
  • the crude product was separated by column chromatography (30%-35% ethyl acetate in petroleum ether) to obtain compound 006-4, MS (ESI) m/z: 263.9 [M+H] + .
  • reaction solution was cooled to room temperature, the reaction solution was added to ice water, stirred for 40 minutes, the supernatant was added to a separatory funnel, and then ethyl acetate was added for extraction (200 mL ⁇ 3), and the organic phases were combined and used Washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was isolated by column chromatography (25%-35% ethyl acetate in petroleum ether) to give compound 006-5. MS(ESI) m/z: 322.9 [M- tBu ] + .
  • Step 1 Synthesis of Compound 007-2:
  • Step 1 Synthesis of Compound 008-2:
  • compound 008-2 (12.00 g, 30.21 mmol, 1 eq) was dissolved in a mixed solution of NN dimethylacetamide (100 mL) and water (10 mL), and dichlorobis[di-tert-butyl] was added.
  • the system was purged with nitrogen three times, The temperature was raised to 130°C for 5 hours.
  • reaction solution was cooled to room temperature, 150 ml of water was added, extracted with ethyl acetate (200 mL ⁇ 3), the organic phases were combined, concentrated under reduced pressure to a concentrated solution volume of about 150 ml, washed with saturated brine (200 mL ⁇ 6), and anhydrous sulfuric acid. Dry over sodium and concentrate under reduced pressure to give crude product.
  • the crude product was isolated by column chromatography (30%-35% ethyl acetate in petroleum ether) to give 008-3, MS (ESI) m/z: 264.1 [M- t Bu] + .
  • Step 1 Synthesis of Compound 009-3:
  • reaction solution was cooled to room temperature, 150 ml of water was added, extracted with ethyl acetate (200 mL ⁇ 3), the organic phases were combined, concentrated under reduced pressure to a concentrated solution volume of about 150 ml, washed with saturated brine (200 mL ⁇ 6), and anhydrous sulfuric acid. Dry over sodium and concentrate under reduced pressure to give crude product.
  • the crude product was isolated by column chromatography (30%-35% ethyl acetate in petroleum ether) to give 009-6, MS (ESI) m/z: 290.1 [M- t Bu] + .
  • 009-6 (4.2g, 12.16mmol, 1eq) was dissolved in tetraethyl titanate (100mL), 004-5A (4.42g, 36.48mmol, 3eq) was added, the system was purged with nitrogen three times, and then heated to 130°C The reaction was carried out for 3 hours.
  • reaction solution was cooled to room temperature, the reaction solution was added to ice water, stirred for 40 minutes, the supernatant was added to a separatory funnel, and then ethyl acetate was added for extraction (200 mL ⁇ 3), and the organic phases were combined and used Washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product.
  • the crude product was isolated by column chromatography (10%-20% ethyl acetate in petroleum ether) to give compound 009-7. MS (ESI) m/z: 449.1 [M+H] + .
  • Step 7 Synthesis of Compound 009-10A:
  • Step 8 Synthesis of Compound 009A:
  • Step 9 Synthesis of Compound 009-9B:
  • Step 10 Synthesis of Compound 009-10B:
  • Step 11 Synthesis of Compound 009B:
  • reaction solution was cooled to room temperature, filtered, 200 mL of water was added to the filtrate, extracted with ethyl acetate (100 mL ⁇ 3), the organic phases were combined, washed with saturated brine (200 mL ⁇ 1), and the organic phase was dried over anhydrous sodium sulfate. , filtered, and the solvent was removed by rotary evaporation.
  • the crude product was separated by flash column chromatography (3%-5% ethyl acetate in petroleum ether) to obtain compound 010-2.
  • reaction solution was cooled to room temperature, 150 ml of water was added, extracted with ethyl acetate (200 mL ⁇ 3), the organic phases were combined, concentrated under reduced pressure to a concentrated solution volume of about 150 mL, washed with saturated brine (200 mL ⁇ 6) , dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product.
  • HEPES hydroxyethylpiperazine ethanethiosulfonic acid
  • EDTA ethylenediaminetetraacetic acid
  • 75 mM KCl 75 mM NaCl
  • 0.01% Brij-35 5 mM dithiothreitol (DTT) and 10 %DMSO (final).
  • the compounds of the present invention have certain inhibitory activity on PTPN11/SHP2-FL.
  • the purpose of this experiment is to verify the inhibitory effect of the compounds of the present invention on the proliferation of KRAS G12C-mutated NCI-H358 human non-small cell lung cancer cells.
  • Cell line NCI-H358 (purchased from Proceedings), RPMI1640 medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • CellTiter-Glo Cell Viability Chemiluminescence Detection Reagent
  • NCI-H358 cells were seeded in a white 96-well plate, 80 ⁇ L of cell suspension per well, which contained 4000 NCI-H358 cells. Cell plates were incubated overnight in a carbon dioxide incubator. The compounds to be tested were diluted 5-fold to the ninth concentration, that is, from 2000 ⁇ M to 5.12 nM, and a double-well experiment was set up. Add 78 ⁇ L of medium to the middle plate, and then transfer 2 ⁇ L of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 ⁇ L of each well to the cell plate. Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.026 nM. The cell plates were placed in a carbon dioxide incubator for 5 days.
  • Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis.
  • the IC50 value can be obtained by curve fitting with four parameters ("log(inhibitor) vs.response in GraphPad Prism") --Variable slope" mode).
  • Table 2 shows the results of the cell activity screening test of the compound H358 of the present invention.
  • the compounds of the present invention have good inhibitory activity on H358 cells.
  • Intravenous and oral vehicles are a certain proportion of hydroxypropyl ⁇ -cyclodextrin aqueous solution or physiological saline solution. Collect whole blood samples within 24 hours, centrifuge at 3000g for 15 minutes, separate the supernatant to obtain plasma samples, add 4 times the volume of acetonitrile solution containing the internal standard to precipitate the protein, centrifuge to take the supernatant, add an equal volume of water, and then centrifuge to take the supernatant.
  • LC-MS/MS analysis method was used to quantitatively analyze the blood drug concentration, and the pharmacokinetic parameters, such as peak concentration, peak time, clearance rate, half-life, area under the drug-time curve, bioavailability, etc., were calculated.
  • the compounds of the present invention can significantly improve the pharmacokinetics single or partial indexes in mice.
  • the purpose of the research project was to use a 5-in-1 probe substrate of CYP isoenzymes to evaluate the inhibition of test articles against human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4).
  • HMM human liver microsomes
  • the compounds of the present invention have weak inhibition on the five CYP isozymes.
  • the full-automatic patch-clamp method was used to detect the effect of Example 1 to be tested on the hERG potassium channel.
  • the cells stably expressing the hERG potassium channel used in the experiment were obtained from CHO-hERE of Aviva Biosciences, and CHO-hERG was cultured in 5% CO 2 at 37°C.
  • CHO hERG medium is shown in Table 5.
  • CHO-hERG cells ready for experiments were cultured for at least two days, and the cell density was above 75%. Before the experiment, cells were digested with TrypLE and then resuspended in extracellular fluid to collect cells.
  • Extracellular fluid needs to be prepared once a month.
  • the intracellular fluid must be aliquoted and stored at -20°C.
  • the composition of intracellular and extracellular fluids is shown in Table 6.
  • Extracellular fluid mM
  • Intracellular fluid mM
  • NaCl 145 - KCl 4 120 KOH - 31.25
  • the compound to be tested and the positive control Amitriptyline were dissolved in DMSO into a stock solution of a certain concentration, then diluted according to different gradients, and finally added to the extracellular fluid according to a certain proportion to be diluted to the concentration to be tested. Visually inspect for the presence or absence of precipitation prior to the start of the experiment. Finally, in the test solution and the positive control Amitriptyline, the maximum concentration of DMSO should not exceed 0.3%.
  • Hold the clamping potential at -80mv first give -50mv voltage stimulation for 80ms to record the cell leakage current value, then depolarize to +20mv, maintain 4800ms, open the hERG channel, and then repolarize to -50mv for 5000ms , elicited the hERG tail current and recorded, and finally, the voltage returned to the clamping potential -80mv and maintained for 3100ms.
  • the above voltage stimulation was repeated every 15000ms.
  • hERG QPatch HTX experiments were performed at room temperature. Whole cell protocols, voltage stimulation protocols and compound detection protocols were established on the software of QPatch Assay Software 5.2 (Sophion Bioscience).
  • I (C) I b +(I fr -I b )*c n /(IC 50 n +c n )
  • C is the compound test concentration
  • n is the slope
  • Curve fitting and inhibition rate calculation are both completed by Qpatch analysis software. If the inhibition rate at the lowest concentration exceeds half inhibition or the inhibition rate at the highest concentration does not reach half inhibition, the corresponding IC 50 of the compound is lower than the lowest concentration or IC 50 value. greater than the highest concentration.
  • Human and animal microsomes were purchased from Corning or Xenotech and stored in a -80°C freezer.
  • T60 incubation plate Prepare two 96-well incubation plates, named T60 incubation plate and NCF60 incubation plate respectively.
  • stop solution 200ng/mL tolbutamide and 200ng/mL labetalol in acetonitrile
  • Test sample MMS(mL/min/kg), H, M Formate salt of compound 004A 19.7, 67.9 Formate salt of compound 004B 33.0, 61.1 Compound 006A 31.2, 100.8 Compound 006B 47.9, 57.3

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Abstract

一种吡嗪硫联苯基类化合物及其应用，具体公开了式(Ⅱ)化合物或其药学上可接受的盐。

Description

吡嗪硫联苯基类化合物及其应用
本申请主张如下优先权：
CN202011008309.7，申请日：2020年09月23日；
CN202110182212.6，申请日：2021年02月09日；
CN202110412812.7，申请日：2021年04月16日；
CN202110590591.2，申请日：2021年05月28日。
技术领域
本发明涉及吡嗪硫联苯基类化合物及其应用，具体涉及式(Ⅱ)化合物或其药学上可接受的盐。
背景技术
酪氨酸激酶的磷酸化作用与酪氨酸磷酸酶的去磷酸化作用足生物体内普遍存在的信号转导机制，它们共同调节细胞内蛋白质的酪氨酸磷酸化水平。Shp2(SH2 domain-containing protein-tyrosine phosphatase-2)就是起去磷酸化作用的一种非跨膜型蛋白酪氨酸磷酸酶，是蛋白酪氨酸磷酸酶(PTP)家族的重要成员之一，其分子由PTPN11基因编码，既可以通过磷酸酶的催化活性来正向调控下游信号转导通路，也可以作为磷酸酶非依赖性的接头蛋白发挥正向调控作用，在特定的条件下亦可发挥负向调控作用，从而广泛参与细胞的分化、迁移等生物学功能的调控及相关的信号转导过程。PTPN11突变被认为是青少年粒单细胞白血病(JMML)的高危因素，同时，因其在不同类型白血病中均存在着Shp2的异常活化和突变而被认为是白血病的原癌基因；在前列腺癌、乳腺癌、胰腺癌、胃癌和神经胶质瘤中，Shp2也被报道呈过度活化状态；在肺癌中Shp2作为癌基因通过调控多种机制促进肿瘤的发生、发展。但在肝癌发生过程中，Shp2却在特定环境的影响下发挥抑癌基因的作用。总之，作为重要的节点分子，Shp2在肿瘤发生、发展的过程中发挥着重要的调控作用，是潜在的治疗靶点。
发明内容
本发明提供了式(Ⅱ)化合物或其药学上可接受的盐，
Figure PCTCN2021119985-appb-000001
其中，
结构单元
Figure PCTCN2021119985-appb-000002
为
Figure PCTCN2021119985-appb-000003
E 1为O或CH 2；
T 1为N或CH；
R 1为
Figure PCTCN2021119985-appb-000004
R 11、R 13和R 14分别独立地为C 1-3烷基；
R 12为H或C 1-3烷基；
R 2为F、Cl、Br或I；
R 3为C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
R 4为H、F、Cl、Br、I或C 1-3烷基，所述C 1-3烷基任选被1、2或3个R b取代；
R a和R b分别独立地为F、Cl、Br、I、OH或NH 2；
n为0、1、2或3；
m为1、2或3；
当n为0时，结构单元
Figure PCTCN2021119985-appb-000005
为
Figure PCTCN2021119985-appb-000006
结构单元
Figure PCTCN2021119985-appb-000007
为
Figure PCTCN2021119985-appb-000008
当n为1、2或3时，结构单元
Figure PCTCN2021119985-appb-000009
为结构单元
Figure PCTCN2021119985-appb-000010
结构单元
Figure PCTCN2021119985-appb-000011
为
Figure PCTCN2021119985-appb-000012
本发明提供了式(Ⅰ)化合物或其药学上可接受的盐，
Figure PCTCN2021119985-appb-000013
其中，
结构单元
Figure PCTCN2021119985-appb-000014
为
Figure PCTCN2021119985-appb-000015
R 1为
Figure PCTCN2021119985-appb-000016
R 11、R 13和R 14分别独立地为C 1-3烷基；
R 12为H或C 1-3烷基；
R 2为F、Cl、Br或I；
R 3为C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
R 4为H、F、Cl、Br、I或C 1-3烷基，所述C 1-3烷基任选被1、2或3个R b取代；
R a和R b分别独立地为F、Cl、Br、I、OH或NH 2；
m为1、2或3。
本发明的一些方案中，上述R 1为
Figure PCTCN2021119985-appb-000017
其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述R 11、R 13和R 14分别独立地为CH 3，其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述R 12为H或CH 3，其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述R 3为CH 3，其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述R 4为F、Cl、Br或I，其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2021119985-appb-000018
为
Figure PCTCN2021119985-appb-000019
Figure PCTCN2021119985-appb-000020
其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2021119985-appb-000021
为
Figure PCTCN2021119985-appb-000022
Figure PCTCN2021119985-appb-000023
其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2021119985-appb-000024
为
Figure PCTCN2021119985-appb-000025
Figure PCTCN2021119985-appb-000026
Figure PCTCN2021119985-appb-000027
其他变量如本发明任意技术方案所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2021119985-appb-000028
为
Figure PCTCN2021119985-appb-000029
Figure PCTCN2021119985-appb-000030
其他变量如本发明任意技术方案所定义。
本发明还有一些方案是由上述各变量任意组合而来。
本发明的一些方案中，上述化合物具有式(Ⅱ-1)的结构
Figure PCTCN2021119985-appb-000031
其中，
R 11、R 12、R 2、n、E 1和结构单元
Figure PCTCN2021119985-appb-000032
如本发明任意技术方案所定义。
本发明的一些方案中，上述化合物具有式(Ⅰ-1)或(Ⅰ-2)的结构
Figure PCTCN2021119985-appb-000033
其中，
R 11、R 12、R 13、R 14、R 2和结构单元
Figure PCTCN2021119985-appb-000034
如本发明任意技术方案所定义。
本发明的一些方案中，上述化合物具有式(Ⅰ-1A)、(Ⅰ-1B)、(Ⅰ-2A)、(Ⅱ-1A)或(Ⅱ-1B)的结构
Figure PCTCN2021119985-appb-000035
Figure PCTCN2021119985-appb-000036
其中，
n、m、E 1、T 1、R 11、R 12、R 13、R 14、R 2、R 3和R 4如本发明任意技术方案所定义。
本发明的一些方案中，上述化合物具有式(Ⅰ-1A-1)、(Ⅰ-2A-1)、(Ⅱ-1A-1)或(Ⅱ-1B-1)的结构
Figure PCTCN2021119985-appb-000037
其中，
m、n、E 1、T 1、R 11、R 12、R 13、R 14、R 2、R 3和R 4如本发明任意技术方案所定义。
本发明还提供了下列化合物或其药学上可接受的盐，
Figure PCTCN2021119985-appb-000038
Figure PCTCN2021119985-appb-000039
本发明的一些方案中，上述化合物为
Figure PCTCN2021119985-appb-000040
Figure PCTCN2021119985-appb-000041
Figure PCTCN2021119985-appb-000042
在本发明的一个技术方案中，本发明上述化合物为：
Figure PCTCN2021119985-appb-000043
在本发明的一个技术方案中，本发明上述化合物为化合物004，
Figure PCTCN2021119985-appb-000044
本发明的一个技术方案中，本发明化合物为化合物004的立体异构体、或所述立体异构体的甲酸盐，所述立体异构体、或其甲酸盐经手性超临界流体色谱分析后保留时间为3.1‐3.5min，优选3.2‐3.4min，更优选约3.3min；所述手性超临界流体色谱分析的条件为：色谱柱:Chiralpak AS‐3(100mm*4.6mm,3μm)；流动相：二氧化碳；[0.05％三乙胺，乙醇％]：40％‐40％。
本发明的一个技术方案中，本发明上述化合物为化合物004的另一立体异构体，所述立体异构体经手性超临界流体色谱分析后保留时间为4.3‐4.7min，优选4.4‐4.6min，更优选约4.5min；所述手性超临界流体色谱分析的条件为：色谱柱:Chiralpak AS‐3(100mm*4.6mm,3μm)；流动相：二氧化碳；[0.05％三乙胺，乙醇％]：40％‐40％。
在本发明的一个技术方案中，本发明上述化合物为
Figure PCTCN2021119985-appb-000045
在本发明的一个技术方案中，本发明化合物为化合物005的立体异构体；所述立体异构体经手性超临界流体色谱分析后保留时间为4.8‐5.2min，优选4.9‐5.1min，更优选约5.0min；所述手性超临界流体色谱分析的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水‐乙醇]；乙醇％:48％‐78％,7min。
本发明的一个技术方案中，本发明化合物为化合物005的另一立体异构体；所述立体异构体经手性超临界流体色谱分析后保留时间为5.6‐6.0min，优选5.7‐5.9min，更优选约5.8min；所述手性超临界流体色谱分析的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水‐乙醇]；乙醇％:48％‐78％,7min。
本发明的一个技术方案中，本发明化合物为
Figure PCTCN2021119985-appb-000046
在发明的一个技术方案中，上述化合物为化合物006的立体异构体，所述立体异构体经手性超临界流体色谱分离后保留时间为4.9-5.3min，优选5.0-5.2min，更优选约5.1min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-甲醇]；甲醇％:40％-40％。
在发明的一个技术方案中，上述化合物为化合物006的另一立体异构体，所述立体异构体经手性超临界流体色谱分离后保留时间为6.6-7.0min，优选6.7-6.9min，更优选约6.8min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-甲醇]；甲醇％:40％-40％。
在本发明的一个技术方案中，本发明化合物为
Figure PCTCN2021119985-appb-000047
在本发明的一个技术方案中，本发明化合物为化合物007的立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为6.6‐7.0min，优选6.7‐6.9min，更优选约6.8min；所述手性超临界流体色谱分离的条件为色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水‐乙醇]；乙醇％:48％‐78％,10min。
在本发明的一个技术方案中，本发明化合物为化合物007的另一立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为7.1-7.5min，优选7.2-7.4min，更优选约7.4min；所述手性超临界流体色谱分离的条件为色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:48％-78％,10min。
在本发明的一个技术方案中，本发明化合物为
Figure PCTCN2021119985-appb-000048
在本发明的一个技术方案中，本发明化合物为化合物008的立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为2.7‐3.1min，优选2.8‐3.0min，更优选约2.9min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水‐乙醇]；乙醇％:45％‐45％。
在本发明的一个技术方案中，本发明化合物为化合物008的另一立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为3.6-4.0min，优选3.7-3.9min，更优选约3.9min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:45％-45％。
在本发明的一个技术方案中，本发明化合物为
Figure PCTCN2021119985-appb-000049
在本发明的一个技术方案中，本发明化合物为化合物010的立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为2.3-2.7min，优选2.4-2.6min，更优选约2.6min；所述手性超临界流体色谱分离的条件为：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:40％-40％。
在本发明的一个技术方案中，本发明化合物为化合物010的另一立体异构体；所述立体异构体经手性超临界流体色谱分离后保留时间为3.2-3.6min，优选3.3-3.5min，更优选约3.4min；所述手性超临界流体色谱分离的条件为：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:40％-40％。
本发明第二方面还提供一种药物组合物，其包含上述任意技术方案所限定的化合物或其药学上可接受的盐和药学上可接受的载体。
本发明还提供一种在需要的受试者中治疗与SHP2相关的疾病的方法，包括向受试者提供有效剂量的上述任意技术方案所限定的化合物或其药学上可接受的盐或药物组合物。
本发明还提供了上述化合物、其异构体或其药学上可接受的盐或药物组合物在制备治疗与SHP2相关疾病的药物中的应用。
技术效果
本发明化合物对蛋白酪氨酸磷酸酶SHP2展现出较好的抑制活性，将会在SHP2异常肿瘤患者中具有优异的治疗效果。
定义与说明
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。
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，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2021119985-appb-000050
和楔形虚线键
Figure PCTCN2021119985-appb-000051
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021119985-appb-000052
和直形虚线键
Figure PCTCN2021119985-appb-000053
表示立体中心的相对构型，用波浪线
Figure PCTCN2021119985-appb-000054
表示楔形实线键
Figure PCTCN2021119985-appb-000055
或楔形虚线键
Figure PCTCN2021119985-appb-000056
或用波浪线
Figure PCTCN2021119985-appb-000057
表示直形实线键
Figure PCTCN2021119985-appb-000058
和直形虚线键
Figure PCTCN2021119985-appb-000059
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，带“*”或“#”的原子为手性原子或手性中心，以(R)或(S)单一对映体形式或富含一种对映体形式存在；例如，
Figure PCTCN2021119985-appb-000060
表示的为
Figure PCTCN2021119985-appb-000061
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，取代基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 PCTCN2021119985-appb-000062
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 PCTCN2021119985-appb-000063
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 PCTCN2021119985-appb-000064
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，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2021119985-appb-000065
直形虚线键
Figure PCTCN2021119985-appb-000066
或波浪线
Figure PCTCN2021119985-appb-000067
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2021119985-appb-000068
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2021119985-appb-000069
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他 基团相连；
Figure PCTCN2021119985-appb-000070
表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括
Figure PCTCN2021119985-appb-000071
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2021119985-appb-000072
仍包括
Figure PCTCN2021119985-appb-000073
这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
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。
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(例如亲核取代反应)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。
除非另有说明，超临界流体色谱的条件，例如，超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:48％-78％,7min，其中7min表示的是乙醇浓 度从48％增加到78％所需要的时间。
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。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2021119985-appb-000074
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
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；eq代表当量、等量；NaCNBH 3代表氰基硼氢化钠；r.t.代表室温；mp代表熔点；DCM代表二氯甲烷；MeOH代表甲醇；SFC代表超临界流体色谱；MMS表示肝固有清除率。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021119985-appb-000075
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面经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，仍然属于本发明的保护范围。
实施例1
Figure PCTCN2021119985-appb-000076
Figure PCTCN2021119985-appb-000077
步骤1：化合物001-2的合成：
将化合物001-1(5g,24.22mmol,1eq)溶解到乙腈(50mL),加入二碘甲烷(9.73g,36.33mmol,2.93mL,1.5eq)，0℃缓慢加入亚硝酸叔丁脂(3.75g,36.33mmol,4.32mL,1.5eq)，混合物升温至20℃搅拌，然后升温至80℃搅拌1小时。将反应液在43℃减压浓缩。将浓缩物溶解在80mL石油醚中，过滤，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。柱层析：粗品经柱层析(石油醚)分离，得化合物001-2。
步骤2：化合物001-3的合成：
在氮气的保护下，将化合物001-2(5g,15.76mmol,1eq)和二甲基氧膦(1.23g,15.76mmol,1eq)溶解到二氧六环(50mL)，一次加入三(二亚苄基丙酮)二钯(721.38mg,787.78μmol,0.05eq)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(455.82mg,787.78μmol,0.05eq)，N,N-二异丙基乙胺(5.09g,39.39mmol,6.86mL,2.5eq)，然后升温至60℃搅拌16小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在30mL水和50mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(30mL，30mL，30mL)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。用高效液相色谱(色谱柱:Phenomenex Genimi NX C18150*40mm*5μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:22％-32％,10min)分离纯化，得化合物001-3。MS(ESI)m/z:269.0[M+H] +。
步骤3：化合物001-4的合成：
在氮气的保护下，将化合物001-3(2.3g,8.60mmol,1eq)和化合物001-3A(2.25g,10.32mmol,1.2eq) 溶解到二氧六环(25mL)，一次加入N,N-二异丙基乙胺(3.33g,25.80mmol,4.49mL,3eq)，三(二亚苄基丙酮)二钯(787.38mg,859.86μmol,0.1eq)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(497.53mg,859.86μmol,0.1eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在50mL水和50mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(50mL，30mL，30mL)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。粗品经柱层析(0-10％甲醇在二氯甲烷中)分离。得到化合物001-4。MS(ESI)m/z:405.2[M+H] +。
步骤4：化合物001-5的合成：
将化合物001-4(0.5g,1.23mmol,1eq)溶解到四氢呋喃(5mL)，-78℃一次加入(滴加)溶于四氢呋喃(5mL)的叔丁醇钾(1M,1.85mL,1.5eq)，混合物在-78℃搅拌1小时。将反应液直接旋干得到粗品。得到的粗品，无需进一步纯化，直接用于下一步反应。得到化合物001-5。MS(ESI)m/z:221.0[M+H] +。
步骤5：化合物001-6的合成：
在氮气保护下，将化合物001-5(270mg,1.22mmol,1eq)和化合物001-5A(382.59mg,1.84mmol,1.5eq)溶解到二氧六环(3mL)，一次加入三(二亚苄基丙酮)二钯(112.05mg,122.36μmol,0.1eq)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(70.80mg,122.36μmol,0.1eq)，N,N-二异丙基乙胺(474.43mg,3.67mmol,639.40μL,3eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在30mL水和30mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(30mL，30mL，30mL)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。粗品经柱层析(0-10％甲醇在二氯甲烷中)(TLC检测DCM：MeOH＝10:1)分离。得到化合物001-6。MS(ESI)m/z:347.9[M+H] +。
步骤6：化合物001-7的合成：
将化合物001-6(40mg,114.88μmol,1eq)和化合物001-6A(47.29mg,172.32μmol,1.5eq)溶解到二甲基乙酰胺(2mL)和水(2mL)的混合溶剂中，一次加入碳酸钾(79.39mg,574.40μmol,5eq)，然后升温至100℃搅拌16小时。将反应液直接旋干得到粗品。得到化合物001-7，直接用于下一步反应。MS(ESI)m/z:586.3[M+H] +。
步骤7：化合物001的合成：
将化合物001-7(45mg,76.77μmol,1eq)溶解到盐酸/二氧六环(4M,479.83μL,25eq)，混合物在20℃搅拌1小时。将反应液直接旋干得到粗品。用高效液相色谱(色谱柱:Phenomenex Gemini-NX C18 75*30mm*3μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:0％-30％,7min)分离纯化，得到化合物001的甲酸盐。MS(ESI)m/z:482.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 1.32-1.35(m,3H),1.71(br d,J＝12.80Hz,1H),1.80-1.90(m,3H),1.95(s,3H),1.99(s,3H),3.09-3.25(m,2H),3.41(br d,J＝4.02Hz,1H),3.87(d,J＝9.29Hz,1H),4.00(d,J＝9.03Hz,1H),4.18-4.38(m,3H),6.95(d,J＝7.53Hz,1H),7.36(m,1H),7.62(s,1H),7.75(m,1H)。
实施例2
Figure PCTCN2021119985-appb-000078
步骤1：化合物002-1的合成：
在氮气的保护下，将化合物001-1(200mg,968.68μmol,1eq)溶解到乙腈(5mL)，一次加入二碘甲烷(73.00mg,774.94μmol,69.52μL,0.8eq)，升温至60℃，缓慢加入亚硝酸叔丁酯(149.83mg,1.45mmol,172.82μL,1.5eq)，然后升温至80℃搅拌1小时。将反应液在43℃减压浓缩。将浓缩物溶解在80mL石油醚中，过滤，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。柱层析：粗品经柱层析(石油醚＝100％)分离。得到化合物002-1。 1H NMR(400MHz,CDCl 3)δppm 7.33(dd,J＝7.15,2.13Hz,1H),6.95-7.05(m,2H),2.40(s,3H)。
步骤2：化合物002-2的合成：
将化合物002-1(2.8g,11.79mmol,1eq)溶解到甲醇(40mL)，一次加入乙酸胺(1.82g,23.57mmol,2eq)，醋酸碘苯(9.49g,29.47mmol,2.5eq)，混合物在20℃搅拌1小时。将反应液直接旋干得到粗品。粗品经柱层析(0-10％甲醇在二氯甲烷中)分离得到化合物002-2。MS(ESI)m/z:269.7[M+H] +。
步骤3：化合物002-3的合成：
在氮气的保护下，将化合物002-2(1g,3.72mmol,1eq)和化合物001-3A(894.37mg,4.10mmol,1.1eq)溶解到二氧六环(10mL)，一次加入二异丙基乙胺(1.44g,11.17mmol,1.95mL,3eq)，三(二亚苄基丙酮)二钯(340.98mg,372.36μmol,0.1eq)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(215.45mg,372.36μmol,0.1eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在50mL水和50mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(50mL，30mL，30mL)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。柱层析：粗品经柱层析(0-10％甲醇在二氯甲烷中)分离。得到化合物002-3。MS(ESI)m/z:406.1[M+H] +。
步骤4：化合物002-4的合成：
在氮气的保护下，将化合物002-4(100mg,246.30μmol,1eq)溶解到四氢呋喃(1.23mL)，-78℃一次加入叔丁醇钾(1M,1.23mL,eq)，混合物在-78℃搅拌1小时。将反应液直接旋干得到粗品。得到的粗品，无需进一步纯化，直接用于下一步反应。得到化合物002-4。MS(ESI)m/z:221.9[M+H]+。
步骤5：化合物002-5的合成：
在氮气的保护下，将化合物002-4(50mg,225.50μmol,1eq)和化合物001-5A(70.51mg,338.25μmol,1.5eq)溶解到二氧六环(3mL)，一次加入二异丙基乙胺(87.43mg,676.51μmol,117.84μL,3eq),三(二亚苄基丙酮)二钯(20.65mg,22.55μmol,0.1eq),4,5-双二苯基膦-9,9-二甲基氧杂蒽(13.05mg,22.55μmol,0.1eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在30mL水和30mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(30mL×3)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。粗品经柱层析(0-10％甲醇在二氯甲烷中)分离。得到化合物002-5。MS(ESI)m/z:348.9[M+H]+。
步骤6：化合物002-6的合成：
在氮气的保护下，将化合物002-5(50mg,143.16μmol,1eq)和化合物001-6A(39.29mg,143.16μmol,1eq)溶解到二甲基乙酰胺(2mL)，水(2mL)，一次加入碳酸钾(59.36mg,429.48μmol,3eq)，然后升温至100℃搅拌16小时。将反应液直接旋干得到化合物002-6。MS(ESI)m/z:587.1[M+H]+。
步骤7：化合物002的合成：
将化合物002-6(50mg,85.15μmol,1eq)溶解到盐酸/甲醇(4M,532.17μL,25eq)，混合物在20℃搅拌1小时。将反应液直接旋干得到粗品。用高效液相色谱(色谱柱:Phenomenex Gemini-NX C18 75*30mm*3μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:0％-30％,7min)分离纯化。得到化合物002的甲酸盐。将002的甲酸盐加入到乙酸乙酯(30mL)中，饱和碳酸氢钠溶液洗，无水硫酸钠干燥，过滤，滤液旋干可得化合物002。MS(ESI)m/z:483.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.96(d,J＝ 7.75Hz,1H),7.63(s,1H),7.37(t,J＝8.00Hz,1H),6.97(d,J＝8.50Hz,1H),4.63(br s,2H),4.20-4.37(m,4H),3.99(m,1H),3.87(m,1H),3.36(s,3H),3.10-3.24(m,1H),1.77-1.91(m,3H),1.70(m,1H),1.32(m,3H)。
实施例3
Figure PCTCN2021119985-appb-000079
步骤1：化合物003-1的合成：
在氮气的保护下，将化合物002-2(1.9g,7.07mmol,1eq)溶解到N，N-二甲基甲酰胺(20mL)，0℃一次加入氢化钠(424.45mg,10.61mmol,60％纯度,1.5eq)，混合物在0℃搅拌10min，然后加入碘甲烷(2.01g,14.15mmol,880.86μL,2eq)升温至25℃搅拌小时。将反应液逐滴滴入10mL冰水中，50mL乙酸乙酯稀释，分液，水相乙酸乙酯洗(50mL)，合并有机相，无水硫酸钠干燥，过滤，滤液减压旋去溶剂。粗品经柱层析(石油醚：乙酸乙酯＝0-50％)分离，得到化合物003-1。MS(ESI)m/z:283.9[M+H] +。
步骤2：化合物003-2的合成：
在氮气的保护下，将化合物003-1(1.5g,5.31mmol,1eq)和化合物001-3A(1.27g,5.84mmol,1.1 eq)溶解到而二氧六环(20mL)，一次加入二异丙基乙胺(2.06g,15.92mmol,2.77mL,3eq)，三(二亚苄基丙酮)二钯(486.08mg,530.81μmol,0.1eq)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(307.14mg,530.81μmol,0.1eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在50mL水和50mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(50mL，30mL，30mL)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。柱层析：粗品经柱层析(0-10％甲醇在二氯甲烷中)分离。得到化合物003-2。MS(ESI)m/z:420.0[M+H] +。
步骤3：化合物003-3的合成：
在氮气的保护下，将化合物003-2(100mg,238.08μmol,1eq)溶解到四氢呋喃(1mL)，-78℃一次加入叔丁醇钾(1M,1.19mL,5eq)，混合物在-78℃搅拌1小时。将反应液直接旋干得到得到化合物003-3。MS(ESI)m/z:235.9[M+H]+。
步骤4：化合物003-4的合成：
在氮气的保护下，将化合物003-3(56mg,237.54μmol,1eq)和化合物001-5A(74.27mg,356.30μmol,1.5eq)溶解到二氧六环(3mL)，一次加入二异丙基乙胺(92.10mg,712.61μmol,124.12uL,3eq),三(二亚苄基丙酮)二钯(21.75mg,23.75μmol,0.1eq),4,5-双二苯基膦-9,9-二甲基氧杂蒽(13.74mg,23.75μmol,0.1eq)，然后升温至110℃搅拌5小时。将反应液冷却到20℃，并且在43℃减压浓缩。将浓缩物溶解在30mL水和30mL乙酸乙酯中，萃取分层，同时水相用乙酸乙酯(30mL×3)萃取三次。合并有机相，用饱和氯化钠溶液(30mL)洗涤一次，最后用无水硫酸钠干燥有机相，过滤，滤液减压浓缩。粗品经柱层析(0-10％甲醇在二氯甲烷中)分离得到化合物003-4。MS(ESI)m/z:362.9[M+H] +。
步骤5：化合物003-5的合成：
在氮气的保护下，将化合物003-4(85mg,233.98μmol,1eq)和化合物001-6A(77.05mg,280.77μmol,1.2eq)溶解到二甲基乙酰胺(2mL),水(2mL)，一次加入碳酸钾(97.01mg,701.93μmol,3eq)，然后升温至100℃搅拌16小时。将反应液直接旋干得到粗品。得到化合物003-5。MS(ESI)m/z:601.1[M+H] +。
步骤6：化合物003的合成：
将化合物003-5(50mg,83.16μmol,1eq)溶解到氯化氢/甲醇(4M,20.79μL,1eq)，混合物在20℃搅拌1小时。将反应液直接旋干得到粗品。送高效液相色谱(Phenomenex Gemini-NX C18 75*30mm*3μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:5％-35％,7min)分离纯化得到化合物003的甲酸盐。将003的甲酸盐加入到乙酸乙酯(30mL)中，饱和碳酸氢钠溶液洗，无水硫酸钠干燥，过滤，滤液旋干可得化合物003。MS(ESI)m/z:497.0[M+H ]+。 1H NMR(400MHz,CD 3OD)δppm 7.92(dd,J＝7.91,1.38Hz,1H), 7.63(s,1H),7.40(t,J＝7.91Hz,1H),7.00(dd,J＝8.03,1.51Hz,1H),4.25-4.38(m,3H),4.02(d,J＝9.29Hz,1H),3.90(d,J＝9.29Hz,1H),3.44(d,J＝4.27Hz,1H),3.36(s,3H),3.07-3.24(m,2H),2.57(s,3H),1.79-1.90(m,3H),1.68-1.76(m,1H),1.35(s,3H)。
实施例4
Figure PCTCN2021119985-appb-000080
Figure PCTCN2021119985-appb-000081
步骤1：化合物004-2的合成：
在氮气的保护下，将化合物004-1(5.0g,19.43mmol,1eq)溶解于无水四氢呋喃中(60mL)中，氮气置换三次后降温至-78℃。向反应混合物中缓慢滴加二异丙基氨基锂(2.0M,10.69mL,1.1eq)的四氢呋喃溶液后混合物在-78℃下搅拌1小时，之后往体系中缓慢滴加化合物004-2A的四氢呋喃溶液并在-78℃下反应30分钟，然后将反应体系缓慢升温至-25℃反应15小时。反应结束后用100mL饱和氯化铵溶液淬灭，乙酸乙酯萃取三次(50mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(石油醚：乙酸乙酯＝0-10％)分离得到化合物004-2。MS(ESI)m/z:388.2[M+Na] +。
步骤2：化合物004-3的合成：
在氮气的保护下，将化合物004-2(6.91g,18.91mmol,1eq)溶于二氧六环(80mL)和甲醇(32mL)中，再加入氢氧化钠水溶液(6M,16mL,5.08eq)后，升温至100℃回流反应15小时。反应结束后冷却至室温，减压除去有机溶剂后用稀盐酸(1.0M)调节pH为3-4，过滤，滤饼用水洗涤，洗涤后的滤饼重新溶解于乙酸乙酯中，无水硫酸钠干燥后旋干得化合物004-3，MS(ESI)m/z:360.1[M+Na] +。
步骤3：化合物004-4的合成：
在氮气的保护下，将化合物004-4(6.10g,18.08mmol,1eq)和多聚磷酸(40mL，1.0eq)加入单口瓶中，120℃下反应1小时。反应结束后冷却至室温，将反应混合物倒入冰水中淬灭，冰浴下缓慢用氢氧化钠水溶液(6M)调节pH至9。乙酸乙酯萃取三次(50mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂后将粗品溶于二氯甲烷(100mL)中。加入二碳酸二叔丁酯(6.12g,28.05mmol,6.44mL,3.0eq)和三乙胺(5.68g,56.10mmol,7.81mL,6.0eq)后反应混合物在25℃下反应2小时。反应结束后加水，分液，水相用乙酸乙酯萃取(50mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂得化合物004-4。MS(ESI)m/z:342.1[M+Na] +。
步骤4：化合物004-5的合成：
在氮气的保护下，将化合物004-4(2g,6.26mmol,1eq)和化合物004-5A(2.28g,18.79mol,3.0eq)加入单口瓶中，随后加入钛酸四乙酯(5mL)100℃回流反应18小时。反应结束后冷却至室温，将混合物倒入冰水中淬灭，加入乙酸乙酯(50mL)后搅拌1小时，分液，水相用乙酸乙酯萃取(50 mL×3)。合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(石油醚：乙酸乙酯＝0-20％)分离，得到化合物004-5。MS(ESI)m/z:445.1[M+Na] +。
步骤5：化合物004-6的合成：
在氮气的保护下，将化合物004-5(2.54g,6.01mmol,1eq)溶于四氢呋喃(25mL)中，降温至-20℃后加入硼氢化钠(455mg,12.02mmol,2.0eq)。反应体系逐渐恢复至25℃反应12小时。反应结束后，冰浴下加水淬灭反应，乙酸乙酯萃取(50mL×3)后合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物004-6。MS(ESI)m/z:447.1[M+Na] +。
步骤6：化合物004-7的合成：
将化合物004-6(34mg,80μmol,1eq)溶于二氯甲烷(5mL)中，加入三氟乙酸(91.2mg,800μmol,59.23μL,10eq)。反应混合物在25℃下反应2小时后加入碳酸钾中和反应体系至中性，旋干溶剂得化合物004-7。MS(ESI)m/z:325.1[M+H] +。
步骤7：化合物004-8的合成：
将化合物004-7(25.9mg,80μmol,1eq)和化合物002-5(27.6mg,80μmol,1eq)溶于N，N-二甲基乙酰胺(4mL)和水(4mL)中，加入碳酸钾(57.38mg,415μmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(10mL)后用乙酸乙酯萃取(10mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物004-8。MS(ESI)m/z:637.1[M+H] +。
步骤8：化合物004的合成：
将化合物004-8(23mg,36.1μmol,1eq)溶于甲醇(2mL)中，加入盐酸甲醇溶液(4M,530μL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得粗品，粗品送制备高效液相色谱(色谱柱：Phenomenex Gemini-NX C18 75*30mm*3μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:48％-78％,7min)分离得到化合物004的甲酸盐。将004的甲酸盐加入到乙酸乙酯(30mL)中，饱和碳酸氢钠溶液洗，无水硫酸钠干燥，过滤，滤液旋干可得化合物004。MS(ESI)m/z:533.2[M+H] +。 1H NMR(400MHz,CDCl 3)δppm 7.95(dd,J＝7.78,1.25Hz,1H),7.68(s,1H),7.28(m,2H),7.04(dd,J＝7.91,1.38Hz,1H),6.91–6.96(m,2H),4.88(s,2H),4.21(m,2H),3.98(s,1H),3.34(s,3H),3.17–3.27(m,2H),3.05-3.14(d,J＝15.56Hz,1H),2.75(d,J＝15.56Hz,1H),1.30-1.34(m,2H)。
步骤9：化合物004-8A和004-8B的合成：
将化合物004-8送手性超临界流体色谱进行分离(色谱柱:Chiralpak AS-3(100mm*4.6mm,3μm)；流动相:二氧化碳；[0.05％三乙胺，乙醇％]:40％-40％)，分离得到两个异构体，异构体1为004-8A(保留时间2.557min)。MS(ESI)m/z:637.1[M+H] +。异构体2为004-8B(保留时间3.028min)，MS(ESI) m/z:637.1[M+H] +。
步骤10：化合物004A的合成：
将化合物004-8A(23mg,36.1μmol,1eq)溶于甲醇(2mL)中，加入盐酸甲醇溶液(4M,530μL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得粗品，粗品送高效液相色谱(色谱柱：Welch Xtimate C18 150*25mm*5μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:10％-40％,7min)分离得到化合物004A的甲酸盐。MS(ESI)m/z:533.2[M+H] +。
1H NMR(400MHz,CD 3OD)δppm 7.95(dd,J＝7.78,1.25Hz,1H),7.64(s,1H),7.50dd,J＝8.28,5.27Hz,1H),7.38(t,J＝7.91,1H),7.15–7.02(m,2H),6.99(dd,J＝8.03,1.51Hz,1H),4.45–4.25(m,3H),3.36(s,3H),3.32–3.22(m,2H),3.20–3.10(m,2H),2.00-1.50(m,4H)。
1H NMR(400MHz,CDCl 3)δppm 7.95(dd,J＝7.78,1.25Hz,1H),7.68(s,1H),7.28(m,2H),7.04(dd,J＝7.91,1.38Hz,1H),6.91–6.96(m,2H),4.88(s,2H),4.21(m,2H),3.98(s,1H),3.34(s,3H),3.17–3.27m,2H),3.05-3.14(d,J＝15.56Hz,1H),2.75(d,J＝15.56Hz,1H),1.30-1.34(m,2H)。
004A的甲酸盐送手性超临界流体色谱进行分析：(色谱柱:Chiralpak AS-3(100mm*4.6mm,3μm)；流动相:二氧化碳；[0.05％三乙胺，乙醇％]:40％-40％)保留时间为3.327min。
步骤11：化合物004B的合成：
将化合物004-8B(23mg,36.1μmol,1eq)溶于甲醇(2mL)中，加入盐酸甲醇溶液(4M,530μL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得粗品，粗品送高效液相色谱(色谱柱：Welch Xtimate C18 150*25mm*5μm；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:10％-70％,7min)分离得到化合物004B的甲酸盐。MS(ESI)m/z:533.2[M+H] +。
1H NMR(400MHz,CD 3OD)δppm 7.95(dd,J＝7.78,1.25Hz,1H),7.64(s,1H),7.54(dd,J＝8.53,5.02Hz,1H),7.38(t,J＝7.91,1H),7.18–7.05(m,2H),6.99(dd,J＝8.03,1.25Hz,1H),4.45–4.25(m,3H),3.36(s,3H),3.32–3.22(m,2H),3.22–3.15(m,2H),2.00–1.50(m,4H)。
1H NMR(400MHz,CDCl 3)δppm 7.95(dd,J＝7.78,1.25Hz,1H),7.68(s,1H),7.28(m,2H),7.04(dd,J＝7.91,1.38Hz,1H),6.91–6.96(m,2H),4.88(s,2H),4.21(m,2H),3.98(s,1H),3.34(s,3H),3.17–3.27(m,2H),3.05-3.14(d,J＝15.56Hz,1H),2.75(m,1H),1.30-1.34(m,2H)。
004B的甲酸盐送手性超临界流体色谱进行分析：(色谱柱:Chiralpak AS-3(100mm*4.6mm,3μm)；流动相:二氧化碳；[0.05％三乙胺，乙醇％]:40％-40％)保留时间为4.498min。
实施例5
Figure PCTCN2021119985-appb-000082
步骤1：化合物005-2的合成：
将化合物005-1(500mg,1.17mmol,1eq)溶于二氯甲烷(5mL)中，加入三氟乙酸(3.0mL)。反应混合物在25℃下反应2小时后加入碳酸钾中和反应体系至中性，旋干溶剂得化合物005-2。MS(ESI)m/z:327.1[M+H] +。
步骤2：化合物005-3的合成：
将化合物005-2(380mg,1.16mmol,1eq)和化合物002-5(405mg,1.16mmol,1eq)溶于N，N-二甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(995mg,5.8mmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(10mL)后用乙酸乙酯萃取(10mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物005-3。MS(ESI)m/z:639.1[M+H] +。
步骤3：化合物005的合成：
将化合物005-3(250mg,391μmol,1eq)溶于甲醇(10mL)中，加入盐酸甲醇溶液(4M,2.5mL, 58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得粗品，粗品送超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:48％-78％,7min。分离得到化合物005A(5.032min)和化合物005B(5.823min)。化合物005A：MS(ESI)m/z:535.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.94(dd,J＝7.78,1.25Hz,1H),7.65(s,1H)7.38–7.34(m,2H),7.00–6.97(m,1H),6.91–6.55(m,2H),4.48–4.40(m,1H),4.37–4.26(m,1H),4.09(s,1H),3.45–3.37(m,2H),3.35(s,3H),1.96–1.93(m,2H),1.85–1.82(m,2H)。化合物005B：MS(ESI)m/z:535.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.94(dd,J＝7.78,1.25Hz,1H),7.65(s,1H),7.38–7.34(m,2H),7.00–6.97(m,1H),6.91–6.66–6.55(m,2H),4.48–4.40(m,1H),4.37–4.26(m,1H),4.09(s,1H),3.45–3.37(m,2H),3.35(s,3H),1.96–1.93(m,2H),1.85–1.82(m,2H)。
实施例6
Figure PCTCN2021119985-appb-000083
步骤1：化合物006-3的合成：
将化合物006-1(7.13g,33.93mmol,1eq)溶于四氢呋喃(160mL)中，待温度降低至-78℃下加入双异丙基氨基锂(2M,22.06mL,1.3eq)，反应体系在-78℃下反应1小时，加入006-2(10g,37.32mmol,1.1eq)，体系在-78℃下再反应1小时，而后在搅拌下缓慢升温至25℃。反应结束后用25mL饱和氯化铵溶液淬灭，乙酸乙酯萃取三次(150mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(石油醚：乙酸乙酯＝0-10％)分离得到化合物006-3。MS(ESI)m/z:296.8[M+H] +。
步骤2：化合物006-4的合成：
在氮气的保护下，将化合物006-3(12.00g,30.21mmol,1eq)溶于N-N二甲基乙酰胺(100mL)和水(10mL)的混合溶液中，加入二氯双[二叔丁基-(4-二甲基氨基苯基)膦]钯(2.14g,3.02mmol,2.14mL,0.1eq)和三乙胺(12.23g,120.82mmol,16.82mL,4eq)，体系抽换氮气三次，升温至130℃反应5小时。将反应液冷却至室温，加入水150ml，用乙酸乙酯萃取(200mL×3)，合并有机相，减压浓缩至浓缩液体积为150mL左右，水洗4次，饱和食盐水洗涤2次，无水硫酸钠干燥，减压浓缩得粗产物。粗品经柱层析(30％～35％乙酸乙酯在石油醚中)分离得到化合物006-4，MS(ESI)m/z:263.9[M+H] +。
步骤3：化合物006-5的合成：
将化合物006-4(8.83g,27.65mmol,1eq)溶于钛酸四乙酯(85ml)中，加入化合物004-5A(10.05g,82.94mmol,3eq)，体系抽换氮气三次，而后升温至130℃反应3小时。反应结束后，将反应液冷却至室温，将反应液加入至冰水中，搅拌40分钟，将上清液加入分液漏斗中，再加入乙酸乙酯萃取(200mL×3)，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得粗产物。粗产物经柱层析(25％～35％乙酸乙酯在石油醚中)分离得到化合物006-5。MS(ESI)m/z:322.9[M- tBu] +。
步骤4：化合物006-6的合成：
将化合物006-5(7.34g,17.37mmol,1eq)溶于四氢呋喃中(70ml)，降温至0℃，搅拌下加入硼氢化钠(1.31g,34.74mmol,2eq)，反应体系逐渐恢复至25℃下反应16小时。向反应液中加入150mL水淬灭未反应完的NaBH 4，乙酸乙酯萃取(200mL×3)，合并有机相用无水硫酸钠干燥，减压浓缩的粗产物。粗产物经SFC纯化得到化合物006-6。MS(ESI)m/z:325.0[M- tBu] +。
步骤5：化合物006-7的合成：
将化合物006-6(1.10g,2.59mmol,1eq)溶于二氯甲烷(10mL)中，加入三氟乙酸(3.84g,33.68mmol,2.49mL,13eq)，混合液在25℃下反应50min。反应结束后，减压浓缩除掉部分三氟乙酸，再向浓缩液中加入30ml水，加入3g碳酸钾除掉多余三氟乙酸，乙酸乙酯萃取(50mL×3)，有机相合并后用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物006-7。MS(ESI)m/z:325.1[M+H] +。
步骤6：化合物006-8的合成：
将化合物006-7(380mg,1.16mmol,1eq)和化合物002-5(405mg,1.16mmol,1eq)溶于N，N-二 甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(995mg,5.8mmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(50mL)后用乙酸乙酯萃取(50mL×3)，合并有机相并用饱和食盐水洗涤(50mL×3)，有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物006-8。MS(ESI)m/z:637.2[M+H] +。
步骤7：化合物006的合成：
将化合物006-8(308mg,483μmol,1eq)溶于甲醇(10mL)中，加入盐酸甲醇溶液(4M,2.5mL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得006粗品，粗品送手性超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-甲醇]；甲醇％:40％-40％。分离得到化合物006A(5.115min)和化合物006B(6.812min)。化合物006A：MS(ESI)m/z:533.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.94(d,J＝7.78Hz,1H),7.65(s,1H),7.37(t,J＝7.91Hz,1H),7.24(dd,J＝7.91,5.14Hz,1H),7.19–7.10(m,1H),7.00–6.93(m,2H),4.39–4.25(m,2H),3.98(s,1H),3.35(s,3H),3.30-3.10(m,3H),2.79(d,J＝15.31Hz,1H),1.90–1.70(m,2H),1.64–1.59(m,1H),1.45–1.35(m,1H)。化合物006B：MS(ESI)m/z:533.1[M+H] +。δppm 7.94(d,J＝7.78Hz,1H),7.65(s,1H),7.37(t,J＝7.91Hz,1H),7.24(dd,J＝7.91,5.14Hz,1H),7.19–7.10(m,1H),7.00–6.93(m,2H),4.39–4.25(m,2H),3.98(s,1H),3.35(s,3H),3.30-3.10(m,3H),2.79(d,J＝15.31Hz,1H),1.90–1.70(m,2H),1.64–1.59(m,1H),1.45–1.35(m,1H)。
实施例7
Figure PCTCN2021119985-appb-000084
Figure PCTCN2021119985-appb-000085
步骤1：化合物007-2的合成：
将化合物007-1(500mg,1.23mmol,1eq)溶于二氯甲烷(5mL)中，加入三氟乙酸(3.0mL)。反应混合物在25℃下反应2小时后加入碳酸钾中和反应体系至中性，旋干溶剂得化合物007-2。MS(ESI)m/z:306.1[M+H] +。
步骤2：化合物007-3的合成：
将化合物007-2(366mg,1.19mmol,1eq)和化合物002-5(420mg,1.20mmol,1eq)溶于N，N-二甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(832mg,5.8mmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(10mL)后用乙酸乙酯萃取(10mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物007-3。MS(ESI)m/z:621.1[M+H] +。
步骤3：化合物007的合成：
将化合物007-3(238mg,391μmol,1eq)溶于甲醇(10mL)中，加入盐酸甲醇溶液(4M,2.5mL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得粗品，粗品送超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:48％-78％,10min。分离得到化合物007A(6.802min)和化合物007B(7.384min)。化合物007A：MS(ESI)m/z:516.1[M+H] +。化合物007A：MS(ESI)m/z:516.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm8.35(d,J＝5.14Hz,1H)7.95(dd,J＝7.91Hz,1.38,1H),7.84(d,J＝7.53Hz,2H),7.63(s,1H),7.37(t,J＝7.91Hz,1H),7.28(dd,J＝7.40,5.14Hz,1H),7.00(dd,J＝7.91,1.38Hz,1H),4.37–4.26(m,2H),4.04(s,1H),3.35(s,3H),3.20–3.30(m,3H),2.97–2.82(m,1H),1.95–1.75(m,2H),1.70–1.60(m,1H),1.45–1.40(m,1H)。化合物007B：MS(ESI)m/z:516.1[M+H] +。 1H NMR(400MHz,CD 3OD)δppm8.35(d,J＝5.14Hz, 1H),7.95(dd,J＝7.91Hz,1.38,1H),7.84(d,J＝7.53Hz,2H),7.63(s,1H),7.37(t,J＝7.91Hz,1H),7.28(dd,J＝7.40,5.14Hz,1H),7.00(dd,J＝7.91,1.38Hz,1H),4.37–4.26(m,2H),4.04(s,1H),3.35(s,3H),3.20–3.30(m,3H),2.97–2.82(m,1H),1.95–1.75(m,2H),1.70–1.60(m,1H),1.45–1.40(m,1H)。
实施例8
Figure PCTCN2021119985-appb-000086
步骤1：化合物008-2的合成：
将化合物006-1(19g,70.92mmol,1.1eq)溶于四氢呋喃(160mL)中，待温度降低至-78℃下加入双异丙基氨基锂(2M,38.6mL,1.2eq)，反应体系在-78℃下反应1小时，加入008-1(13.6g,64.50mmol,1.0eq)，体系在-78℃下再反应1小时，而后在搅拌下缓慢升温至25℃。反应结束后用250mL饱和氯化铵溶液淬灭，乙酸乙酯萃取三次(150mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(石油醚：乙酸乙酯＝0-10％)分离得到化合物008-2。MS(ESI)m/z:297.9[M- tBu] +。
步骤2：化合物008-3的合成：
在氮气的保护下，将化合物008-2(12.00g,30.21mmol,1eq)溶于N-N二甲基乙酰胺(100mL)和水(10mL)的混合溶液中，加入二氯双[二叔丁基-(4-二甲基氨基苯基)膦]钯(2.14g,3.02mmol,2.14mL,0.1eq)和三乙胺(12.23g,120.82mmol,16.82mL,4eq)，体系抽换氮气三次，升温至130℃反应5小时。将反应液冷却至室温，加入水150ml，用乙酸乙酯萃取(200mL×3)，合并有机相，减压浓缩至浓缩液体积为150ml左右，饱和食盐水洗涤(200mL×6)，无水硫酸钠干燥，减压浓缩得粗产物。粗品经柱层析(30％～35％乙酸乙酯在石油醚中)分离得到008-3，MS(ESI)m/z:264.1[M- tBu] +。
步骤3：化合物008-4的合成：
将008-3(18g,56.36mmol,1eq)溶于钛酸四乙酯(180mL)中，加入004-5A(20.49g,169.08mmol,3eq)，体系抽换氮气三次，而后升温至130℃反应3小时。反应结束后，将反应液冷却至室温，将反应液加入至冰水中，搅拌40分钟，将上清液加入分液漏斗中，再加入乙酸乙酯萃取(200mL×3)，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得粗产物。粗产物经柱层析(25％～35％乙酸乙酯在石油醚中)分离得到化合物008-4。MS(ESI)m/z:323.2[M-Boc] +。
步骤4：化合物008-5的合成：
将008-4(18g,42.60mmol,1eq)溶于四氢呋喃中(70mL)，降温至0℃，搅拌下加入硼氢化钠(3.22g,85.20mmol,2eq)，反应体系逐渐恢复至25℃下反应16小时。向反应液中加入150mL水淬灭未反应完的NaBH 4，乙酸乙酯萃取(200mL×3)，合并有机相用无水硫酸钠干燥，减压浓缩的粗产物。粗产物经SFC纯化(色谱柱：DAICEL CHIRALPAK AD(250mm*50mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:25％-25％。分离得到化合物008-5。MS(ESI)m/z:325.2[M-Boc] +。
步骤5：化合物008-6的合成：
将008-5(800mg,1.88mmol,1eq)溶于二氯甲烷(10mL)中，加入三氟乙酸(3.84g,33.68mmol,2.49mL,13eq)，混合液在25℃下反应50分钟。反应结束后，减压浓缩除掉部分三氟乙酸，再向浓缩液中加入30ml水，加入3g碳酸钾除掉多余三氟乙酸，乙酸乙酯萃取(50mL×3)，有机相合并后用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物008-6。MS(ESI)m/z:325.2[M+H] +。
步骤6：化合物008-7的合成：
将化合物008-6(570mg,1.76mmol,1eq)和化合物002-5(767mg,2.20mmol,1.2eq)溶于N，N-二甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(607mg,4.39mmol,2.5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(50mL)后用乙酸乙酯萃取(50mL×3)，合并有机相并用饱和食盐水洗涤(50mL×3)，有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物008-7。MS(ESI) m/z:637.2[M+H] +。
步骤7：化合物008的合成：
将化合物008-7(1.04g,1.63mmol,1eq)溶于甲醇(10mL)中，加入盐酸甲醇溶液(4M,2.5mL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得008粗品，粗品送超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:45％-45％。分离得到化合物008A(2.921min)和化合物008B(3.872min)。化合物008A：MS(ESI)m/z:533.1[M+H]+。 1H NMR(400MHz,CD 3OD)δppm 7.94(dd,J＝7.91,1.38Hz,1H),7.63(s,1H),7.38(t,J＝7.91Hz,1H),7.30–7.20(m,2H),7.00–6.95(m,2H),4.35–4.28(m,2H),4.03(s,1H),3.36(s,3H),3.32–3.25(m,2H),3.21(d,J＝16.06Hz,1H),2.85(d,J＝16.06Hz,1H),1.92–1.77(m,2H),1.65–1.60(m,1H),1.50–1.43(m,1H)。化合物008B：MS(ESI)m/z:533.1[M+H] +。 1H NMR(400MHz,DMSO-d6)δppm 7.84(dd,J＝7.91,1.38Hz,1H),7.63(s,1H),7.42(t,J＝7.91Hz,1H),7.30–7.20(m,2H),6.99(t,J＝8.53Hz,1H),6.86(dd,J＝8.53,1.51Hz,1H),4.25–4.18(m,2H),3.89(s,1H),3.36(s,3H),3.32–3.15(m,2H),3.10(d,J＝16.06Hz,1H),2.65(d,J＝16.06Hz,1H),1.80–1.65(m,2H),1.60–1.50(m,1H),1.20–1.05(m,1H)。
实施例9
Figure PCTCN2021119985-appb-000087
Figure PCTCN2021119985-appb-000088
步骤1：化合物009-3的合成：
0℃下将叔丁醇钾(17.43g,155.36mmol,3.5eq)溶于乙二醇二甲醚(500mL)中，抽换氮气三次后在0℃下缓慢滴加009-2(13.00g,66.58mmol,1.5eq)的乙二醇二甲醚(500mL)溶液。控制温度不能高于5℃。混合物在0℃下搅拌反应1小时后在0℃下加入异丙醇(3.73g,62.14mmol,4.76mL,1.4eq)，混合物继续搅拌反应30分钟。再加入009-1(10g,44.39mmol,1eq)的DME(500mL)溶液。混合物在0℃下搅拌反应1小时后恢复至25℃继续搅拌反应12小时。反应结束后用200mL饱和氯化铵溶液淬灭，乙酸乙酯萃取(500mL*3)，有机相合并后用无水硫酸钠干燥，旋干。粗品通过快速柱层析纯化，(石油醚：乙酸乙酯＝20:1-10:1)得到化合物009-3。
步骤2：化合物009-5的合成：
将化合物009-3(6.3g,26.66mmol,1eq)溶于四氢呋喃(250mL)中，待温度降低至-78℃下加入双异丙基氨基锂(2M,22.06mL,1.3eq)，反应体系在-78℃下反应1小时，加入009-4(7.50g,27.99mmol,1.05eq)的四氢呋喃溶液50mL，体系在-78℃下再反应1小时，而后在搅拌下缓慢升温至25℃。反应结束后用250mL饱和氯化铵溶液淬灭，乙酸乙酯萃取三次(450mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。粗品经柱层析(石油醚：乙酸乙酯＝10-20％)分离得到化合物009-5。MS(ESI)m/z:369.0[M- tBu] +。
步骤3：化合物009-6的合成：
在氮气的保护下，将化合物009-5(5.1g,12.05mmol,1eq)溶于N-N二甲基乙酰胺(200mL)和水(20mL)的混合溶液中，加入二氯双[二叔丁基-(4-二甲基氨基苯基)膦]钯(0.85g,1.2mmol,0.1eq)和三乙胺(4.88g,48.19mmol,6.71mL,4eq)，体系抽换氮气三次，升温至130℃反应5小时。将反应液冷却至室温，加入水150ml，用乙酸乙酯萃取(200mL×3)，合并有机相，减压浓缩至浓缩液体积为150ml左右，饱和食盐水洗涤(200mL×6)，无水硫酸钠干燥，减压浓缩得粗产物。粗品经柱层析(30％～35％乙酸乙酯在石油醚中)分离得到009-6，MS(ESI)m/z:290.1[M- tBu] +。
步骤4：化合物009-7的合成：
将009-6(4.2g,12.16mmol,1eq)溶于钛酸四乙酯(100mL)中，加入004-5A(4.42g,36.48mmol,3eq)，体系抽换氮气三次，而后升温至130℃反应3小时。反应结束后，将反应液冷却至室温，将反应液加入至冰水中，搅拌40分钟，将上清液加入分液漏斗中，再加入乙酸乙酯萃取(200mL×3)，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得粗产物。粗产物经柱层析(10％～20％乙酸乙酯在石油醚中)分离得到化合物009-7。MS(ESI)m/z:449.1[M+H] +。
步骤5：化合物009-8的合成：
将009-7(4.5g,10.03mmol,1eq)溶于四氢呋喃中(70mL)，降温至0℃，搅拌下加入硼氢化钠(379.51mg,10.03mmol,1eq)，反应体系逐渐恢复至25℃下反应4小时。向反应液中加入150mL水淬灭未反应完的NaBH 4，乙酸乙酯萃取(200mL×3)，合并有机相用无水硫酸钠干燥，减压浓缩的粗产物。粗产物经高效液相色谱纯化(色谱柱：Phenomenex Genimi NX C18(150mm*40mm,5μm)；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:42％-72％)，分离得到化合物009-8A和化合物009-8B。MS(ESI)m/z:451.1[M+H] +步骤6：化合物009-9A的合成：
将009-8A(0.4g,0.89mmol,1eq)溶于二氯甲烷(10mL)中，加入三氟乙酸(3.84g,33.68mmol,2.49mL,13eq)，混合液在25℃下反应50分钟。反应结束后，减压浓缩除掉部分三氟乙酸，再向浓缩液中加入30ml水，加入3g碳酸钾除掉多余三氟乙酸，乙酸乙酯萃取(50mL×3)，有机相合并后用无 水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物009-9A。MS(ESI)m/z:351.1[M+H] +。
步骤7：化合物009-10A的合成：
将化合物009-9A(300mg,0.860mmol,1eq)和化合物002-5(300mg,0.860mmol,1eq)溶于N，N-二甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(0.591mg,4.3mmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(50mL)后用乙酸乙酯萃取(50mL×3)，合并有机相并用饱和食盐水洗涤(50mL×3)，有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物009-10A。MS(ESI)m/z:351.1[M+H] +。
步骤8：化合物009A的合成：
将化合物009-10A(40mg,60.3μmol,1eq)溶于甲醇(5mL)中，加入盐酸甲醇溶液(4M,2.5mL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得009A粗品，粗品经制备高效液相色谱(色谱柱：Phenomenex Gemini-NX C18(75mm*30mm,3μm)；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:5％-35％)，7min。分离得到化合物009A的甲酸盐：MS(ESI)m/z:560.0[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.98–7.86(m,1H),7.51(s,1H),7.48–7.34(m,2H),7.00–6.97(m,1H),6.91–6.55(m,2H),4.71–4.65(m,1H),4.61(s,2H),4.35(s,1H),3.37(s,3H),3.10–3.02(m,1H),2.44–2.34(m,1H),2.27–2.15(m,2H),2.12–2.01(m,3H),1.91–1.83(m,1H),1.64–1.57(m,1H)。
步骤9：化合物009-9B的合成：
将009-8B(0.4g,0.89mmol,1eq)溶于二氯甲烷(10mL)中，加入三氟乙酸(3.84g,33.68mmol,2.49mL,13eq)，混合液在25℃下反应50分钟。反应结束后，减压浓缩除掉部分三氟乙酸，再向浓缩液中加入30ml水，加入3g碳酸钾除掉多余三氟乙酸，乙酸乙酯萃取(50mL×3)，有机相合并后用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物009-9B。MS(ESI)m/z:351.1[M+H] +。
步骤10：化合物009-10B的合成：
将化合物009-9B(300mg,0.860mmol,1eq)和化合物002-5(300mg,0.860mmol,1eq)溶于N，N-二甲基乙酰胺(10mL)和水(10mL)中，加入碳酸钾(0.591mg,4.3mmol,5eq)后100℃下反应48小时。反应结束后冷却至室温，加入水(50mL)后用乙酸乙酯萃取(50mL×3)，合并有机相并用饱和食盐水洗涤(50mL×3)，有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(二氯甲烷：甲醇＝0-10％)分离，得到化合物009-10B。MS(ESI)m/z:351.1[M+H] +。
步骤11：化合物009B的合成：
将化合物009-10B(20mg,60.3μmol,1eq)溶于甲醇(5mL)中，加入盐酸甲醇溶液(4M,2.5mL,58.5eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得009B粗品，粗品经制备高效液相色谱(色谱柱：Phenomenex Gemini-NX C18(75mm*30mm,3μm)；流动相:[水(0.225％甲酸)-乙腈]；乙腈％:5％- 35％)，7min。分离得到化合物009B的甲酸盐，MS(ESI)m/z:5,60.0[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.98–7.86(m,1H),7.51(s,1H),7.48–7.34(m,2H),7.00–6.97(m,1H),6.91–6.55(m,2H),4.71–4.65(m,1H),4.61(s,2H),4.35(s,1H),3.37(s,3H),3.10–3.02(m,1H),2.44–2.34(m,1H),2.27–2.15(m,2H),2.12–2.01(m,3H),1.91–1.83(m,1H),1.64–1.57(m,1H)。
实施例10
Figure PCTCN2021119985-appb-000089
Figure PCTCN2021119985-appb-000090
步骤1：化合物010-2的合成：
将化合物010-1(8.5g,41.06mmol,1eq)溶于三氯甲烷(100mL)中，加入N-溴代丁二酰亚胺(7.31g,41.06mmol,1eq)，过氧化苯甲酰(596.75mg,2.46mmol,0.06eq)，体系抽换氮气三次，而后缓慢升温至80℃反应5hr。反应结束后将反应液冷却至室温，过滤，滤液中加水200mL，乙酸乙酯萃取(100mL×3)，合并有机相，用饱和食盐水(200mL×1)洗涤后有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂。粗品经快速柱层析(3％～5％乙酸乙酯在石油醚中)分离得到化合物010-2。
步骤2：化合物010-3的合成：
将化合物006-1(4.35g,20.67mmol,1eq)溶于四氢呋喃(120mL)中，体系抽换氮气三次，待温度降低至-78℃下加入双异丙基氨基锂(2M,12.40mL,1.2eq)，反应体系在-78℃下反应1小时，加入010-2(6.5g,22.73mmol,1.1eq)的四氢呋喃溶液(30mL)。体系在-78℃下再反应1小时，而后在搅拌下缓慢升温至25℃继续反应10小时。反应结束后用100mL饱和氯化铵溶液淬灭，乙酸乙酯萃取三次(100mL×3)，合并有机相，无水硫酸钠干燥，过滤，旋蒸除去溶剂。柱层析：粗品经柱层析(10％～15％乙酸乙酯在石油醚中)分离得到化合物010-3。MS(ESI)m/z＝358.9[M- tBu] +。
步骤3：化合物010-4的合成：
将化合物010-3(4.41g,10.62mmol,1eq)溶于N-N二甲基乙酰胺(40mL)和水(4mL)的混合溶液中，加入二氯双[二叔丁基-(4-二甲基氨基苯基)膦]钯(751.94mg,1.06mmol,751.94μL,0.1eq)和三乙胺(4.30g,42.48mmol,5.91mL,4eq)，体系抽换氮气三次，升温至130℃反应5小时。反应结束后，将反应液冷却至室温，加入水150ml，用乙酸乙酯萃取(200mL×3)，合并有机相，减压浓缩至浓缩液体积为150mL左右，饱和食盐水洗涤(200mL×6)，无水硫酸钠干燥，减压浓缩得粗产物。粗品经柱层析(15％～25％乙酸乙酯在石油醚中)分离得到化合物010-4，MS(ESI)m/z:＝282.1[M- tBu] +。
步骤4：化合物010-5的合成：
将化合物010-4(2.35g,6.97mmol,1eq)溶于钛酸四乙酯(30mL)中，加入004-5A(2.53g,20.90mmol,3eq)，体系抽换氮气三次，而后升温至130℃反应3小时。反应结束后，将反应液冷却至室温，将反应液加入冰水中，搅拌40分钟，将上清液加入分液漏斗中，再加入乙酸乙酯萃取(100mL×3)，合并有机相，用饱和食盐水洗涤，无水硫酸钠干燥，减压浓缩得粗产物。粗产物经柱层析(25％～35％乙酸乙酯在石油醚中)分离得到化合物010-5。MS(ESI)m/z:341.1[M-Boc] +。
步骤5：化合物010-6的合成：
将化合物010-5(2.68g,6.08mmol,1eq)溶于四氢呋喃中(30mL)，降温至0℃，搅拌下加入硼氢化钠(460.30mg,12.17mmol,2eq)，反应体系逐渐恢复至25℃下反应16小时。向反应液中加入100mL水淬灭未反应完的NaBH 4，乙酸乙酯萃取(100mL×3)，合并有机相并用无水硫酸钠干燥，减压浓缩得粗产物。粗产物经柱层析(45％～55％乙酸乙酯在石油醚中)，分离得到化合物010-6。MS(ESI)m/z:343.1[M-Boc] +。
步骤6：化合物010-7的合成：
将化合物010-6(435.50mg,984.04μmol,1eq)溶于二氯甲烷(5mL)中，加入三氟乙酸(1.57g,13.78mmol,1.02mL,14eq)，混合液在25℃下反应50分钟。反应结束后，减压浓缩除掉部分三氟乙酸，再向浓缩液中加入30mL水，加入3g碳酸钾除掉多余三氟乙酸，乙酸乙酯萃取(50mL×3)，有机相合并后用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到化合物010-7。MS(ESI)m/z:343.1[M+H] +。
步骤7：化合物010-8的合成：
将化合物001-7(330.00mg,963.66μmol,1eq)和化合物002-5(403.88mg,1.16mmol,1.2eq)溶于N,N-二甲基乙酰胺(7mL)和水(7mL)中，加入碳酸钾(399.55mg,2.89mmol,3eq)后80℃下反应16小时。反应结束后冷却至室温，加入水(50mL)后用乙酸乙酯萃取(50mL×3)，合并有机相并用饱和食盐水洗涤(50mL×6)，有机相用无水硫酸钠干燥，过滤，旋蒸除去溶剂得到粗产物。粗产物经柱层析(4％～8％二氯甲烷在甲醇中)，分离得到化合物010-8。MS(ESI)m/z:655.2[M+H] +。
步骤8：化合物010的合成：
将化合物010-8(160mg,244.18μmol,1eq)溶于甲醇(2mL)中，加入盐酸甲醇溶液(4M,1.86mL,30.43eq)。反应混合物在25℃下反应1小时后直接旋干溶剂得010粗品，粗品送超临界流体色谱(色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-乙醇]；乙醇％:40％-40％。分离得到化合物010A(保留时间：2.561min)和化合物010B(保留时间：3.389min)。化合物010A：MS(ESI)m/z:551.0[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.94(dd,J＝7.91,1.38Hz,1H),7.61(s,1H),7.35(t,J＝7.91Hz,1H),7.30–7.23(m,1H),7.18–7.10(m,1H),7.00–6.95(m,1H),4.35–4.28(m,2H),3.94(s,1H),3.36(s,3H),3.30–3.11(m,3H),2.82–2.75(m,1H),1.90–1.71(m,2H),1.64–1.55(m,1H),1.45–1.37(m,1H)。化合物010B：MS(ESI)m/z:551.0[M+H] +。 1H NMR(400MHz,CD 3OD)δppm 7.94(dd,J＝7.91,1.38Hz,1H),7.61(s,1H),7.35(t,J＝7.91Hz,1H),7.30–7.23(m,1H),7.18–7.10(m,1H),7.00–6.95(m,1H),4.35–4.28(m,2H),3.94(s,1H),3.36(s,3H),3.30–3.11(m,3H),2.82–2.75(m,1H),1.9–1.71(m,2H),1.64–1.55(m,1H),1.45–1.37(m,1H)。
生物测试
实验例1：体外评价
反应缓冲液：
60mM羟乙基哌嗪乙硫磺酸(HEPES)(pH 7.4)，1mM乙二胺四乙酸(EDTA)，75mM KCl，75mM NaCl，0.01％Brij-35,5mM二硫苏糖醇(DTT)和10％DMSO(最终)。
酶：PTPN11/SHP2-FL(RBC生产，没有CAS号)
重组人PTPN11全长(Genbank登录号#NM_002834；aa 2-597，
同种型1(规范))在大肠杆菌中表达，具有N-端StrepII-TEV，C-端组氨酸的标签。Mw＝71.93kDa。
活化肽：H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide(基于出版物)
底物：DiFMUP[6,8-二氟-7-羟基-4-甲基香豆素]
测定中的最终浓度：
0.35μM活化肽
100μM DiFMUP(6,8-二氟-4-甲基伞形酮磷酸酯)
步骤：
1.在新制备的反应缓冲液中制备指定的酶/肽和底物；
2.将酶/肽溶液加入到反应孔中；
3.通过声学技术将化合物在100％DMSO中提供到酶溶液中(Echo550；纳升范围)，在室温下孵育30分 钟；
4.将底物溶液加入到反应孔中以引发反应；
5.监测酶活性(Ex/Em 355/460)，作为室温下荧光底物荧光信号增加60分钟的时程测量；
6.数据分析：取时间过程测量的线性部分的斜率×(信号/分钟)，并计算相对于DMSO对照的％酶活性；减去酶基础活性(无肽)的背景斜率。
本发明化合物体外筛选试验结果如标1所示。
表1.本发明化合物体外筛选试验结果
化合物编号 PTPN11/SHP2-FL(IC 50nM)
化合物001的甲酸盐 21.8
化合物002的甲酸盐 12.6
化合物003的甲酸盐 72.4
化合物004的甲酸盐 2.84
化合物004A的甲酸盐 3.21
化合物004B的甲酸盐 2.61
化合物005A 5.35
化合物005B 3.66
化合物006A 2.95
化合物006B 2.22
化合物008A 2.78
化合物008B 2.43
化合物010A 6.01
化合物010B 5.81
结论：本发明化合物对PTPN11/SHP2-FL有一定的抑制活性。
实验例2：化合物H358细胞活性评价
实验目的：
本实验旨在验证本发明化合物对KRAS G12C突变的NCI-H358人非小细胞肺癌细胞增殖抑制效果。
实验材料：
细胞株NCI-H358(购自普诺赛)、RPMI1640培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。
实验方法：
将NCI-H358细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含4000个NCI-H358细胞。细胞板置于二氧化碳培养箱中过夜培养。将待测化合物用排枪进5倍稀释至第9个浓度，即从2000μM稀释至5.12nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养5天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入25μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。加入化合物的细胞板结束孵育后，采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。
本发明化合物H358细胞活性筛选试验结果如表2所示。
表2.本发明化合物体外筛选试验结果
化合物编号 H358(IC 50nM)
化合物004A的甲酸盐 56.5
化合物004B的甲酸盐 19.2
化合物006A 30
化合物006B 18
化合物010A 11.9
化合物010B 7.9
结论：本发明化合物对H358细胞的抑制活性良好。
实验例3：化合物药代动力学评价
实验目的：测试化合物在CD-1小鼠体内药代动力学
实验材料：CD-1小鼠(雄性,32-33g)
实验操作：
以标准方案测试化合物静脉注射及口服给药后的啮齿类动物药代特征，实验中候选化合物配成澄清溶液，给予小鼠单次静脉注射及口服给药。静注及口服溶媒为一定比例的羟丙基β环糊精水溶液或生理盐水溶液。收集24小时内的全血样品，3000g离心15分钟，分离上清得血浆样品，加入4倍体积含内标的乙腈溶液沉淀蛋白，离心取上清液加入等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。
本发明化合物药代动力学测试结果如表3所示。
表3 药代动力学测试结果
Figure PCTCN2021119985-appb-000091
结论：本发明化合物可以显著提高小鼠药代动力学单项或部分指标。
实验例4：人肝微粒体CYP抑制实验
研究项目的目的是采用CYP同工酶的5合1探针底物来评价供试品对人肝微粒体细胞色素P450同工酶(CYP1A2、CYP2C9、CYP2C19、CYP2D6和CYP3A4)的抑制性。
混合人肝微粒体(HLM)购自Corning Inc.(Steuben,New York,USA)或者XenoTech,LLC.(Lenexa,KS,USA)或者其他的供应商，使用前都储存在低于-70℃条件下。
将稀释好的系列浓度的供试品工作液加入到含有人肝微粒体、探针底物和循环体系的辅助因子的孵育体系中，不含供试品而含有溶剂的对照作为酶活性对照(100％)。探针底物生成的代谢产物在样品中的浓度采用液相色谱-串联质谱(LC-MS/MS)方法进行测定。使用SigmaPlot(V.11)对供试品平均百分比活性对浓度作非线性回归分析。通过三参数或四参数反曲对数方程来计算IC 50值。
测试结果如表4所示：
表4
Figure PCTCN2021119985-appb-000092
Figure PCTCN2021119985-appb-000093
结论：本发明化合物对五个CYP同工酶抑制程度均较弱。
实验例5：hERG钾离子通道的抑制试验
1.实验目的：
用全自动膜片钳的方法检测待测实施例1对hERG钾离子通道的影响。
2.实验方法
2.1.细胞培养
实验所用的稳定表达hERG钾离子通道的细胞来自于Aviva Biosciences的CHO-hERE，CHO-hERG培养于5％CO 2，37℃的环境下。CHO hERG培养液见表5。
表5 CHO hERG培养液
Reagent Supplier Catalog Number Volume(mL)
F12Hams Invitrogen 31765-092 500
FBS Invitrogen 10099-141 50
G418/Geneticin Invitrogen 10131-027 1
Hygromycin B Invitrogen 10687-010 1
2.2.细胞的前期准备
准备用于实验的CHO-hERG细胞至少培养两天以上，且细胞密度达到75％以上。实验开始之前，用TrypLE消化细胞，然后用细胞外液重悬收集细胞。
2.3.细胞内外液的配制
细胞外液需每个月配制一次。细胞内液须分装冻存在-20℃。细胞内外液成分见表6。
表6 细胞内外液成分
组成成分 细胞外液(mM) 细胞内液(mM)
NaCl 145 -
KCl 4 120
KOH - 31.25
CaCl 2 2 5.374
MgCl 2 1 1.75
Glucose 10 -
Na 2ATP - 4
HEPES 10 10
EGTA - 10
pH 7.4with NaOH 7.2with KOH
渗透压 295mOsm 285mOsm
2.4.化合物的配制
将待测化合物和阳性对照Amitriptyline用DMSO溶解成一定浓度的储备液，然后按照不同的梯度稀释，最后按一定的比例加入细胞外液中，稀释成待测浓度。在实验开始前用肉眼检查看有无沉淀。最后，待测溶液和阳性对照Amitriptyline中，DMSO的浓度最高不能超过0.3％。
2.5.电压刺激方案
保持钳制电位在-80mv，首先是给予-50mv的电压刺激，持续80ms以记录细胞漏电流值，随后去极化至+20mv，维持4800ms，打开hERG的通道，然后复极化至-50mv维持5000ms，引出hERG尾电流并记录，最后，电压恢复至钳制电位-80mv，维持3100ms。以上电压刺激，每15000ms重复一次。
2.6.QPatch HTX全细胞膜片钳记录
hERG QPatch HTX实验是在室温下进行的。在QPatch Assay Software 5.2(Sophion Bioscience)的软件上建立全细胞方案，电压刺激方案和化合物检测方案。
首先进行30次重复设定电压刺激，该区段为后续分析的基线区域，随后加入5μL细胞外液，重复三次。依次加入各个化合物的作用浓度，仍旧以5μL加入体积重复三次。每一测试浓度孵育细胞至少不低于5mins。整个记录过程中，各项指标需达到数据分析接收标准，若未达到该标准，则该细胞不计入分析范围，化合物将重新进行测试，以上记录过程由均由Qpatch分析软件自动化操作。每一化合物测试浓度依次为0.24μM、1.20μM、6.00μM、30.00μM，每一浓度至少重复两个细胞。
2.7.数据分析
在每一个完整电流记录中，基于峰值电流在阴性对照中所占的百分比，可以计算出每一化合物作用浓度的抑制百分比。利用标准希式方程拟合得到量效关系曲线，具体方程如下：
I (C)＝I b+(I fr-I b)*c n/(IC 50 n+c n)
C为化合物测试浓度，n为斜率
曲线拟合和抑制率计算均由Qpatch分析软件分析完成，若最低浓度下抑制率超过半数抑制或最高浓度下抑制率未达到半数抑制，则该化合物相应的IC 50低于最低浓度或IC 50值大于最高浓度。
2.8.测试结果
实施例化合物hERG IC50值结果见表7。
表7 实施例化合物hERG IC 50值结果
供试样品 hERG IC 50(nM)
化合物004A的甲酸盐 8.20
化合物004B的甲酸盐 9.82
化合物006A 10.3
化合物006B 14.0
结论：本发明化合物对hERG抑制不明显。
实验例6：体外微粒体稳定性实验
6.1、实验材料：
6.1.1肝微粒体
人和动物微粒体购买于Corning或Xenotech，储存于-80℃冰箱。
6.1.2还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)，供应商：Chem-impex international，货号：00616
6.1.3对照化合物：睾酮，双氯芬酸，普罗帕酮
6.2实验步骤
6.2.1工作液的配制
储备液：10mM DMSO溶液
工作浓度配制：100％乙腈稀释到100μM(有机相含量：99％ACN，1％DMSO)
6.2.2实验步骤
准备2块96孔孵育板，分别命名为T60孵育板和NCF60孵育板。
在T60孵育板和NCF60孵育板上分别加入445μL微粒体工作液(肝微粒体蛋白浓度为0.56mg/mL)，然后将上述孵育板放置于37℃水浴锅中预孵育大约10分钟。
预孵育结束后，在T60孵育板和NCF60孵育板上分别加入5μL供试品或对照化合物工作液，混匀。在NCF60孵育板上每孔添加50μL磷酸钾盐缓冲液启动反应；在T0终止板中加入180μL的终止液(含200ng/mL tolbutamide和200ng/mL labetalol的乙腈溶液)和6uL的NADPH再生体系工作液，从T60孵育板中取出54μL样品至T0终止板(T0样品产生)。在T60孵育板上每孔添加44μL NADPH再生体系工作液启动反应。在Blank板中只添加54μL微粒体工作液、6uL的NADPH再生体系工作液和180μL的终止液。因此，在供试品或对照化合物的样品中，化合物、睾酮、双氯芬酸和普罗帕酮的反应终浓度为1μM，肝微粒体的浓度为0.5mg/mL，DMSO和乙腈在反应体系中的终浓度分别为0.01％(v/v)和0.99％(v/v)。
孵育适当时间(如5、15、30、45和60分钟)后，分别在每个终止板的样品孔中加入180μL的终止液(含200ng/mL tolbutamide和200ng/mL labetalol的乙腈溶液)，之后从T60孵育板中取 出60μL样品以终止反应。
所有样品板摇匀并在3220×g离心20分钟，然后每孔取80μL上清液稀释到240μL纯水中用于液相色谱串联质谱分析。
本发明化合物MMS结果如表8所示。
表8 本发明化合物MMS结果
供试样品 MMS(mL/min/kg),H,M
化合物004A的甲酸盐 19.7,67.9
化合物004B的甲酸盐 33.0,61.1
化合物006A 31.2,100.8
化合物006B 47.9,57.3

Claims (22)

  1. 式(Ⅱ)化合物或其药学上可接受的盐，
    Figure PCTCN2021119985-appb-100001
    其中，
    结构单元
    Figure PCTCN2021119985-appb-100002
    为
    Figure PCTCN2021119985-appb-100003
    E 1为O或CH 2；
    T 1为N或CH；
    R 1为
    Figure PCTCN2021119985-appb-100004
    R 11、R 13和R 14分别独立地为C 1-3烷基；
    R 12为H或C 1-3烷基；
    R 2为F、Cl、Br或I；
    R 3为C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
    R 4为H、F、Cl、Br、I或C 1-3烷基，所述C 1-3烷基任选被1、2或3个R b取代；
    R a和R b分别独立地为F、Cl、Br、I、OH或NH 2；
    n为0、1、2或3；
    m为1、2或3；
    当n为0时，结构单元
    Figure PCTCN2021119985-appb-100005
    为
    Figure PCTCN2021119985-appb-100006
    结构单元
    Figure PCTCN2021119985-appb-100007
    为
    Figure PCTCN2021119985-appb-100008
    当n为1、2或3时，结构单元
    Figure PCTCN2021119985-appb-100009
    为结构单元
    Figure PCTCN2021119985-appb-100010
    结构单元
    Figure PCTCN2021119985-appb-100011
    为
    Figure PCTCN2021119985-appb-100012
  2. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，R 11、R 13和R 14分别独立地为CH 3。
  3. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，R 12为H或CH 3。
  4. 根据权利要求1～3任意一项所述的化合物或其药学上可接受的盐，其中，R 1为
    Figure PCTCN2021119985-appb-100013
  5. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，R 3为CH 3。
  6. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，R 4为F、Cl、Br或I。
  7. 根据权利要求1所述的化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2021119985-appb-100014
    为
    Figure PCTCN2021119985-appb-100015
  8. 根据权利要求7所述的化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2021119985-appb-100016
    为
    Figure PCTCN2021119985-appb-100017
  9. 根据权利要求8所述的化合物或其药学上可接受的盐，其中，结构单元
    Figure PCTCN2021119985-appb-100018
    为
    Figure PCTCN2021119985-appb-100019
    Figure PCTCN2021119985-appb-100020
  10. 根据权利要求1所述的化合物或其药学上可接受的盐，其化合物具有式(Ⅱ-1)的结构
    Figure PCTCN2021119985-appb-100021
    其中，
    R 11、R 12、R 2、n、E 1和结构单元
    Figure PCTCN2021119985-appb-100022
    如权利要求1所定义。
  11. 根据权利要求1所述的化合物或其药学上可接受的盐，其化合物具有式(Ⅱ-1A)或(Ⅱ-1B)的结构，
    Figure PCTCN2021119985-appb-100023
    其中，n、m、T 1、R 11、R 12、R 2和R 4如权利要求1-10所定义。
  12. 根据权利要求1所述的化合物或其药学上可接受的盐，其化合物具有式(Ⅰ-1A)、(Ⅰ-1B)或(Ⅰ-2A)的结构，
    Figure PCTCN2021119985-appb-100024
    Figure PCTCN2021119985-appb-100025
    其中，n、R 11、R 12、R 13、R 14、R 2、R 3和R 4如权利要求1-10所定义。
  13. 根据权利要求11-12所述的化合物或其药学上可接受的盐，其化合物具有式(Ⅰ-1A-1)、(Ⅰ-2A-1)、(Ⅱ-1A-1)或(Ⅱ-1B-1)的结构
    Figure PCTCN2021119985-appb-100026
    其中，m、n、E 1、T 1、R 11、R 12、R 13、R 14、R 2、R 3和R 4如权利要求11-12所定义。
  14. 下列化合物或其药学上可接受的盐，
    Figure PCTCN2021119985-appb-100027
    Figure PCTCN2021119985-appb-100028
  15. 根据权利要求14所述的化合物或其药学上可接受的盐，其化合物为
    Figure PCTCN2021119985-appb-100029
    Figure PCTCN2021119985-appb-100030
  16. 下式化合物的立体异构体、或所述立体异构体的药学上可接受的盐，
    Figure PCTCN2021119985-appb-100031
    其中，所述立体异构体经手性超临界流体色谱分析后保留时间为3.1-3.5min，优选3.2-3.4min，更优选3.3min；所述手性超临界流体色谱分析的条件为：色谱柱:Chiralpak AS-3(100mm*4.6mm,3μm)；流动相：二氧化碳；[0.05％三乙胺，乙醇％]：40％-40％。
  17. 下式化合物的立体异构体、或所述立体异构体的药学上可接受的盐，
    Figure PCTCN2021119985-appb-100032
    其中，所述化合物经手性超临界流体色谱分析后保留时间为4.3-4.7min，优选4.4-4.6min，更优选4.5min；所述手性超临界流体色谱分析的条件为：色谱柱:Chiralpak AS-3(100mm*4.6mm,3μm)；流动相：二氧化碳；[0.05％三乙胺，乙醇％]：40％-40％。
  18. 下式化合物的立体异构体，或所述异构体的药学上可接受的盐，
    Figure PCTCN2021119985-appb-100033
    其中，所述化合物经手性超临界流体色谱分离后保留时间为4.9-5.3min，优选5.0-5.2min，更优选5.1min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-甲醇]；甲醇％:40％-40％。
  19. 下式化合物的立体异构体、或所述立体异构体的药学上可接受的盐，
    Figure PCTCN2021119985-appb-100034
    其中，所述化合物经手性超临界流体色谱分离后保留时间为6.6-7.0min，优选6.7-6.9min，更优选6.8min；所述手性超临界流体色谱分离的条件为：色谱柱：DAICEL CHIRALPAK AS(250mm*30mm,10μm)；流动相:[0.1％氨水-甲醇]；甲醇％:40％-40％。
  20. 一种药物组合物，其包含权利要求1‐19任意一项所述的化合物或其药学上可接受的盐和药学上可接受的载体。
  21. 根据权利要求1～19任意一项所述化合物或其药学上可接受的盐或根据权利要求20所述的药物组合物在制备治疗与SHP2相关的疾病的药物中的应用。
  22. 根据权利要求21所述的应用，其中所述SHP2相关的疾病为实体瘤。
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