WO2022017519A1 - 喹唑啉类化合物 - Google Patents

喹唑啉类化合物 Download PDF

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WO2022017519A1
WO2022017519A1 PCT/CN2021/108256 CN2021108256W WO2022017519A1 WO 2022017519 A1 WO2022017519 A1 WO 2022017519A1 CN 2021108256 W CN2021108256 W CN 2021108256W WO 2022017519 A1 WO2022017519 A1 WO 2022017519A1
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mmol
compound
added
stirred
reduced pressure
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English (en)
French (fr)
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吴立方
孙飞
丁照中
陈曙辉
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Medshine Discovery Inc
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Medshine Discovery Inc
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Priority to CN202180061375.7A priority Critical patent/CN116234807B/zh
Priority to US18/005,347 priority patent/US12552787B2/en
Priority to EP21846493.1A priority patent/EP4186903A4/en
Publication of WO2022017519A1 publication Critical patent/WO2022017519A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention relates to a class of quinazoline compounds and their pharmaceutically acceptable salts, as well as the application of the compounds or their pharmaceutically acceptable salts in the preparation of medicaments for treating cancer.
  • Mutations in the RAS protein occur in approximately 20% to 30% of cancer cases and are the most commonly mutated oncogenes in pancreatic, colorectal, and non-small cell lung cancers.
  • the RAS protein acts as a molecular switch, which is activated when it binds to GTP and inactive when it binds to GDP. Mutations in the RAS protein reduce its ability to hydrolyze GTP with GTPases, allowing the molecular switch to remain active GTP-bound at all times, driving unexamined oncogenic downstream signaling, such as through the RAS-RAF-MEK-ERK pathway and the RAS-PI3K- The PDK1-AKT pathway, etc., promotes the survival and proliferation of cancer cells.
  • SOS1 protein is a class of guanine nucleotide exchange factor (GEF), and studies have shown that this protein can catalyze the binding of KRAS to GTP, thereby promoting the activation of KRAS. Therefore, targeting and inhibiting the SOS1 protein, the interaction of SOS1-KRAS, regardless of the mutation of KRAS, has become a potential tumor therapy target. In addition, studies have shown that combined MEK inhibition results in deep pathway blockade and tumor degeneration in vivo, and is a potential treatment for most KRAS-driven cancers.
  • GEF guanine nucleotide exchange factor
  • the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof,
  • Ring A is selected from phenyl and 5-membered heteroaryl
  • R 1 is selected from H and NH 2 ;
  • R 2 is selected from C 1-3 alkyl, said C 1-3 alkyl optionally substituted 3, 4 or 5 R a;
  • R 3 is selected from H and F
  • R 4 is selected from C 3-8 cycloalkyl and 3-8 membered heterocycloalkyl, the C 3-8 cycloalkyl and 3-8 membered heterocycloalkyl are independently optionally substituted by 1, 2, 3 or 4 R b substitutions;
  • R 5 is selected from H, Cl and CH 3 ;
  • R a is independently selected from D, F, Cl, Br, I, OH, CN, NH 2 , CH 3 , -CH 2 NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OCH 3 , - COOH, -COOCH 3 and cyclopropyl;
  • R is independently selected from F, Cl, Br, I, OH, CN, NH 2, -OCH 3 , and -COOH.
  • the present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 is selected from H and NH 2 ;
  • R 2 is selected from C 1-3 alkyl, said C 1-3 alkyl optionally substituted 3, 4 or 5 R a;
  • R 3 is selected from H and F
  • R 4 is selected from C 3-8 cycloalkyl and 3-8 membered heterocycloalkyl, the C 3-8 cycloalkyl and 3-8 membered heterocycloalkyl are independently optionally substituted by 1, 2, 3 or 4 R b substitutions;
  • R a is independently selected from D, F, Cl, Br, I, OH, CN, NH 2 , CH 3 , -CH 2 NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OCH 3 , - COOH, -COOCH 3 and cyclopropyl;
  • R b is independently selected from H, F, Cl, Br, I, OH, CN, NH 2 , C 1-3 alkyl, -CH 2 NH 2 , -NHCH 3 , -N(CH 3 ) 2 , - OCH 3 , -COOH, -COOCH 3 and cyclopropyl, C 1-3 alkyl and cyclopropyl are each independently optionally substituted with 1, 2 or 3 R;
  • R is independently selected from F, Cl, Br, I, OH, CN, NH 2 and -COOH.
  • R 1 is selected from NH 2 and other variables are as defined herein.
  • R 1 is selected from H, and other variables are as defined in the present invention.
  • R 2 is selected from CF 3 .
  • Other variables are as defined in the present invention.
  • R 2 is selected from CF 3 , CHF 2 , Other variables are as defined in the present invention.
  • R 4 is selected from cyclopropyl, bicyclo[2.2.2]octane, 5-6 membered heterocycloalkyl and 8 membered heterocycloalkyl, said cyclopropyl, bicyclo[ 2.2.2] Octane, 5-6 membered heterocycloalkyl and 8 membered heterocycloalkyl are each independently optionally substituted with 1, 2, 3 or 4 Rb, other variables are as defined herein.
  • R 4 is selected from cyclopropyl, bicyclo [2.2.2] octane, and 5-6 membered heterocycloalkyl, cyclopropyl, bicyclo [2.2.2] octane and
  • the 5-6 membered heterocycloalkyl groups are each independently optionally substituted with 1, 2, 3 or 4 R b , other variables are as defined herein.
  • R 4 is selected from the above-described said Each independently is optionally substituted with 1, 2, 3 or 4 R b and other variables are as defined herein.
  • R 4 is selected from the above-described said Each independently is optionally substituted with 1, 2, 3 or 4 R b and other variables are as defined herein.
  • R 4 is selected from the above-described said Each independently is optionally substituted with 1, 2, 3 or 4 R b and other variables are as defined herein.
  • the above R b are independently selected from H, F, Cl, Br, I, OH, CN, NH 2 , CH 3 , -CH 2 F, -CHF 2 , -CH 2 CN, -CH 2 OH, -CH 2 OCH 3 , -CH 2 CH 3 , -CH 2 NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -OCH 3 , -COOH, -COOCH 3 , Other variables are as defined in the present invention.
  • R b are each independently selected from H, F, Cl, Br, I, OH, CN, NH 2 , CH 3 , -CH 2 CH 3 , -CH 2 NH 2 , -NHCH 3, -N (CH 3) 2 , -OCH 3, -COOH, -COOCH 3, cyclopropyl, and -CH 2 F, additional variables are defined as the present invention.
  • R 4 is selected from the above-described Other variables are as defined in the present invention.
  • R 4 is selected from the above-described Other variables are as defined in the present invention.
  • R 4 is selected from the above-described Other variables are as defined in the present invention.
  • the above-mentioned ring A is selected from phenyl and thienyl, and other variables are as defined in the present invention.
  • the above-mentioned compounds are selected from
  • Ring A, R 1 , R 2 , R 3 , R 4 , R 5 and R b are as defined in the present invention.
  • the above-mentioned compounds are selected from
  • R 1 , R 2 , R 3 , R b and R 5 are as defined in the present invention.
  • the above-mentioned compounds are selected from
  • R 1 , R 2 , R 3 and R 5 are as defined in the present invention.
  • the above-mentioned compounds are selected from
  • R b is as defined in the present invention.
  • the present invention also provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from
  • the above-mentioned compounds are selected from
  • the present invention also provides the use of the above compound or a pharmaceutically acceptable salt thereof in the preparation of a medicine for inhibiting SOS1 protein.
  • the present invention also provides the use of the above compounds or their pharmaceutically acceptable salts in the preparation of medicaments for the treatment of lung cancer, pancreatic cancer and/or rectal cancer.
  • the compounds of the present invention have good KRAS(G12C)-SOS1 binding inhibitory activity.
  • 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.
  • 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).
  • D in the present invention refers to deuterium ( 2 H).
  • 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.
  • 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 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.
  • 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 positioned and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly 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 -OCH3 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;
  • the number of atoms in a ring is generally defined as the number of ring members, eg, "5-7 membered ring” refers to a “ring” of 5-7 atoms arranged around it.
  • 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.
  • C 1-3 alkoxy refers to those alkyl groups containing from 1 to 3 carbon atoms attached to the remainder of the molecule through an oxygen atom.
  • the C 1-3 alkoxy group includes C 1-2 , C 2-3 , C 3 and C 2 alkoxy and the like.
  • Examples of C 1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • the term “5-membered heteroaryl” and “5-membered heteroaromatic ring” can be used interchangeably in the present invention, and the term “5-membered heteroaryl” refers to a 5-membered ring atom having a conjugated ⁇ -electron system The monocyclic group of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) p , p is 1 or 2).
  • a 5-membered heteroaryl group can be attached to the remainder of the molecule through a heteroatom or a carbon atom.
  • the 5-membered heteroaryl include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl, and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.) etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazole) base, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2, 4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-
  • C 3-8 cycloalkyl means a saturated cyclic hydrocarbon group consisting of 3 to 8 carbon atoms, which includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, paracyclic and bridge ring.
  • the C 3-8 cycloalkyl includes C 3-6 , C 3-5 , C 4-8 , C 4-6 , C 4-5 , C 5-8 or C 5-6 cycloalkyl and the like; It can be one price, two price or multiple price.
  • C3-8 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2]bicyclooctane, and the like.
  • a heteroatom may occupy the position of attachment of the heterocycloalkyl to the rest of the molecule.
  • the 3-8 membered heterocycloalkyl includes 3-6 membered, 3-5 membered, 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl and the like.
  • 3-8 membered heterocycloalkyl examples include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2- piperidinyl and 3-piperidyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl,
  • a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule.
  • the 5-6 membered heterocycloalkyl includes 5- and 6-membered heterocycloalkyl.
  • 5-6 membered heterocycloalkyl examples include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.) , tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1 -piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazole Alkyl, 1,2-oxazinyl, 1,2-thiazinyl, hex
  • C n-n + m or C n -C n + m to n + m comprises n number of any one particular case of carbon, such as C 1-12 include C 1, C 2, C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , also including any one 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
  • 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 enumerated below, embodiments formed in 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. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuK ⁇ radiation, and the scanning mode is: After collecting the relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.
  • SXRD single crystal X-ray diffraction method
  • the cultured single crystal is collected by Bruker D8 venture diffractometer
  • the light source is CuK ⁇ radiation
  • the scanning mode is: After collecting the 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.
  • DMSO dimethyl sulfoxide
  • DMF stands for N,N-dimethylformamide
  • DCM stands for dichloromethane
  • dioxane or 1,4-dioxane stands for 1,4-dioxane ring
  • Ts represents 4-methylbenzyl
  • Boc represents tert-butoxycarbonyl, which is a protective group for amino
  • Cbz represents benzyloxycarbonyl, which is a protective group for amino
  • CO 2 represents carbon dioxide
  • One pressure unit; LC-MS stands for liquid chromatography; HPLC stands for liquid chromatography; mg stands for milligram; ⁇ g stands for microgram; ng stands for nanogram; ⁇ L stands for microliter; mL stands for milliliter; mm stands for millimeter; ⁇ m stands for micrometer; mM stands for micromol/liter, ⁇ M stands for micromol/liter; nM stands for sodium mol/liter; h stands for hours; min stands for
  • Figure 1 is a graph of tumor growth curves.
  • Figure 2 is a graph showing the change in body weight of the tested animals.
  • the present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention.
  • 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 enumerated below, embodiments formed in 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. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.
  • Step A Compound A (2.3 g, 11.37 mmol) was dissolved in methanol (10 mL), then tetrahydropyran-4-amine (AA-1) (1 g, 9.89 mmol) was added and the reaction mixture was Stir at 25 degrees Celsius for 12 hours, then add sodium methoxide (1.86 g, 11.37 mmol) and continue stirring at 25 degrees Celsius for 2 hours. The reaction was quenched with concentrated hydrochloric acid (12 mol/L, 2 mL), filtered, and the filter cake was washed with water (10 mL). The filter cake was collected and dried under reduced pressure to obtain intermediate AA-2.
  • AA-1 tetrahydropyran-4-amine
  • Step B Intermediate AA-2 (800 mg, 3.16 mmol) was dissolved in acetonitrile (10 mL) followed by triethylamine (479.47 mg, 4.74 mmol) followed by p-toluenesulfonyl chloride (632.35 mg, 3.32 Millimoles). The reaction mixture was stirred at 25 degrees Celsius for 2 hours. Filter and wash the filter cake with water (10 mL). After the filter cake was collected, vacuum-dried to obtain intermediate AA-3, which was directly used in the next reaction.
  • Step C Intermediate AA-3 (250 mg, 613.59 ⁇ mol), acetamide (26.24 mg, 613.59 ⁇ mol) and potassium phosphate (143.27 mg, 674.95 ⁇ mol) were added to 1,4-dioxane ( 2 mL), under nitrogen protection, was added 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (35.50 mg, 61.36 ⁇ mol) and bis-bis[(1,2 , 3)-1-phenyl-2-propene]dipalladium(II) (16.08 mg, 30.68 ⁇ mol). After three nitrogen purges, the reaction mixture was stirred at 110 degrees Celsius for 2 hours.
  • Step D Crude intermediate AA-4 (60 mg) was added to methanolic ammonia (7 mol/L, 1 mL), then sealed and stirred at room temperature for 72 hours. After concentration under reduced pressure, the residue was dissolved in methanol (3 mL), and then an aqueous sodium hydroxide solution (1 mol/L, 2 mL) was added. The reaction mixture was stirred at 50 degrees Celsius for 30 minutes. After natural cooling, the mixture was diluted with water (10 mL), and then extracted with dichloromethane (40 mL/time, extracted 5 times). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dried under reduced pressure to obtain intermediate AA.
  • intermediate AD Referring to the preparation process of intermediate AA, replace tetrahydropyran-4-amine (AA-1) in step A with 4-aminobicyclo[2.2.2]octan-1-ol (AD -1).
  • 1 H NMR 400 MHz, CD 3 OD
  • ⁇ 8.64 (s, 1H), 6.28 (s, 1H), 2.50-2.46 (m, 6H), 2.33 (s, 3H), 1.94-1.78 (m, 6H) .
  • Step A AF-1 (22.3 g, 220.57 mmol) and phthalic anhydride (32.67 g, 220.57 mmol) were added to glacial acetic acid (200 mL), then the reaction mixture was stirred at 110 °C for 3 Hour. After concentration under reduced pressure to remove acetic acid, water (200 mL) was added, and the mixture was stirred at 15°C for 1 hour. After filtration, the filter cake was collected and dried under reduced pressure to obtain AF-2.
  • Step B AF-2 (10 g, 43.25 mmol) and N,N-dimethylformamide (158.07 mg, 2.16 mmol) were added to toluene (30 mL) at room temperature followed by the slow addition of dichloro Sulfoxide (5.40 g, 45.41 mmol). After the addition was complete, the reaction mixture was stirred at 110 degrees Celsius for 3 hours. The solvent was removed by concentration under reduced pressure to obtain AF-3, which was directly used in the next reaction.
  • Step C AF-3 (20.5 g, 82.12 mmol) and 2,6-lutidine (10.56 g, 98.54 mmol) were added to tetrahydrofuran (200 mL) followed by dry palladium on carbon (10%, 1.02 grams). The reaction mixture was stirred under hydrogen 45 psi pressure at 30 degrees Celsius for 20 hours. Additional palladium on carbon (10%, 1.02 g) was then added and stirring was continued for 24 hours at 30 degrees Celsius under hydrogen 45 psi pressure. After filtration, the filtrate was concentrated under reduced pressure.
  • Step D AF-4 (7.54 g, 35.04 mmol) was dissolved in dichloromethane (45 mL) followed by the slow dropwise addition of bis(2-methoxyethyl)aminosulfur trifluoride (18.60 g, 84.09 g mmol) in dichloromethane (20 mL). After the addition was complete, the reaction mixture was stirred at 20 degrees Celsius for 48 hours. The reaction was quenched with saturated aqueous sodium carbonate (100 mL) at 10 degrees Celsius and stirred for 1 hour. Water (100 mL) was then added, followed by extraction with dichloromethane (100 mL/time, 3 extractions). The combined organic phases were dried over anhydrous sodium sulfate.
  • Step E AF-5 (7.1 g, 29.93 mmol) was added to 2-(2-aminoethylamino)ethanol (21.30 mL). The reaction mixture was stirred at 80 degrees Celsius for 2 hours. Methanol (150 ml) was added, and the mixture was distilled at 90°C under atmospheric pressure, and the fraction was cooled in a dry ice bath. To the fractions was added hydrogen chloride in methanol (4 mol/L, 15 mL). After concentration under reduced pressure, AF-6 was obtained.
  • 1 H NMR (400 MHz, CD 3 OD) ⁇ 6.04-5.71 (m, 1H), 1.24 (s, 4H).
  • Step G AF-7 (5.17 g, 19.95 mmol) and 4-methylbenzenesulfonyl chloride (4.58 g, 24.02 mmol) were added to acetonitrile (50 mL) followed by triethylamine (3.03 g, 29.92 mmol). The reaction mixture was stirred at 20 degrees Celsius for 2 hours. After dilution with water (50 mL), it was filtered. The filter cake was washed with water (30 mL/time, washed 3 times) and dried in vacuo to obtain AF-8.
  • Step H Combine AF-8 (7.41 g, 17.92 mmol), acetamide (1.06 g, 17.92 mmol), potassium phosphate (4.19 g, 19.72 mmol), bis-bis[(1,2,3)- 1-Phenyl-2-propene]dipalladium(II) (187.90 mg, 358.50 ⁇ mol) and Xantphos (414.87 mg, 717.00 ⁇ mol) were added in one portion to 1,4-dioxane (35 mL). The reaction mixture was under nitrogen and stirred at 115 degrees Celsius for 12 hours.
  • Step I AF-9 (4.22 g, 14.05 mmol) was added to ammonia in methanol (7 mol/L, 34 mL). The reaction mixture was stirred at 15 degrees Celsius for 3 days. After concentration under reduced pressure, an aqueous sodium hydroxide solution (1 mol/L, 30 mL) was added to the residue, followed by stirring at 50 degrees Celsius for 30 minutes. After natural cooling, the solution was washed with tert-butyl methyl ether (70 mL/time, washed 3 times). The aqueous phase was acidified with concentrated hydrochloric acid (12 mol/L), adjusted to pH about 4, filtered, and the filter cake was collected and dried under reduced pressure to obtain intermediate AF.
  • Step A Under nitrogen protection, AG-1 (3.00 g, 14.00 mmol) and Intermediate A (3.40 g, 16.80 mmol) were dissolved in methanol (30 mL), and the reaction mixture was stirred at 20°C for 12 hours . Subsequently, a methanol solution of sodium methoxide (3.63 g, 16.80 mmol, mass fraction: 25%) was added, and the reaction was continued to be stirred at 20 degrees Celsius for 12 hours. Methanol was removed by concentration under reduced pressure, the residue was diluted with water (100 mL), and washed with methyl tert-butyl ether (100 mL/time, washed twice).
  • Step B AG-2 (3.15 g, 8.60 mmol) and p-toluenesulfonyl chloride (1.97 g, 10.32 mmol) were added to acetonitrile (32 mL) followed by triethylamine (1.30 g, 12.90 mmol) .
  • the reaction mixture was stirred at 20 degrees Celsius for 2 hours. After concentration under reduced pressure, the residue was diluted with water (50 mL) and stirring was continued at 20°C for 2 hours. After filtration, the filter cake was dried under reduced pressure to obtain AG-3.
  • Step C Combine AG-3 (1.3 g, 2.50 mmol), acetamide (147.50 mg, 2.50 mmol), potassium phosphate (583.07 mg, 2.75 mmol) and 4,5-bis(diphenylphosphonium)- 9,9-Dimethylxanthene (288.98 mg, 499.43 ⁇ mol) was added to 1,4-dioxane (10 mL). After 3 nitrogen purges, additional bis(phenylpropenylpalladium chloride) (130.88 mg, 249.71 ⁇ mol) was added. The reaction mixture was stirred at 110 degrees Celsius for 4 hours.
  • Step D AG-4 (0.67 g, 1.64 mmol) was added to ammonia methanol solution (7 mol/L, 50 mL) and stirred at 20°C for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol/L, 20 mL), and then washed with methyl tert-butyl ether (30 mL/time, washed 3 times). The aqueous phase was adjusted to pH 4 by adding concentrated hydrochloric acid, and then filtered under reduced pressure. The filter cake was collected and dried under reduced pressure to obtain intermediate AG.
  • Step A AH-1 (500 mg, 2.5 mmol) and Intermediate A (605.64 mg, 3.0 mmol) were added to methanol (5 mL) followed by sodium methoxide in methanol (539.49 mg, 3.0 mmol) , quality score: 30%).
  • the reaction mixture was stirred at 10-20 degrees Celsius for 12 hours, then adjusted to pH 4-5 with hydrochloric acid (2 mol/L), and extracted with dichloromethane (50 mL/time, twice). The organic phases were combined, washed once with brine (20 mL), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AH-2.
  • Step B AH-2 (880 mg, 2.5 mmol) was added to acetonitrile (10 mL), followed by triethylamine (379.05 mg, 12.90 mmol) and p-toluenesulfonyl chloride at 0-10 degrees Celsius (499.91 mg, 2.62 mmol).
  • the reaction mixture was stirred at 20-30 degrees Celsius for 1 hour, then dichloromethane (50 mL) was added for extraction once.
  • the organic phase was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure.
  • Step C AH-3 (900 mg, 1.78 mmol) was added to 1,4-dioxane (10 mL) followed by acetamide (110.19 mg, 1.87 mmol) under nitrogen protection, Potassium phosphate (414.84 mg, 1.95 mmol), bis(phenylpropenylpalladium chloride) (93.12 mg, 177.67 ⁇ mol) and 4,5-bis(diphenylphosphonium)-9,9-dimethyloxa Anthracene (205.60 mg, 355.33 ⁇ mol). The reaction mixture was stirred at 110°C for 2 hours, and after natural cooling, ethyl acetate (50 mL) was added to dilute.
  • Step D AH-4 (510 mg, 1.30 mmol) was added to ammonia methanol solution (7 mol/L, 50 mL), and stirred at 20-30 degrees Celsius for 12 hours. After concentration under reduced pressure, the residue was subjected to preparative HPLC (chromatographic column: Welch Ultimate XB-CN, 250 mm ⁇ 50 mm ⁇ 10 ⁇ m; mobile phase: A phase: n-hexane; B phase: 0.1% ammonia water ethanol solution, 5% ⁇ 45% ; 15 min) was purified to give intermediate AH.
  • preparative HPLC chromatographic column: Welch Ultimate XB-CN, 250 mm ⁇ 50 mm ⁇ 10 ⁇ m; mobile phase: A phase: n-hexane; B phase: 0.1% ammonia water ethanol solution, 5% ⁇ 45% ; 15 min
  • Step A AI-1 (2 g, 9.38 mmol), acetic acid (563.29 mg, 9.38 mmol) and ammonium formate (22.36 g, 354.56 mmol) were added to methanol (500 mL) followed by cyanoboron Sodium hydride (1.18 g, 18.76 mmol). The reaction mixture was stirred at 20-30 degrees Celsius for 72 hours. After concentration under reduced pressure, ethyl acetate (200 mL) was added to dissolve the residue. The solution was washed with saturated brine (50 mL/time, washed twice), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AI-2.
  • Step B AI-2 (1.4 g, 6.53 mmol) and Intermediate A (1.58 g, 7.84 mmol) were added to methanol (20 mL) followed by sodium methoxide in methanol (1.69 g, 7.84 mmol) , mass fraction: 25%), the reaction mixture was stirred at 10 to 20 degrees Celsius for 12 hours. The pH value was adjusted to 4-5 with hydrochloric acid (2 mol/L), and then extracted with dichloromethane (50 mL/time, extracted twice). The organic phases were combined, washed once with saturated brine (20 mL), and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AI-3.
  • Step C To a solution of AI-3 (1.55 g, 4.23 mmol) in acetonitrile (20 mL) was added triethylamine (642.09 mg, 6.35 mmol) and p-toluenesulfonyl chloride (887.15 mg) at 0-10 degrees Celsius , 4.65 mmol). The reaction mixture was stirred at 20-30 degrees Celsius for 1 hour, then extracted with dichloromethane (50 mL). The organic phase was washed once with saturated brine (20 mL), and dried over anhydrous sodium sulfate.
  • Step D Under nitrogen, combine AI-4 (1.77 g, 3.4 mmol), acetamide (210.87 mg, 3.57 mmol), potassium phosphate (793.87 mg, 3.74 mmol), bis(phenylpropenyl chloride) palladium) (178.20 mg, 340.00 ⁇ mol) and 4,5-bis(diphenylphosphonium)-9,9-dimethylxanthene (393.46 mg, 679.99 ⁇ mol) were added to 1,4-dioxanthene in turn six rings (20 ml). The reaction mixture was stirred at 110°C for 2 hours, and after natural cooling, ethyl acetate (50 mL) was added for dilution.
  • Step E AI-5 (710 mg, 1.74 mmol) was added to ammonia methanol solution (7 mol/L, 20 mL), and the reaction was stirred at 20-30 degrees Celsius for 12 hours. After concentration under reduced pressure, the residue was subjected to preparative HPLC (chromatographic column: Welch Ultimate XB-SiOH, 250mm ⁇ 50mm ⁇ 10 ⁇ m; mobile phase: A phase: n-hexane; B phase: 0.1% ammonia water ethanol solution, 1% ⁇ 30% ; 15 min) purification to give intermediate AI.
  • preparative HPLC chromatographic column: Welch Ultimate XB-SiOH, 250mm ⁇ 50mm ⁇ 10 ⁇ m; mobile phase: A phase: n-hexane; B phase: 0.1% ammonia water ethanol solution, 1% ⁇ 30% ; 15 min
  • Step A AJ-1 (0.5 g, 2.5 mmol) and Intermediate A (606.15 mg, 3 mmol) were dissolved in methanol (6 mL), then sodium methoxide (620.41 mg, 2.87 mmol) was added and the reaction The mixture was stirred at 25 degrees Celsius for 12 hours. After concentration under reduced pressure to remove methanol, water (30 mL) was added to the residue, followed by washing with methyl tert-butyl ether (40 mL). The aqueous phase was adjusted to pH 5-6 with 1 molar hydrochloric acid. After filtration, the filter cake was concentrated under reduced pressure to obtain AJ-2.
  • Step B AJ-2 (470 mg, 1.33 mmol) was dissolved in acetonitrile (6 mL) followed by the addition of p-toluenesulfonyl chloride (254.28 mg, 1.33 mmol) and triethylamine (202.45 mg, 2.0 mmol) .
  • the reaction mixture was stirred at 25 degrees Celsius for 5 hours.
  • Dichloromethane (60 mL) was added for extraction, and the organic phase was washed with water (20 mL/time, washed twice), then washed with saturated brine (20 mL/time, washed twice), dried over anhydrous sodium sulfate, and filtered. After that, the filtrate was concentrated under reduced pressure.
  • Step C Combine AJ-3 (0.5 g, 987.02 ⁇ mol), acetamide (69.96 mg, 1.18 mmol), potassium phosphate (230.47 mg, 1.09 mmol) and 4,5-bis(diphenylphosphonium)- 9,9-Dimethylxanthene (114.22 mg, 197.41 ⁇ mol) was successively added to 1,4-dioxane (2 mL), and after nitrogen replacement 3 times, bis(phenylpropenyl chloride) was added.
  • palladium 51.73 mg, 98.70 micromoles. The reaction mixture was stirred at 110 degrees Celsius for 2 hours.
  • Step D AJ-4 (250 mg, 635.43 ⁇ mol) was dissolved in ammonia methanol solution (7 mol/L, 1.82 mL) and stirred at 20° C. for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol/L, 30 mL), and then washed with methyl tert-butyl ether (30 mL/time, washed three times). Dilute hydrochloric acid (2 mol/L) was added to the aqueous phase to adjust the pH to about 4, and then extracted with dichloromethane (30 mL/time, twice).
  • Step A To a saturated aqueous solution of sodium bicarbonate (100 mL) of AK-1 (2.50 g, 16.28 mmol), a solution of triphosgene (4.02 g, 13.56 mmol) in toluene (50 mL) was added. , the reaction mixture was stirred at 25 °C for 12 h. It was concentrated under reduced pressure, methanol (100 mL) and ethyl acetate (100 mL) were added to the residue, followed by stirring well. After filtration, the filtrate was concentrated under reduced pressure to obtain AK-2.
  • Step B AK-2 (2.30 g, 16.07 mmol) was dissolved in dichloromethane (60 mL) followed by triethylamine (4.88 g, 48.20 mmol), p-toluenesulfonyl chloride (3.37 g, 17.67 mmol) mol) and 4-dimethylaminopyridine (98.15 mg, 0.80 mmol).
  • the reaction mixture was stirred at 25°C for 12 hours, then diluted with water (40 mL) and extracted with dichloromethane (50 mL/time, 2 extractions). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AK-3.
  • Step C AK-3 (3.50 g, 11.77 mmol) was dissolved in N,N-dimethylformamide (60 mL) and sodium azide (2.12 g, 32.61 mmol) was added. The reaction mixture was stirred at 80°C for 1 hour, diluted with saturated sodium carbonate (100 mL), and extracted with ethyl acetate (50 mL/time, extracted twice). The organic phases were combined and the solution was used directly in the next step.
  • Step D To a mixture of AK-4 in ethyl acetate (100 mL) was added methanol (30 mL). After sufficient nitrogen replacement, palladium on carbon (0.20 g, 10% content) was added. The reaction mixture was stirred at 25°C under a hydrogen balloon for 12 hours and then filtered. After the filtrate was concentrated under reduced pressure, ethyl acetate (100 mL) was added to the residue, followed by stirring at 30 degrees Celsius for 30 minutes. It was filtered again, and the filtrate was concentrated under reduced pressure to obtain AK-5.
  • Step E Dissolve AK-5 (1.6 g, 11.26 mmol) and Intermediate A (2.5 g, 12.38 mmol) in methanol (20 mL), stir for 3 hours, then add sodium methoxide (30% purity, 2.23 g, 12.38 mmol). After the addition was complete, the reaction mixture was stirred at 30 degrees Celsius for 12 hours. Water (100 mL) was added to dilute the reaction solution, and then washed with ethyl acetate (50 mL/time, washed twice). The aqueous phase was adjusted to pH about 4 with hydrochloric acid (1 mol/L) and extracted with ethyl acetate (100 mL/time, 3 times). The combined organic phases were washed once with saturated brine (200 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AK-6.
  • Step F Dissolve AK-6 (2.4 g, 8.16 mmol) in acetonitrile (60 mL), then add p-toluenesulfonyl chloride (1.71 g, 8.97 mmol) and triethylamine (1.24 g, 12.23 mmol, 1.7 ml). The reaction mixture was stirred at 30 degrees Celsius for 14 hours. After concentration under reduced pressure, methyl tert-butyl ether (40 mL) and water (40 mL) were added to the residue, followed by stirring for 30 minutes. After filtration, the filter cake was collected and dried under reduced pressure to obtain AK-7.
  • Step G Under nitrogen, combine AK-7 (250 mg, 557.48 ⁇ mol), acetamide (32.93 mg, 557.48 ⁇ mol), potassium phosphate (130.17 mg, 613.23 ⁇ mol), 4,5-bis(diphenyl) phosphonium)-9,9-dimethylxanthene (9.68 mg, 16.72 ⁇ mol) and bis(phenylpropenylpalladium chloride) (4.38 mg, 8.36 ⁇ mol) were added sequentially to 1,4-dioxane ring (2 mL). The reaction mixture was stirred at 110 degrees Celsius for 24 hours.
  • Step H AK-8 (110 mg, 635.43 ⁇ mol) was dissolved in ammonia methanol solution (7 mol/L, 20 mL) and stirred at 20°C for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol/L, 30 mL), and then washed with methyl tert-butyl ether (30 mL/time, washed three times). Dilute hydrochloric acid (2 mol/L) was added to the aqueous phase to adjust the pH to about 4, and then extracted with dichloromethane (30 mL/time, twice).
  • Step B AL-2 (3.36 g, 10.41 mmol) and difluoromethylsulfonylbenzene (2 g, 10.41 mmol) were added to tetrahydrofuran (30 mL), cooled to -78°C under nitrogen protection, Lithium bis(trimethylsilyl)amide (1 mol/L, 20.81 mL) was then added dropwise. After the dropwise addition was complete, the reaction mixture was stirred at minus 78 degrees Celsius for 1 hour. The reaction was quenched by adding water (50 mL) and extracted with ethyl acetate (50 mL/time, extracted twice).
  • Step C AL-3 (500 mg, 971.62 ⁇ mol), sodium acetate (6.38 g, 77.73 mmol) and acetic acid (10 mL) were added to N,N-dimethylformamide (10 mL), followed by Magnesium powder (472.30 mg, 19.43 mmol) was added portionwise. After the addition was complete, the reaction mixture was stirred at 25°C for 4 hours. After filtration, the filtrate was adjusted to a pH of about 7 with saturated aqueous sodium bicarbonate solution, and then extracted with ethyl acetate (30 mL/time, extracted twice). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude AL-4, which was directly used in the next reaction.
  • Step D Crude AL-4 (188 mg) was dissolved in tetrahydrofuran (2 mL), followed by addition of hydrochloric acid in dioxane (4 mol/L, 250 ⁇ L). The reaction mixture was stirred at 25 degrees Celsius for 1 hour. After concentration under pressure, the residue was washed once with methyl tert-butyl ether (2 mL) and once with petroleum ether (2 mL). After drying under reduced pressure, AL-5 was obtained.
  • Step E AL-5 (100 mg, 326 ⁇ mol), Intermediate A (75.79 mg, 374.92 ⁇ mol) and sodium methoxide (25% purity, 70.45 mg, 326 ⁇ mol) were added to methanol (3 mL) , the reaction mixture was stirred at 25 °C for 12 h. Water (20 mL) was added to dilute the reaction solution, and then washed with ethyl acetate (20 mL/time, washed twice). The aqueous phase was adjusted to pH about 4 with 1 molar hydrochloric acid and then extracted with ethyl acetate (20 mL/time, 3 extractions). The combined organic phases were washed once with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AL-6.
  • Step F Dissolve AL-6 (250 mg, 591.88 ⁇ mol) in acetonitrile (5 mL), then add p-toluenesulfonyl chloride (124.13 mg, 651.07 ⁇ mol) and triethylamine (119.40 mg, 1.18 mmol) .
  • the reaction mixture was stirred at 30 degrees Celsius for 14 hours.
  • Step G Under nitrogen, combine AL-7 (300 mg, 520.32 ⁇ mol), acetamide (30.74 mg, 520.32 ⁇ mol), potassium phosphate (132.54 mg, 624.38 ⁇ mol), 4,5-bis(diphenyl) phosphonium)-9,9-dimethylxanthene (15.06 mg, 26.02 ⁇ mol) and bis(phenylpropenylpalladium chloride) (13.64 mg, 26.02 ⁇ mol) were added sequentially to 1,4-dioxane ring (3 mL). The reaction mixture was stirred at 110 degrees Celsius for 24 hours.
  • Step H AL-8 (160 mg, 345.25 ⁇ mol) was dissolved in methanolic ammonia (7 mol/L, 10 mL) and stirred at 20°C for 18 hours. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol/L, 20 mL), and then washed with methyl tert-butyl ether (30 mL/time, washed three times). Dilute hydrochloric acid (2 mol/L) was added to the aqueous phase to adjust the pH to about 4, and then extracted with dichloromethane (30 mL/time, twice).
  • Step I AL-9 (120 mg, 278.81 micromoles) was dissolved in dichloromethane (5 mL) followed by the addition of hydrobromic acid in acetic acid (250 microliters). The reaction mixture was stirred at 25 degrees Celsius for 5 hours and quenched by the addition of methanol (1 mL). After concentration under reduced pressure, the residue was washed with petroleum ether (5 mL/time, washed twice). After drying under reduced pressure, AL-10 was obtained.
  • Step J Acetyl chloride (15.90 mg, 202.52 ⁇ mol) was dissolved in tetrahydrofuran (0.5 mL) until use.
  • AL-10 60 mg, 202.52 ⁇ mol
  • triethylamine 40.91 mg, 405.04 ⁇ mol
  • the reaction mixture was stirred at 0 degrees Celsius for 2 hours.
  • Step A To a solution of B-1 (3.00 g, 13.69 mmol) in tetrahydrofuran (120 mL) was added cesium carbonate (4.46 g, 13.69 mmol) and ( ⁇ )-tert-butylsulfinamide (1.66 g, 13.69 Millimoles). The reaction mixture was stirred at 20°C for 0.5 hours, then water (100 mL) was added to quench the reaction and extracted twice with 100 mL of ethyl acetate (50 mL each). After liquid separation, the organic phase was dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the crude product B-2, which was directly used in the next reaction.
  • Step C To a solution of B-3 (3.70 g, 10.56 mmol) in tetrahydrofuran (30 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 7.92 mL). The mixture was stirred at 20°C for 3 hours, then petroleum ether (60 mL) was added, filtered through a Buchner funnel, and the collected filter cake was dried in vacuo to obtain a crude product of B-4, which was directly used in the next reaction.
  • Step D Dissolve B-4 (2.50 g, 10.15 mmol) in ethanol (50 mL), add reduced iron powder (2.84 g, 50.77 mmol), ammonium chloride (2.72 g, 50.77 mmol) and water (10 mL). The reaction mixture was stirred at 60 degrees Celsius for 3 hours. After naturally cooling to room temperature, sodium carbonate aqueous solution (100 mL) was added to quench, and then extracted twice with 100 mL ethyl acetate (50 mL/time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate B, which was directly used in the next reaction.
  • Step A To a solution of C-1 (10.00 g, 45.46 mmol) in tetrahydrofuran (100 mL) was added cesium carbonate (14.87 g, 45.64 mmol) and (R)-tert-butylsulfinamide (5.53 g, 45.64 g Millimoles). The reaction mixture was stirred at 20°C for 0.5 hours, then water (100 mL) was added to quench the reaction and extracted twice with 200 mL of ethyl acetate (100 mL each). The combined organic phases were washed once with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude C-2, which was directly used in the next reaction.
  • Step B Vinylmagnesium bromide (1 mol/L, 55.48 mL, 55.48 mmol) was added dropwise to a solution of C-2 (14.90 g, 46.23 mmol) in tetrahydrofuran (300 mL) at -78°C. After the addition, the reaction mixture was stirred at -78°C for 1 hour, then naturally heated to 0°C, quenched with saturated aqueous ammonium chloride solution (100 mL), and extracted twice with 200 mL of ethyl acetate (100 mL/time) .
  • Step C To a solution of C-3 (6.20 g, 17.70 mmol) in tetrahydrofuran (100 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 8.85 mL). The mixture was stirred at 20°C for 1 hour, then petroleum ether (20 mL) was added, filtered through a Buchner funnel, and the collected filter cake was vacuum-dried to obtain crude C-4, which was directly used in the next reaction.
  • Step D Dissolve C-4 (4.3 g, 15.21 mmol) in ethanol (100 mL), add reduced iron powder (4.25 g, 76.07 mmol), ammonium chloride (4.07 g, 76.07 mmol) and water (10 mL). The reaction mixture was stirred at 60 degrees Celsius for 3 hours. After naturally cooling to room temperature, sodium carbonate aqueous solution (100 mL) was added to quench, and the mixture was extracted three times with 300 mL ethyl acetate (100 mL/time). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate C, which was directly used in the next reaction.
  • Step A Compound D-1 (25 g, 130.13 mmol) was added to concentrated sulfuric acid (40 mL) followed by dropwise addition of fuming nitric acid (19.87 g, 315.33 mmol) at 0 degrees Celsius. After the addition, the reaction mixture was naturally warmed to 20°C, stirred for 2 hours, then slowly poured into ice water (200 mL), and extracted with dichloromethane (100 mL/time, extracted 3 times). The extracted organic phases were combined, washed once with 1 mol/L aqueous sodium hydroxide solution (100 mL), and then the aqueous phase was extracted with dichloromethane (50 mL/time, twice extracted).
  • Step B D-2 (26 g, 109.65 mmol) was added to tetrahydrofuran (200 mL) followed by (R)-tert-butylsulfinamide (13.29 g, 109.65 mmol) and cesium carbonate (35.73 g) , 109.65 mmol).
  • the reaction mixture was stirred at 2 degrees Celsius for 30 minutes. After adding water (300 mL) to dilute, it was extracted with ethyl acetate (300 mL/time, extracted twice). The organic phases were combined, washed with saturated brine (300 ml/time, washed once), and then dried with anhydrous sodium sulfate.
  • Step C Under nitrogen protection, vinylmagnesium bromide solution (1 mol/L, 88.16 mL) was slowly added to dimethylzinc solution (1 mol/L, 101.38 mL) at 20°C, stirring after the addition 3 hours. Subsequently, the temperature was lowered to -78 degrees Celsius, and a solution of D-3 (15 g, 44.08 mmol) in tetrahydrofuran (100 mL) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at minus 78 degrees Celsius for 30 minutes. The reaction was quenched with saturated aqueous ammonium chloride solution (200 mL), and extracted with ethyl acetate (300 mL/time, extracted twice).
  • Step D To a solution of D-4 (2.5 g, 6.79 mmol) in tetrahydrofuran (20 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 3.4 mL). The reaction mixture was stirred at 20 degrees Celsius for 1 hour. After adding petroleum ether (80 mL) to dilute, the filter cake was collected after filtration to obtain D-5, which was directly used in the next reaction.
  • Step E D-5 (2 g, 7.57 mmol) was added to a mixed solvent of ethanol (50 mL) and water (10 mL), followed by reduced iron powder (2.11 g, 37.85 mmol) and ammonium chloride (2.02 g, 37.85 mmol). After the reaction system was replaced with nitrogen three times, it was stirred at 60 degrees Celsius for 3 hours. After cooling, the reaction mixture was filtered. The filtrate was neutralized with saturated aqueous sodium carbonate solution (30 mL), and extracted with ethyl acetate (30 mL/time, extracted twice). The combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain Intermediate D.
  • Step A E-1 (6.42 g, 30.57 mmol) was added to dimethyl sulfoxide (50 mL) followed by potassium fluoride (3.55 g, 61.14 mmol) and cuprous iodide (11.64 g, 61.14 mmol) and finally ethyl (trimethylsilyl)difluoroacetate (12 g, 61.14 mmol) was added.
  • the reaction mixture was stirred at 60 degrees Celsius for 15 hours under nitrogen protection. After natural cooling, tert-butyl methyl ether (200 mL) and water (300 mL) were added to dilute the reaction solution, and stirring was continued for 10 minutes, followed by filtration.
  • the filter cake was washed with tert-butyl methyl ether (30 mL/time, washed 3 times).
  • the filtrates were combined, and the organic phase was separated, washed with water (300 mL) and saturated brine (300 mL) in turn, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: petroleum ether) to obtain E-2.
  • Step B E-2 (3.8 g, 18.43 mmol) was added to a mixed solvent of tetrahydrofuran (30 mL) and ethanol (5 mL), cooled to 0 °C, and sodium borohydride (1.40 g, 36.86 mmol) was added. ). After the addition was complete, the reaction mixture was stirred at 15-20 degrees Celsius for 2 hours. The reaction was quenched with saturated aqueous ammonium chloride solution (50 mL), then water (50 mL) was added to dilute the reaction solution, and the mixture was extracted with tert-butyl methyl ether (50 mL/time, extracted twice).
  • Step C E-3 (2.37 g, 14.44 mmol) was added to dry dichloromethane (30 mL) followed by imidazole (1.08 g, 15.88 mmol) followed by 4-dimethylaminopyridine (176.36 mg) , 1.44 mmol). With stirring, tert-butyldiphenylchlorosilane (4.17 g, 15.16 mmol) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at 10-15 degrees Celsius for 16 hours. Water (100 mL) was added to dilute the reaction solution. The organic phase was washed once with saturated brine (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure.
  • Step D E-4 (1.5 g, 3.73 mmol) was added to dry tetrahydrofuran (20 mL) under nitrogen. After cooling to minus 65 degrees Celsius, lithium diisopropylamide solution (1 mol/L, 6.71 mL) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at minus 65 degrees Celsius for 1 hour. N,N-Dimethylformamide (1.63 g, 22.36 mmol) was then added and stirring was continued at minus 65 degrees Celsius for 30 minutes. Water (40 mL) was added to dilute the reaction solution, and the mixture was extracted with tert-butyl methyl ether (30 mL/time, extracted twice). The combined organic phases were washed once with saturated brine (50 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain crude E-5, which was directly used in the next reaction.
  • Step E E-5 (1.6 g, 3.72 mmol) was added to tetrahydrofuran (20 mL) followed by (R)-tert-butylsulfinamide (495.41 mg, 4.09 mmol) followed by cesium carbonate (1.33 mmol) g, 4.09 mmol).
  • the reaction mixture was stirred at 10-15 degrees Celsius for 16 hours.
  • the reaction solution was diluted with water (40 mL), and then extracted with tert-butyl methyl ether (30 mL/time, extracted twice). The combined organic phases were washed once with saturated brine (40 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure.
  • Step F E-6 (2.6 g, 4.87 mmol) was added to dry tetrahydrofuran (50 mL) under nitrogen protection. After cooling to minus 60 to minus 65 degrees Celsius, vinylmagnesium bromide solution (1 mol/L, 9.74 mL) was slowly added dropwise. After the addition, the reaction mixture was stirred at minus 65 degrees Celsius for 10 minutes, and then naturally rose to 0 degrees Celsius, and the stirring was continued for 50 minutes. The reaction was slowly quenched with saturated aqueous ammonium chloride (50 mL), then diluted with water (60 mL), and extracted with tert-butyl methyl ether (60 mL/time, extracted twice).
  • Step G E-7 (930 mg, 1.66 mmol) was added to tetrahydrofuran (5 mL) followed by hydrogen chloride in 1,4-dioxane (4 mol/L, 1.24 mL). The reaction mixture was stirred at 10-15 degrees Celsius for 2 hours. Petroleum ether (40 mL) was then added to dilute, and stirring was continued for 30 minutes at 10-15 degrees Celsius. After filtration, the filter cake was dried under reduced pressure to obtain Intermediate E.
  • Step B To a solution of F-2 (5.00 g, 22.22 mmol) in tetrahydrofuran (50 mL) was added dropwise n-butyllithium (2.5 mol/L, 13.33 mL, 33.33 mmol) at -78°C under nitrogen protection ). After the addition was complete, the reaction mixture was stirred at -78°C for 1 hour, then N,N-dimethylformamide (3.25 g, 44.44 mmol) was added and stirring was continued at -78°C for 1 hour. The reaction mixture was poured into dilute hydrochloric acid (1 mol/L, 100 mL), and then extracted with ethyl acetate (30 mL/time, extracted three times). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain crude F-3, which was directly used in the next reaction.
  • n-butyllithium 2.5 mol/L, 13.33 mL, 33.33 m
  • Step C To a solution of F-3 (3.20 g, 18.38 mmol) in tetrahydrofuran (60 mL) was added cesium carbonate (5.99 g, 18.38 mmol) and (R)-tert-butylsulfinamide (2.23 g, 18.38 g Millimoles). The reaction mixture was stirred at 0°C for 1 hour, then diluted with water (200 mL), and extracted with ethyl acetate (100 mL/time, 2 extractions). The combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • Step D Vinylmagnesium bromide solution (1 mol/L, 3.61 mmol) was added dropwise to dimethylzinc toluene solution (1 mol/L, 4.15 mmol) at 20 degrees Celsius under nitrogen protection. After the addition was complete, the reaction mixture was stirred at 20°C for 4 hours, then cooled to -78°C, after which a solution of F-4 (0.50 g, 1.80 mmol) in tetrahydrofuran (10 mL) was slowly added dropwise. After completion, continue stirring at -78 degrees Celsius for 0.5 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride (200 mL), then extracted with ethyl acetate (300 mL/time, twice).
  • Step E To a solution of F-5 (0.40 g, 1.31 mmol) in tetrahydrofuran (5 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 0.98 mL). After the mixture was stirred at 15-20 degrees Celsius for 2 hours, petroleum ether (40 ml) was added, and stirring was continued for 30 minutes. After filtration, collect the filter cake. After drying under reduced pressure, Intermediate F was obtained.
  • Step A Intermediate AF (200 mg, 748.42 ⁇ mol) was added to acetonitrile (10 mL) followed by potassium phosphate (397.16 mg, 1.87 mmol) and hexachlorotripolyphosphazene (260.19 mg, 748.42 ⁇ mol) ). The reaction mixture was stirred at 15 degrees Celsius for 1 hour. Intermediate E (369.80 mg, 448.42 micromoles) was then added and stirring continued for 1 hour. Then, ammonia water (25%, 1 mL) was added and stirred for 1 hour; then saturated aqueous potassium carbonate solution (30 mL) was added and stirred for 16 hours. Extracted with ethyl acetate (30 mL/time, extracted twice).
  • Step B 7-1 (360 mg, 509.28 micromoles) was added to methanol (10 mL) followed by ammonium fluoride (188.63 mg, 5.09 mmol). The reaction mixture was stirred at 70 degrees Celsius for 2 hours. After concentration under reduced pressure, the residue was separated and purified by preparative HPLC (chromatographic column: Waters Xbridge 150 ⁇ 25 mm ⁇ 5 ⁇ m; mobile phase: A phase: ammonia water; B phase: acetonitrile; 22%-52%; 9 minutes) to obtain the compound 7.
  • preparative HPLC chromatographic column: Waters Xbridge 150 ⁇ 25 mm ⁇ 5 ⁇ m; mobile phase: A phase: ammonia water; B phase: acetonitrile; 22%-52%; 9 minutes
  • Step A Intermediate AG (270 mg, 1.14 mmol), hexachlorocyclotriphosphazene (394.99 mg, 1.14 mmol) and potassium phosphate (602.92 mg, 2.84 mmol) were added to acetonitrile (10 mL). The reaction mixture was stirred at 20 degrees Celsius for 1 hour, then Intermediate F (446.67 mg, 1.19 mmol) was added and stirring was continued at 20 degrees Celsius for 2 hours. After filtration, the filtrate was concentrated under reduced pressure.
  • Step B 9-1 (156 mg, 279.77 ⁇ mol) was dissolved in 1,4-dioxane (3 mL) followed by addition of hydrochloric acid in dioxane (4 mol/L, 1 mL). The reaction mixture was stirred at 40°C for 2 hours and concentrated under reduced pressure to give 9-2.
  • Step C Acetic acid (7.29 mg, 121.47 ⁇ mol), N,N-diisopropylethylamine (52.33 mg, 404.90 ⁇ mol) and 2-(7-azobenzotriazole)-N,N , N,N-Tetramethylurea hexafluorophosphate (46.19 mg, 121.47 ⁇ mol) was added to N,N-dimethylformamide (4 mL). After the reaction mixture was stirred at 20 degrees Celsius for 15 minutes, additional 9-2 (40 mg, 80.98 micromoles) was added. The reaction mixture was continued to stir at 20 degrees Celsius for 1 hour.
  • Step A Intermediate AH (200 mg, 554.93 ⁇ mol), potassium phosphate (294.48 mg, 1.39 mmol) and hexachlorocyclotriphosphazene (192.93 mg, 554.93 ⁇ mol) were added to acetonitrile (3 mL), After the reaction mixture was stirred at 20-30 degrees Celsius for 1 hour, Intermediate F (145.07 mg, 610.42 micromoles) was added and stirring was continued for 12 hours. Ammonia water (mass fraction: 25%, 0.5 ml) was added and stirred for 1 hour, and then potassium carbonate aqueous solution (mass fraction: 20%, 20 ml) was added and stirred for 1 hour. Extract once with dichloromethane (50 mL).
  • Step B 11-1 (110 mg, 202.36 ⁇ mol) was added to 1,4-dioxane (3 mL), followed by dioxane hydrochloride solution (4 mol/L, 1 mL). The reaction mixture was stirred at 30 degrees Celsius for 2 hours. After concentration under reduced pressure, 11-2 was obtained. LC-MS (ESI) m/z: 444.2 [M+H] + .
  • Step C Acetic acid (18.77 mg, 312.55 ⁇ mol), 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (118.84 mg, 312.55 ⁇ mol) mol) and N,N-diisopropylethylamine (107.72 mg, 833.46 ⁇ mol) were added to N,N-dimethylformamide (1 mL), and the reaction mixture was stirred at 20-30 °C for 15 min. 11-2 (100 mg, 208.37 ⁇ mol) was added and stirring was continued at 20-30 degrees Celsius for 0.5 hours.
  • Step A Intermediate AI (200 mg, 534.14 ⁇ mol), potassium phosphate (283.46 mg, 1.34 mmol) and hexachlorocyclotriphosphazene (185.70 mg, 554.93 ⁇ mol) were added to acetonitrile (7 mL), The reaction mixture was stirred at 20-30 degrees Celsius for 1 hour. Additional Intermediate F (139.63 mg, 587.55 micromoles) was added and stirring was continued for 12 hours. Subsequently, ammonia water (mass fraction: 25%, 0.5 ml) was added and stirred for 1 hour, and potassium carbonate aqueous solution (mass fraction: 20%, 20 ml) was added and stirred for 1 hour.
  • Step B 12-1 (200 mg, 358.68 micromoles) was added to 1,4-dioxane (3 mL) followed by dioxane hydrochloride solution (4 mol/L, 1 mL). The reaction mixture was stirred at 30 degrees Celsius for 2 hours. After the liquid was concentrated under reduced pressure, 12-2 was obtained. LC-MS (ESI) m/z: 458.2 [M+H] + .
  • Step C Acetic acid (32.28 mg, 537.50 ⁇ M), 2-(7-azobenzotriazole)-N,N,N,N-tetramethylurea hexafluorophosphate (204.37 mg, 537.50 ⁇ M) mol) and N,N-diisopropylethylamine (185.24 mg, 1.43 mmol) were added to N,N-dimethylformamide (1 mL), and the reaction mixture was stirred at 20-30 degrees Celsius for 15 minutes. Additional 12-2 (177 mg, 358.33 micromoles) was added and stirring was continued for 0.5 h.
  • Step A Intermediate AJ (146 mg, 405.10 ⁇ mol) was dissolved in acetonitrile (4 mL), hexachlorocyclotriphosphazene (140.84 mg, 405.10 ⁇ mol) and potassium phosphate (257.97 mg, 1.22 mmol) were added . The reaction mixture was stirred at 25 degrees Celsius for 1 hour, then Intermediate F (81.50 mg, 405.10 micromoles) was added and stirring was continued at 25 degrees Celsius for 1 hour. After filtration, the filtrate was concentrated under reduced pressure.
  • Step B 13-1 (50 mg, 91.98 ⁇ mol) was dissolved in 1,4-dioxane (4 mL), followed by addition of hydrochloric acid in dioxane (4 mol/L, 1 mL). The reaction mixture was stirred at 40 degrees Celsius for 1 hour. After concentration under reduced pressure, 13-2 was obtained. LC-MS (ESI) m/z: 444.2 [M+H] + .
  • Step C Acetic acid (8.26 mg, 137.52 ⁇ mol), N,N-diisopropylethylamine (47.4 mg, 366.72 ⁇ mol) and 2-(7-azobenzotriazole)-N,N , N,N-Tetramethylurea hexafluorophosphate (52.29 mg, 137.52 ⁇ mol) was added to N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25 degrees Celsius for 15 minutes, then 13-2 (44 mg, 91.68 micromoles) was added, and after the addition was complete, stirring was continued at 25 degrees Celsius for 1 hour.
  • KRAS (G12C) protein was expressed and purified by Wuhan Pujian Biotechnology Co., Ltd., SOS1 exchange domin (564-1049) protein (Human recombinant) was purchased from Cytoskeleton, Mab Anti 6HIS-XL665 and Mab Anti GST-Eu cryptate were purchased from Cisbio.
  • the multi-plate reader Nivo5 was purchased from PerkinElmer.
  • the experimental buffer was prepared and used immediately.
  • Hepes 4-Hydroxyethylpiperazineethanesulfonic acid (Hepes): 5mM; Sodium chloride (NaCl): 150mM; Ethylenediaminetetraacetic acid (EDTA): 10mM; CO-630 (Igepal): 0.0025%; Fluorine Potassium chloride (KF): 100 mM; dithiothreitol (DTT): 1 mM; bovine serum albumin (BSA): 0.05%.
  • the IC 50 value can be obtained by curve fitting with four parameters (Log(inhibitor) vs.response in GraphPad Prism --Variable slope mode).
  • Table 1 provides the inhibitory activity of compounds of the invention on KRAS(G12C) and SOS1 binding.
  • the compounds of the present invention have desirable KRAS(G12C)-SOS1 binding inhibitory activity.
  • DLD-1 50,000 cells/well
  • RPMI-1640 medium 10% fetal bovine serum
  • the compounds of the present invention have significant inhibitory activity on the phosphorylation level of ERK kinase in DLD-1 cells.
  • EMEM medium fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Vicente, and low melting point agarose was purchased from Sigma. Almar blue reagent was purchased from Invitrogen.
  • the A427 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision Multilabel Analyzer (PerkinElmer).
  • the compound to be tested was diluted 3-fold to the ninth concentration, that is, from 6mM to 0.9 ⁇ M, and a double-well experiment was set up. For example, add 198 ⁇ L of medium to the middle plate, and then according to the corresponding position, transfer 2 ⁇ L of the compound diluted in each well to the first middle plate, then add 100 ⁇ L of medium to the second middle plate, and take the first middle plate. Add 100 ⁇ L of the mixed compound, and transfer 40 ⁇ L per well to the cell plate after mixing. Compound concentrations transferred to cell plates ranged from 30 [mu]M to 4.5 nM. The cell plates were placed in a carbon dioxide incubator for an additional 7 days.
  • the IC 50 value can be obtained by curve fitting with four parameters (“log(inhibitor) vs. response--Variable slope” mode).
  • Table 4 provides the inhibitory activity of the compounds of the present invention on the proliferation of A427 cells. Among them, "A” indicates that the IC 50 value is less than or equal to 100 nM; “B” indicates that the IC 50 value is greater than 100 nM and less than or equal to 500 nM; “C” indicates that the IC 50 value is greater than 500 nM.
  • CONCLUSION The compounds of the present invention exhibited significant inhibitory cell proliferation activity in A427 cells.
  • the purpose of this experiment was to evaluate the pharmacokinetic behavior of compounds after single intravenous injection or gavage administration in mice.
  • the compound is formulated into a clear solution of 0.2 mg/mL, vehicle: 5% DMSO/95% (10% hydroxypropyl- ⁇ -cyclodextrin) aqueous solution;
  • the compound is formulated at 0.3 mg/mL mL of suspension, vehicle: 5% DMSO/95% (10% hydroxypropyl- ⁇ -cyclodextrin) in water.
  • LC-MS/MS high performance liquid chromatography-tandem mass spectrometry
  • the retention time of compound and internal standard (diclofenac), chromatogram acquisition and chromatogram integration were processed by software Analyst (Applied Biosystems), and data statistics were processed by software Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems).
  • the unit of analyte concentration in the sample is ng/mL, with three significant figures reserved. All values expressed in percentages (such as % deviation and % coefficient of variation, etc.) are reserved to one decimal place.
  • Each calibration curve contains at least 6 concentration levels.
  • the preparation of calibration standards requires stock solutions from different sources from those of quality control samples.
  • Calibration standards whose calculated concentrations deviate from the indicated values by more than ⁇ 15.0% (the lower limit of quantitation by ⁇ 20.0%) should be rejected for regression analysis. Rejected calibration standards should be less than 25%, and each calibration curve should contain at least 6 calibration standards that meet the acceptance criteria. If the lower limit of quantitation and the upper limit of quantitation calibration standard need to be rejected, the upper and lower limit of quantification of the analysis batch will be raised and lowered accordingly.
  • Plasma concentrations were processed using the non-compartmental model of the pharmacokinetic software of WinNonlin TM Version 6.3 (Pharsight, Mountain View, CA), and the pharmacokinetic parameters were calculated using the linear log trapezoidal method.
  • the pharmacokinetic parameters to be calculated include but are not limited to (data permitting) T 1/2 , Vd ss , CL, AUC 0-24h in IV group; C max , T max , AUC 0-24h in PO group, oral biologic Utilization (F%).
  • mice with an intravenous injection dose of 1 mg/Kg and an oral gavage dose of 3 mg/Kg are shown in Table 5 below.
  • the compounds of the present invention exhibit good pharmacokinetic properties in mouse PK studies.
  • KRAS G12C mutation-dependent cell lines Human pancreatic cancer MIA PaCa-2 cells are KRAS G12C mutation-dependent cell lines.
  • KRAS protein G12C mutation leads to continuous activation of KRAS to activate downstream signaling pathways such as ERK and AKT, and promote the growth and proliferation of tumor cells. Maintaining the KRAS protein in an inactive state bound to GDP inhibits tumor growth.
  • mice were subcutaneously inoculated with MIA PaCa-2 cells to establish a subcutaneously transplanted tumor model of human pancreatic cancer.
  • the test is divided into each test compound group and vehicle control group, each group of 6 animals, intragastric administration, the vehicle control group is administered once a day, each test compound group is administered twice a day, with an interval of 12 hours, A total of 18 days were administered.
  • the efficacy was evaluated according to the relative tumor inhibition rate (TGI), and the safety was evaluated according to the changes in animal body weight and death.
  • TGI tumor inhibition rate
  • the experimental animals were BALB/c nude mice, female, and the mice were 6-7 weeks old when the tumor cells were inoculated, and were purchased from Shanghai Bikai Laboratory Animal Co., Ltd.
  • the experimental animals were kept in an independent ventilation box with constant temperature and humidity.
  • the temperature of the rearing room was 21-24 °C
  • the humidity was 40-60%
  • the ventilation was 10-20 times/hour
  • the light and dark alternating time of day and night was 12 hours/12 hours; cobalt was continuously supplied.
  • the rat raising box is a polysulfone rat box, used after autoclaving, with a size of 325mm ⁇ 210mm ⁇ 180mm; the bedding material is autoclaved corncob, 5 animals per box, and the IACUC approval number, experiment number, experiment number are marked on the cage card. Start time, project leader, experimental staff, animal source, group and animal number, etc.; experimental animals are marked by ear number.
  • MIA PaCa-2 cells were cultured in DEME medium containing 10% fetal bovine serum and 2.5% HS. MIA PaCa-2 cells in exponential growth phase were collected, resuspended in phosphate buffered saline (PBS) to an appropriate concentration and mixed 1:1 with matrix (Matrigel) for subcutaneous tumor inoculation in mice. The right side of the mice was subcutaneously inoculated with 5 ⁇ 10 6 MIA PaCa-2 cells.
  • PBS phosphate buffered saline
  • Mesatrigel matrix
  • tumor volume calculation formula: tumor volume (mm3) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter, and b represents the short diameter).
  • StudyDirector TM software was used to collect data, including the measurement of the long and short diameters of the tumor and the weighing of the animal body weight. The raw data is directly imported into the software after being measured by the balance and vernier caliper, and any changes in the data will be recorded in this software.
  • the growth curve of the tumor is shown in Figure 1, and the body weight change curve of the test animals is shown in Figure 2. It can be seen from the experimental results that the single drug of the compound of the present invention has a significant inhibitory effect on tumor proliferation in the human pancreatic cancer MIA PaCa-2 cell line subcutaneous xenografted BALB/c nude mouse animal model, and no weight loss of the test animals was found. It is shown that the compounds of the present invention have good safety after administration.

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Abstract

喹唑啉类化合物及其药学上可接受的盐，具体涉及式(II)化合物及其药学上可接受的盐。

Description

喹唑啉类化合物
本申请主张如下优先权：
CN202010723467.4，申请日2020年07月24日；
CN202110653818.3，申请日2021年06月11日。
技术领域
本发明涉及一类喹唑啉类化合物及其药学上可接受的盐，以及该化合物或其药学上可接受的盐在制备治疗癌症药物中的应用。
背景技术
RAS蛋白的突变发生于约20％～30％的癌症病例中，是胰腺癌、结直肠癌和非小细胞肺癌中最常见的突变致癌基因。RAS蛋白充当了分子开关的角色，其与GTP结合处于激活状态，与GDP结合处于失活状态。RAS蛋白的突变降低了其与GTP酶水解GTP的能力，使分子开关一直保持活性GTP结合状态，驱动未经检查的致癌下游信号传导，如通过RAS-RAF-MEK-ERK通路和RAS-PI3K-PDK1-AKT通路等，促进癌细胞的存活于增殖。
SOS1蛋白是一类鸟嘌呤核苷酸交换因子(GEF)，研究表明，这类蛋白可催化KRAS与GTP的结合，从而促进KRAS的激活。因此，靶向抑制SOS1蛋白，SOS1-KRAS的相互作用，而与KRAS是何种突变无关，成为有潜力的肿瘤治疗靶点。此外，研究还表明，联合MEK抑制可导致体内深度通路阻滞和肿瘤退变，是一种潜在的治疗大多数KRAS驱动的癌症的方法。
发明内容
本发明提供式(II)化合物或其药学上可接受的盐，
Figure PCTCN2021108256-appb-000001
其中，
环A选自苯基和5元杂芳基；
R 1选自H和NH 2；
R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2、3、4或5个R a取代；
R 3选自H和F；
R 4选自C 3-8环烷基和3-8元杂环烷基，所述C 3-8环烷基和3-8元杂环烷基分别独立地任选被1、2、3或4个R b取代；
R 5选自H、Cl和CH 3；
R a分别独立地选自D、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、 -COOH、-COOCH 3和环丙基；
R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、C 1-3烷基、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、C 1- 3烷氧基、-COOH、-COO-C 1-3烷基、-C(＝O)-C 1-3烷基、-S(＝O) 2-C 1-3烷基和环丙基，所述C 1-3烷基、C 1- 3烷氧基和环丙基分别独立地任选被1、2或3个R取代；
R分别独立地选自F、Cl、Br、I、OH、CN、NH 2、-OCH 3和-COOH。
本发明还提供式(I)化合物或其药学上可接受的盐，
Figure PCTCN2021108256-appb-000002
其中，
R 1选自H和NH 2；
R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2、3、4或5个R a取代；
R 3选自H和F；
R 4选自C 3-8环烷基和3-8元杂环烷基，所述C 3-8环烷基和3-8元杂环烷基分别独立地任选被1、2、3或4个R b取代；
R a分别独立地选自D、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3和环丙基；
R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、C 1-3烷基、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3和环丙基，C 1-3烷基和环丙基分别独立地任选被1、2或3个R取代；
R分别独立地选自F、Cl、Br、I、OH、CN、NH 2和-COOH。
在本发明的一些方案中，上述R 1选自NH 2，其他变量如本发明所定义。
在本发明的一些方案中，上述R 1选自H，其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自CF 3、
Figure PCTCN2021108256-appb-000003
其他变量如本发明所定义。
在本发明的一些方案中，上述R 2选自CF 3、CHF 2、
Figure PCTCN2021108256-appb-000004
其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自环丙基、双环[2.2.2]辛烷、5-6元杂环烷基和8元杂环烷基，所述环丙基、双环[2.2.2]辛烷、5-6元杂环烷基和8元杂环烷基分别独立地任选被1、2、3或4个Rb取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自环丙基、双环[2.2.2]辛烷和5-6元杂环烷基，所述环丙基、双环[2.2.2]辛烷和5-6元杂环烷基分别独立地任选被1、2、3或4个R b取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000005
Figure PCTCN2021108256-appb-000006
所述
Figure PCTCN2021108256-appb-000007
分别独立地任选被1、2、3或4个R b取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000008
所述
Figure PCTCN2021108256-appb-000009
分别独立地任选被1、2、3或4个R b取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000010
所述
Figure PCTCN2021108256-appb-000011
Figure PCTCN2021108256-appb-000012
分别独立地任选被1、2、3或4个R b取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2F、-CHF 2、-CH 2CN、-CH 2OH、-CH 2OCH 3、-CH 2CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3、
Figure PCTCN2021108256-appb-000013
其他变量如本发明所定义。
在本发明的一些方案中，上述R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3、环丙基和-CH 2F，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000014
Figure PCTCN2021108256-appb-000015
Figure PCTCN2021108256-appb-000016
Figure PCTCN2021108256-appb-000017
其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000018
Figure PCTCN2021108256-appb-000019
Figure PCTCN2021108256-appb-000020
其他变量如本发明所定义。
在本发明的一些方案中，上述R 4选自
Figure PCTCN2021108256-appb-000021
Figure PCTCN2021108256-appb-000022
其他变量如本发明所定义。
在本发明的一些方案中，上述环A选自苯基与噻吩基，其他变量如本发明所定义。
在本发明的一些方案中，上述化合物选自
Figure PCTCN2021108256-appb-000023
Figure PCTCN2021108256-appb-000024
其中，环A、R 1、R 2、R 3、R 4、R 5和R b如本发明所定义。
在本发明的一些方案中，上述化合物选自
Figure PCTCN2021108256-appb-000025
Figure PCTCN2021108256-appb-000026
其中，R 1、R 2、R 3、R b和R 5如本发明所定义。
在本发明的一些方案中，上述化合物选自
Figure PCTCN2021108256-appb-000027
其中，R 1、R 2、R 3和R 5如本发明所定义。
在本发明的一些方案中，上述化合物选自
Figure PCTCN2021108256-appb-000028
其中，R b如本发明所定义。
本发明还提供化合物或其药学上可接受的盐，其中，化合物选自
Figure PCTCN2021108256-appb-000029
Figure PCTCN2021108256-appb-000030
在本发明的一些方案中，上述化合物选自
Figure PCTCN2021108256-appb-000031
Figure PCTCN2021108256-appb-000032
Figure PCTCN2021108256-appb-000033
本发明还提供上述化合物或其药学上可接受的盐在制备抑制SOS1蛋白的药物中的应用。
本发明还提供上述化合物或其药学上可接受的盐在制备治疗肺癌、胰腺癌和/或直肠癌药物中的应用。
本发明还有一些技术方案是由上述各变量自由组合而来。
技术效果
本发明的化合物具有良好的KRAS(G12C)-SOS1结合抑制活性。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物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 PCTCN2021108256-appb-000034
和楔形虚线键
Figure PCTCN2021108256-appb-000035
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021108256-appb-000036
和直形虚线键
Figure PCTCN2021108256-appb-000037
表示立体中心的相对构型，用波浪线
Figure PCTCN2021108256-appb-000038
表示楔形实线键
Figure PCTCN2021108256-appb-000039
或楔形虚线键
Figure PCTCN2021108256-appb-000040
或用波浪线
Figure PCTCN2021108256-appb-000041
表示直形实线键
Figure PCTCN2021108256-appb-000042
或直形虚线键
Figure PCTCN2021108256-appb-000043
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)。
除非另有说明，本发明所述D是指氘( 2H)。
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。
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。
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 PCTCN2021108256-appb-000044
直形虚线键
Figure PCTCN2021108256-appb-000045
或波浪线
Figure PCTCN2021108256-appb-000046
表示。例如-OCH3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 PCTCN2021108256-appb-000047
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 PCTCN2021108256-appb-000048
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 PCTCN2021108256-appb-000049
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 PCTCN2021108256-appb-000050
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2021108256-appb-000051
仍包括
Figure PCTCN2021108256-appb-000052
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，环上原子的数目通常被定义为环的元数，例如，“5-7元环”是指环绕排列5-7个原子的“环”。
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，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，本发明术语“5元杂芳基”与“5元杂芳环”可以互换使用，术语“5元杂芳基”表示由5个环原子组成的具有共轭π电子体系的单环基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子。其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O) p，p是1或2)。5元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5元杂芳基的实例包括但不限吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包 括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1，2，3-三唑基、2H-1，2，3-三唑基、1H-1，2，4-三唑基和4H-1，2，4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)。除非另有规定，“C 3-8环烷基”表示由3至8个碳原子组成的饱和环状碳氢基团，其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。所述C 3-8环烷基包括C 3-6、C 3-5、C 4-8、C 4-6、C 4-5、C 5-8或C 5-6环烷基等；其可以是一价、二价或者多价。C 3-8环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基、环庚基、降冰片烷基、[2.2.2]二环辛烷等。
除非另有规定，术语“3-8元杂环烷基”本身或者与其他术语联合分别表示由3至8个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，碳、氮和硫杂原子可任选被氧化(即C(＝O)、NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“3-8元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述3-8元杂环烷基包括3-6元、3-5元、4-6元、5-6元、4元、5元和6元杂环烷基等。3-8元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1，2-噁嗪基、1，2-噻嗪基、六氢哒嗪基、高哌嗪基、高哌啶基或二氧杂环庚烷基等。
除非另有规定，术语“5-6元杂环烷基”本身或者与其他术语联合分别表示由5至6个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，碳、氮和硫杂原子可任选被氧化(即C(＝O)、NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“5-6元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述5-6元杂环烷基包括5元和6元杂环烷基。5-6元杂环烷基的实例包括但不限于吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1，2-噁嗪基、1，2-噻嗪基、六氢哒嗪基、高哌嗪基或高哌啶基等。
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。
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 D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2021108256-appb-000053
/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：DMSO代表二甲基亚砜；DMF代表N，N-二甲基甲酰胺；DCM代表二氯甲烷；dioxane或1，4-dioxane代表1，4-二氧六环；Ts代表4-甲基本磺酰基；Boc代表叔丁氧羰基，是氨基的一种保护基；Cbz代表苄氧羰基，是氨基的一种保护基；CO 2代表二氧化碳；Bar代表巴，是一个压强单位；LC-MS代表液质联用色谱；HPLC代表液相色谱；mg代表毫克；μg代表微克；ng代表纳克；μL代表微升；mL代表毫升；mm代表毫米；μm代表微米；mM代表微摩尔/升，μM代表微摩尔/升；nM代表钠摩尔/升；h代表小时；min代表分钟；HPLC代表高效液相色谱法。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021108256-appb-000054
软件命名，市售化合物采用供应商目录名称。
附图说明
图1为肿瘤生长曲线图。
图2为受试动物体重变化曲线图。
具体实施方式
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本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。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。
中间体AA
Figure PCTCN2021108256-appb-000055
步骤A：将化合物A(2.3克，11.37毫摩尔)溶解在甲醇(10毫升)中，然后加入四氢吡喃-4-胺 (AA-1)(1克，9.89毫摩尔)，反应混合物在25摄氏度下搅拌12小时，然后加入甲醇钠(1.86克，11.37毫摩尔)，继续在25摄氏度下搅拌2小时。用浓盐酸(12摩尔/升，2毫升)淬灭反应，过滤，用水(10毫升)洗涤滤饼。收集滤饼，减压干燥后得到中间体AA-2。 1H NMR(400MHz，DMSO-d 6)δ＝10.81(s，1H)，8.26(s，1H)，5.72(s，1H)，4.81(tt，J＝3.9，12.0Hz，1H)，3.97(dd，J＝3.8，11.1Hz，2H)，3.81(s，3H)，3.50-3.39(m，2H)，1.90-1.88(m，2H)，1.72(dd，J＝2.0，11.9Hz，2H)。LC-MS(ESI)m/z：254.3[M+H] +。
步骤B：将中间体AA-2(800毫克，3.16毫摩尔)溶解在乙腈(10毫升)中，随后依次加入三乙胺(479.47毫克，4.74毫摩尔)和对甲苯磺酰氯(632.35毫克，3.32毫摩尔)。反应混合物在25摄氏度下搅拌2小时。过滤，并用水(10毫升)洗涤滤饼。滤饼收集后，真空干燥，得到中间体AA-3，直接用于下一步反应。 1H NMR(400MHz，DMSO-d 6)δ＝8.32(s，1H)，7.82(d，J＝8.4Hz，2H)，7.51(d，J＝8.1Hz，2H)，6.01(s，1H)，4.77(tt，J＝3.8，12.0Hz，1H)，3.96(dd，J＝3.9，11.3Hz，2H)，3.66(s，3H)，3.46(s，2H)，2.44(s，3H)，1.93-1.91(m，2H)，1.73(d，J＝10.0Hz，2H)。LC-MS(ESI)m/z：408.2[M+H] +。
步骤C：将中间体AA-3(250毫克，613.59微摩尔)，乙酰胺(26.24毫克，613.59微摩尔)和磷酸钾(143.27毫克，674.95微摩尔)加入到1，4-二氧六环(2毫升)中，氮气保护下，再加入4，5-双(二苯基膦)-9，9-二甲基氧杂蒽(35.50毫克，61.36微摩尔)和二-双[(1，2，3)-1-苯基-2-丙烯]二钯(II)(16.08毫克，30.68微摩尔)。氮气置换三次后，反应混合物在110摄氏度下搅拌2小时。自然冷却至室温后，加压浓缩，剩余物经过薄层层析法(展开剂：乙酸乙酯/石油醚＝1/1)纯化，得到中间体AA-4。 1H NMR(400MHz，CDCl 3)δ＝10.80(s，1H)，8.26(s，1H)，7.74(s，1H)，5.12(tt，J＝4.6，11.7Hz，1H)，4.11(dd，J＝4.2，11.4Hz，2H)，3.89(s，3H)，3.63-3.48(m，2H)，2.21(s，3H)，1.89-1.79(m，4H)。LC-MS(ESI)m/z：295.1[M+H] +。
步骤D：将中间体AA-4粗品(60毫克)加入到氨的甲醇溶液(7摩尔/升，1毫升)中，然后密封室温搅拌72小时。减压浓缩后，将剩余物溶解在甲醇(3毫升)中，然后加入氢氧化钠水溶液(1摩尔/升，2毫升)。反应混合物在50摄氏度下搅拌30分钟。自然冷却后，混合物用水(10毫升)稀释，然后用二氯甲烷萃取(40毫升/次，萃取5次)。合并的有机相用无水硫酸钠干燥，过滤后，滤液减压浓缩干燥得到中间体AA。 1H NMR(400MHz，DMSO-d 6)δ＝11.83(s，1H)，8.52(s，1H)，6.20(s，1H)，5.05-4.86(m，1H)，4.00(dd，J＝3.7，11.4Hz，2H)，3.54-3.40(m，2H)，2.25(s，3H)，1.97-1.95(m，2H)，1.83-1.77(m，2H)。LC-MS(ESI)m/z：262.2[M+H] +。
中间体AB
Figure PCTCN2021108256-appb-000056
Figure PCTCN2021108256-appb-000057
中间体AB的制备参考中间体AA的制备流程，将步骤A中的四氢吡喃-4-胺(AA-1)替换成3-甲基四氢呋喃-3-胺(AB-1)。 1H NMR(400MHz，CD 3OD)δ＝8.63(s，1H)，6.34(s，1H)，4.44(d，J＝9.4Hz，1H)，4.05-3.95(m，3H)，2.63-2.50(m，2H)，2.35(s，3H)，1.68(s，3H)。LC-MS(ESI)m/z：262.3[M+H] +。
中间体AC
Figure PCTCN2021108256-appb-000058
中间体AC的制备参考中间体AA的制备流程，将步骤A中的四氢吡喃-4-胺(AA-1)替换成1-(氟甲基)环丙烷胺(AC-1)。 1H NMR(400MHz，DMSO-d 6)δ＝11.83(br s，1H)，8.35(s，1H)，6.16(s，1H)，4.62(s，1H)，4.50(s，1H)，2.23(s，3H)，1.30-1.23(m，4H)。LC-MS(ESI)m/z：250.1[M+H] +。
中间体AD
Figure PCTCN2021108256-appb-000059
中间体AD的制备参考中间体AA的制备流程，将步骤A中的四氢吡喃-4-胺(AA-1)替换成4-氨基双环[2.2.2]辛烷-1-醇(AD-1)。 1H NMR(400MHz，CD 3OD)δ＝8.64(s，1H)，6.28(s，1H)，2.50-2.46(m，6H)，2.33(s，3H)，1.94-1.78(m，6H)。LC-MS(ESI)m/z：302.1[M+H] +。
中间体AE
Figure PCTCN2021108256-appb-000060
中间体AE的制备参考中间体AA的制备流程，将步骤A中的四氢吡喃-4-胺(AA-1)替换成1-甲基环丙烷胺(AE-1)。 1H NMR(400MHz，CDCl 3)δ＝10.40-10.12(m，1H)，8.52(s，1H)，6.45(s，1H)，2.36(s，3H)，1.52(s，3H)，1.05(dd，J＝2.7，11.6Hz，4H).LC-MS(ESI)m/z：232.2[M+H] +。
中间体AF
Figure PCTCN2021108256-appb-000061
步骤A：将AF-1(22.3克，220.57毫摩尔)和邻苯二甲酸酐(32.67克，220.57毫摩尔)加入到冰醋酸(200毫升)中，随后，该反应混合物在110摄氏度下搅拌3小时。减压浓缩除去醋酸后，加入水(200毫升)，并在15摄氏度下搅拌1小时。过滤，收集滤饼，减压干燥后，得到AF-2。 1H NMR(400MHz，DMSO-d 6)δ＝13.60-12.40(m，1H)，7.95-7.82(m，4H)，1.66-1.59(m，2H)，1.47-1.40(m，2H)。
步骤B：室温下，将AF-2(10克，43.25毫摩尔)和N，N-二甲基甲酰胺(158.07毫克，2.16毫摩尔)加入到甲苯(30毫升)中，随后缓慢加入二氯亚砜(5.40克，45.41毫摩尔)。加完后，该反应混合物在110摄氏度下搅拌3小时。减压浓缩除去溶剂，得到AF-3，直接用于下一步反应。
步骤C：将AF-3(20.5克，82.12毫摩尔)和2，6-二甲基吡啶(10.56克，98.54毫摩尔)加入到四氢呋喃(200毫升)中，随后加入干钯碳(10％，1.02克)。反应混合物在氢气45psi压力、30摄氏度下搅拌20小时。随后补加钯碳(10％，1.02克)，继续在氢气45psi压力、30摄氏度下搅拌24小时。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V(石油醚/乙酸乙酯＝10/1～3/1)+20％二氯甲烷)分离纯化，得到AF-4。 1H NMR(400MHz，CDCl 3)δ＝8.88(s，1H)，7.89-7.82(m，2H)，7.77-7.71(m，2H)，1.86-1.79(m，2H)，1.72-1.65(m，2H)。
步骤D：将AF-4(7.54克，35.04毫摩尔)溶解在二氯甲烷(45毫升)中，随后缓慢滴加双(2-甲氧基乙基)氨基三氟化硫(18.60克，84.09毫摩尔)的二氯甲烷(20毫升)溶液。加完后，反应混合物在20摄氏度搅拌48小时。在10摄氏度下，用饱和碳酸钠水溶液(100毫升)淬灭反应，并搅拌1小时。然后加入水(100毫升)，再用二氯甲烷萃取(100毫升/次，萃取3次)。合并的有机相用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V石油醚/乙酸乙酯＝15/1～10/1)分离纯化，得到AF-5。 1H NMR(400MHz，CDCl 3)δ＝7.88(dd，J＝3.2，5.6Hz，2H)，7.76(dd，J＝3.2，5.6Hz，2H)，6.07-5.69(m，1H)，1.47-1.41(m，2H)，1.27-1.20(m，2H)。
步骤E：将AF-5(7.1克，29.93毫摩尔)加入到2-(2-氨基乙胺基)乙醇(21.30毫升)中。该反应混合物在80摄氏度下搅拌2小时。再加入甲醇(150毫升)，在90摄氏度，常压蒸馏，馏分用干冰浴冷却。向馏分中加入氯化氢的甲醇溶液(4摩尔/升，15毫升)。减压浓缩后，得到AF-6。 1H NMR(400MHz，CD 3OD)δ＝6.04-5.71(m，1H)，1.24(s，4H)。
步骤F：将AF-6(4克，27.86毫摩尔)和化合物A(6.76克，33.43毫摩尔)加入到甲醇(40毫升)中，随后加入甲醇钠的甲醇溶液(25％，5.42克)。反应混合物在10摄氏度搅拌17小时后，再加入甲醇钠的甲醇溶液(25％，7.22克)，继续搅拌2小时。减压浓缩除去甲醇，剩余物用浓盐酸(12摩尔/升)酸化至pH＝6～7。用水(100毫升)稀释后，再用乙酸乙酯萃取(100毫升/次，萃取3次)。合并的有机相用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物在叔丁基甲醚(100毫升)中搅拌1小时，过滤，收集滤饼，得到AF-7。 1H NMR(400MHz，DMSO-d 6)δ＝10.93(s，1H)，8.09(s，1H)，6.32-5.98(m，1H)，5.68(s，1H)，3.80(s，3H)，1.41-1.23(m，4H)。
步骤G：将AF-7(5.17克，19.95毫摩尔)和4-甲基苯磺酰氯(4.58克，24.02毫摩尔)加入到乙腈(50毫升)中，随后再加入三乙胺(3.03克，29.92毫摩尔)。反应混合物在20摄氏度搅拌2小时。加入水(50毫升)稀释后，过滤。滤饼用水洗涤(30毫升/次，洗涤3次)，真空干燥，得到AF-8。 1H NMR(400MHz，DMSO-d 6)δ＝8.15(s，1H)，7.84(d，J＝8.4Hz，2H)，7.52(d，J＝8.4Hz，2H)，6.31-5.98(m， 2H)，3.67(s，3H)，2.44(s，3H)，1.43-1.30(m，4H)。
步骤H：将AF-8(7.41克，17.92毫摩尔)，乙酰胺(1.06克，17.92毫摩尔)，磷酸钾(4.19克，19.72毫摩尔)，二-双[(1，2，3)-1-苯基-2-丙烯]二钯(II)(187.90毫克，358.50微摩尔)和Xantphos(414.87毫克，717.00微摩尔)一次加入到1，4-二氧六环(35毫升)中。反应混合物经氮气保护，在115摄氏度下搅拌12小时。自然冷却后，加入水(100毫升)稀释反应液，再用二氯甲烷萃取(100毫升/次，萃取3次)。合并的有机相用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V二氯甲烷/乙酸乙酯＝10/1～5/1)分离纯化，得到AF-9。 1H NMR(400MHz，CDCl 3)δ＝10.79(br s，1H)，8.23(s，1H)，7.69(s，1H)，6.40-6.03(m，1H)，3.89(s，3H)，2.21(s，3H)，1.57-1.50(m，2H)，1.19-1.11(m，2H)。
步骤I：将AF-9(4.22克，14.05毫摩尔)加入到氨的甲醇溶液(7摩尔/升，34毫升)中。反应混合物在15摄氏度下搅拌3天。减压浓缩后，向剩余物中加入氢氧化钠水溶液(1摩尔/升，30毫升)，然后在50摄氏度搅拌30分钟。自然冷却后，该溶液用叔丁基甲醚洗涤(70毫升/次，洗涤3次)。水相用浓盐酸(12摩尔/升)酸化，调节至pH约为4，过滤，收集滤饼，减压干燥，得到中间体AF。 1H NMR(400MHz，DMSO-d 6)δ＝11.85(br s，1H)，8.36(s，1H)，6.38-6.05(m，2H)，2.24(s，3H)，1.50-1.29(m，4H)。
中间体AG
Figure PCTCN2021108256-appb-000062
步骤A：氮气保护下，将AG-1(3.00克，14.00毫摩尔)和中间体A(3.40克，16.80毫摩尔)溶解于甲醇(30毫升)中，反应混合物在20摄氏度下搅拌反应12小时。随后加入甲醇钠的甲醇溶液(3.63克，16.80毫摩尔，质量分数：25％)，在20摄氏度下继续搅拌反应12小时。减压浓缩除掉甲醇，剩余物用水(100毫升)稀释，再用甲基叔丁基醚洗涤(100毫升/次，洗涤两次)。水相用盐酸(1摩尔/升)调节pH值6～7后过滤，滤液减压浓缩，得到AG-2。 1H NMR(400MHz，CDCl 3)δ＝10.43(s，1H)，8.32-8.22(m，1H)，5.88(s，1H)，3.94-3.90(m，3H)，3.30(ddd，J＝3.4，9.8，13.7Hz，2H)，1.72(s，3H)，1.66(br s，5H)，1.46(s，9H)。
步骤B：将AG-2(3.15克，8.60毫摩尔)和对甲苯磺酰氯(1.97克，10.32毫摩尔)加入到乙腈(32毫升)中，随后加入三乙胺(1.30克，12.90毫摩尔)。反应混合物在20摄氏度下搅拌反应2小 时。减压浓缩后，剩余物用水(50毫升)稀释，并在20摄氏度下继续搅拌2小时。过滤后，滤饼减压干燥，得到AG-3。 1H NMR(400MHz，CDCl 3)δ＝8.25-8.31(m，1H)，7.72-7.84(m，2H)，7.31(d，J＝8.13Hz，2H)，5.95-6.01(m，1H)，3.61-3.73(m，2H)，3.15-3.25(m，2H)，2.40(s，3H)，2.17-2.27(m，2H)，2.05-2.16(m，2H)，1.59-1.63(m，3H)，1.57(s，3H)，1.34-1.42(m，9H)。
步骤C：将AG-3(1.3克，2.50毫摩尔)，乙酰胺(147.50毫克，2.50毫摩尔)，磷酸钾(583.07毫克，2.75毫摩尔)和4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(288.98毫克，499.43微摩尔)加入到1，4-二氧六环(10毫升)中。氮气置换3次后，再加入双(苯丙烯基氯化钯)(130.88毫克，249.71微摩尔)。反应混合物在110摄氏度搅拌4小时。自然冷却后，加水(30毫升)稀释，再用二氯甲烷萃取(30毫升/次，萃取2次)。有机相用饱和食盐水(30毫升)洗涤一次，经无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V二氯甲烷/石油醚＝10/1～3/1)纯化后，得到AG-4。LC-MS(ESI)m/z：408.3[M+H] +。
步骤D：将AG-4(0.67克，1.64毫摩尔)加入到氨甲醇溶液(7摩尔/升，50毫升)中，在20摄氏度下搅拌3天。减压浓缩后，剩余物加入氢氧化钠溶液(1摩尔/升，20毫升)稀释，再用甲基叔丁基醚洗涤(30毫升/次，洗涤3次)。水相加入浓盐酸调节pH等于4后，减压抽滤。收集滤饼，减压干燥后得到中间体AG。 1H NMR(400MHz，CDCl 3)δ＝8.64(s，1H)，6.60-6.49(m，1H)，3.95-3.74(m，2H)，3.36-3.26(m，2H)，2.49(s，3H)，2.40-2.20(m，4H)，1.85-1.77(m，3H)，1.75(s，1H)，1.48(s，9H)。
中间体AH
Figure PCTCN2021108256-appb-000063
步骤A：将AH-1(500毫克，2.5毫摩尔)和中间体A(605.64毫克，3.0毫摩尔)加入到甲醇(5毫升)中，随后加入甲醇钠的甲醇溶液(539.49毫克，3.0毫摩尔，质量分数：30％)。反应混合物在10～20摄氏度下搅拌12小时，然后用盐酸(2摩尔/升)调节pH值4～5，再用二氯甲烷萃取(50毫升/次，萃取2次)。有机相合并后，用食盐水(20毫升)洗涤一次，然后用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AH-2。 1H NMR(400MHz，CDCl 3)δ＝10.51(s，1H)，7.97(s，1H)，5.88(s，1H)，3.95-3.86(m，1H)，3.83(s，3H)，3.81-3.74(m，1H)，3.73-3.67(m，1H)，3.65-3.60(m，1H)，2.21(m，1H)，2.08-1.96(m，1H)，1.42(s，9H)，1.24(s，3H)。
步骤B：在0～10摄氏度下，将AH-2(880毫克，2.5毫摩尔)加入到乙腈(10毫升)中，随后依 次加入三乙胺(379.05毫克，12.90毫摩尔)和对甲苯磺酰氯(499.91毫克，2.62毫摩尔)。反应混合物在20～30摄氏度下搅拌1小时，然后加入二氯甲烷(50毫升)萃取一次。有机相用饱和食盐水(20毫升)洗涤，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V二氯甲烷/石油醚＝10/1～2/1)分离纯化后，得到AH-3。 1H NMR(400MHz，CDCl 3)δ＝8.13(br s，1H)，7.85(d，J＝8.4Hz，2H)，7.38(d，J＝8.2Hz，2H)，6.14(s，1H)，5.50-5.36(m，1H)，4.20-4.05(m，1H)，3.89-3.77(m，4H)，3.50-3.37(m，1H)，2.48(s，3H)，2.36-2.26(m，1H)，2.13-2.06(m，1H)，1.48(s，9H)，1.31(d，J＝5.0Hz，3H)。
步骤C：将AH-3(900毫克，1.78毫摩尔)加入到1，4-二氧六环(10毫升)中，随后在氮气保护下，依次加入乙酰胺(110.19毫克，1.87毫摩尔)，磷酸钾(414.84毫克，1.95毫摩尔)，双(苯丙烯基氯化钯)(93.12毫克，177.67微摩尔)和4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(205.60毫克，355.33微摩尔)。反应混合物在110摄氏度搅拌2小时，自然冷却后，加入乙酸乙酯(50毫升)稀释。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V/V石油醚/乙酸乙酯/乙醇＝30/3/1至9/3/1)分离纯化，得到AH-4。 1H NMR(400MHz，CDCl 3)δ＝10.81(s，1H)，8.17(s，1H)，7.76(s，1H)，5.55-5.44(m，1H)，4.17-3.99(m，1H)，3.90-3.74(m，4H)，3.70-3.42(m，1H)，2.35-2.25(m，1H)，2.23-2.19(m，3H)，2.15-2.02(m，1H)，1.48(s，9H)，1.38-1.27(m，3H)。
步骤D：将AH-4(510毫克，1.30毫摩尔)加入到氨甲醇溶液(7摩尔/升，50毫升)中，在20～30摄氏度下搅拌12小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Welch Ultimate XB-CN，250mm×50mm×10μm；流动相：A相：正己烷；B相：0.1％的氨水乙醇溶液，5％～45％；15分钟)纯化得到中间体AH。 1H NMR(400MHz，CDCl 3)δ＝11.81(br s，1H)，8.49-8.26(m，1H)，6.18(s，1H)，5.38-5.20(m，1H)，3.98(m，1H)，3.71(m，1H)，3.56(m，1H)，2.57-2.53(m，1H)，2.24(s，3H)，2.07-1.92(m，1H)，1.40(s，9H)，1.21(d，J＝5.6Hz，3H)。
中间体AI
Figure PCTCN2021108256-appb-000064
步骤A：将AI-1(2克，9.38毫摩尔)，乙酸(563.29毫克，9.38毫摩尔)和甲酸铵(22.36克，354.56毫摩尔)加入到甲醇(500毫升)中，随后加入氰基硼氢化钠(1.18克，18.76毫摩尔)。反应混合物在20～30摄氏度下搅拌72小时。减压浓缩后，加入乙酸乙酯(200毫升)溶解剩余物。该溶液用 饱和食盐水洗涤(50毫升/次，洗涤2次)，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AI-2。 1H NMR(400MHz，CDCl 3)δ＝4.12-3.88(m，1H)，3.76(td，J＝3.2，10.8Hz，1H)，3.62-3.44(m，1H)，3.36-3.23(m，1H)，2.34-2.12(m，2H)，1.78(td，J＝9.8，13.0Hz，2H)，1.46(s，9H)，1.28(d，J＝6.4Hz，3H)。
步骤B：将AI-2(1.4克，6.53毫摩尔)和中间体A(1.58克，7.84毫摩尔)加入到甲醇(20毫升)中，然后加入甲醇钠的甲醇溶液(1.69克，7.84毫摩尔，质量分数：25％)，反应混合物在10～20摄氏度下搅拌12小时。用盐酸(2摩尔/升)调节pH值4～5，再用二氯甲烷萃取(50毫升/次，萃取2次)。有机相合并后，用饱和食盐水(20毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AI-3。 1H NMR(400MHz，CDCl 3)δ＝10.47-10.41(m，1H)，8.01(s，1H)，5.93-5.82(m，1H)，4.96-4.81(m，1H)，3.91-3.78(m，4H)，3.72-3.59(m，1H)，3.52-3.38(m，1H)，2.29-2.19(m，1H)，1.92-1.83(m，1H)，1.81-1.69(m，2H)，1.41(s，9H)，1.27-1.23(m，3H)。
步骤C：在0～10摄氏度下，向AI-3(1.55克，4.23毫摩尔)的乙腈(20毫升)溶液中加入三乙胺(642.09毫克，6.35毫摩尔)和对甲苯磺酰氯(887.15毫克，4.65毫摩尔)。反应混合物在20～30摄氏度下搅拌反应1小时，然后用二氯甲烷(50毫升)萃取。有机相用饱和食盐水(20毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，剩余物经硅胶柱层析(洗脱剂：V/V二氯甲烷/石油醚＝10/1～2/1)纯化后，得到AI-4。 1H NMR(400MHz，CDCl 3)δ＝8.21-8.16(m，1H)，7.89-7.81(m，2H)，7.40(d，J＝8.2Hz，2H)，6.16-6.05(m，1H)，5.27-4.84(m，1H)，3.98-3.72(m，5H)，3.54-3.39(m，1H)，2.49(s，3H)，2.40-2.26(m，1H)，2.01-1.80(m，2H)，1.63-1.57(m，1H)，1.50(s，9H)，1.33-1.28(m，3H)。
步骤D：在氮气保护下，将AI-4(1.77克，3.4毫摩尔)，乙酰胺(210.87毫克，3.57毫摩尔)，磷酸钾(793.87毫克，3.74毫摩尔)，双(苯丙烯基氯化钯)(178.20毫克，340.00微摩尔)和4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(393.46毫克，679.99微摩尔)依次加入到1，4-二氧六环(20毫升)中。反应混合液在110摄氏度下搅拌2小时，自然冷却后，加入乙酸乙酯(50毫升)稀释。过滤后，滤液减压浓缩，剩余物经硅胶柱层析(洗脱剂：V/V/V石油醚/乙酸乙酯/乙醇＝30/10/1至9/3/1)纯化后，得到AI-5。 1H NMR(400MHz，CDCl 3)δ＝10.88-10.66(m，1H)，8.28-8.08(m，1H)，7.81-7.65(m，1H)，5.00-4.83(m，1H)，3.93-3.82(m，4H)，3.75-3.64(m，1H)，3.49(ddd，J＝6.0，8.6，14.2Hz，1H)，2.36-2.23(m，1H)，2.20(s，3H)，1.96-1.87(m，1H)，1.86-1.75(m，1H)，1.67-1.55(m，1H)，1.47(s，9H)，1.33-1.26(m，3H)。
步骤E：将AI-5(710毫克，1.74毫摩尔)加入到氨甲醇溶液(7摩尔/升，20毫升)中，在20～30摄氏度下搅拌反应12小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Welch Ultimate XB-SiOH，250mm×50mm×10μm；流动相：A相：正己烷；B相：0.1％的氨水乙醇溶液，1％～30％；15分钟)纯化，得到中间体AI。 1H NMR(400MHz，CDCl 3)δ＝11.81(br s，1H)，8.69-8.47(m，1H)，6.24-6.07(m，1H)，5.19-4.66(m，1H)，3.94-3.82(m，1H)，3.67(ddd，J＝2.4，7.2，13.8Hz，1H)，3.47-3.38(m，1H)，2.27-2.12(m，5H)，1.86(ddd，J＝2.8，5.8，12.4Hz，1H)，1.72-1.56(m，1H)，1.43(s，9H)，1.19(d，J＝6.4Hz，3H)。
中间体AJ
Figure PCTCN2021108256-appb-000065
步骤A：将AJ-1(0.5克，2.5毫摩尔)和中间体A(606.15毫克，3毫摩尔)溶解在甲醇(6毫升)中，然后加入甲醇钠(620.41毫克，2.87毫摩尔)，反应混合物在25摄氏度下搅拌12小时。减压浓缩除去甲醇后，向剩余物中加入水(30毫升)，再用甲基叔丁基醚(40毫升)洗涤。水相用1摩尔的盐酸调至pH值5～6。过滤后，滤饼减压浓缩，得到AJ-2。 1H NMR(400MHz，CDCl 3)δ＝8.13(s，1H)，6.02-5.86(m，1H)，5.93(s，1H)，4.30(m，1H)，3.93(s，3H)，3.69-3.37(m，3H)，2.35(m，1H)，1.78(m，1H)，1.63(s，3H)，1.48(s，9H)。
步骤B：将AJ-2(470毫克，1.33毫摩尔)溶解在乙腈(6毫升)中，然后加入对甲苯磺酰氯(254.28毫克，1.33毫摩尔)和三乙胺(202.45毫克，2.0毫摩尔)。反应混合液在25摄氏度下搅拌5小时。加入二氯甲烷(60毫升)萃取，有机相用水洗涤(20毫升/次，洗涤2次)，再用饱和食盐水洗涤(20毫升/次，洗涤2次)，经无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V石油醚/乙酸乙酯＝5/1～1/1)纯化后，得到AJ-3。
步骤C：将AJ-3(0.5克，987.02微摩尔)，乙酰胺(69.96毫克，1.18毫摩尔)，磷酸钾(230.47毫克，1.09毫摩尔)和4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(114.22毫克，197.41微摩尔)依次加入到1，4-二氧六环(2毫升)中，氮气置换3次后，再加入双(苯丙烯基氯化钯)(51.73毫克，98.70微摩尔)。反应混合物在110摄氏度搅拌2小时。自然冷却后，加入水(30毫升)稀释，再用二氯甲烷萃取(30毫升/次，萃取2次)。有机相合并后，用饱和食盐水(30毫升)洗涤一次，经无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V二氯甲烷/石油醚＝10/1至3/1)纯化后，得到AJ-4。 1H NMR(400MHz，CDCl 3)δ＝10.77(s，1H)，8.31-8.17(m，1H)，7.70(s，1H)，4.37-4.15(m，1H)，3.92(s，3H)，3.65(d，J＝8.66Hz，1H)，3.55-3.39(m，2H)，2.42-2.29(m，2H)，2.24(s，3H)，1.65(s，3H)，1.50(s，9H)。
步骤D：将AJ-4(250毫克，635.43微摩尔)溶于氨甲醇溶液(7摩尔/升，1.82毫升)中，在20摄氏度搅拌3天。减压浓缩后，剩余物加入氢氧化钠溶液(1摩尔/升，30毫升)稀释，再用甲基叔丁基醚洗涤(30毫升/次，洗涤三次)。水相加入稀盐酸(2摩尔/升)调节pH值约为4，再用二氯甲烷萃取(30毫升/次，萃取两次)。有机相合并后，用饱和食盐水(30毫升)洗涤一次，经无水硫酸钠干燥， 过滤后，滤液减压浓缩，得到中间体AJ。 1H NMR(400MHz，DMSO-d 6)δ＝11.84(s，1H)，8.43(s，1H)，6.14(d，J＝4.25Hz，1H)，3.49-3.40(m，1H)，3.43(d，J＝11.63Hz，3H)，2.25(s，2H)，1.53(d，J＝4.88Hz，3H)，1.42(s，12H).
中间体AK
Figure PCTCN2021108256-appb-000066
步骤A：向AK-1(2.50克，16.28毫摩尔)的饱和的碳酸氢钠水溶液(100毫升)中，加入三光气(4.02克，13.56毫摩尔)的甲苯溶液(50毫升)，加完后，反应混合物在25摄氏度下搅拌12小时。减压浓缩，向剩余物中加入甲醇(100毫升)和乙酸乙酯(100毫升)，然后充分搅拌。过滤后，滤液减压浓缩，得到AK-2。 1H NMR(DMSO-d 6，400MHz)δ＝4.94(br s，1H)，4.50(t，J＝8.4Hz，1H)，4.27(br s，1H)，4.10-4.00(m，2H)，3.41(s，1H)，2.99(dd，J＝4.0，12.0Hz，1H)，2.12(ddd，J＝5.2，8.4，13.6Hz，1H)，1.60-1.50(m，1H)。
步骤B：将AK-2(2.30克，16.07毫摩尔)溶解在二氯甲烷(60毫升)中，随后加入三乙胺(4.88克，48.20毫摩尔)，对甲苯磺酰氯(3.37克，17.67毫摩尔)和4-二甲氨基吡啶(98.15毫克，0.80毫摩尔)。反应混合物在25摄氏度下搅拌12小时，然后加入水(40毫升)稀释，再用二氯甲烷萃取(50毫升/次，萃取2次)。合并有机相，用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AK-3。 1H NMR(DMSO-d 6，400MHz)δ＝7.80(d，J＝8.4Hz，2H)，7.51(d，J＝8.0Hz，2H)，5.2-5.0(m，1H)，4.50(t，J＝8.6Hz，1H)，4.10-4.00(m，2H)，3.60(d，J＝13.2Hz，1H)，3.20-3.10(m，1H)，2.44(s，3H)，2.40-2.30(m，1H)，1.81(ddd，J＝2.0，3.4，14.8Hz，1H)。
步骤C：将AK-3(3.50克，11.77毫摩尔)溶解在N，N-二甲基甲酰胺(60毫升)中，然后加入叠氮钠(2.12克，32.61毫摩尔)。反应混合物在80摄氏度下搅拌1小时后，用饱和碳酸钠(100毫升) 稀释，再用乙酸乙酯萃取(50毫升/次，萃取2次)。合并有机相，溶液直接用于下一步。
步骤D：向AK-4的乙酸乙酯溶液(100毫升)混合液中加入甲醇(30毫升)。充分置换氮气后，再加入钯碳(0.20克，10％含量)。反应混合物在25摄氏度下，氢气球氛围中搅拌12小时，然后过滤。滤液减压浓缩后，向剩余物中加入乙酸乙酯(100毫升)，并在30摄氏度下，搅拌30分钟。再次过滤，滤液减压浓缩，得到AK-5。 1H NMR(400MHz，DMSO-d 6)δ＝4.47(t，J＝8.2Hz，1H)，4.22-4.06(m，2H)，3.64-3.53(m，2H)，2.73-2.61(m，1H)，1.80-1.65(m，2H)，1.56(ddd，J＝5.8，8.8，12.6Hz，1H)，1.03(t，J＝7.2Hz，1H)。
步骤E：将AK-5(1.6克，11.26毫摩尔)和中间体A(2.5克，12.38毫摩尔)溶解在甲醇(20毫升)中，搅拌3小时，然后再加入甲醇钠(纯度30％，2.23克，12.38毫摩尔)。加完后，反应混合物在30摄氏度下搅拌12小时。加入水(100毫升)稀释反应液，再用乙酸乙酯洗涤(50毫升/次，洗涤取2次)。水相用盐酸(1摩尔/升)调至pH值约为4，然后用乙酸乙酯萃取(100毫升/次，萃取3次)。合并的有机相用饱和食盐水(200毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AK-6。
步骤F：将AK-6(2.4克，8.16毫摩尔)溶解在乙腈(60毫升)中，然后加入对甲苯磺酰氯(1.71克，8.97毫摩尔)和三乙胺(1.24克，12.23毫摩尔，1.7毫升)。反应混合液在30摄氏度下搅拌14小时。减压浓缩后，向剩余物中加入甲基叔丁基醚(40毫升)和水(40毫升)，并搅拌30分钟。过滤后，收集滤饼，减压干燥，得到AK-7。 1H NMR(400MHz，CDCl 3)δ＝8.16(s，1H)，7.88(d，J＝8.4Hz，2H)，7.41(d，J＝8.4Hz，2H)，6.17(s，1H)，4.96-4.82(m，1H)，4.66-4.50(m，2H)，4.31-4.18(m，2H)，3.91-3.83(m，3H)，3.43(dd，J＝5.6，12.7Hz，1H)，2.51(s，3H)，2.47-2.35(m，1H)，2.10(td，J＝9.6，13.8Hz，1H)。
步骤G：氮气保护下，将AK-7(250毫克，557.48微摩尔)，乙酰胺(32.93毫克，557.48微摩尔)，磷酸钾(130.17毫克，613.23微摩尔)，4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(9.68毫克，16.72微摩尔)和双(苯丙烯基氯化钯)(4.38毫克，8.36微摩尔)依次加入到1，4-二氧六环(2毫升)中。反应混合物在110摄氏度搅拌24小时。自然冷却后，加入水(40毫升)稀释，再用乙酸乙酯萃取(30毫升/次，萃取2次)。有机相合并后，用无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：乙酸乙酯)纯化后，得到AK-8。 1H NMR(400MHz，CDCl 3)δ＝10.81(br s，1H)，8.21(s，1H)，7.76(s，1H)，4.81-4.70(m，1H)，4.68-4.53(m，2H)，4.26-4.11(m，2H)，3.92(s，3H)，3.58-3.41(m，1H)，2.48(ddd，J＝2.4，6.6，13.6Hz，1H)，2.25(s，3H)，2.12-1.96(m，1H)。
步骤H：将AK-8(110毫克，635.43微摩尔)溶于氨甲醇溶液(7摩尔/升，20毫升)中，在20摄氏度搅拌3天。减压浓缩后，剩余物加入氢氧化钠溶液(1摩尔/升，30毫升)稀释，再用甲基叔丁基醚洗涤(30毫升/次，洗涤三次)。水相加入稀盐酸(2摩尔/升)调节pH值约为4，再用二氯甲烷萃取(30毫升/次，萃取两次)。有机相合并后，用饱和食盐水(30毫升)洗涤一次，经无水硫酸钠干燥，过滤后，滤液减压浓缩，得到中间体AK。LC-MS(ESI)m/z：303.1[M+H] +。
中间体AL
Figure PCTCN2021108256-appb-000067
步骤A：将AL-1(15克，68.42毫摩尔)溶解在二氯甲烷(100毫升)中，随后加入钛酸四乙酯(46.82克，205.26毫摩尔，42.56毫升)。反应混合物在30摄氏度下搅拌12小时。缓慢加入饱和碳酸氢钠水溶液(200毫升)淬灭反应，继续搅拌30分钟。减压过滤，滤饼用二氯甲烷洗涤(100毫升/次，洗涤2次)。分离有机相，减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V石油醚/乙酸乙酯＝10/1～3/1)纯化，得到AL-2。
步骤B：将AL-2(3.36克，10.41毫摩尔)和二氟甲基磺酰苯(2克，10.41毫摩尔)加入到四氢呋喃(30毫升)中，在氮气保护下冷却至零下78摄氏度，随后滴加双(三甲基硅基)胺基锂(1摩尔/升，20.81毫升)。滴加完成后，反应混合物在零下78摄氏度下搅拌1小时。加入水(50毫升)淬灭反应，用乙酸乙酯萃取(50毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥后，过滤，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V石油醚/乙酸乙酯＝1/1)纯化，得到AL-3。LC-MS(ESI)m/z：515.1[M+H] +。
步骤C：将AL-3(500毫克，971.62微摩尔)，乙酸钠(6.38克，77.73毫摩尔)和醋酸(10毫升)加入到N，N-二甲基甲酰胺(10毫升)中，随后分批加入镁粉(472.30毫克，19.43毫摩尔)。加完后，反应混合物在25摄氏度下搅拌4小时。过滤后，滤液用饱和碳酸氢钠水溶液调节pH值约为7，再用乙酸乙酯萃取(30毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥后，过滤，滤液减压浓缩，得到AL-4粗品，直接用于下一步反应。
步骤D：将AL-4粗品(188毫克)溶解在四氢呋喃(2毫升)中，然后加入盐酸二氧六环溶液(4 摩尔/升，250微升)。反应混合物在25摄氏度搅拌1小时。加压浓缩后，剩余物用甲基叔丁基醚(2毫升)洗涤一次，再用石油醚(2毫升)洗涤一次。减压干燥后，得到AL-5。 1H NMR(400MHz，DMSO-d 6)δ＝9.29(s，2H)，7.51-7.27(m，5H)，6.68-6.25(m，1H)，5.17-5.05(m，2H)，3.76-3.47(m，4H)，2.38-2.06(m，2H)。
步骤E：将AL-5(100毫克，326微摩尔)，中间体A(75.79毫克，374.92微摩尔)和甲醇钠(纯度25％，70.45毫克，326微摩尔)加入到甲醇(3毫升)中，反应混合物在25摄氏度下搅拌12小时。加入水(20毫升)稀释反应液，再用乙酸乙酯洗涤(20毫升/次，洗涤2次)。水相用1摩尔每升的盐酸调至pH值约为4，然后用乙酸乙酯萃取(20毫升/次，萃取3次)。合并的有机相用饱和食盐水(20毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到AL-6。
步骤F：将AL-6(250毫克，591.88微摩尔)溶解在乙腈(5毫升)中，然后加入对甲苯磺酰氯(124.13毫克，651.07微摩尔)和三乙胺(119.40毫克，1.18毫摩尔)。反应混合液在30摄氏度下搅拌14小时。加入水(20毫升)稀释，再用乙酸乙酯萃取(15毫升/次，萃取2次)。有机相合并后，用无水硫酸钠干燥。过滤后，滤液加压浓缩。剩余物经薄层色谱法(展开剂：V/V石油醚/乙酸乙酯＝2/1)纯化，得到AL-7。
步骤G：氮气保护下，将AL-7(300毫克，520.32微摩尔)，乙酰胺(30.74毫克，520.32微摩尔)，磷酸钾(132.54毫克，624.38微摩尔)，4，5-双(二苯基磷)-9，9-二甲基氧杂蒽(15.06毫克，26.02微摩尔)和双(苯丙烯基氯化钯)(13.64毫克，26.02微摩尔)依次加入到1，4-二氧六环(3毫升)中。反应混合物在110摄氏度搅拌24小时。自然冷却后，加入水(40毫升)稀释，再用乙酸乙酯萃取(30毫升/次，萃取2次)。有机相合并后，用无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：乙酸乙酯)纯化后，得到AL-8。LC-MS(ESI)m/z：464.2[M+H] +。
步骤H：将AL-8(160毫克，345.25微摩尔)溶于氨甲醇溶液(7摩尔/升，10毫升)中，在20摄氏度搅拌18小时。减压浓缩后，剩余物加入氢氧化钠溶液(1摩尔/升，20毫升)稀释，再用甲基叔丁基醚洗涤(30毫升/次，洗涤三次)。水相加入稀盐酸(2摩尔/升)调节pH值约为4，再用二氯甲烷萃取(30毫升/次，萃取两次)。有机相合并后，用饱和食盐水(30毫升)洗涤一次，经无水硫酸钠干燥，过滤后，滤液减压浓缩，得到AL-9。LC-MS(ESI)m/z：431.2[M+H] +。
步骤I：将AL-9(120毫克，278.81微摩尔)溶解在二氯甲烷(5毫升)中，随后加入氢溴酸的醋酸溶液(250微升)。反应混合物在25摄氏度下搅拌5小时，加入甲醇(1毫升)淬灭反应。减压浓缩后，剩余物用石油醚洗涤(5毫升/次，洗涤2次)。减压干燥后，得到AL-10。
步骤J：将乙酰氯(15.90毫克，202.52微摩尔)溶解在四氢呋喃(0.5毫升)中备用。将AL-10(60毫克，202.52微摩尔)和三乙胺(40.91毫克，405.04微摩尔)加入到四氢呋喃(3毫升)中，冷却至0摄氏度后，滴加前述乙酰氯的四氢呋喃溶液。加完后，反应混合物在0摄氏度下搅拌2小时。加入饱和碳酸氢钠水溶液(5毫升)稀释反应液，再用乙酸乙酯萃取(15毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥，过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：乙酸乙酯)纯化，得到中间体AL。LC-MS(ESI)m/z：339.1[M+H] +。
中间体B
Figure PCTCN2021108256-appb-000068
步骤A：向B-1(3.00克，13.69毫摩尔)的四氢呋喃(120毫升)溶液中加入碳酸铯(4.46克，13.69毫摩尔)和(±)-叔丁基亚磺酰胺(1.66克，13.69毫摩尔)。反应混合物在20摄氏度搅拌0.5小时，然后加入水(100毫升)淬灭反应，再用100毫升乙酸乙酯萃取两次(50毫升/次)。分液后，有机相用无水硫酸钠干燥，过滤后减压浓缩得到B-2粗品，直接用于下一步反应。
步骤B：在零下78摄氏度下，向B-2(4.40克，13.65毫摩尔)的四氢呋喃(100毫升)溶液中滴加乙烯基溴化镁(1摩尔/升，16.38毫升，16.38毫摩尔)。加完后，反应混合物在零下78摄氏度搅拌1小时，然后自然升温至0摄氏度，用饱和氯化铵水溶液(100毫升)淬灭，再用100毫升乙酸乙酯萃取两次(50毫升/次)。分液后，有机相用无水硫酸钠干燥，过滤后减压浓缩。剩余物经硅胶柱层析(洗脱剂：石油醚/乙酸乙酯＝5/1～3/1)纯化后得到B-3。
步骤C：向B-3(3.70克，10.56毫摩尔)的四氢呋喃(30毫升)溶液中加入氯化氢的1，4-二氧六环溶液(4摩尔/升，7.92毫升)。混合液在20摄氏度搅拌3小时，随后加入石油醚(60毫升)，经布氏漏斗过滤，收集的滤饼并真空干燥后得到B-4粗品，直接用于下一步反应。 1H NMR(400MHz，DMSO-d 6)δ＝9.16-8.81(m，3H)，8.75(s，1H)，8.55(s，1H)，8.44(br s，1H)，6.22-6.06(m，1H)，5.53-5.28(m，3H)。
步骤D：将B-4(2.50克，10.15毫摩尔)溶解在乙醇(50毫升)中，依次加入还原铁粉(2.84克，50.77毫摩尔)、氯化铵(2.72克，50.77毫摩尔)和水(10毫升)。反应混合物在60摄氏度下搅拌3小时。自然冷却至室温后，加入碳酸钠水溶液(100毫升)淬灭，再用100毫升乙酸乙酯萃取两次(50毫升/次)。有机相合并后，用无水硫酸钠干燥，过滤后，滤液减压浓缩，得到中间体B，直接用于下一步反应。LC-MS(ESI)m/z：217.2[M+H] +。
中间体C
Figure PCTCN2021108256-appb-000069
Figure PCTCN2021108256-appb-000070
步骤A：向C-1(10.00克，45.46毫摩尔)的四氢呋喃(100毫升)溶液中加入碳酸铯(14.87克，45.64毫摩尔)和(R)-叔丁基亚磺酰胺(5.53克，45.64毫摩尔)。反应混合物在20摄氏度搅拌0.5小时，然后加入水(100毫升)淬灭反应，再用200毫升乙酸乙酯萃取两次(100毫升/次)。合并的有机相用饱和食盐水洗涤一次(100毫升)，经无水硫酸钠干燥，过滤后滤液减压浓缩得到C-2粗品，直接用于下一步反应。
步骤B：在零下78摄氏度下，向C-2(14.90克，46.23毫摩尔)的四氢呋喃(300毫升)溶液中滴加乙烯基溴化镁(1摩尔/升，55.48毫升，55.48毫摩尔)。加完后，反应混合物在零下78摄氏度搅拌1小时，然后自然升温至0摄氏度，用饱和氯化铵水溶液(100毫升)淬灭，再用200毫升乙酸乙酯萃取两次(100毫升/次)。合并的有机相用饱和食盐水洗涤一次(100毫升)，经无水硫酸钠干燥，过滤后减压浓缩。剩余物经硅胶柱层析(洗脱剂：石油醚/乙酸乙酯＝5/1～3/1)纯化后得到C-3。 1H NMR(400MHz，CDCl 3)δ＝8.37(s，2H)，7.89(s，1H)，5.90-5.87(m，1H)，5.47-5.26(m，2H)，5.08(dd，J＝4.2，7.2Hz，1H)，3.55(d，J＝3.9Hz，1H)，1.21(s，9H)。
步骤C：向C-3(6.20克，17.70毫摩尔)的四氢呋喃(100毫升)溶液中加入氯化氢的1，4-二氧六环溶液(4摩尔/升，8.85毫升)。混合液在20摄氏度搅拌1小时，随后加入石油醚(20毫升)，经布氏漏斗过滤，收集的滤饼经真空干燥后得到C-4粗品，直接用于下一步反应。
步骤D：将C-4(4.3克，15.21毫摩尔)溶解在乙醇(100毫升)中，依次加入还原铁粉(4.25克，76.07毫摩尔)、氯化铵(4.07克，76.07毫摩尔)和水(10毫升)。反应混合物在60摄氏度下搅拌3小时。自然冷却至室温后，加入碳酸钠水溶液(100毫升)淬灭，再用300毫升乙酸乙酯萃取三次(100毫升/次)。有机相合并后，用无水硫酸钠干燥，过滤后，滤液减压浓缩，得到中间体C，直接用于下一步反应。 1H NMR(400MHz，CDCl 3)δ＝6.99(s，1H)，6.87(s，1H)，6.81(s，1H)，6.01-5.97(m，1H)，5.29(dd，J＝1.3，17.1Hz，1H)，5.17(dd，J＝1.1，10.2Hz，1H)，4.51(d，J＝5.6Hz，1H)，4.20-3.38(m，2H)。LC-MS(ESI)m/z：217.2[M+H] +。
中间体D
Figure PCTCN2021108256-appb-000071
步骤A：将化合物D-1(25克，130.13毫摩尔)加入到浓硫酸(40毫升)中，随后在0摄氏度下滴加发烟硝酸(19.87克，315.33毫摩尔)。加完后，反应混合物自然升温至20摄氏度后，继续搅拌2小时，然后缓慢倒入冰水(200毫升)中，再用二氯甲烷萃取(100毫升/次，萃取3次)。将萃取的有机相合并后，用1摩尔/升的氢氧化钠水溶液(100毫升)洗涤一次，然后，水相再用二氯甲烷萃取(50毫升/次，萃取2次)。所有的有机相合并后，减压浓缩除去溶剂，得到化合物D-2，直接用于下一步反应。 1H NMR(400MHz，CDCl 3)δ＝10.43(s，1H)，8.96(dd，J＝2.8，5.6Hz，1H)，8.78(dd，J＝2.4，5.6Hz，1H)。
步骤B：将D-2(26克，109.65毫摩尔)加入到四氢呋喃(200毫升)中，随后加入(R)-叔丁基亚磺酰胺(13.29克，109.65毫摩尔)和碳酸铯(35.73克，109.65毫摩尔)。反应混合物在2摄氏度下搅拌30分钟。加入水(300毫升)稀释后，用乙酸乙酯萃取(300毫升/次，萃取2次)。有机相合并后，用饱和食盐水洗涤(300毫升/次，洗涤1次)，然后用无水硫酸钠干燥。过滤后，滤液减压浓缩，剩余物经硅胶柱层析(洗脱剂：V/V，石油醚/乙酸乙酯＝10/1～5/1)纯化，得到D-3。 1H NMR(400MHz，CDCl 3)δ＝9.08(dd，J＝2.8，5.4Hz，1H)，8.95(s，1H)，8.68(dd，J＝2.8，5.6Hz，1H)，1.34(s，9H)。
步骤C：在20摄氏度，氮气保护下，将乙烯基溴化镁溶液(1摩尔/升，88.16毫升)缓慢加入到二甲基锌溶液(1摩尔/升，101.38毫升)中，加完后搅拌3小时。随后，降温至零下78摄氏度，缓慢滴加D-3(15克，44.08毫摩尔)的四氢呋喃(100毫升)溶液。加完后，反应混合物在零下78摄氏度下搅拌30分钟。用饱和氯化铵水溶液(200毫升)淬灭反应，再用乙酸乙酯萃取(300毫升/次，萃取2次)。有机相合并后，用饱和食盐水洗涤(300毫升/次，洗涤1次)，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V，乙酸乙酯/石油醚＝5/1～3/1)纯化，得到D-4。 1H NMR(400MHz，CDCl 3)δ＝8.53(ddd，J＝2.8，5.6，18.0Hz，2H)，5.98(ddd，J＝6.8，10.0，17.0Hz，1H)，5.50-5.30(m，3H)，3.70(d，J＝5.6Hz，1H)，1.29(s，9H).LC-MS(ESI)m/z：369.1[M+H] +。
步骤D：向D-4(2.5克，6.79毫摩尔)的四氢呋喃(20毫升)溶液中加入氯化氢的1，4-二氧六环溶液(4摩尔/升，3.4毫升)。反应混合物在20摄氏度下搅拌1小时。加入石油醚(80毫升)稀释后，过滤后收集滤饼，得到D-5，直接用于下一步反应。
步骤E：将D-5(2克，7.57毫摩尔)加入到乙醇(50毫升)和水(10毫升)的混合溶剂中，随后加入还原铁粉(2.11克，37.85毫摩尔)和氯化铵(2.02克，37.85毫摩尔)。反应体系用氮气置换3次后，在60摄氏度搅拌3小时。冷却后，反应混合物过滤。滤液用饱和碳酸钠水溶液(30毫升)中和，再用乙酸乙酯萃取(30毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到中间体D。
中间体E
Figure PCTCN2021108256-appb-000072
步骤A：将E-1(6.42克，30.57毫摩尔)加入到二甲基亚砜(50毫升)中，随后加入氟化钾(3.55克，61.14毫摩尔)和碘化亚铜(11.64克，61.14毫摩尔)，最后加入(三甲基硅基)二氟乙酸乙酯(12克，61.14毫摩尔)。反应混合物在氮气保护下，与60摄氏度搅拌15小时。自然冷却后，加入叔丁基甲醚(200毫升)和水(300毫升)稀释反应液，并继续搅拌10分钟，然后过滤。滤饼用叔丁基甲醚洗涤(30毫升/次，洗涤3次)。滤液合并，分离得到有机相，依次用水(300毫升)和饱和食盐水(300毫升)各洗涤一次，再用无水硫酸钠干燥。过滤，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：石油醚)分离纯化，得到E-2。 1H NMR(400MHz，CDCl 3)δ＝7.75-7.65(m，1H)，7.44-7.38(m，1H)，7.28(s，1H)，4.36(q，J＝7.2Hz，2H)，1.36(t，J＝7.2Hz，3H)。
步骤B：将E-2(3.8克，18.43毫摩尔)加入到四氢呋喃(30毫升)和乙醇(5毫升)的混合溶剂中，冷却至0摄氏度后，加入硼氢化钠(1.40克，36.86毫摩尔)。加完后，反应混合物在15～20摄氏度下搅拌2小时。用饱和氯化铵水溶液(50毫升)淬灭反应，随后加入水(50毫升)稀释反应液，再 用叔丁基甲醚萃取(50毫升/次，萃取2次)。合并的有机相用饱和食盐水洗涤(50毫升/次，洗涤2次)，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经硅胶柱层析(洗脱剂：V/V石油醚/乙酸乙酯＝10/1)分离纯化，得到E-3。
步骤C：将E-3(2.37克，14.44毫摩尔)加入到无水二氯甲烷(30毫升)中，随后依次加入咪唑(1.08克，15.88毫摩尔)和4-二甲氨基吡啶(176.36毫克，1.44毫摩尔)。搅拌下，缓慢滴加叔丁基二苯基氯硅烷(4.17克，15.16毫摩尔)。加完后，反应混合物在10～15摄氏度下搅拌16小时。加入水(100毫升)稀释反应液.有机相用饱和食盐水(100毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余经硅胶柱层析(洗脱剂：V/V，石油醚/乙酸乙酯＝25/1)分离纯化，得到E-4。 1H NMR(400MHz，CDCl 3)δ＝7.61-7.55(m，5H)，7.45-7.36(m，7H)，7.18(dd，J＝1.2，5.0Hz，1H)，3.99(t，J＝12.0Hz，2H)，1.02(s，9H)。
步骤D：氮气保护下，将E-4(1.5克，3.73毫摩尔)加入到无水四氢呋喃(20毫升)中。冷却至零下65摄氏度后，缓慢滴加二异丙基氨基锂溶液(1摩尔/升，6.71毫升)。加完后，反应混合物在零下65摄氏度下搅拌1小时。随后加入N，N-二甲基甲酰胺(1.63克，22.36毫摩尔)，然后在零下65摄氏度下继续搅拌30分钟。加入水(40毫升)稀释反应液，用叔丁基甲醚萃取(30毫升/次，萃取2次)。合并的有机相用饱和食盐水(50毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩，得到E-5粗品，直接用于下一步反应。
步骤E：将E-5(1.6克，3.72毫摩尔)加入到四氢呋喃(20毫升)中，随后依次加入(R)-叔丁基亚磺酰胺(495.41毫克，4.09毫摩尔)和碳酸铯(1.33克，4.09毫摩尔)。反应混合物在10～15摄氏度下搅拌16小时。用水(40毫升)稀释反应液，再用叔丁基甲醚萃取(30毫升/次，萃取2次)。合并的有机相用饱和食盐水(40毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：V/V，石油醚/乙酸乙酯＝20/1)分离纯化，得到E-6。 1H NMR(400MHz，CDCl 3)δ＝8.62(d，J＝0.6Hz，1H)，7.75(s，1H)，7.56(td，J＝1.4，8.0Hz，4H)，7.52(d，J＝1.4Hz，1H)，7.487.42(m，2H)，7.40-7.36(m，4H)，3.99(t，J＝11.8Hz，2H)，1.27(s，9H)，1.02(s，9H)。LC-MS(ESI)m/z：534.2[M+H] +。
步骤F：在氮气保护下，将E-6(2.6克，4.87毫摩尔)加入到无水四氢呋喃(50毫升)中。冷却至零下60至零下65摄氏度间，缓慢滴加乙烯基溴化镁溶液(1摩尔/升，9.74毫升)。加完后，反应混合物在零下65摄氏度下搅拌10分钟后，自然升至0摄氏度，继续搅拌50分钟。用饱和氯化铵水溶液(50毫升)缓慢淬灭反应，然后加入水(60毫升)稀释，再用叔丁基甲醚萃取(60毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：V/V，石油醚/乙酸乙酯＝5/1)分离纯化，得到E-7。
步骤G：将E-7(930毫克，1.66毫摩尔)加入到四氢呋喃(5毫升)中，随后加入氯化氢的1，4-二氧六环溶液(4摩尔/升，1.24毫升)。反应混合物在10～15摄氏度下搅拌2小时。随后加入石油醚(40毫升)稀释，并在10～15摄氏度下继续搅拌30分钟。过滤后，滤饼减压干燥，得到中间体E。 1H NMR(400MHz，CD 3OD)δ＝7.80(s，1H)，7.58(d，J＝6.6Hz，4H)，7.52-7.31(m，8H)，6.17(ddd，J＝6.8，10.4，17.2Hz，1H)，5.58-5.49(m，2H)，5.29(d，J＝6.8Hz，1H)，4.03(t，J＝12.2Hz，2H)，1.01(s，9H)。
中间体F
Figure PCTCN2021108256-appb-000073
步骤A：0摄氏度下，向F-1(10.00克，49.26毫摩尔)的二氯甲烷(100毫升)溶液中滴加二乙胺基三氟化硫(15.88克，98.52毫摩尔)。反应混合物于0摄氏度搅拌1小时，然后缓慢加入饱和碳酸氢钠水溶液至pH＝8，再用二氯甲烷萃取(50毫升/次，萃取三次)，合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得F-2粗品，直接用于下一步反应。
步骤B：在零下78摄氏度、氮气保护下，向F-2(5.00克，22.22毫摩尔)的四氢呋喃(50毫升)溶液中滴加正丁基锂(2.5摩尔每升，13.33毫升，33.33毫摩尔)。加完后，反应混合物在零下78摄氏度搅拌1小时，然后加入N，N-二甲基甲酰胺(3.25克，44.44毫摩尔)，再于零下78摄氏度继续搅拌1小时。反应混合物倒入稀盐酸(1摩尔/升，100毫升)中，然后用乙酸乙酯萃取(30毫升/次，萃取三次)。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩得F-3粗品，直接用于下一步反应。
步骤C：向F-3(3.20克，18.38毫摩尔)的四氢呋喃(60毫升)溶液中加入碳酸铯(5.99克，18.38毫摩尔)和(R)-叔丁基亚磺酰胺(2.23克，18.38毫摩尔)。反应混合物在0摄氏度下搅拌1小时，然后加入水(200毫升)稀释，再用乙酸乙酯萃取(100毫升/次，萃取2次)。合并的有机相用无水硫酸钠干燥，过滤后减压浓缩。剩余物经制备HPLC(色谱柱：Phenomenex luna C18，250×50mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，40-70％；18分钟)得到F-4。 1H NMR(400MHz，DMSO-d 6)δ＝8.72(s，1H)，8.19(t，J＝7.0Hz，1H)，7.89(t，J＝7.0Hz，1H)，7.52(t，J＝7.8Hz，1H)，7.48-7.14(m，1H)，1.21(s，9H)。
步骤D：在20摄氏度、氮气保护下，向二甲基锌甲苯溶液(1摩尔/升，4.15毫摩尔)中滴加乙烯基溴化镁溶液(1摩尔/升，3.61毫摩尔)。加完后，反应混合物在20摄氏度搅拌4小时，然后冷却至-78摄氏度，随后，缓慢滴加F-4(0.50克，1.80毫摩尔)的四氢呋喃(10毫升)溶液。完成后，继续在零下78摄氏度搅拌0.5小时。反应混合物用饱和氯化铵水溶液(200毫升)淬灭，然后用乙酸乙酯萃取(300毫升/次，萃取两次)。合并的有机相，用饱和氯化钠(300毫升/次)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经柱硅胶层析(洗脱剂：V/V，石油醚/乙酸乙酯＝5/1～3/1)纯 化，得到F-5。 1H NMR(400MHz，DMSO-d 6)δ＝7.70(t，J＝7.0Hz，1H)，7.56(t，J＝7.0Hz，1H)，7.42-7.07(m，2H)，6.04-5.95(m，1H)，5.35-5.08(m，3H)，1.13(s，9H)。
步骤E：向F-5(0.40克，1.31毫摩尔)的四氢呋喃(5毫升)溶液中加入氯化氢的1，4-二氧六环溶液(4摩尔/升，0.98毫升)。混合液在15～20摄氏度下搅拌2小时后，加入石油醚(40毫升)，继续搅拌30分钟。过滤后，收集的滤饼。减压干燥后，得到中间体F。
化合物1
Figure PCTCN2021108256-appb-000074
将中间体AA(20毫克，76.55微摩尔)，六氯三聚磷腈(29.27毫克，84.20微摩尔)和磷酸钾(40.62毫克，191.37微摩尔)加入到乙腈(2毫升)中，在20～25摄氏度下搅拌1小时，然后加入中间体B(16.55毫克，76.55微摩尔)，继续搅拌1小时。减压浓缩除去溶剂后，剩余物经制备HPLC分离纯化(柱子：Unisil 3-100 C18 Ultra 150×50mm×3μm，流动相：A相：0.225％三氟乙酸水溶液；B相：乙腈，20％～40％，10分钟)得到化合物1。 1H NMR(400MHz，CD 3OD)δ＝9.09(s，1H)，6.93(d，J＝6.5Hz，2H)，6.85(s，1H)，6.42(s，1H)，6.24-6.12(m，2H)，5.44-5.22(m，3H)，4.12(d，J＝11.8Hz，2H)，3.63(t，J＝11.8Hz，2H)，2.39(s，3H)，2.14-2.04(m，2H)，1.93(d，J＝8.6Hz，2H)。LC-MS(ESI)m/z：460.4[M+H] +。
化合物2
Figure PCTCN2021108256-appb-000075
Figure PCTCN2021108256-appb-000076
化合物2的制备参考实施例1的制备流程，将中间体AA替换成中间体AB。
化合物2由制备HPLC分离纯化(分离条件：柱子：Unisil 3-100 C18 Ultra 150×50mm×3μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，18％～38％，10分钟)得到。 1H NMR(400MHz，DMSO-d 6)δ＝9.00(s，1H)，8.87(s，1H)，8.28(s，1H)，6.84-6.73(m，3H)，6.22-6.07(m，3H)，5.63(br s，2H)，5.36-5.26(m，2H)，4.36(dd，J＝4.9，9.0Hz，1H)，3.96-3.82(m，3H)，2.26(s，3H)，1.59(s，3H)。LC-MS(ESI)m/z：460.3[M+H] +。
化合物3
Figure PCTCN2021108256-appb-000077
化合物3的制备参考实施例1的制备流程，将中间体AA替换成中间体AC。
化合物3由制备HPLC分离纯化(分离条件：柱子：Phenomenex luna C18 150×25mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，12％～42％，10分钟)得到。 1H NMR(400MHz，DMSO-d 6)δ＝9.21(s，2H)，6.90-6.64(m，3H)，6.29-6.02(m，3H)，5.90-5.52(m，2H)，5.42-5.22(m，2H)，4.92-4.37(m，2H)，2.28(s，3H)，1.31(br s，4H)。LC-MS(ESI)m/z：448.1[M+H] +。
化合物4
Figure PCTCN2021108256-appb-000078
化合物4的制备参考实施例1的制备流程，将中间体AA替换成中间体AD。
化合物4由制备HPLC分离纯化(分离条件：柱子：Phenomenex luna C18 150×25mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，12％～42％，10分钟)得到。 1H NMR(400MHz，DMSO-d 6)δ＝8.95-9.07(m，1H)，8.73(s，1H)，6.83-6.73(m，3H)，6.20-6.10(m，2H)，6.03(s，1H)，5.63(br s，2H)，5.35-5.24(m，2H)，3.16(s，1H)，2.42-2.35(m，6H)，2.24(s，3H)，1.75-1.68(m，6H)。LC-MS(ESI)m/z：500.1[M+H] +。
化合物5
Figure PCTCN2021108256-appb-000079
化合物5的制备参考实施例1的制备流程，将中间体AA替换成中间体AE，将中间体B替换成中间体C。
化合物5由制备HPLC分离纯化(分离条件：柱子：Unisil 3-100 C18 Ultra 150×50mm×3μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，18％～38％，10分钟)得到。 1H NMR(400MHz，CD 3OD)δ＝9.32(s，1H)，6.99-6.92(m，2H)，6.88(s，1H)，6.39-6.14(m，3H)，5.42-5.27(m，2H)，2.45(s，3H)，1.60(s，3H)，1.24(t，J＝3.1Hz，2H)，1.17-1.05(m，2H)。LC-MS(ESI)m/z：430.2[M+H] +。
化合物6
Figure PCTCN2021108256-appb-000080
将中间体AF(60毫克，224.52微摩尔)加入到乙腈(5毫升)中，随后加入磷酸钾(119.15毫克，561.31微摩尔)和六氯三聚磷腈(78.06毫克，224.52微摩尔)。反应混合物在15摄氏度下搅拌1小时。随后加入中间体D(52.58毫克，224.52微摩尔)，并继续搅拌1小时。然后加入氨水(25％，1毫升)，搅拌1小时；再加入饱和碳酸钾水溶液(30毫升)搅拌16小时。用乙酸乙酯萃取(20毫升/次，萃取2次)。有机相合并后，减压浓缩。剩余物经制备HPLC(色谱柱：Unisil 3-100 C18 Ultra 150×50mm×3μm；流动相：A相：0.225％甲酸水溶液；B相：乙腈，13％-43％；10分钟)分离纯化，得到化合物6。 1H NMR(400MHz，DMSO-d 6)δ＝9.35-9.06(m，2H)，6.83(dd，J＝2.6，5.6Hz，1H)，6.78(dd，J＝2.8，5.4Hz，1H)，6.51-6.10(m，4H)，5.64-5.37(m，2H)，5.36-5.18(m，2H)，2.25(s，3H)，1.56-1.27(m，4H)。LC-MS(ESI)m/z：484.2[M+H] +。
化合物7
Figure PCTCN2021108256-appb-000081
步骤A：将中间体AF(200毫克，748.42微摩尔)加入到乙腈(10毫升)中，随后加入磷酸钾(397.16毫克，1.87毫摩尔)和六氯三聚磷腈(260.19毫克，748.42微摩尔)。反应混合物在15摄氏度下搅拌1小时。随后加入中间体E(369.80毫克，448.42微摩尔)，并继续搅拌1小时。然后加入氨水(25％，1毫升)，搅拌1小时；再加入饱和碳酸钾水溶液(30毫升)搅拌16小时。用乙酸乙酯萃取(30毫升/次，萃取2次)。有机相合并后，减压浓缩。剩余物经制备HPLC(色谱柱：Welch Ultimate XB-SiOH 250×70×10μm；流动相：A相：正己烷；B相：0.1％氨乙醇溶液，1％-35％；20分钟)分离纯化，得到7-1。
步骤B：将7-1(360毫克，509.28微摩尔)加入到甲醇(10毫升)中，随后加入氟化铵(188.63毫克，5.09毫摩尔)。反应混合物在70摄氏度下搅拌2小时。减压浓缩后，剩余物经制备HPLC(层析柱：Waters Xbridge 150×25mm×5μm；流动相：A相：氨水；B相：乙腈；22％-52％；9分钟)分离纯化，得到化合物7。 1H NMR(400MHz，CD 3OD)δ＝9.12(s，1H)，7.67-7.52(m，1H)，7.18(s，1H)，6.51(dd，J＝1.0，6.4Hz，1H)，6.38(s，1H)，6.33-6.05(m，2H)，5.48-5.34(m，2H)，3.90(t，J＝13.2Hz，2H)，2.42(s，3H)，1.66-1.27(m，4H)。LC-MS(ESI)m/z：469.1[M+H] +。
化合物8
Figure PCTCN2021108256-appb-000082
Figure PCTCN2021108256-appb-000083
将中间体AF(700毫克，2.62毫摩尔)，六氯三聚磷腈(867.31毫克，2.50毫摩尔)和磷酸钾(1.32克，6.24毫摩尔)加入到乙腈(20毫升)中，在155摄氏度下搅拌1小时，然后加入中间体F(593毫克，2.50毫摩尔)，继续搅拌1小时。减压浓缩除去溶剂后，剩余物经制备HPLC分离纯化(色谱柱：Waters Xbridge BEH C18 250×50mm×10μm；流动相：A相：氨水；B相：乙腈，35％-65％；20分钟)得到化合物8。 1H NMR(400MHz，CD 3OD)δ＝9.21(s，1H)，7.70-7.50(m，2H)，7.39-7.25(m，1H)，7.20-6.84(m，1H)，6.56-5.95(m，4H)，5.42-5.20(m，2H)，2.33(s，3H)，1.69-1.31(m，4H)。LC-MS(ESI)m/z：451.2[M+H] +。
化合物9
Figure PCTCN2021108256-appb-000084
步骤A：将中间体AG(270毫克，1.14毫摩尔)，六氯环三磷腈(394.99毫克，1.14毫摩尔)和磷酸钾(602.92毫克，2.84毫摩尔)加入到乙腈(10毫升)中。反应混合物在20摄氏度搅拌1小时，随后加入中间体F(446.67毫克，1.19毫摩尔)，继续在20摄氏度下搅拌2小时。过滤后，滤液减压浓缩。剩余物经制备HPLC(色谱柱：Welch Ultimate XB-SiOH，250mm×70mm×3μm；流动相：A相：正己烷；B相：0.1％的氨水乙醇溶液，1％～35％；20分钟)纯化，得到9-1。 1H NMR(400MHz，CDCl3)δ＝9.02-8.91(m，1H)，7.76-7.51(m，2H)，7.33(t，J＝7.8Hz，1H)，7.20-6.84(m，1H)，6.55-6.43(m，1H)，6.36-6.30(m，1H)，5.47-5.23(m，2H)，4.61(br s，1H)，3.90-3.79(m，2H)，3.63(d，J＝7.2Hz，2H)，2.58-2.42(m，2H)，2.38(s，3H)，1.84(s，2H)，1.55-1.38(m，9H)，1.20(s，3H)。
步骤B：将9-1(156毫克，279.77微摩尔)溶于1，4-二氧六环(3毫升)中，随后加入盐酸二氧六环溶液(4摩尔/升，1毫升)。反应混合物在40摄氏度下搅拌2小时后，减压浓缩得到9-2。LC-MS(ESI)m/z：458.0[M+H] +。
步骤C：将醋酸(7.29毫克，121.47微摩尔)，N，N-二异丙基乙胺(52.33毫克，404.90微摩尔)和2-(7-偶氮苯并三氮唑)-N，N，N，N-四甲基脲六氟磷酸酯(46.19毫克，121.47微摩尔)加入到N，N-二甲基甲酰胺(4毫升)中。反应混合物在20摄氏度搅拌15分钟后，再加入9-2(40毫克，80.98微摩尔)。反应混合物在20摄氏度继续搅拌1小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Phenomenex Synergi C18，150mm×25mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：10％～10％乙腈；10分钟)纯化，得到化合物9。 1H NMR(400MHz，CDCl 3)δ＝9.22-9.06(m，1H)，8.84(s，1H)，7.72-7.64(m，1H)，7.60-7.52(m，1H)，7.39-7.09(m，2H)，6.44-6.32(m，1H)，6.23(ddd，J＝6.1，10.4，16.9Hz，1H)，6.07(s，1H)，5.36-5.13(m，2H)，4.06-3.90(m，1H)，3.61(dd，J＝5.2，13.6Hz，1H)，3.19(td，J＝6.6，13.6Hz，2H)，2.64-2.53(m，1H)，2.34-2.23(m，3H)，2.19(d，J＝1.6Hz，3H)，2.00(s，3H)，1.79-1.68(m，3H)。LC-MS(ESI)m/z：500.2[M+H] +。
化合物10
Figure PCTCN2021108256-appb-000085
将9-2(75毫克，151.84微摩尔)和2，6-二甲基吡啶(56.94毫克，531.43微摩尔)加入到二氯甲烷 (1毫升)中，混合物冷却至0摄氏度，随后加入氯甲酸甲酯(12.91毫克，136.65微摩尔)。加完后，反应混合物在20摄氏度搅拌反应1小时。再次冷却至0摄氏度后，加入水(20毫升)淬灭反应，然后用乙酸乙酯萃取(20毫升/次，萃取两次)。合并的有机相用水(20毫升)洗一次，饱和食盐水(20毫升)洗涤一次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经制备HPLC(色谱柱：Unisil 3-100 C18 Ultra，150mm×50mm×3μm；流动相：A相：0.225％甲酸水溶液；B相：13％～43％乙腈；15分钟)纯化，得到化合物10。 1H NMR(400MHz，DMSO-d 6)δ＝9.23-8.96(m，1H)，8.87-8.77(m，1H)，7.74-7.51(m，2H)，7.40-7.08(m，2H)，6.46-6.35(m，1H)，6.30-6.16(m，1H)，6.11-6.03(m，1H)，5.38-5.12(m，2H)，3.77-3.70(m，2H)，3.59(s，3H)，3.28(br s，2H)，2.46-2.37(m，2H)，2.31-2.22(m，2H)，2.19(s，3H)，1.73(s，3H)。LC-MS(ESI)m/z：516.2[M+H] +。
化合物11
Figure PCTCN2021108256-appb-000086
步骤A：将中间体AH(200毫克，554.93微摩尔)，磷酸钾(294.48毫克，1.39毫摩尔)和六氯环三磷腈(192.93毫克，554.93微摩尔)加入到乙腈(3毫升)中，反应混合物在20～30摄氏度搅拌1小时后，加入中间体F(145.07毫克，610.42微摩尔)，然后继续搅拌12小时。再加入氨水(质量分数：25％，0.5毫升)搅拌1小时，随后加入碳酸钾水溶液(质量分数：20％，20毫升)继续搅拌1小时。用二氯甲烷(50毫升)萃取一次。有机相用饱和食盐水(10毫升)洗涤一次，再用无水硫酸钠干 燥。过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：V/V二氯甲烷/甲醇＝10/1)分离纯化后，得到11-1。LC-MS(ESI)m/z：544.3[M+H] +。
步骤B：将11-1(110毫克，202.36微摩尔)加入到1，4-二氧六环(3毫升)中，随后加入盐酸二氧六环溶液(4摩尔/升，1毫升)。反应混合物在30摄氏度下搅拌2小时。减压浓缩后，得到11-2。LC-MS(ESI)m/z：444.2[M+H] +。
步骤C：将醋酸(18.77毫克，312.55微摩尔)，2-(7-偶氮苯并三氮唑)-N，N，N，N-四甲基脲六氟磷酸酯(118.84毫克，312.55微摩尔)和N，N-二异丙基乙胺(107.72毫克，833.46微摩尔)加入到N，N-二甲基甲酰胺(1毫升)中，反应混合物在20～30摄氏度搅拌15分钟。再加入11-2(100毫克，208.37微摩尔)，继续在20～30摄氏度搅拌0.5小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Phenomenex Synergi C18，150mm×25mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：13％～46％乙腈；11分钟)纯化，得到11。 1H NMR(400MHz，MeOD)δ＝9.24(m，1H)，7.68(t，J＝7.2Hz，1H)，7.61(t，J＝7.0Hz，1H)，7.43-7.31(m，1H)，7.21-6.86(m，1H)，6.54(m，1H)，6.41-6.23(m，2H)，5.86-5.67(m，1H)，5.44(d，J＝10.4Hz，1H)，5.33(d，J＝17.4Hz，1H)，4.56-4.41(m，1H)，4.28-3.99(m，1H)，3.77-3.59(m，1H)，2.73-2.62(m，1H)，2.47(d，J＝2.8Hz，3H)，2.34-2.20(m，1H)，2.19-2.03(m，3H)，1.43-1.33(m，3H).LC-MS(ESI)m/z：486.3[M+H] +。
化合物12
Figure PCTCN2021108256-appb-000087
步骤A：将中间体AI(200毫克，534.14微摩尔)，磷酸钾(283.46毫克，1.34毫摩尔)和六氯环三磷腈(185.70毫克，554.93微摩尔)加入到乙腈(7毫升)中，反应混合物在20～30摄氏度搅拌1小时。再加入中间体F(139.63毫克，587.55微摩尔)，继续搅拌12小时。随后加入氨水(质量分数：25％，0.5毫升)搅拌1小时，在加入碳酸钾水溶液(质量分数：20％，20毫升)继续搅拌1小时。用二氯甲烷(50毫升)萃取一次后，有机相用饱和食盐水(10毫升)洗涤依次，再用无水硫酸钠干燥。过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：V/V二氯甲烷/甲醇＝10/1)分离纯化后，得到12-1。 1H NMR(400MHz，CDCl3)δ＝8.61-8.42(m，1H)，7.44(td，J＝6.6，12.9Hz，2H)，7.16-7.05(m，1H)，6.98-6.64(m，1H)，6.49-6.38(m，1H)，6.37-6.23(m，1H)，6.16-5.99(m，1H)，5.29-5.07(m，2H)，5.01-4.82(m，1H)，3.84-3.69(m，1H)，3.64-3.53(m，1H)，3.28(ddd，J＝6.0，8.6，14.4Hz，1H)，2.37-2.29(m，3H)，2.24-2.08(m，1H)，1.90-1.82(m，1H)，1.80-1.71(m，1H)，1.66-1.57(m，1H)，1.42-1.34(m，9H)，1.18-1.10(m，3H)。LC-MS(ESI)m/z：558.3[M+H] +。
步骤B：将12-1(200毫克，358.68微摩尔)加入到1，4-二氧六环(3毫升)中，随后加入盐酸二氧六环溶液(4摩尔/升，1毫升)。反应混合物在30摄氏度下搅拌2小时。液减压浓缩后，得到12-2。LC-MS(ESI)m/z：458.2[M+H] +。
步骤C：将醋酸(32.28毫克，537.50微摩尔)，2-(7-偶氮苯并三氮唑)-N，N，N，N-四甲基脲六氟磷酸酯(204.37毫克，537.50微摩尔)和N，N-二异丙基乙胺(185.24毫克，1.43毫摩尔)加入到N，N-二甲基甲酰胺(1毫升)中，反应混合物在20～30摄氏度搅拌15分钟。再加入12-2(177毫克，358.33微摩尔)，继续搅拌0.5小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Phenomenex Synergi C18，150mm×25mm×10μm；流动相：A相：0.225％甲酸水溶液；B相：15％～54％乙腈；13分钟)纯化，得到化合物12。 1H NMR(400MHz，MeOD)δ＝9.33-9.20(m，1H)，7.76-7.68(m，1H)，7.67-7.57(m，1H)，7.42-7.31(m，1H)，7.18-6.87(m，1H)，6.59-6.50(m，1H)，6.39-6.24(m，2H)，5.51-5.41(m，1H)，5.38-5.27(m，1H)，5.06-4.91(m，3H)，4.44-4.28(m，1H)，2.54-2.44(s，3H)，2.35-2.22(m，1H)，2.22-2.14(m，4H)，2.09-1.92(m，1H)，1.85-1.72(m，1H)，1.50-1.29(m，3H)。LC-MS(ESI)m/z：500.3[M+H] +。
化合物13
Figure PCTCN2021108256-appb-000088
Figure PCTCN2021108256-appb-000089
步骤A：将中间体AJ(146毫克，405.10微摩尔)溶于乙腈(4毫升)中，加入六氯环三磷腈(140.84毫克，405.10微摩尔)和磷酸钾(257.97毫克，1.22毫摩尔)。反应混合物在25摄氏度搅拌1小时，然后加入中间体F(81.50毫克，405.10微摩尔)，继续在25摄氏度搅拌1小时。过滤后，滤液减压浓缩。剩余物经制备HPLC(色谱柱：Welch Ultimate XB-SiOH，250mm×50mm×10μm；流动相：A相：正己烷；B相：0.1％的氨水乙醇，15％～55％；15分钟)纯化，得到13-1。LC-MS(ESI)m/z：544.3[M+H] +。
步骤B：将13-1(50毫克，91.98微摩尔)溶于1，4-二氧六环(4毫升)中，然后加入盐酸二氧六环溶液(4摩尔/升，1毫升)。反应混合物在40摄氏度搅拌1小时。减压浓缩后，得到13-2。LC-MS(ESI)m/z：444.2[M+H] +。
步骤C：将醋酸(8.26毫克，137.52微摩尔)，N，N-二异丙基乙胺(47.4毫克，366.72微摩尔)和2-(7-偶氮苯并三氮唑)-N，N，N，N-四甲基脲六氟磷酸酯(52.29毫克，137.52微摩尔)加入到N，N-二甲基甲酰胺(2毫升)中。反应混合物在25摄氏度搅拌15分钟，然后加入13-2(44毫克，91.68微摩尔)，加完后，在25摄氏度继续搅拌1小时。减压浓缩后，剩余物经制备HPLC(色谱柱：Phenomenex Synergi C18，150mm×50mm×3μm；流动相：A相：0.225％甲酸水溶液；B相：8％～38％乙腈；15分钟)纯化，得到化合物13。 1H NMR(400MHz，DMSO-d 6)δ＝9.19-9.02(m，1H)，8.87(d，J＝15.6Hz，1H)，7.73-7.54(m，2H)，7.40-7.10(m，2H)，6.46-6.34(m，1H)，6.31-6.16(m，1H)，6.10(d，J＝3.4Hz，1H)，5.34(dd，J＝10.8Hz，1H)，5.20(dd，J＝17.0Hz，1H)，4.50-4.40(m，1H)，3.73-3.50(m，4H)，2.80-2.70(m，1H)，2.27-2.18(m，3H)，2.02-1,93(m，3H)，1.63-1.52(m，3H)。LC-MS(ESI)m/z：486.2[M+H] +。
化合物14
Figure PCTCN2021108256-appb-000090
将中间体AK(50毫克，165.41微摩尔)溶于乙腈(5毫升)中，加入六氯环三磷腈(60.38毫克，173.68微摩尔)和磷酸钾(140.44毫克，661.63微摩尔)。反应混合物在30摄氏度搅拌3小时，然后加入中间体F(39.31毫克，165.41微摩尔)，继续在30摄氏度搅拌12小时。过滤后，滤液减压浓缩。剩余物经薄层色谱法(展开剂：乙酸乙酯)纯化，得到化合物14。LC-MS(ESI)m/z：486.2[M+H] +。
化合物15
Figure PCTCN2021108256-appb-000091
将中间体AL(50毫克，147.79微摩尔)溶于乙腈(5毫升)中，加入六氯环三磷腈(51.38毫克，147.79微摩尔)和磷酸钾(94.11毫克，443.37微摩尔)。反应混合物在30摄氏度搅拌3小时，然后加入中间体F(35.10毫克，147.79微摩尔)，继续在30摄氏度搅拌12小时。过滤后，滤液减压浓缩。 剩余物经薄层色谱法(展开剂：乙酸乙酯)纯化，得到化合物15。LC-MS(ESI)m/z：522.2[M+H] +。
生物学测试：
实验例1.KRAS(G12C)和SOS1结合实验
1、实验材料：
KRAS(G12C)蛋白由武汉普健生物科技有限公司表达纯化，SOS1 exchange domin(564-1049)protein(Human recombinant)购自Cytoskeleton，Mab Anti 6HIS-XL665和Mab Anti GST-Eu cryptate购自Cisbio。多功能酶标仪Nivo5购自于PerkinElmer。
2、1×缓冲液(1×buffer)配制：
本实验缓冲液采用现配现用。
配制方法：4-羟乙基哌嗪乙磺酸(Hepes)：5mM；氯化钠(NaCl)：150mM；乙二胺四乙酸(EDTA)：10mM；CO-630(Igepal)：0.0025％；氟化钾(KF)：100mM；二硫苏糖醇(DTT)：1mM；牛血清白蛋白(BSA)：0.05％。
3、实验方法：
1)用DMSO将待测化合物用排枪进行5倍稀释至第8个浓度，即从1mM稀释至0.064μM。
2)用1×buffer将待测化合物各梯度稀释成DMSO为2％的工作液，5μL/孔加到对应孔中，对应浓度梯度为20μM至0.00128nM，设置双复孔实验。1000转，离心1分钟。
3)用1×buffer配制KRAS(G12C)(200nM)和Mab Anti GST-Eu cryptate(1ng/μL)的混和工作液，将该混合工作液放置25℃中孵育5分钟，2.5μL/孔加入到对应孔。
4)用1×buffer配制SOS1(80nM)和MabAnti 6HIS-XL665(8g/μL)的混和工作液，2.5μL/孔加入到对应孔中，空白孔(Blank)中加入2.5μL Mab Anti 6HIS-XL665(8g/μL)稀释液，此时化合物终浓度梯度为10μM稀释至0.64nM，KRAS(G12C)(500nM)，MAb Anti GST-Eu cryptate(0.25ng/μL)，SOS1(20nM)，Mab Anti 6HIS-XL665(2g/μL)，反应体系置于25度反应60分钟。反应结束后采用多标记分析仪读取HTRF。
4、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，IC 50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)。表1提供了本发明的化合物对KRAS(G12C)和SOS1结合的抑制活性。
表1 发明的化合物对KRAS(G12C)和SOS1结合的抑制活性
样品 IC 50(nM)
化合物1 32
化合物2 26
化合物3 25
化合物4 21
化合物7 13
化合物8 13
结论：本发明的化合物有理想的KRAS(G12C)-SOS1结合抑制活性。
实验例2.HTRF检测DLD-1细胞中pERK的表达水平实验
通过均相时间分辨荧光法(HTRF)检测KRAS G13D突变的DLD-1细胞ERK蛋白磷酸化程度，以化合物的IC 50和IC 90值为指标，来评价化合物对RAS信号通路中的ERK磷酸化程度的抑制活性。1、实验步骤和方法：
1)种DLD-1(50000细胞/孔)细胞到96孔板，每孔90微升RPMI-1640培养基(10％胎牛血清)，放入37℃，5％二氧化碳培养箱培养过夜。
2)取10微升的10×化合物工作液按表2所示加入到细胞培养板中。在溶媒对照和阳性对照中加入10微升DMSO-细胞培养液混合液。DMSO终浓度为0.25％。将96孔细胞板放回培养箱中培养1.5小时。
表2 400×本发明化合物存储板(μM)
Figure PCTCN2021108256-appb-000092
3)去掉细胞上清液，立刻加入50微升1×裂解液，室温震荡孵育30分钟。
4)使用移液枪吹打混匀，从96孔板中转移16微升细胞裂解液至HTRF 384孔检测板。
5)加入4微升预先混合的抗体溶液，封板，于4摄氏度孵育。用HTRF读板器检测665纳米和620纳米下的荧光发射强度。
2、数据分析：
1)设置Envision仪器的荧光波长：665nm和620nm，进行荧光定量；
2)计算每孔受体和供体发射信号：Ratio＝Signal 665nM/Signal 620nM；
3)将原始数据去除背景数值后，与DMSO处理组进行比较，计算得出相对的pERK水平；
4)利用GraphPad 8.0软件，根据Log[inhibitor]vs Response-variance slope进行IC 50的计算。表3提供了本发明的化合物对pERK的抑制活性。
表3 本发明化合物在DLD-1细胞中对p-ERK的抑制活性
样品 IC 50(nM)
化合物6 23
化合物8 37
化合物9 36
化合物10 87
化合物13 26
化合物15 25
结论：本发明化合物在DLD-1细胞中对ERK激酶的磷酸化水平有显著抑制活性。
实验例3.A427细胞(KRAS G12D)体外抗增殖实验
1、实验材料：
EMEM培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特，低熔点琼脂糖购自Sigma。Almar blue试剂购自Invitrogen。A427细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
2、实验方法：
1)将A427细胞种于96孔U型板中，先将低熔点琼脂糖配成2％的母液，使用时先将琼脂糖母液在微波炉中加热使其完全融化，之后至于42℃水浴锅中使琼脂糖保持液体状态。将凝胶加入含血清的培养基中配成凝胶浓度为0.6％作为底层胶，按照每孔50μL铺到96孔U型板中。待底层胶凝固后，再将2％凝胶加入到含细胞的培养基中，配成凝胶浓度为0.4％的含细胞的上层胶，细胞密度为4×10 4细胞/毫升，按照每孔75μL加到铺有底层胶的96孔U型板中，细胞密度为3000个每孔。待上层胶凝固后，细胞板置于二氧化碳培养箱中过夜培养。
2)加化合物当天，在铺好细胞的96孔U型板中加入85μL液体培养基。将待测化合物用排枪进行3倍稀释至第9个浓度，即从6mM稀释至0.9uM，设置双复孔实验。向中间板中加入97μL培养基，再按照对应位置，转移2.5μL每孔的梯度稀释化合物至中间板，混匀后转移40μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是30μM至4.5nM。细胞板置于二氧化碳培养箱中培养7天，第8天，将待测化合物用排枪进行3倍稀释至第九个浓度，即从6mM稀释至0.9μM，设置双复孔实验。像中间板中加入198μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至第一块中间板中，再向第二块中间板中加入100μL培养基，取第一块中间板中的混匀化合物100μL加入，混匀后转移40μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是30μM至4.5nM。细胞板置于二氧化碳培养箱中再培养7天。化合物与细胞共孵育14天，向细胞板中加入每孔20μL的Almar blue检测试剂，将加染料的板子置于水平摇床上震荡15分钟，再将板子至于室温孵育至5小时使发光信号稳定。采用多标记分析仪读数。
3、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，IC 50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)。表4提 供了本发明的化合物对A427细胞增殖的抑制活性。其中，“A”表示IC 50数值小于或等于100nM；“B”表示IC 50数值大于100nM，同时小于或等于500nM；“C”表示IC 50数值大于500nM。
表4 本发明化合物在A427细胞中的抑制细胞增殖活性
样品 IC 50(nM)
化合物8 A
化合物13 A
化合物15 A
结论：本发明化合物在在A427细胞中表现出显著的抑制细胞增殖活性。
实验例4.小鼠药代动力学研究
本实验旨在评价化合物在小鼠体内单次静脉注射或灌胃给药后的药代动力学行为。静脉注射给药，化合物配制成0.2mg/mL的澄清溶液，溶媒：5％DMSO/95％(10％羟丙基-β-环糊精)水溶液；灌胃给药，化合物配制成0.3mg/mL的混悬液，溶媒：5％DMSO/95％(10％羟丙基-β-环糊精)水溶液。
化合物在血浆中的浓度由高效液相色谱-串联质谱(LC-MS/MS)进行测定。化合物和内标(双氯芬酸)的保留时间、色谱图采集和色谱图的积分采用软件Analyst(Applied Biosystems)进行处理，数据的统计采用软件Watson LIMS(Thermo Fisher Scientific)或Analyst(Applied Biosystems)进行处理。样品中分析物浓度单位为ng/mL，保留3位有效数字，所有以百分数表示的数值(如：％偏差和％变异系数等)均保留到小数点后一位。每条校正曲线至少包含6个浓度水平。校正标样的配制需采用和质控样品不同来源的储备液。校正标样算得的浓度与标示值的偏差超出±15.0％(定量下限超出±20.0％)回归分析中应拒绝该标样。被拒绝的校正标样应小于25％，且每条校正曲线至少包含6个符合接受标准的校正标样。如定量下限和定量上限校正标样需要拒绝时，该分析批的定量上限和下限将相应的提高和降低。
采用WinNonlin TM Version 6.3(Pharsight，Mountain View，CA)药动学软件的非房室模型处理血浆浓度，使用线性对数梯形法方法计算药动学参数。需计算的药动学参数包含但不局限于(数据允许)IV组的T 1/2、Vd ss、CL、AUC 0-24h；PO组的C max、T max、AUC 0-24h、口服生物利用度(F％)。
本发明实施例在静脉注射给药剂量1mg/Kg和口服灌胃给药剂量3mg/Kg下的小鼠体内的药代动力学相关参数如下表5所示。
表5 本发明化合物在小鼠中经静脉注射和口服灌胃给药后的药代动力学参数
Figure PCTCN2021108256-appb-000093
结论：本发明的化合物在小鼠PK研究中表现良好的药代动力学性质。
实验例5.评价受试化合物在人源胰腺癌MIA PaCa-2细胞株皮下异种移植BALB/c裸小鼠动物模型中的抗肿瘤作用
人源胰腺癌MIA PaCa-2细胞为KRAS G12C突变依赖细胞株，KRAS蛋白G12C突变导致KRAS持续活化从而激活下游ERK、AKT等信号通路，促进肿瘤细胞的生长增殖。使KRAS蛋白维持在与GDP结合形式的失活状态，从而抑制肿瘤的生长。
BALB/c裸小鼠皮下接种MIA PaCa-2细胞，建立人源胰腺癌皮下移植肿瘤模型。试验分为各受试化合物组和溶媒对照组，每组6只动物，灌胃给药，溶媒对照组为每天给药1次，各受试化合物组为每天2次给药，间隔12小时，共给药18天。根据相对肿瘤抑制率(TGI)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。
实验动物为BALB/c裸鼠，雌性，肿瘤细胞接种时的小鼠周龄为6～7周，购于上海必凯实验动物有限公司。实验动物均饲养于恒温恒湿的独立通风盒内，饲养室温度21～24℃，湿度40～60％，10～20次/小时换气，昼夜明暗交替时间12小时/12小时；持续供给钴60放射灭菌的鼠全价颗粒饲料，不限量自由摄取，饮用自来水(高压蒸汽灭菌后使用)，饮水瓶不间断供水，自由摄取。饲养鼠盒为聚砜鼠盒，高压灭菌后使用，规格为325mm×210mm×180mm；垫料是高压灭菌玉米芯，每盒5只动物，笼卡上标明IACUC批准号、实验编号、实验开始时间、课题负责人、实验人员、动物来源、组别和动物号等；实验动物打耳号进行标记。
MIA PaCa-2细胞培养在含10％胎牛血清和2.5％HS的DEME培养液中。收集指数生长期的MIA PaCa-2细胞，磷酸盐缓冲液(PBS)重悬至适合浓度并与基质(Matrigel)1∶1混合后用于小鼠皮下肿瘤接种。小鼠右侧皮下接种5×10 6MIA PaCa-2细胞。
待肿瘤体积生长到70～80mm 3左右(接种细胞后第13天)，开始给药。当单只小鼠体重下降大于20％，按照动物福利对其实施安乐死。
肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低(体重每周测量2次)情况，眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算公式：肿瘤体积(mm3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。实验中使用StudyDirector TM软件收集数据，包括肿瘤的长短径的测量和动物体重的称量。原始数据由天平和游标卡尺测量后直接导入软件，数据的任何变动都将被记录在此软件中。
肿瘤的生长曲线如图1所示，受试动物的体重变化曲线如图2所示。从实验结果可知，本发明的化合物单药在人源胰腺癌MIA PaCa-2细胞株皮下异种移植BALB/c裸小鼠动物模型中有显著的抑制肿瘤增殖作用，未发现受试动物体重降低，表明本发明的化合物给药后有良好的安全性。

Claims (13)

  1. 式(II)化合物或其药学上可接受的盐，
    Figure PCTCN2021108256-appb-100001
    其中，
    环A选自苯基和5元杂芳基；
    R 1选自H和NH 2；
    R 2选自C 1-3烷基，所述C 1-3烷基任选被1、2、3、4或5个R a取代；
    R 3选自H和F；
    R 4选自C 3-8环烷基和3-8元杂环烷基，所述C 3-8环烷基和3-8元杂环烷基分别独立地任选被1、2、3或4个R b取代；
    R 5选自H、Cl和CH 3；
    R a分别独立地选自D、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3和环丙基；
    R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、C 1-3烷基、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、C 1- 3烷氧基、-COOH、-COO-C 1-3烷基、-C(＝O)-C 1-3烷基、-S(＝O) 2-C 1-3烷基和环丙基，所述C 1-3烷基、C 1- 3烷氧基和环丙基分别独立地任选被1、2或3个R取代；
    R分别独立地选自F、Cl、Br、I、OH、CN、NH 2、-OCH 3和-COOH。
  2. 根据权利要求1所述的化合物或其药学上可接受盐，其中，R 1选自NH 2。
  3. 根据权利要求1所述的化合物或其药学上可接受盐，其中，R 2选自CF 3、CHF 2、
    Figure PCTCN2021108256-appb-100002
    Figure PCTCN2021108256-appb-100003
  4. 根据权利要求1所述的化合物或其药学上可接受盐，其中，R 4选自环丙基、双环[2.2.2]辛烷、5-6元杂环烷基和8元杂环烷基，所述环丙基、双环[2.2.2]辛烷、5-6元杂环烷基和8元杂环烷基分别独立地任选被1、2、3或4个R b取代。
  5. 根据权利要求4所述的化合物或其药学上可接受盐，其中，R 4选自
    Figure PCTCN2021108256-appb-100004
    Figure PCTCN2021108256-appb-100005
    所述
    Figure PCTCN2021108256-appb-100006
    Figure PCTCN2021108256-appb-100007
    分别独立地任选被1、2、3或4个R b取代。
  6. 根据权利要求1所述的化合物或其药学上可接受盐，其中，R b分别独立地选自H、F、Cl、Br、I、OH、CN、NH 2、CH 3、-CH 2F、-CHF 2、-CH 2CN、-CH 2OH、-CH 2OCH 3、-CH 2CH 3、-CH 2NH 2、-NHCH 3、-N(CH 3) 2、-OCH 3、-COOH、-COOCH 3、
    Figure PCTCN2021108256-appb-100008
  7. 根据权利要求5或6所述的化合物或其药学上可接受盐，其中，R 4选自
    Figure PCTCN2021108256-appb-100009
    Figure PCTCN2021108256-appb-100010
  8. 根据权利要求1所述的化合物或其药学上可接受盐，其中，环A选自苯基与噻吩基。
  9. 根据权利要求1～7任意一项所述的化合物或其药学上可接受的盐，其中，化合物选自
    Figure PCTCN2021108256-appb-100011
    Figure PCTCN2021108256-appb-100012
    其中，环A、R 1、R 2、R 3、R 4、R 5和R b如权利要求1～8任意一项所定义。
  10. 根据权利要求9所述的化合物或其药学上可接受的盐，其中，化合物选自
    Figure PCTCN2021108256-appb-100013
    Figure PCTCN2021108256-appb-100014
    其中，R 1、R 2、R 3、R b和R 5如权利要求9所定义。
  11. 化合物或其药学上可接受的盐，其中，化合物选自
    Figure PCTCN2021108256-appb-100015
    Figure PCTCN2021108256-appb-100016
  12. 根据权利要求11所述的化合物或其药学上可接受的盐，其中，化合物选自
    Figure PCTCN2021108256-appb-100017
  13. 根据权利要求1～12任意一项所述化合物或其药学上可接受的盐在制备治疗肺癌、胰腺癌和/或直肠癌药物中的应用。
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WO2023011540A1 (zh) * 2021-08-03 2023-02-09 苏州信诺维医药科技股份有限公司 稠环化合物、药物组合物及其应用
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WO2024083120A1 (zh) * 2022-10-18 2024-04-25 南京明德新药研发有限公司 苄氨基喹啉类化合物及其制备方法
WO2024211712A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024211663A1 (en) 2023-04-07 2024-10-10 Revolution Medicines, Inc. Condensed macrocyclic compounds as ras inhibitors
WO2024216048A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of ras inhibitors, compositions containing the same, and methods of use thereof
WO2024216016A1 (en) 2023-04-14 2024-10-17 Revolution Medicines, Inc. Crystalline forms of a ras inhibitor

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