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WO2024012456A1 - 哌嗪桥取代的杂环并嘧啶类化合物 - Google Patents

哌嗪桥取代的杂环并嘧啶类化合物 Download PDF

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Publication number
WO2024012456A1
WO2024012456A1 PCT/CN2023/106806 CN2023106806W WO2024012456A1 WO 2024012456 A1 WO2024012456 A1 WO 2024012456A1 CN 2023106806 W CN2023106806 W CN 2023106806W WO 2024012456 A1 WO2024012456 A1 WO 2024012456A1
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Prior art keywords
compound
pharmaceutically acceptable
crude product
ethyl acetate
add
Prior art date
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PCT/CN2023/106806
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English (en)
French (fr)
Inventor
张杨
付志飞
俞晨曦
罗妙荣
陈健
孙继奎
胡伯羽
高娜
黎健
陈曙辉
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Medshine Discovery Inc
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Medshine Discovery Inc
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Priority to CN202380050686.2A priority Critical patent/CN119421885A/zh
Priority to AU2023308487A priority patent/AU2023308487A1/en
Priority to KR1020257002488A priority patent/KR20250033237A/ko
Priority to EP23838947.2A priority patent/EP4556481A1/en
Priority to JP2025501760A priority patent/JP2025523101A/ja
Publication of WO2024012456A1 publication Critical patent/WO2024012456A1/zh
Priority to MX2025000472A priority patent/MX2025000472A/es
Anticipated expiration legal-status Critical
Priority to ZA2025/00635A priority patent/ZA202500635B/en
Ceased legal-status Critical Current

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/08—Bridged 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 piperazine bridge-substituted heterocyclic pyrimidine compounds or pharmaceutically acceptable salts thereof, specifically to compounds represented by formula (III-1) or pharmaceutically acceptable salts thereof.
  • NRAS, HRAS, and KRAS mutations in the RAS family cause cancers in nearly a quarter of all human cancers, making them among the most common genetic mutations associated with cancer. It covers almost all cancer types and causes 1 million deaths worldwide every year.
  • KRAS is the most common oncogene (85% of all RAS mutations) and is present in 90% of pancreatic cancers, 30-40% of colon cancers, and 15-20% of lung cancers (mostly non-small cell lung cancers). ).
  • G12C, G12D, and G12R are the most common KRAS mutations among patients.
  • RAS Ranosine diphosphate family proteins
  • RAS protein regulates multiple downstream pathways including RAF-MEK-ERK, PI3K/Akt/mTOR through switching between two expression forms, thereby affecting cell growth and differentiation, as well as tumor occurrence and development. .
  • mutant KRAS has a high affinity for guanosine triphosphate (GTP), and there are factors such as small catalytic sites and smooth protein surfaces that are difficult to target, the development of small molecule inhibitors has always been challenging, creating KRAS The "incurable” legend.
  • Mirati's breakthrough in KRAS G12D non-covalent inhibitors tumors with KRAS G12D mutations have gradually entered the field of precision medicine.
  • the present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof,
  • X is selected from CH, C-Rx, N and N + -O - ; preferably, X is selected from N and N + -O - ; Rx is selected from F, Cl, Br;
  • R 1 is selected from described Each independently optionally substituted by 1, 2, 3 or 4 R a ;
  • R 3 is selected from H and D
  • Each R a is independently selected from F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, C 1-3 alkoxy, C 1-3 haloalkyl, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2- 4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are independently optionally substituted by 1, 2 or 3 R;
  • Each R is independently selected from F, Cl, Br, I, CH2F , CHF2 and CF3 .
  • the present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof,
  • X is selected from CH, N and N + -O - ; preferably, X is selected from N and N + -O - ;
  • R 1 is selected from described Each independently optionally substituted by 1, 2, 3 or 4 R a ;
  • R 3 is selected from H and D
  • Each R a is independently selected from F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are each independently optionally substituted by 1, 2 or 3 R;
  • Each R is independently selected from F, Cl, Br, I, CH2F , CHF2 and CF3 .
  • the present invention also provides compounds represented by formula (III-1) or pharmaceutically acceptable salts thereof,
  • X is selected from N and N + -O - ;
  • R 1 is selected from described Each independently optionally substituted by 1, 2, 3 or 4 R a ;
  • R 3 is selected from H and D
  • Each R a is independently selected from F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are each independently optionally substituted by 1, 2 or 3 R;
  • Each R is independently selected from F, Cl, Br, I, CH2F , CHF2 and CF3 .
  • the present invention also provides compounds represented by formula (III-2) or pharmaceutically acceptable salts thereof,
  • R 1 is selected from phenyl, pyridyl and naphthyl, which are independently optionally substituted by 1, 2, 3 or 4 R a ;
  • R 2 is selected from described Each independently optionally substituted by 1, 2 or 3 R c ;
  • R 3 is selected from H and D
  • Each R a is independently selected from F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are each independently optionally substituted by 1, 2 or 3 R;
  • R b is selected from H, CN, CH 3 and OCH 3 ;
  • Each R c is independently selected from F, Cl, Br, I, CH 2 F, CHF 2 , CF 3 and CH 2 CF 3 ;
  • Each R is independently selected from F, Cl, Br, I, CH2F , CHF2 and CF3 .
  • the present invention also provides compounds represented by formula (II) or pharmaceutically acceptable salts thereof,
  • R 1 is selected from phenyl, pyridyl and naphthyl, which are independently optionally substituted by 1, 2, 3 or 4 R a ;
  • R 2 is selected from described Each independently optionally substituted by 1, 2 or 3 R c ;
  • R 3 is selected from H and D
  • Each R a is independently selected from F, Cl, Br, I, OH, NH 2 , C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are each independently optionally substituted by 1, 2 or 3 R;
  • R b is selected from H, CN, CH 3 and OCH 3 ;
  • Each R c is independently selected from F, Cl, Br, I, CH 2 F, CHF 2 , CF 3 and CH 2 CF 3 ;
  • Each R is independently selected from F, Cl, Br, I, CH2F , CHF2 and CF3 .
  • the compound or a pharmaceutically acceptable salt thereof is selected from,
  • X, R 1 and R 3 are as defined in the present invention.
  • each R a is independently selected from F, Cl, OH, NH 2 , CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , OCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , and cyclopropyl, the CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , OCH 3 , OCH 2 CH 3 , OCH(CH 3 ) 2 , and cyclopropyl are each independently optionally substituted by 1, 2 or 3 R, and other variables are as defined in the present invention.
  • each R a is independently selected from F, Cl, OH, NH 2 , CH 3 , CHF 2 , CF 3 , CH 2 CF 3 , CH(CH 3 )CF 3 , OCH 3.
  • Other variables are as defined in the present invention.
  • said R1 is selected from Other variables are as defined in the present invention.
  • said R1 is selected from described Each is independently optionally substituted by 1, 2, 3 or 4 Ra , and other variables are as defined in the present invention.
  • said R1 is selected from Other variables are as defined in the present invention.
  • said R1 is selected from Other variables are as defined in the present invention.
  • said R1 is selected from Other variables are as defined in the present invention.
  • said R 2 is selected from Other variables are as defined in the present invention.
  • said R 2 is selected from Other variables are as defined in the present invention.
  • said R 2 is selected from Other variables are as defined in the present invention.
  • the R 3 is selected from H and D, and other variables are as defined in the invention.
  • X is selected from N, and other variables are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of
  • R 1 , R 2 and R 3 are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 1 , R 2 and R 3 are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 1 and R 3 are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 1 and R 3 are as defined in the present invention.
  • the compound or a pharmaceutically acceptable salt thereof is selected from,
  • R 1 and R 3 are as defined in the present invention.
  • compound 3 is prepared from compound 2-4A through the steps described in Examples 2 and 3, wherein, by LCMS (chromatographic column: Agilent Poroshell 120EC-C18 2.7um 3.0*30mm , mobile phase: A: water (0.037% formic acid)-B: acetonitrile (0.0187% formic acid); B: 5%-95%) shows that the retention time of 2-4A is 0.776min, and its isomer retention time is 0.801min.
  • LCMS chromatographic column: Agilent Poroshell 120EC-C18 2.7um 3.0*30mm
  • mobile phase A: water (0.037% formic acid)-B: acetonitrile (0.0187% formic acid); B: 5%-95%) shows that the retention time of 2-4A is 0.776min, and its isomer retention time is 0.801min.
  • Example 3 of the present invention Compound 3 is prepared from Compound 3-2A through the steps described in Example 3, wherein 3-2A is analyzed by SFC (chromatographic column: Cellulose 2 100mm*4.6mm, 3 ⁇ m, mobile phase : [A (supercritical carbon dioxide), B (methanol (0.05% diethanolamine))]; B: 40%) shows a retention time of 4.964 min, and the retention time of its enantiomer is 8.382 min.
  • SFC chromatographic column: Cellulose 2 100mm*4.6mm, 3 ⁇ m, mobile phase : [A (supercritical carbon dioxide), B (methanol (0.05% diethanolamine))]; B: 40%
  • Example 3 of the present invention compound 3 was analyzed by SFC (chromatographic column: Chiralcel OJ-3 100*4.6mm, 3 ⁇ m; mobile phase: A (supercritical carbon dioxide) and B (ethanol, containing 0.05% diethylamine); Gradient: B%: 40%-40%) shows a retention time of 3.761 min.
  • the present invention also provides the compounds shown and pharmaceutically acceptable salts thereof,
  • the compound 3 was analyzed by SFC (chromatographic column: Chiralcel OJ-3 100*4.6mm, 3 ⁇ m; mobile phase: A (supercritical carbon dioxide) and B (ethanol, containing 0.05% diethylamine); gradient: B%: 40%-40%) showed a retention time of 3.761min.
  • the present invention also provides the compounds shown and pharmaceutically acceptable salts thereof,
  • the compound 3 was analyzed by chiral HPLC (chromatographic column: FLM Chiral NQ, 150*4.6mm, 3 ⁇ m; mobile phase: A: (n-hexane) and B: (ethanol, containing 0.2% diethylamine, v/v ); gradient: B%: isocratic 30%; elution time: 60min; column temperature: 35°C) shows a retention time of 17.387min.
  • the three stereoisomers of compound 3 are analyzed by chiral HPLC (chromatographic column: FLM Chiral NQ, 150*4.6mm, 3 ⁇ m; mobile phase: A: (n-hexane) and B (Ethanol, containing 0.2% diethylamine, v/v); Gradient: B%: Isocratic 30%; Elution time: 60min; Column temperature: 35°C) shows retention times of 22.587min, 26.205min and 44.765min .
  • the invention provides compound 7A2 or a pharmaceutically acceptable salt thereof; said compound 7A2 has one of the following chemical structures, which is analyzed by SFC (column: ChiralPak IG-3, 50*4.6 mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min).
  • SFC column: ChiralPak IG-3, 50*4.6 mm, 3 ⁇ m
  • mobile phase A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min).
  • the retention time under the conditions is 2.236min.
  • the present invention also provides compounds represented by the following formula or pharmaceutically acceptable salts thereof,
  • the present invention provides compounds represented by the following formula or pharmaceutically acceptable salts thereof,
  • the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of
  • the present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in preparing drugs for treating solid tumors with KRAS G12D mutations.
  • the present invention provides a pharmaceutical composition, which contains a compound represented by any one of the general chemical formulas of Formula I, Formula II, Formula III-1, and Formula III-2 of the present invention or a pharmaceutically acceptable salt thereof, and any pharmaceutical Acceptable carrier.
  • the present invention provides the compounds represented by any of the general chemical formulas of Formula I, Formula II, Formula III-1 and Formula III-2 of the present invention or pharmaceutically acceptable salts thereof for preparing entities for treating KRAS G12D mutations.
  • the present invention also provides the use of the aforementioned pharmaceutical composition for treating solid tumors with KRAS G12D mutations; preferably, the solid tumors are selected from colon cancer and pancreatic cancer.
  • the invention also provides the following synthesis methods:
  • R 3 is selected from H, F and Cl;
  • R 1a is selected from R 1 is selected from Synthesis route 4:
  • R 3 is selected from H, F and Cl;
  • R 1a is selected from R 1 is selected from
  • the invention also provides the following test methods:
  • control compound stock solution 1mM
  • concentration of the test compound stock solution 10mM
  • GP2D cells are seeded in a transparent 96-well cell culture plate. Each well contains 80 ⁇ L of cell suspension. Each well contains 8,000 cells. The cell plate is placed in a carbon dioxide incubator and incubated at 37°C overnight;
  • This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated GP2D human colon cancer cells.
  • Cell line GP2D DMEM culture medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.
  • GP2D cells were seeded in a 96-well U-bottom cell culture plate, with 80 ⁇ L of cell suspension per well containing 2,000 GP2D cells. thin The cell plate was placed in a carbon dioxide incubator and cultured overnight. Use a row gun to dilute the compound to be tested 5 times to the 8th concentration, that is, from 200 ⁇ M to 2.56 nM, and set up a double well experiment. Add 78 ⁇ L of culture medium to the middle plate, then transfer 2 ⁇ L of the gradient dilution compound per well to the middle plate according to the corresponding position, mix well, and transfer 20 ⁇ L of each well to the cell plate. The concentration of compounds transferred into the cell plate ranged from 1 ⁇ M to 0.0128 nM.
  • the cell plate was cultured in a carbon dioxide incubator for 5 days. After the cell plate with the compound added has finished incubating, add 100 ⁇ L of cell viability chemiluminescence detection reagent per well to the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Take multi-label analyzer readings.
  • IC 50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs.” in GraphPad Prism. response--Variable slope" mode).
  • Test method 4 In vivo pharmacokinetic experiment
  • test compound was mixed with 10% dimethyl sulfoxide/60% polyethylene glycol 400/30% aqueous solution, vortexed and ultrasonicated to prepare a clear solution of approximately 1 mg/mL, which was filtered through a microporous membrane and set aside for later use.
  • Male SD mice aged 7 to 10 weeks were selected and the candidate compound solution was administered intravenously at a dose of 3 mg/kg.
  • Candidate compound solutions are administered orally at a dose of approximately 30 mg/kg.
  • Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight Company, USA).
  • Cell culture Human colon cancer GP2D cells are cultured in monolayer in vitro.
  • the culture conditions are DMEM/F12 medium plus 20% fetal bovine serum, 1% double antibody, and cultured in a 37°C 5% carbon dioxide incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the required number, collect the cells, count them, resuspend them in an appropriate amount of PBS, and add Matrigel at a ratio of 1:1 to obtain a cell suspension with a cell density of 25x 10 6 cells/mL.
  • Tumor diameter was measured twice weekly using vernier calipers.
  • Relative tumor proliferation rate T/C (%) TRTV/CRTV ⁇ 100% (TRTV: RTV of treatment group; CRTV: RTV of negative control group).
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of administration in a certain treatment group - Average tumor volume at the beginning of administration in this treatment group)/(Average tumor volume at the end of treatment in the solvent control group - Average tumor volume at the beginning of treatment in the solvent control group) Tumor volume)] ⁇ 100%.
  • the compound of the present invention has a good binding effect with KRAS G12D protein and can significantly inhibit KRAS G12D enzyme and GP2D cell p-ERK.
  • the compound of the present invention has good cell proliferation inhibitory activity on KRAS G12D mutated cells and has excellent tumor inhibitory effect. .
  • the compounds of the present invention have better pharmacokinetic characteristics.
  • the term "pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue. , without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts refers to salts of compounds of the present invention prepared from compounds having specific substituents found in 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 pure solution or in a suitable inert solvent.
  • 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.
  • Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.
  • the pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid groups or bases.
  • 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, diastereoisomers isomer, the (D)-isomer, the (L)-isomer, as well as their racemic mixtures and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which belong to the present invention. within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
  • the compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound.
  • compounds can be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterated drugs can be replaced by heavy hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce side effects and increase drug stability. , enhance efficacy, extend drug biological half-life and other advantages. All variations in the isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the specific atom is normal and the substituted compound is stable.
  • oxygen it means that two hydrogen atoms are replaced.
  • Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it may or may not be substituted. Unless otherwise specified, the type and number of substituents may be arbitrary on the basis of chemical achievability.
  • any variable e.g., R
  • its definition in each instance is independent.
  • said group may optionally be substituted by up to two R's, with independent options for R in each case.
  • substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • the direction of connection is arbitrary, for example, The middle linking group L is -MW-.
  • -MW- can be connected to ring A and ring B in the same direction as the reading order from left to right. You can also connect ring A and ring B in the opposite direction to the reading order from left to right.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • connection mode of the chemical bond is non-positioned and there are H atoms at the connectable site, when the chemical bond is connected, the number of H atoms at the site will be reduced correspondingly with the number of connected chemical bonds and become the corresponding valence. group.
  • the chemical bond connecting the site to other groups can be a straight solid line bond straight dashed key or wavy lines express.
  • the straight solid line bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in represents the nitrogen atom in the group The two ends are connected to other groups;
  • the wavy lines in indicate that the phenyl group is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;
  • use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center
  • using straight solid line keys and straight dotted keys Represent the relative configuration of the three-dimensional center with a wavy line
  • wedge-shaped solid line key or wedge-shaped dotted key or use tilde Represents a straight solid line key or straight dotted key
  • Use straight solid keys and straight dotted keys Indicates the relative configuration of the stereocenter, representing mixture. represent mixture, represent
  • Certain compounds of the present invention may exist as atropisomers, which are conformational isomers that occur when rotation about a single bond in the molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule.
  • the compounds disclosed in the present invention include all atropisomers, which may be pure individual atropisomers, enriched in one of the atropisomers, or non-specific mixtures thereof. If the rotational potential energy around a single bond is high enough and the interconversion between conformations is slow enough, this can allow separation of isomers. For example, and is a pair of atropisomers, in which the phenyl Indicates that the three-dimensional orientation of this side is outward, Indicates that the three-dimensional orientation of this side is inward.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C5 , C6 , C7 , C8 , C9 , C10 , C11 , and C12 , also include any range from n to n+m, for example, C1-12 includes C1-3 , 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.; similarly, n yuan to n The +m member indicates that the number of atoms in the ring is n to n+m.
  • a 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, and a 9-membered ring.
  • 3-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.
  • C 1-3 alkyl is used to mean 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 (such as methyl), divalent (such as methylene) or multivalent (such as methine) .
  • Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n - propyl and isopropyl), and the like.
  • halogen or halogen by itself or as part of another substituent means a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.
  • C 1-3 alkoxy means those alkyl groups containing 1 to 3 carbon atoms that are 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 groups, etc.
  • Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C 2-4 alkenyl is used to mean a straight or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond. Can be located anywhere on the group.
  • the C 2-4 alkenyl group includes C 2-3 , C 4 , C 3 and C 2 alkenyl groups, etc.; the C 2-4 alkenyl group can be monovalent, divalent or multivalent. Examples of C 2-4 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.
  • C 2-4 alkynyl is used to mean a straight-chain or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond. Can be located anywhere on the group.
  • the C 2-4 alkynyl group includes C 2-3 , C 4 , C 3 and C 2 alkynyl groups, etc. It can be monovalent, bivalent or polyvalent. Examples of C 2-4 alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.
  • N + -O - is used to represent "N( ⁇ O)", representing the oxide of N.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives and preferred embodiments include, but are not limited to, 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 involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • single crystal X-ray diffraction uses a Bruker D8 venture diffractometer to collect diffraction intensity data on the cultured single crystal.
  • the light source is CuK ⁇ radiation, and the scanning mode is: ⁇ / ⁇ scanning. After collecting relevant data, the direct method is further used. (Shelxs97)
  • the absolute configuration can be confirmed by analyzing the crystal structure.
  • Boc represents tert-butyryl carbonate
  • Fmoc represents 9-fluorenylmethoxycarbonyl
  • TIPS represents triisopropylsilyl
  • PMB represents p-methoxybenzyl
  • Tf represents trifluoromethanesulfonyl
  • DCE represents dichloroethane
  • THF represents tetrahydrofuran
  • H 2 O represents water
  • FA represents formic acid
  • ACN represents acetonitrile
  • PE represents petroleum ether
  • EA represents ethyl acetate
  • DEA diethanolamine
  • IPA represents isopropyl alcohol
  • DBU represents 1,8-diazaza Bispiro[5.4.0]undec-7-ene
  • the second-generation Grubbs catalyst represents the compound with CAS number 246047-72-3
  • the proportion of eluent in column chromatography represents the volume ratio
  • M in concentration represents mol/L.
  • the molecular docking process was performed using Maestro ( Performed with Glide SP [1] in version 2017-2) and default options.
  • the crystal structure PDB:6UT0 of KRAS_G12C in the PDB database was selected, Cys12 was simulated and mutated into Asp12, and after energy optimization, it was used as a docking template.
  • hydrogen atoms were added using the Protein Preparation Wizard module of Maestro [2] and the OPLS3 force field was used.
  • LigPrep was used to generate the three-dimensional structure of the molecule and energy minimization was performed [3]
  • the confgen module was used to sample the small molecule conformation.
  • step 1
  • step 1
  • Step 12 Preparation of intermediate 1-3A-2
  • reaction solution was diluted with ethyl acetate (30 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
  • intermediate 2-9A was analyzed in SFC (chromatographic column: (S, S) Whelk-01 100 ⁇ 4.6mm ID, 5.0 ⁇ m, mobile phase: The retention time in A: supercritical carbon dioxide, B: IPA (0.05% DEA), gradient: 40% B, flow rate: 2.5mL/min) is 3.276 min, and the ee value is 98.3%;
  • Compound 2-9B is analyzed in SFC ( Chromatographic column: (S, S) Whelk-01 100 ⁇ 4.6mm ID, 5.0 ⁇ m, mobile phase: A: supercritical carbon dioxide, B: IPA (0.05% DEA), gradient: 40% B, flow rate: 2.5mL/min ) in which the retention time is 3.879min and the ee value is 96.4%.
  • the crude product was purified by preparative SFC (chromatographic column: DAICEL CHIRALCEL OD (250mm*50mm, 10 ⁇ m); mobile phase: [A (supercritical carbon dioxide), B (ethanol containing 0.1% ammonia)]; B: 40%) to obtain the product 3-2A and 3-2B.
  • reaction solution was cooled to 25°C, insoluble matter was filtered off, concentrated to remove most of the toluene, diluted with ethyl acetate (30 mL), washed with water (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product.
  • reaction solution was diluted with ethyl acetate (200 mL), washed with saturated brine (40 mLx2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • compound 5-4 (17.8g, 93.41mmol) was dissolved in phosphorus oxychloride (234.25g, 1.53mol), and then reacted at 95°C for 3 hours.
  • the reaction system was concentrated under reduced pressure, tetrahydrofuran (360 mL) was added, and then amine thiocyanate (21.33 g, 280.23 mmol) was added in batches at 20°C, followed by reaction at 40°C for 16 hours. Add water (300 mL) to the reaction system, extract the aqueous phase with ethyl acetate (300 mL*3), and separate the layers.
  • compound 5-6 (3.5g, 14.25mmol) and compound 5-6A (3.98g, 14.67mmol) were dissolved in dioxane (70mL), water (7mL) and ethanol (14mL), and phosphoric acid was added Potassium (9.07g, 42.74mmol) and chlorine (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl) [2-(2'-amino -1,1'-biphenyl)]palladium (II) (1.68g, 2.14mmol), and then reacted at 105°C for 1.5 hours.
  • compound 5-7 (2.1g, 5.92mmol) was dissolved in ethanol (63mL), silver sulfate (2.21g, 7.10mmol) and iodine element (1.58g, 6.22mmol) were added at 0°C, and then the temperature was slowly raised to React at 10°C for 2 hours.
  • Add saturated sodium sulfite solution (70 mL) and ethyl acetate (100 mL) to the reaction system, filter and separate the layers. The aqueous phase was extracted with ethyl acetate (100mL*3) and separated. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product.
  • compound 5-10 (0.15g, 163.89 ⁇ mol) and copper iodide (156.07mg, 819.47 ⁇ mol) were dissolved in N,N-dimethylformamide (3.75mL), and compound 5-10A (314.86 mg, 1.64mmol), react at 80°C for 2 hours.
  • compound 5-11 (0.06g, 69.98 ⁇ mol) was dissolved in tetrahydrofuran (1.2mL) and water (0.4mL), potassium monopersulfate (82.38mg, 489.89 ⁇ mol) was added at 0°C, and the temperature was slowly raised to 10 °C reaction for 4 hours. Add potassium monopersulfate (35.31 mg, 209.95 ⁇ mol) and react at 10°C for 2 hours. Slowly add saturated sodium sulfite solution (5mL), extract the aqueous phase with ethyl acetate (10mL*3), and separate the liquids.
  • compound 5-12 (0.05g, 56.22 ⁇ mol) and compound 2-7 (13.93mg, 84.33 ⁇ mol) were dissolved in tetrahydrofuran (1.5mL), and sodium tert-butoxide (10.81mg, 112.44 ⁇ mol) was added at 0°C. The temperature was slowly raised to 10°C and reacted for 3 hours. Water (5 mL) and ethyl acetate (5 mL) were added to the reaction system, filtered, and the filtrate was separated. The aqueous phase was extracted with ethyl acetate (5mL*3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-13. MS(ESI)m/z:974.3[M+1] + .
  • step 1
  • Compound 6-1A1 Analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B% :5%-5%, 3min), the retention time is 1.463min, and the ee value is 97.49%.
  • Compound 6-1A2 Analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B% :5%-5%, 3min), the retention time is 1.983min, and the ee value is 95.66%.
  • Compound 6-1B (0.2g, 205.45 ⁇ mol) was passed through preparative SFC (column: ChiralPak IH, 250*30mm, 10 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (methanol, containing 0.1% ammonia); gradient: B%: 40%-40%, 11 min) were separated and purified to obtain compound 6-1B1 and compound 6-1B2 respectively.
  • Compound 6-1B1 Analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B% :5%-5%, 3min), the retention time is 1.330min, and the ee value is 94.45%.
  • Compound 6-1B2 Analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B% :5%-5%, 3min), the retention time is 1.495min, and the ee value is 98.01%.
  • compound 6-1A1 (0.07g, 71.91 ⁇ mol) was dissolved in dichloromethane (3.5mL), trifluoroacetic acid (1.07g, 9.42mmol) was added, and then reacted at 20°C for 3 hours. Concentrate under reduced pressure to obtain crude product. The crude product was passed through high performance liquid chromatography (column: Waters Xbridge BEH C18100*30mm*10 ⁇ m; mobile phase: A (acetonitrile) and B (water, containing 10mM ammonium bicarbonate); gradient: B%: 35%-70%) Isolate and purify to obtain compound 6A1.
  • compound 6-1A2 (0.07g, 71.91 ⁇ mol) was dissolved in dichloromethane (3.5mL), trifluoroacetic acid (1.07g, 9.42mmol) was added, and then reacted at 20°C for 3 hours. Concentrate under reduced pressure to obtain crude product. The crude product was passed through high performance liquid chromatography (column: Waters Xbridge BEH C18100*30mm*10 ⁇ m; mobile phase: A (acetonitrile) and B (water, containing 10mM ammonium bicarbonate); gradient: B%: 35%-70%) Isolate and purify to obtain compound 6A2.
  • compound 6-1B1 (0.06g, 61.64 ⁇ mol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (921.00mg, 8.08mmol) was added, and then reacted at 20°C for 2 hours. Concentrate under reduced pressure to obtain crude product. The crude product was separated by high performance liquid chromatography (column: Waters :30%-70%), purified to obtain compound 6B1.
  • compound 6-1B2 (0.06g, 61.64 ⁇ mol) was dissolved in dichloromethane (3mL), trifluoroacetic acid (921.00mg, 8.08mmol) was added, and then reacted at 20°C for 2 hours. Concentrate under reduced pressure to obtain crude product. The crude product was separated by high performance liquid chromatography (column: Waters :25%-65%), purified to obtain compound 6B2.
  • step 1
  • the crude product was separated by high performance liquid chromatography (column: Phenomenex Luna C18 75*30mm*3 ⁇ m; mobile phase: A (acetonitrile) and B (water, containing 0.04% formic acid); gradient: B%: 15%-55%) , and then further pass through the preparation of SFC (column: DAICEL CHIRALPAK IG (250mm*30mm, 10 ⁇ m); mobile phase: A (supercritical CO 2 ) and B (ethanol, containing 0.1% ammonia); gradient: B%: 50%-50 %, 11 min) were separated and purified to obtain compound 7A1 and compound 7A2 respectively.
  • Compound 7A1 Analytical SFC (column: ChiralPak IG-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5 %-5%, 3min), the retention time is 1.693min, and the ee value is 97.92%.
  • Compound 7A2 Analytical SFC (column: ChiralPak IG-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropyl alcohol, containing 0.1% isopropylamine); gradient: B%: 5 %-5%, 3min), the retention time is 2.236min, and the ee value is 98.26%.
  • the crude product was separated by high performance liquid chromatography (column: Phenomenex Luna C18 75*30mm*3 ⁇ m; mobile phase: A (acetonitrile) and B (water, containing 0.04% formic acid); gradient: B%: 15%-55%) , and then further pass through the preparation of SFC (column: DAICEL CHIRALPAK IG (250mm*30mm, 10 ⁇ m); mobile phase: A (supercritical CO 2 ) and B (ethanol, containing 0.1% ammonia); gradient: B%: 55%-55 %, 7 min) were separated and purified to obtain compound 7B1 and compound 7B2 respectively.
  • Compound 7B1 Analytical SFC (column: ChiralPak IG-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropyl alcohol, containing 0.1% isopropylamine); gradient: B%: 5 %-5%, 3min), the retention time is 1.745min, and the ee value is 98.56%.
  • Compound 7B2 Analytical SFC (column: ChiralPak IG-3, 50*4.6mm, 3 ⁇ m; mobile phase: A (supercritical CO 2 ) and B (isopropyl alcohol, containing 0.1% isopropylamine); gradient: B%: 5 The retention time under the condition of %-5%, 3min) is 2.343min, and the ee value is 98.62%.
  • step 1
  • Dissolve 8-7D (21 mg, 29.43 ⁇ mol) in dichloromethane (0.6 mL), add trifluoroacetic acid (0.2 mL), and stir at 15°C for 0.5 hours. Add ammonia solution dropwise to the reaction solution until the pH is 9, and concentrate under reduced pressure.
  • the crude product was separated and purified by high performance liquid phase preparative chromatography (chromatographic column: Phenomenex Luna 100*30mm*3 ⁇ m; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; running gradient: B%: 1%-30%, Run 8min), The formate salt of 8D was obtained.
  • GP2D cells are seeded in a transparent 96-well cell culture plate. Each well contains 80 ⁇ L of cell suspension. Each well contains 8,000 cells. The cell plate is placed in a carbon dioxide incubator and incubated at 37°C overnight;
  • the compound of the present invention has significant GP2D p-ERK inhibitory effect.
  • This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated GP2D human colon cancer cells.
  • Cell line GP2D and DMEM culture medium were purchased from GIBCO, FBS was purchased from Hyclone, and L-glutamine was purchased from Invitrogen.
  • 96-well plates were purchased from Ultra Low Cluster.
  • the 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and the 2104EnVision plate reader was purchased from PerkinElmer.
  • GP2D cells were cultured under DMEM+10% FBS+2mM L-glutamine culture conditions in a 37°C, 5% CO 2 incubator. Passage regularly and use cells in the logarithmic growth phase for plating. GP2D cells were seeded in a 96-well U-bottom cell culture plate, with 135 ⁇ L of cell suspension per well containing 6,000 GP2D cells. The culture plate was cultured overnight in an incubator at 37°C, 5% CO 2 , and 100% relative humidity. Use a row gun to dilute the compound to be tested 5 times to the 8th concentration, that is, from 200 ⁇ M to 2.56 nM, and set up a double well experiment.
  • IC 50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs.” in GraphPad Prism. response--Variable slope" mode).
  • the compound of the present invention has significant anti-proliferative activity on KRAS G12D mutated GP2D cells.
  • This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated AsPC-1 human pancreatic cancer cells.
  • Cell line AsPC-1 and RPMI-1640 culture medium were purchased from GIBCO, and FBS was purchased from Hyclone.
  • 96-well plates were purchased from Ultra Low Cluster, The 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and the 2104EnVision plate reader was purchased from PerkinElmer.
  • AsPC-1 cells were cultured according to RPMI-1640+10% FBS culture conditions in a 37°C, 5% CO 2 incubator. Passage regularly and use cells in the logarithmic growth phase for plating. AsPC-1 cells were seeded in a 96-well U-bottom cell culture plate, and each well of 135 ⁇ L cell suspension contained 500 AsPC-1 cells. The culture plate was cultured overnight in an incubator at 37°C, 5% CO 2 , and 100% relative humidity. Use a row gun to dilute the compound to be tested 5 times to the 8th concentration, that is, from 200 ⁇ M to 2.56 nM, and set up a double well experiment.
  • IC 50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs.” in GraphPad Prism. response--Variable slope" mode).
  • the compound of the present invention has significant anti-proliferative activity on AsPC-1 cells with KRAS G12D mutation.
  • This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated PANC04.03 human pancreatic cancer cells.
  • Cell line PANC04.03 and RPMI-1640 culture medium were purchased from GIBCO, FBS was purchased from Hyclone, and human insulin was purchased from Yeasen.
  • 96-well plates were purchased from Ultra Low Cluster, The 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and the 2104 EnVision plate reader was purchased from PerkinElmer.
  • PANC04.03 cells were cultured according to the culture conditions of RPMI-1640+15% FBS+5ug/ml human insulin in a 37°C, 5% CO 2 incubator. Passage regularly and use cells in the logarithmic growth phase for plating. PANC04.03 cells were seeded in a 96-well U-bottom cell culture plate, with 135 ⁇ L of cell suspension per well containing 2,000 PANC04.03 cells. The culture plate was cultured overnight in an incubator at 37°C, 5% CO 2 , and 100% relative humidity. Use a volute gun to dilute the compound to be tested 5 times to the 8th concentration, that is, dilute it from 200 ⁇ M to 2.56 nM, set up double well experiment.
  • IC 50 can be obtained by curve fitting with four parameters ("log(inhibitor) vs.” in GraphPad Prism. response--Variable slope" mode).
  • the compound of the present invention has significant anti-proliferative activity on KRAS G12D mutated PANC04.03 cells.
  • This experiment aims to investigate the pharmacokinetic characteristics of the compound of the present invention under oral and intravenous injection in CD-1 mice.
  • test compound was mixed with 10% dimethyl sulfoxide + 90% (10% hydroxypropyl- ⁇ -cyclodextrin (HP- ⁇ -CD) aqueous solution), vortexed and sonicated to prepare 0.6, 3.0 and 10.0 respectively. mg/mL clear solution.
  • Male CD-1 mice aged 7 to 10 weeks were selected, and the candidate compound solution was administered intravenously (i.v.) at a dose of 3 mg/kg (administration concentration 0.6 mg/mL).
  • candidate compound solutions are administered orally (p.o.) at a dose of 30 mg/kg (administration concentration 3.0 mg/mL) or 100 mg/kg (administration concentration 10.0 mg/mL).
  • Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight Company, USA).
  • the compound of the present invention has good pharmacokinetic characteristics in mice.
  • Cell culture Human colon cancer GP2D cells are cultured in monolayer in vitro.
  • the culture conditions are DMEM/F12 medium plus 20% fetal bovine serum and cultured in a 37°C 5% carbon dioxide incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the required number, collect the cells, count them, resuspend them in an appropriate amount of PBS, and add Matrigel at a ratio of 1:1 to obtain a cell suspension with a cell density of 25x 10 6 cells/mL.
  • Tumor diameter was measured twice weekly using vernier calipers.
  • TGI tumor inhibitory effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%).
  • Relative tumor proliferation rate T/C (%) TRTV/CRTV ⁇ 100% (TRTV: RTV of treatment group; CRTV: RTV of negative control group).
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • the average tumor volume, V t is the average tumor volume at a certain measurement, and TRTV and CRTV take the data on the same day.
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of administration in a certain treatment group - Average tumor volume at the beginning of administration in this treatment group)/(Average tumor volume at the end of treatment in the solvent control group - Average tumor volume at the beginning of treatment in the solvent control group) Tumor volume)] ⁇ 100%.
  • the compound of the present invention has excellent tumor inhibitory effect.
  • Cell culture Human pancreatic cancer PANC04.03 cells were cultured in monolayer in vitro. The culture conditions were RPMI-1640+15% FBS+10 units/mL insulin, 37°C, and 5% carbon dioxide incubator. Passage was performed twice a week with routine digestion treatment with trypsin-EDTA. When the cell saturation is 80%-90% and the number reaches the required number, collect the cells, count them, and resuspend them in an appropriate amount of PBS to obtain a cell suspension with a cell density of 25x 10 6 cells/mL.
  • mice When the average tumor volume reaches 190mm3 , random groups are divided according to the tumor volume, with 6 animals in each group.
  • the dosage of the blank group is 0, and the dosage of the test group is 30mg/kg, 100mg/kg, and 150mg/kg.
  • the dosage volume is 10 ⁇ L/g, administered orally, twice a day for 28 days.
  • Tumor diameter was measured twice weekly using vernier calipers.
  • Relative tumor proliferation rate T/C (%) TRTV/CRTV ⁇ 100% (TRTV: RTV of treatment group; CRTV: RTV of negative control group).
  • the relative tumor volume (RTV) is calculated based on the results of tumor measurement.
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI (%) [1-(Average tumor volume at the end of administration in a certain treatment group - Average tumor volume at the beginning of administration in this treatment group)/(Average tumor volume at the end of treatment in the solvent control group - Average tumor volume at the beginning of treatment in the solvent control group) Tumor volume)] ⁇ 100%.
  • the compound of the present invention has excellent tumor inhibitory effect.

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Abstract

公开了一类哌嗪桥取代的杂环并嘧啶类化合物或其药学上可接受的盐，具体公开了式(III-1)所示化合物或其药学上可接受的盐。

Description

哌嗪桥取代的杂环并嘧啶类化合物
本发明主张如下优先权：
申请号：CN202210822828.X，申请日：2022年7月12日；
申请号：CN202211262711.7，申请日：2022年10月14日；
申请号：CN202211407253.1，申请日：2022年11月10日；
申请号：CN202310041534.8，申请日：2023年1月11日；
申请号：CN202310041762.5，申请日：2023年1月12日；
申请号：CN202310066868.0，申请日：2023年1月19日；
申请号：CN202310800516.3，申请日：2023年6月30日。
技术领域
本发明涉及一类哌嗪桥取代的杂环并嘧啶类化合物或其药学上可接受的盐，具体涉及式(III-1)所示化合物或其药学上可接受的盐。
背景技术
RAS家族中的NRAS，HRAS和KRAS突变引起的癌症占所有人类癌症的近四分之一，使其成为与癌症相关的最常见基因突变之一。几乎覆盖了所有的癌症类型，每年在全球造成100万人死亡。其中，KRAS是最常见的致癌基因(所有RAS突变的85％)，存在于90％的胰腺癌中，30-40％的结肠癌中，15-20％的肺癌中(大多为非小细胞肺癌)。根据存在的特定突变，G12C、G12D和G12R是病友们最常见的KRAS突变。除此之外，还有G12A、G12S、G12V等。
RAS(Rat Sarcoma)家族蛋白广泛表达于各类真核生物，有两种表现形式：非激活状态下GDP(二磷酸鸟苷)结合形式和激活状态下的GTP(三磷酸鸟苷)结合形式。RAS蛋白正是通过两种表现形式之间的切换，来调控包括RAF-MEK-ERK、PI3K/Akt/mTOR在内的多个下游通路，从而影响细胞的生长和分化，以及肿瘤的发生和发展。
由于突变型KRAS对三磷酸鸟苷(GTP)具有较高的亲和力，又存在催化位点小、蛋白表面光滑等难以靶向的因素，使小分子抑制剂的开发一直备受挑战，造就了KRAS的“不可成药”传奇。随着Mirati公司在KRAS G12D非共价抑制剂上的突破，KRAS G12D突变的肿瘤开始逐渐迈入精准医疗领域。
发明内容
本发明提供了式(III-1)所示化合物或其药学上可接受的盐，
其中，
X选自CH、C-Rx、N和N+-O-；优选的，X选自N和N+-O-；Rx选自F、Cl、Br；
R1选自所述分别独立地任选被1、2、3或4个Ra取代；
R3选自H和D；
各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C1-3卤代烷基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
本发明提供了式(III-1)所示化合物或其药学上可接受的盐，
其中，
X选自CH、N和N+-O-；优选的，X选自N和N+-O-；
R1选自所述分别独立地任选被1、2、3或4个Ra取代；
R3选自H和D；
各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
本发明还提供了式(III-1)所示化合物或其药学上可接受的盐，
其中，
X选自N和N+-O-；
R1选自所述分别独立地任选被1、2、3或4个Ra取代；
R3选自H和D；
各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
本发明还提供了式(III-2)所示化合物或其药学上可接受的盐，
其中，
R1选自苯基、吡啶基和萘基，所述苯基、吡啶基和萘基分别独立地任选被1、2、3或4个Ra取代；
R2选自 所述 分别独立地任选被1、2或3个Rc取代；
R3选自H和D；
各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
Rb选自H、CN、CH3和OCH3；
各Rc分别独立地选自F、Cl、Br、I、CH2F、CHF2、CF3和CH2CF3；
各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
本发明还提供了式(II)所示化合物或其药学上可接受的盐，
其中，
R1选自苯基、吡啶基和萘基，所述苯基、吡啶基和萘基分别独立地任选被1、2、3或4个Ra取代；
R2选自 所述 分别独立地任选被1、2或3个Rc取代；
R3选自H和D；
各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
Rb选自H、CN、CH3和OCH3；
各Rc分别独立地选自F、Cl、Br、I、CH2F、CHF2、CF3和CH2CF3；
各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自，
其中，X、R1和R3如本发明所定义。
在本发明的一些方案中，所述各Ra分别独立地选自F、Cl、OH、NH2、CH3、CH2CH3、CH(CH3)2、OCH3、OCH2CH3、OCH(CH3)2、和环丙基，所述CH3、CH2CH3、CH(CH3)2、OCH3、OCH2CH3、OCH(CH3)2、和环丙基分别独立地任选被1、2或3个R取代，其他变量如本发明所定义。
在本发明的一些方案中，所述各Ra分别独立地选自F、Cl、OH、NH2、CH3、CHF2、CF3、CH2CF3、CH(CH3)CF3、OCH3、OCF3、 其他变量如本发明所定义。
在本发明的一些方案中，所述R1选自 其他变量如本发明所定义。
在本发明的一些方案中，所述R1选自所述 分别独立地任选被1、2、3或4个Ra取代，其他变量如本发明所定义。
在本发明的一些方案中，所述R1选自 其他变量如本发明所定义。
在本发明的一些方案中，所述R1选自 其他变量如本发明所定义。
在本发明的一些方案中，所述R1选自 其他变量如本发明所定义。
在本发明的一些方案中，所述R2选自其他变量如本发明所定义。
在本发明的一些方案中，所述R2选自 其他变量如本发明所定义。
在本发明的一些方案中，所述R2选自其他变量如本发明所定义。
在本发明的一些方案中，所述R3选自H和D，其他变量如本发明所定义。
在本发明的一些方案中，所述X选自N，其他变量如本发明所定义。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自
其中，R1、R2和R3如本发明所定义。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自，
其中，R1、R2和R3如本发明所定义；
条件是，R2不为
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自，
其中，R1和R3如本发明所定义。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自，
其中，R1和R3如本发明所定义。
在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自，
其中，R1和R3如本发明所定义。
在本发明的实施例2和3中，化合物3是由化合物2-4A经过实施例2以及3中所述步骤制备得到，其中，通过TLC薄层层析(石油醚:丙酮＝5:1)展开两次，较小的Rf值对应的产物为化合物2-4A，较大的Rf值对应的产物为其异构体。
在本发明的实施例2和3中，化合物3是由化合物2-4A经过实施例2以及3中所述步骤制备得到，其中，通过TLC薄层层析(石油醚:丙酮＝5:1)展开两次，化合物2-4A的Rf值为0.5，其异构体的Rf值为0.55。
在本发明的实施例2和3中，化合物3是由化合物2-4A经过实施例2以及3中所述步骤制备得到，其中，通过LCMS(色谱柱：Agilent Poroshell 120EC-C18 2.7um 3.0*30mm，流动相：A:水(0.037％甲酸)-B:乙腈(0.0187％甲酸)；B:5％-95％)显示2-4A保留时间为0.776min，其异构体保留时间为0.801min。
本发明的实施例3中，化合物3是由化合物3-2A经过实施例3中所述步骤制备得到，其中，3-2A通过SFC分析(色谱柱:Cellulose 2 100mm*4.6mm,3μm，流动相:[A(超临界二氧化碳),B(甲醇(0.05％二乙醇胺))]；B:40％)显示保留时间为4.964min，其对映异构体的保留时间为8.382min。
本发明的实施例3中，化合物3通过SFC分析(色谱柱：Chiralcel OJ-3 100*4.6mm,3μm；流动相：A(超临界二氧化碳)和B(乙醇，含0.05％二乙胺)；梯度：B％:40％-40％)显示保留时间3.761min。
本发明还提供了所示化合物及其药学上可接受的盐，
其中，该化合物3经过SFC分析(色谱柱：Chiralcel OJ-3 100*4.6mm,3μm；流动相：A(超临界二氧化碳)和B(乙醇，含0.05％二乙胺)；梯度：B％:40％-40％)显示保留时间3.761min。
本发明还提供了所示化合物及其药学上可接受的盐，
其中，该化合物3经过手性HPLC分析(色谱柱：FLM Chiral NQ，150*4.6mm,3μm；流动相：A:(正己烷)和B：(乙醇，含有0.2％二乙胺，v/v)；梯度：B％:等度30％；洗脱时间：60min；柱温：35℃)显示保留时间17.387min。
在本发明的一些方案中，其中，化合物3的三个立体异构体经过手性HPLC分析(色谱柱：FLM Chiral NQ，150*4.6mm,3μm；流动相：A:(正己烷)和B：(乙醇，含有0.2％二乙胺，v/v)；梯度：B％:等度30％；洗脱时间：60min；柱温：35℃)显示保留时间为22.587min，26.205min和44.765min。
在发明的一个技术方案中，本发明提供化合物7A2或其药学上可接受盐；所述化合物7A2具有如下结构中的一种化学结构，其在分析SFC(柱子：ChiralPak IG-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为2.236min。
本发明还提供了下式所示化合物或其药学上可接受的盐，



本发明提供了下式所示化合物或其药学上可接受的盐，
















在本发明的一些方案中，所述化合物或其药学上可接受的盐，其化合物选自
本发明提供了所述化合物或其药学上可接受的盐，在制备治疗KRASG12D突变的实体瘤药物中的应用。
本发明提供一种药物组合物，其包含本发明式I、式II、式III-1、式III-2中任一化学通式所示化合物或其药学上可接受的盐，以及任意药学上可接受的载体。
本发明提供了本发明式I、式II、式III-1、式III-2中任一化学通式所示所述化合物或其药学上可接受的盐在制备用于治疗KRAS G12D突变的实体瘤药物中的应用；优选地，所述实体瘤选自结肠癌和胰腺癌。
本发明还提供了前述药物组合物用于治疗KRAS G12D突变的实体瘤药物中的应用；优选地，所述实体瘤选自结肠癌和胰腺癌。
本发明还提供了下列合成方法：
合成路线1：
合成路线2：
合成路线3：
其中，R3选自H、F和Cl；R1a选自R1选自合成路线4：
其中，R3选自H、F和Cl；R1a选自R1选自
本发明还提供了下列测试方法：
测试方法1.KRASG12D抑制活性测试
1.实验目的：
通过TR-FRET的方法，筛选出能有效抑制KRASG12D与GTP结合的化合物。
2.耗材和仪器：
表1.耗材和仪器

3.试剂准备：
a.储存试剂：
1)KRAS核苷酸交换缓冲液
取20mL 1000mM HEPES，20mL 500mM EDTA，10mL 5M氯化钠，100％0.1mL吐温20，949.9mL水，配制成1L溶液，用过滤法消毒，4℃条件下储存。
2)KRAS实验缓冲液
取20mL 1000mM HEPES，10mL 1000mM氯化镁，30mL 5M氯化钠，100％0.05mL吐温20，939.95mL水，配制成1L溶液，用过滤法消毒，4℃条件下储存。
3)KRAS/Bodipy GDP/Tb-SA混合液
取9.5μL 95μM KRASG12D蛋白，440.5μL KRAS核苷酸交换缓冲液混合，室温下孵育1小时后，与8.4μL17.9μM Tb-SA，1.8μL 5mM Bodipy GDP，9539.8μL KRAS实验缓冲液，配制成1L溶液，混合后室温下静置6小时，储存至-80℃条件下。
b.实验试剂：
1)KRAS酶溶液
取73.3μL KRAS/Bodipy GDP/Tb-SA混合液，2126.7μL KRAS实验缓冲液，配制成2200μL溶液。
2)SOS/GTP混合液
取1.59μL 166μM SOS蛋白，198μL 100mM GTP，2000.41μL KRAS实验缓冲液，配制成2200μL溶液。
4.实验流程：
1)对照化合物母液浓度为1mM，待测化合物母液浓度为10mM。转移9μL对照化合物和待测化合物至384-LDV板内；
2)使用Bravo将LDV板上的化合物进行10点3倍稀释；
3)使用ECHO将LDV板上的化合物转移9nL至实验板；
4)使用Dragonfly自动加样仪依次向实验板每孔中加入3μL 3nM Kras/0.5nM TB-SA/30nM BodipyGDP混合液和3μL Ras buffer，以1000rpm/min，将实验板离心1分钟；
5)实验板在室温中孵育1小时；
6)使用Dragonfly自动加样仪在实验板每孔加入3μL 120nM SOS/9mM GTP混合液，以1000rpm/min，将实验板离心1分钟；
7)实验板在室温中孵育1小时；
8)使用Envision读板并记录数据；
9)使用Excel和Xlfit进行数据分析，计算待测化合物IC50。
测试方法2.GP2D细胞p-ERK抑制测试
1.实验目的：
通过HTRF的方法，筛选出能有效抑制GP2D细胞p-ERK的化合物。
2.实验流程：
1).GP2D细胞种于透明96孔细胞培养板中，80μL细胞悬液每孔，每孔包含8000个细胞，细胞板放入二氧化碳培养箱，37度过夜孵育；
2).取2μL化合物加入78μL细胞培养基，混匀后，取20μL化合物溶液加入到对应细胞板孔中，细胞板放回二氧化碳培养箱继续孵育1小时；
3).结束孵育后，弃掉细胞上清加入50μL 1X细胞裂解液每孔，室温摇晃孵育30分钟；
4).使用detection buffer将Phospho-ERK1/2 Eu Cryptate antibody和Phospho-ERK1/2d2 antibody稀释20倍；
5).取16μL细胞裂解物上清每孔到新的384白色微孔板中，再加入2μL Phospho-ERK1/2 Eu Cryptate antibody稀释液和2μL Phospho-ERK1/2 d2 antibody稀释液，常温孵育至少4小时；
6).孵育结束后使用多标记分析仪读取HTRF excitation:320nm,emission:615nm，665nm；
7).计算待测化合物IC50。
测试方法3.GP2D 3D CTG实验
1.实验目的：
本实验旨在验证本发明化合物对KRAS G12D突变的GP2D人结肠癌细胞的增殖抑制效果。
2.实验材料：
细胞株GP2D、DMEM培养基，盘尼西林/链霉素抗生素购自维森特，胎牛血清购自Biosera。CellTiter-3D Cell Viability Assay(3D细胞活率化学发光检测试剂)试剂购自Promega。
3.实验方法：
将GP2D细胞种于96孔U底细胞培养板中，80μL细胞悬液每孔，其中包含2000个GP2D细胞。细 胞板置于二氧化碳培养箱中过夜培养。将待测化合物用排枪进5倍稀释至第8个浓度，即从200μM稀释至2.56nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是1μM至0.0128nM。细胞板置于二氧化碳培养箱中培养5天。加入化合物的细胞板结束孵育后，向细胞板中加入每孔100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。
测试方法4.体内药代动力学实验
1.实验目的：
本实验旨在考察本发明化合物在SD小鼠口服及静脉注射下的药代动力学特征。
2.实验方法：
受试化合物与10％二甲基亚砜/60％聚乙二醇400/30％水溶液混合，涡旋并超声，制备得到约1mg/mL澄清溶液，微孔滤膜过滤后备用。选取7至10周龄的雄性SD小鼠，静脉注射给予候选化合物溶液，剂量为3mg/kg。口服给予候选化合物溶液，剂量约30mg/kg。收集一定时间的全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件(美国Pharsight公司)计算药代参数。
测试方法5.体内药效学实验
1.实验目的：
人结肠癌GP2D细胞裸小鼠皮下移植肿瘤Balb/c Nude小鼠模型的体内药效学研究。
2.实验方法：
细胞培养：人结肠癌GP2D细胞体外单层培养，培养条件为DMEM/F12培养基中加20％胎牛血清，1％双抗，37℃5％二氧化碳孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，重悬于适量PBS中，1:1加入基质胶，获取细胞密度为25x 106cells/mL的细胞悬液。
细胞接种：将0.2mL(5×106cells/mouse个)Mia PaCa-2细胞(加基质胶，体积比为1:1)皮下接种于每只小鼠的右后背。
实验操作：肿瘤平均体积达到约190mm3时，根据肿瘤体积进行随机分组，每组6只，空白组给药剂量为0，测试组给药剂量分别为30mg/kg、100mg/kg，给药体积10μL/g，口服给药，给药22天，每天两次。
3.肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即D0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。
TGI(％)，反映肿瘤生长抑制率。TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
技术效果
本发明化合物与KRASG12D蛋白有较好的结合作用，可显著抑制KRASG12D酶、GP2D细胞p-ERK，本发明化合物对KRASG12D突变的细胞具有良好的细胞增殖抑制活性，具有优异的肿瘤抑制效果。此外，本发明化合物具有较好的药代动力学特征。
相关定义
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。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。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，在手性HPLC(手性高效液相色谱)分析和SFC(超临界流体色谱)分析中，化合物 的保留时间可能会因为测定仪器等因素而产生差异。对任何特定的化合物，化合物的保留时间可能存在测量误差。因此，在确定每个化合物时，应该将此误差考虑在内，在误差内也属于本申请的范围。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR)0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键直形虚线键或波浪线表示。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；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；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；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 这4种连接方式，即使-N-上画出了H原子，但是仍包括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。
除非另有说明，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键或直形虚线键例如，使用直形实线键和直形虚线键表示立体中心的相对构型，代表的混合物。代表 的混合物，代表
本发明的某些化合物可以以阻转异构体存在，其是构象异构体，当由于与分子的其它部分的空间相互作用而阻止或大大减缓绕分子中单键的旋转时出现。本发明公开的化合物包括所有的阻转异构体，可以是纯的单独的阻转异构体、或者是富含其中一种阻转异构体、或者是各自的非特异性混合物。如果围绕单键的旋转势能足够高，并且构象之间的相互转化足够慢，则可以允许分离异构体。例如， 与为一对阻转异构体，其中，苯基上的表示该侧立体朝向为向外，表示该侧立体朝向为向内。
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用表示，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。
除非另有规定，Cn-n+m或Cn-Cn+m包括n至n+m个碳的任何一种具体情况，例如C1-12包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、和C12，也包括n至n+m中的任何一个范围，例如C1-12包括C1- 3、C1-6、C1-9、C3-6、C3-9、C3-12、C6-9、C6-12、和C9-12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。
除非另有规定，术语“C1-3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C1-3烷基包括C1-2和C2-3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)。C1- 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“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟(F)、氯(Cl)、溴(Br)或碘(I)原子。
除非另有规定，术语“C1-3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C1-3烷氧基包括C1-2、C2-3、C3和C2烷氧基等。C1-3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，“C2-4烯基”用于表示直链或支链的包含至少一个碳-碳双键的由2至4个碳原子组成的碳氢基团，碳-碳双键可以位于该基团的任何位置上。所述C2-4烯基包括C2-3、C4、C3和C2烯基等；所述C2-4烯基可以是一价、二价或者多价。C2-4烯基的实例包括但不限于乙烯基、丙烯基、丁烯基、丁间二烯基等。
除非另有规定，“C2-4炔基”用于表示直链或支链的包含至少一个碳-碳三键的由2至4个碳原子组成的碳氢基团，碳-碳三键可以位于该基团的任何位置上。所述C2-4炔基包括C2-3、C4、C3和C2炔基等。其可以是一价、二价或者多价。C2-4炔基的实例包括但不限于乙炔基、丙炔基、丁炔基等。
除非另有规定，在本发明式III中，当X为N+-O-时，“N+-O-”用于表示“N(→O)”，代表N的氧化物。
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：φ/ω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。Boc代表碳酸叔丁酰基；Fmoc代表9-芴基甲氧羰基；TIPS代表三异丙基硅基；PMB代表对甲氧基苄基；Tf代表三氟甲磺酰基；DCE代表二氯乙烷；THF代表四氢呋喃；H2O代表水；FA代表甲酸；ACN代表乙腈；PE代表石油醚；EA代表乙酸乙酯；DEA代表二乙醇胺；IPA代表异丙醇；DBU代表1,8-二偶氮杂双螺环[5.4.0]十一-7-烯；二代Grubbs催化剂代表CAS号为246047-72-3的化合物；柱色谱法中洗脱剂的比例代表的为体积比；浓度中M代表mol/L。
化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。
附图说明
图1.化合物A与和KRASG12D蛋白的结合模式图；
图2.化合物B与和KRASG12D蛋白的结合模式图；
图3.化合物C与和KRASG12D蛋白的结合模式图；
图4.化合物D与和KRASG12D蛋白的结合模式图；
图5.化合物E与和KRASG12D蛋白的结合模式图；
图6.化合物F与和KRASG12D蛋白的结合模式图；
图7.化合物G与和KRASG12D蛋白的结合模式图；
图8.化合物H与和KRASG12D蛋白的结合模式图；
图9.化合物I与和KRASG12D蛋白的结合模式图；
图10.化合物J与和KRASG12D蛋白的结合模式图；
图11.化合物K与和KRASG12D蛋白的结合模式图。
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。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
计算例1
分子对接过程是通过使用Maestro(版本2017-2)中的Glide SP[1]和默认选项进行的。选取PDB数据库中KRAS_G12C的晶体结构PDB:6UT0，将Cys12模拟突变为Asp12，经过能量优化后，作为对接模板。为了准备蛋白质，使用Maestro[2]的蛋白质准备向导模块添加氢原子，并使用OPLS3力场。对于配体的准备，使用LigPrep生成了分子的三维结构，并进行了能量最小化[3]，使用confgen模块对小分子构象进行采样。以6UT0的配体作为质心生成了边长为的正方体对接网格。在分子对接过程中放置参考化合物。分析蛋白质受体与配体的相互作用类型，分析蛋白质受体与配体的相互作用类型，然后根据计算得到的docking scrore以及结合模式选择并保存了合理对接构象，如图1至图11所示。
[1]Glide,LLC,New York,NY,2017.
[2]Maestro,LLC,New York,NY,2017.
[3]LigPrep,LLC,New York,NY,2017.
结论：本发明化合物与KRASG12D有较好的结合。
中间体Int-3A和Int-3B的合成
步骤1：
氮气保护，将Int3-1(3g,9.56mmol)和1-1B(1.83g,8.60mmol)溶于二氯甲烷(30mL)，-40℃加入三乙胺(2.90g,28.67mmol)，-40℃反应0.5小时。向反应体系中加入水(20mL)，水相使用二氯甲烷(20mL*4)萃取，分液。有机相使用饱和食盐水(20mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过柱层析(流动相：石油醚：乙酸乙酯＝20:1～1.5:1)分离，纯化得到中间体Int3-2。MS m/z:488.9，490.9[M+1]+。
步骤2：
氮气保护，将中间体Int3-2(0.8g,1.63mmol)和中间体2-7(269.91mg,1.63mmol)溶于N,N-二甲基甲酰胺(8mL)和四氢呋喃(8mL)，加入碳酸铯(1.33g,4.08mmol)和三乙烯二胺(54.97mg,490.06μmol)，25℃反应15小时。向反应体系中加入水(10mL)，水相使用乙酸乙酯(10mL*5)萃取，分液。有机相使用饱和食盐水(10mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过柱层析(流动相：石油醚：乙酸乙酯＝25:1～1:1)分离，纯化得到中间体Int3-3。MS m/z:618.0[M+1]+。
步骤3：
将中间体Int3-3通过制备SFC(色谱柱：REGIS(S,S)WHELK-O1(250mm*25mm,10μm)；流动相：A(超临界CO2)和B(异丙醇，含0.1％氨水)；梯度：B％:60％-60％,10min)分离得到中间体Int-3A和Int-3B。中间体Int-3A:SFC分析方法：色谱柱REGIS(S,S)WHELK-O1(50mm*4.6mm,3.5μm)，流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，保留时间为1.780min，ee值95.37％。1H NMR(400MHz,CDCl3)δ＝7.32(dd,J＝9.2,2.0Hz,1H),5.02(s,1H),4.94(s,1H),4.38-4.25(m,4H),4.24-4.17(m,1H),3.66-3.48(m,3H),3.34-3.20(m,2H),2.93(d,J＝17.2Hz,1H),2.75(dd,J＝8.8,4.0Hz,1H),2.47(d,J＝18.4Hz,1H),1.95(t,J＝5.6Hz,2H),1.89-1.75(m,3H),1.60-1.55(m,2H),1.52(s,9H),0.75(d,J＝3.2Hz,1H),0.52-0.45(m,1H).MS m/z:618.0[M+1]+。
中间体Int-3B：SFC分析方法：色谱柱REGIS(S,S)WHELK-O1(50mm*4.6mm,3.5μm)，流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，保留时间为1.914min，ee值96.62％。1H NMR(400MHz,CDCl3)δ＝7.33(dd,J＝9.2,2.0Hz,1H),5.18-4.85(m,2H),4.52-4.09(m,6H),3.73-3.44(m,3H),3.43-3.10(m,2H),2.88-2.66(m,1H),2.62-2.36(m,1H),2.02-1.67(m,6H),1.52(s,9H),1.27(d,J＝4.4Hz,1H),0.89-0.65(m,1H),0.63-0.40(m,1H).MS m/z:618.0[M+1]+。
中间体Int-4的合成
步骤1：
将Int4-1(100g,397.67mmol)溶于醋酸(300mL)中，冷却至0℃，随后加入浓硫酸(390.03g,3.98mol)，再逐滴加入亚硝酸钠(54.87g,795.34mmol)的水(50mL)溶液，在0℃反应1小时，此时悬浊液变澄清，随后逐滴加入碘化钾(132.03g,795.34mmol)的水(50mL)溶液，在0℃下反应1小时。加入5000mL水搅拌10分钟，随后抽滤，并用水冲洗5次，加入500mL饱和硫代硫酸钠的水溶液搅拌过夜。随后再次抽滤，用水(500mL*4)洗涤5次，抽滤，滤饼为中间体Int4-2。1H NMR(400MHz,CDCl3)δ＝8.37(d,J＝2.0Hz,1H),8.24(d,J＝2.0Hz,1H)。
步骤2：
将中间体Int4-2(126.15g,348.15mmol)溶于乙醇(500mL)与水(200mL)中，随后加入铁粉(38.88g, 696.29mmol)和氯化铵(37.25g,696.29mmol)，在80℃下反应2小时。抽滤，滤液减压浓缩，使用(500mL*2)乙酸乙酯萃取，饱和(500mL*6)食盐水洗涤，无水硫酸钠干燥，减压浓缩得到粗品。粗品用快速过柱仪过柱(洗脱剂：乙酸乙酯/石油醚，乙酸乙酯比例为0-20％)得中间体Int4-3。MS(ESI)m/z:331.8，333.8[M+1]+。
步骤3：
将中间体Int4-3(46g,138.40mmol)溶于N,N-二甲基甲酰胺(300mL)中，在0℃加入钠氢(16.61g,415.21mmol,60％)，并在0℃搅拌0.5小时，再加入中间体Int4-3A(65.03g,415.21mmol)，缓慢升温至25℃下反应2小时。向反应液中缓慢加入1000mL水使其完全析出，抽滤，并使用(250mL*4)水淋洗滤饼，滤饼50℃下真空干燥得中间体Int4-4。1H NMR(400MHz,CDCl3)δ＝7.09(d,J＝8.53Hz,4H),6.94-6.97(m,1H),6.88(d,J＝8.53Hz,4H),6.80(d,J＝2.76Hz,1H),4.50(s,4H),3.81(s,6H)。
步骤4：
将中间体Int4-4(78.66g,137.36mmol)溶于N,N-二甲基甲酰胺(280mL)中，加入碘化亚铜(130.80g,686.80mmol)，置换氮气后加热至100℃，再加入中间体5-10A(211.11g,1.10mol)反应0.5小时。加入500mL乙酸乙酯，500mL水，分液。有机相使用(1L*5)清水洗涤5次，再使用(1L*3)的饱和食盐水进行洗涤；水相使用1L的乙酸乙酯再次进行萃取，再次分液将有机相使用(1L*5)的清水洗涤，再使用(1L*3)的饱和氯化钠溶液进行洗涤。所有有机相减压浓缩得到粗品，向得到的粗品中加入300mL甲醇搅拌，抽滤，滤饼为中间体Int4-5。1H NMR(400MHz,CDCl3)δ＝7.05-7.13(m,4H),6.96(d,J＝3.01Hz,1H),6.84-6.92(m,4H),6.80(d,J＝2.76Hz,1H),4.50(s,4H),3.81(s,6H)。
步骤5：
将中间体Int4-5(0.098g,190.38μmol)、双联频那醇硼酸酯(483.44mg,1.90mmol)和醋酸钾(56.05mg,571.14μmol)加入无水二氧六环(2.5mL)中，随后进行三次氮气置换。加入1,1-双(二苯基膦)二荗铁二氯化钯(41.79mg,57.11μmol)，再次进行三次氮气置换，随后升温至110℃反应20小时。加入50mL水，用150mL乙酸乙酯进行萃取，分液，取有机相并使用(50mL*3)饱和食盐水进行洗涤，减压浓缩得粗品，粗品用快速柱层析分离(洗脱剂：乙酸乙酯：石油醚：0～10％)得中间体Int-4。MS(ESI)m/z:562.2[M+1]+。
中间体Int-5的合成
将Int5-1(3g,13.37mmol),频哪醇硼酸酯(5.09g,20.05mmol)和醋酸钾(2.62g,26.73mmol)溶于N，N-二甲基甲酰胺(30mL)中，氮气置换三次，加入1,1-双(二苯膦基)二茂铁二氯化钯(II)二氯甲烷复合物(1.09g,1.34mmol)，加热至100℃，搅拌6小时。反应液倒入150mL水中，乙酸乙酯萃取(20mL*3)，合并有机相，饱和食盐水洗(15mL),无水硫酸钠干燥，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至3:1),得到Int-5。1H NMR(400MHz,CDCl3)δ7.05(dd,J＝4.0,2.8Hz,1H),6.87(dd,J＝7.6,2.4Hz,1H),3.16-4.46(m,2H),1.38(s,12H)。
实施例1

步骤1：中间体1-1A-2的制备
将中间体1-1A-1(120g,709mmol)溶于叔丁醇(1200mL)和水(1200mL)中，随后依次加入二水合锇酸钾(10.4g，28.3mmol)和N-甲基吗啉氧化物(249g,2.13mol)。将反应液在45℃下搅拌16小时。减压浓缩，除去多余的溶剂，乙酸乙酯(500mL*2)萃取，饱和亚硫酸溶液(1000mL)洗。合并的有机层用饱和食盐水(500mL*3)洗涤，经无水硫酸钠干燥，过滤并减压浓缩得到粗品产物。粗品通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1)纯化，得到1-1A-2。1H NMR(400MHz,CDCl3)δ4.23(t,J＝3.6Hz,2H),3.55-3.58(m,2H),3.35-3.32(m,2H),2.87-2.83(m,2H),1.45(s,9H)。
步骤2：中间体1-1A-3的制备
将中间体1-1A-2(107g，526mmol)溶于二氯甲烷(1700mL)中，冷却至0℃，随后加入二醋酸碘苯(254g，789mmol)。将反应体系转移至25℃并搅拌3小时。加入饱和碳酸氢钠溶液(500mL)淬灭反应体系，并加入二氯甲烷(100mL)搅拌0.5小时，随后有机相用无水硫酸钠干燥，过滤并减压浓缩以得到粗品产物。25℃下加入甲基叔丁基醚(200mL)，并搅拌10分钟，过滤并减压下浓缩以得到粗品中间体1-1A-3。
步骤3：中间体1-1A-4的制备
将中间体1-1A-3(200g)溶于四氢呋喃(600mL)中，冷却至-78℃，随后向反应体系中加入乙烯基溴化镁(1M，1.79L)。随即将反应体系升至25℃并搅拌16小时。10℃下加入饱和氯化铵溶液(1000mL)淬灭反应体系，并用乙酸乙酯(500mL)萃取。有机相用饱和食盐水(500mL*3)洗涤，无水硫酸钠干燥，过滤并减压浓缩得到粗品产物。粗品通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1)纯化，得到中间体1-1A-4。1H NMR(400MHz,CDCl3)δ5.82-5.89(m,2H),5.32(t,J＝11.6Hz,2H),5.16-5.19(m,2H),4.45(s,2H), 3.60-3.70(m,1H),3.37(s,2H),3.25(s,1H),2.95(d,J＝8.8Hz,1H),1.48(s,9H)。
步骤4：中间体1-1A-5的制备
将中间体1-1A-4(80.0g,310mmol)溶于二氯甲烷(1000mL)中，随后将反应体系转移至0℃并加入DBU(23.6g,155mmol)和2,2,2-三氯乙腈(269g,1.87mol)，将反应体系转移至25℃并搅拌16小时。减压浓缩，残余物用乙酸乙酯(500mL*2)萃取，水洗(100mL*2)，饱和食盐水(100mL*3)洗，无水硫酸钠干燥，过滤并减压浓缩得到粗品产物。粗品产物通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1)纯化，得到中间体1-1A-5。1H NMR(400MHz,CDCl3)δ8.37(s,2H),5.81-5.87(m,2H),5.45(s,2H),5.39-5.43(m,2H),5.25-5.30(m,2H),3.61-3.81(m,4H),1.48(s,9H)。
步骤5：中间体1-1A-6的制备
将中间体1-1A-5A(32.1g,238mmol)溶于DCE(850mL)中，随后加入1,5-环辛二烯氯化铱二聚体(12.3g,18.3mmol)。将反应体系冷却至0℃，随后将中间体1-1A-5(100g,183.1mmol)溶于DCE(1.00L)中并转移至上述反应体系，将反应升至25℃继续搅拌16小时。减压浓缩除去多余的溶剂，得到粗品产物。粗品通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1，石油醚/乙酸乙酯＝10:1)纯化，得到中间体1-1A-6。1H NMR(400MHz,CDCl3)δ7.52-7.55(m,2H),7.30(t,J＝7.2Hz,2H),7.22(t,J＝7.2Hz,1H),5.94-6.03(m,2H),5.10(t,J＝19.2Hz,2H),4.99(d,J＝10.4Hz,2H),3.51-3.61(m,4H),3.33(t,J＝13.6Hz,2H),1.48(s,15H)。
步骤6：中间体1-1A-7的制备
将1-1A-6(36.0g,50.4mmol)溶于甲苯(900mL)中，随后加入二代Grubbs催化剂(2.14g，2.52mmol)。反应体系升温至125℃并搅拌16小时。过滤，弃去滤饼，滤液减压浓缩得到粗品产物。粗品通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1，石油醚/乙酸乙酯＝10:1)纯化，得到中间体1-1A-7。MS:m/z＝329.2,[M+1]+。1H NMR(400MHz,CDCl3)δ7.60(t,J＝1.2Hz,2H),7.31(t,J＝7.2Hz,2H),7.22(s,1H),5.96(t,J＝9.2Hz,2H),3.60-3.65(m,2H),3.46-3.53(m,2H),3.09-3.14(m,2H),1.42(s,9H),1.25(d,J＝6.0Hz,6H)。
步骤7：中间体1-1A-8的制备
将中间体1-1A-7(26.8g,81.6mmol)溶于甲醇(201mL)中，随后加入氯化氢/甲醇(4M,67.3mL)。将反应体系升至35℃搅拌16小时。将反应混合物的pH值调节至12，并用乙酸乙酯(30.0mL)萃取，经无水硫酸钠干燥，过滤并减压浓缩得到1-1A-8。MS:m/z＝229.2,[M+1]+。1H NMR(400MHz,CDCl3)δ7.62(t,J＝7.2Hz,2H),7.31(t,J＝7.6Hz,2H),7.21(s,1H),6.01(s,2H),3.42(s,2H),2.89-2.93(m,2H),2.30-2.34(m,2H),1.23(s,6H)。
步骤8：中间体1-1A-9的制备
将中间体1-1A-8(18.6g,79.4mmol)溶于THF(190mL)中，随后加入氯甲酸-9-芴基甲酯(20.5g,79.4mmol)、碳酸钠(25.2g,238.2mmol)。将混合物在0℃下搅拌1小时。乙酸乙酯(50.0mL*2)萃取和水洗(200.0mL)。合并有机相，用饱和食盐水洗(150.0mL)，无水硫酸钠干燥，过滤并减压浓缩得到中间体1-1A-9。 MS:m/z＝451.3,[M+1]+。1H NMR(400MHz,CDCl3)δ7.76(d,J＝13.6Hz,2H),7.54-7.61(m,4H),7.24-7.40(m,7H),5.93-6.01(m,2H),4.34-4.40(m,2H),4.21(s,1H),3.70(t,J＝2Hz,2H),3.55-3.59(m,2H),3.15-3.23(m,2H),1.27(d,J＝2.4Hz,6H)。
步骤9：中间体1-1A-10的制备
将中间体1-1A-9(9.52g,21.1mmol)溶于三氟醋酸(192mL)，加热至75℃搅拌16小时。加入水(20.0mL)，并调节pH至9，再加入二氯甲烷(20.0mL)萃取，无水硫酸钠干燥，过滤并减压浓缩得到粗品产物。粗品在25℃下用正庚烷(6mL)搅拌2小时得到1-1A-10的三氟醋酸盐。MS:m/z＝333.1,[M+1]+。1H NMR(400MHz,CDCl3)δ7.77(d,J＝7.6Hz,2H),7.56(d,J＝7.2Hz,2H),7.41(t,J＝7.6Hz,2H),7.33(t,J＝6Hz,2H),6.18-6.27(m,2H),4.38-4.42(m,2H),4.23(s,1H),3.88(d,J＝2.0Hz,2H),3.82(d,J＝2.4Hz,1H),3.72(d,J＝2.0Hz,1H),3.21-3.60(m,2H)。
步骤10：中间体1-1A-11的制备
将中间体1-1A-10的三氟醋酸盐(1.00g,2.92mmol)溶于四氢呋喃(10.0mL)中，随后依次加入二碳酸二叔丁酯(764mg,3.50mmol)、三乙胺(885mg,8.75mmol)并在25℃下搅拌1小时。加入乙酸乙酯(10.0mL*2)和水(10.0mL)萃取。合并有机相，并用饱和食盐水(15.0mL)洗，无水硫酸钠干燥，过滤并减压浓缩得到粗品产物。粗品通过柱色谱法(石油醚/乙酸乙酯＝1/0至0/1，石油醚：乙酸乙酯＝3:1)纯化，得到中间体1-1A-11。MS:m/z＝433.2,[M+1]+。
步骤11：中间体1-1A的制备
将中间体1-1A-11(5.69g，12.59mmol)溶于乙醇(60.0mL)中，随后加入二甲胺(34.4g，251.8mmol)。将反应体系在25℃下搅拌3小时。直接减压浓缩，残余物用乙酸乙酯(40.0mL)和10％柠檬酸(40.0mL)萃取，将水相的pH值调至9，过滤并用乙酸乙酯(40.0mL*2)萃取，合并有机相，无水硫酸钠干燥，过滤并减压浓缩得到中间体1-1A。MS:m/z＝211.2,[M+1]+。1H NMR(400MHz,CDCl3)δ6.22(d,J＝10Hz,2H),4.40(d,J＝38.8Hz,2H),2.89-3.01(m,2H),2.40(d,J＝13.2Hz,2H),1.49(s,9H)。
步骤12：中间体1-3A-2的制备
将中间体1-3A-1(4g,17.02mmol)溶于AcOH(30mL)中，冰浴降温至0℃，随后加入浓硫酸(17.03g,170.21mmol)，接着滴加亚硝酸钠(1.76g,25.53mmol)的水溶液(5mL)，继续搅拌0.25小时，接着滴加碘化钾(4.24g,25.53mmol)的水溶液(5mL)，移至25℃下反应0.5小时。往反应液中加入水(50mL)，过滤，固体用饱和硫代硫酸钠溶液(2*40mL)洗，水洗(40mL)，固体用二氯甲烷(50mL)溶解，无水硫酸钠干燥、过滤、浓缩得中间体1-3A-2。
步骤13：中间体1-3A-3的制备
将中间体1-3A-2(2.85g,8.24mmol)，碘化亚铜(3.14g,16.48mmol)溶于N,N-二甲基甲酰胺(45mL)中，接着加入氟磺酰基二氟乙酸甲酯(6.33g,32.96mmol)，在80℃下反应1小时。继续反应0.75小时。将反应液 冷却至25℃，滤去不溶物，乙酸乙酯(100mL)淋洗，水洗(3*200mL)，无水硫酸钠干燥，浓缩。粗品通过柱色谱法(石油醚/乙酸乙酯＝30/1至20/1)纯化，得到中间体1-3A-3。
步骤14：中间体1-3A-4的制备
将中间体1-3A-3(1.66g,5.76mmol)，铁粉(1.13g,20.17mmol)，氯化铵(1.54g,28.82mmol)溶于乙醇(20mL)和水(10mL)中，升温至60℃反应2.5小时。往反应液中加入乙酸乙酯(80mL)稀释，滤去不溶物，水洗(2*50mL)，无水硫酸钠干燥、过滤、浓缩得中间体1-3A-4。MS:m/z＝257.9,[M+1]+。
步骤15：中间体1-3A的制备
将中间体1-3A-4(1.4g,5.43mmol)，联硼酸频那醇酯(2.07g,8.14mmol,1.5eq)，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(443.12mg,542.61μmol)，醋酸钾(1.60g,16.28mmol)溶于二氧六环(30mL)中，氮气保护下在85℃下反应16小时。将反应液冷却至25℃，滤去不溶物，乙酸乙酯(50mL)淋洗，浓缩。粗品通过柱色谱法(石油醚/乙酸乙酯＝20/1至10/1)纯化得中间体1-3A。MS:m/z＝306.0,[M+1]+。
步骤16：中间体1-2的制备
将中间体1-1(900mg,3.56mmol)溶于二氯甲烷(10mL)中，冷却至0℃，随后依次加入N,N-二异丙基乙胺(1.38g,10.69mmol)和1-1A(749.60mg,3.56mmol)，0℃下反应1小时。直接减压浓缩得粗品1-2。MS:m/z＝426.0,[M+1]+。
步骤17：中间体1-3的制备
将中间体1-2(220mg,516.10μmol)和1-2A溶于乙腈(10mL)中，随后加入N,N-二异丙基乙胺(200.10mg,1.55mmol)，将反应体系升至80℃并搅拌16小时。将反应液减压浓缩除去大部分溶剂，接着加入乙酸乙酯(10mL)和水洗(5mL)萃取，无水硫酸钠干燥，浓缩。粗品经硅胶柱层析(PE/EA＝1/1到DCM/MeOH＝20/1)分离得中间体1-3。MS:m/z＝549.1,[M+1]+。
步骤18：中间体1-4的制备
将中间体1-3A(0.2g,364.29μmol)，中间体1-3(222.27mg,728.58μmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(29.75mg,36.43μmol)，碳酸铯(356.08mg,1.09mmol)溶于二氧六环(5mL)和水(1.25mL)中，氮气保护下于90℃反应17小时。将反应液冷却至25℃，滤去不溶物，乙酸乙酯(50mL)淋洗，无水硫酸钠干燥，浓缩。粗品经硅胶柱层析(DCM/MeOH＝50/1-20/1)分离得中间体1-4。MS:m/z＝692.2,[M+1]+。
步骤19：化合物1的制备
将中间体1-4(92mg,133.01μmol)溶于二氯甲烷(4mL)中，在20℃下加入三氟乙酸(1.54g,13.51mmol,1mL)，继续反应0.5小时。将反应液浓缩干，二氯甲烷(2mL)溶解，加入碳酸氢钠固体(0.5g)充分搅拌，再加入乙酸乙酯(5mL)继续搅拌5分钟，滤去不溶物，浓缩。粗品经制备HPLC(色谱柱:Boston Green ODS 150*30mm*5μm；流动性:[水(甲酸)-乙腈]；乙腈％:10％-40％,6min)分离得化合物1。MS(ESI)m/z: 592.3[M+1]+。1H NMR(400MHz,CD3OD)δ9.01(s,1H),6.58(d,J＝14Hz,1H),6.41(s,1H),6.34(s,2H),5.56(d,J＝51Hz,1H),4.79–4.75(m,2H),4.62–4.61(m,2H),4.72(s,2H),4.03–3.82(m,5H),3.31–3.30(m,1H),2.59–2.54(m,2H),2.38–2.29(m,4H)。
实施例2

步骤1：中间体2-2的制备
将中间体2-1(50g,418.09mmol)，溶于二氯甲烷(500mL)中，加入三乙胺(84.61g,836.17mmol,)，接着加入二碳酸二叔丁酯(100.37g,459.90mmol)，在25℃下反应16小时。反应液加入100mL水，分液，有机相用无水硫酸钠干燥，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝100/1-50/1)分离得中间体2-2。MS:m/z＝206.1,[M+Na]+。
步骤2：中间体2-3的制备
将中间体2-2溶于无水(25g,136.43mmol)四氢呋喃中(350mL)中，加入3,7-二丙基-3,7-二氮杂二环[3.3.1]壬烷(37.31g,177.36mmol)，冷却至-65℃，再缓慢滴加仲丁基锂(1.3M,157.42mL)，反应1小时后，滴加入氯甲酸甲酯(15.73g,166.44mmol)，在-65℃下反应2小时。反应液滴加饱和饱和氯化铵(20mL)萃灭，乙酸乙酯(300mL*2)萃取，合并有机相，用饱和食盐水(150mL)洗，无水硫酸钠干燥，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝50/1-25/1)分离得中间体2-3。MS:m/z＝186.0[M-tBu+H]+。
步骤3：中间体2-4A的制备
将中间体2-3(12g,49.73mmol)，溶于THF(120mL)中，再加入3-氯-2-氯甲基丙烯(24.87g,198.94mmol)，冷却至-40℃，缓慢滴加双三甲基硅基胺基锂(1M,99.47mL)，逐渐升温至20℃下反应2小时。反应液滴加饱和氯化铵(20mL)淬灭，乙酸乙酯(150mL*2)萃取，合并有机相，用饱和食盐水(80mL)洗，无水硫酸钠干燥，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝20/1-10/1)分离得中间体2-4A。TLC薄层层析(石油醚:丙酮＝5:1)展开两次，2-4A的Rf值为0.5，其异构体的Rf值为0.55。2-4A在LCMS(色谱柱：Agilent Poroshell120EC-C18 2.7um 3.0*30mm，流动相：A:H2O(0.037％FA)-B:ACN(0.0187％FA)；B:5％-95％)上的保留时间为0.776min，其异构体的保留时间为0.801min。MS:m/z＝274.0[M-tBu+H]+。
步骤4：中间体2-5的制备
将中间体2-4A(3.6g,10.92mmol)，溶于氯化氢/乙酸乙酯(4M,27.29mL)中，20℃下反应2小时。反应液减压浓缩得粗品中间体2-5，无需纯化直接投下一步。MS:m/z＝230.1,[M+H]+。
步骤5：中间体2-6的制备
将中间体2-5(2.9g,10.90mmol)，溶于甲醇(100mL)中，再加入碳酸钾(4.52g,32.69mmol)，20℃下反应2小时。反应液加二氯甲烷(80mL)，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝10/1-5/1)分离得中间体2-6。MS:m/z＝194.1,[M+H]+。
步骤6：中间体2-7的制备
将中间体2-6(1.6g,8.28mmol)，溶于无水四氢呋喃(20mL)中，再加入四氢锂铝(628.51mg,16.56
mmol)，0℃下反应2小时。反应液滴加乙酸乙酯(10mL)稀释，滴加水(0.63mL)，无水硫酸钠干燥，过滤，浓缩得粗品中间体2-7，无需纯化直接投下一步。MS:m/z＝166.1[M+H]+。
步骤7：中间体2-8的制备
将中间体1-2(700mg,1.64mmol)和中间体2-7(407.00mg,2.46mmol)，溶于无水甲苯(20mL)中，再加入叔丁醇钠(473.45mg,4.93mmol)，0℃下反应2小时，再在20℃下反应2小时。反应液加入乙酸乙酯(50mL),加水10mL淬灭，分相，有机相饱和食盐水洗(10mL)，无水硫酸钠干燥，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝5/1-2/1)分离得中间体2-8。MS:m/z＝555.1,[M+H]+。
步骤8：中间体2-9A和2-9B的制备
将中间体2-8(0.1g,180.17μmol,1eq)，中间体1-3A(82.45mg,270.26μmol)溶于二氧六环(2mL)和水(0.5mL)中，再加入甲磺酸[正丁基二(1-金刚烷基)膦](2-氨基-1,1'-联苯-2-基)钯(II)(13.12mg,18.02μmol)，碳酸铯(146.75mg,450.42μmol)，90℃下反应16小时。反应液加乙酸乙酯(30mL)稀释，饱和食盐水洗(10mL)，无水硫酸钠干燥，过滤，浓缩。粗品经硅胶柱层析(PE/EA＝1/50-1/100)后，再经制备SFC分离(色谱柱:REGIS(S,S)WHELK-O1(250mm*25mm,10μm)；流动相:A:CO2,B:[0.1％NH3H2O IPA]；B％:45％-45％)得中间体2-9A和2-9B。其中中间体2-9A在分析SFC(色谱柱:(S,S)Whelk-01 100×4.6mm I.D.,5.0μm,流动相: A:超临界二氧化碳,B:IPA(0.05％DEA),梯度:40％B,流速:2.5mL/min)中的保留时间为3.276min，ee值为98.3％；化合物2-9B在分析SFC(色谱柱:(S,S)Whelk-01 100×4.6mm I.D.,5.0μm,流动相:A:超临界二氧化碳,B:IPA(0.05％DEA),梯度:40％B,流速:2.5mL/min)中的保留时间为3.879min，ee值为96.4％。
步骤9：化合物2A的制备
将中间体2-9A(30mg,43.00μmol)溶于二氯甲烷(1.5mL)中，再加入三氟乙酸(147.08mg,1.29mmol)，20℃下反应2小时。反应液用二氯甲烷(20mL)稀释，加入饱和碳酸氢钠(10mL)，分相，水相用二氯甲烷(10mL)萃取，合并有机相，无水硫酸钠干燥，过滤，浓缩。粗产物经制备HPLC(色谱柱:O-Welch C18150*30mm*5μm；流动相:[H2O(FA)-ACN]；B％:1％-41％,10min)纯化得化合物2A的甲酸盐。LCMS:MS(ESI)m/z:598.2[M+H]+。1H NMR(400MHz,CD3OD)δ9.07(s,1H),8.52(br s,1H),6.59(br d,J＝14.05Hz,1H),6.38-6.48(m,1H),6.33(s,2H),5.10-5.26(m,2H),4.59-4.72(m,3H),4.49(d,J＝11.04Hz,1H),4.27(br s,2H),4.08(br d,J＝15.31Hz,1H),3.94(br d,J＝11.04Hz,2H),3.62(br d,J＝15.81Hz,1H),3.53(d,J＝10.54Hz,1H),3.00-3.14(m,2H),2.75(br d,J＝16.31Hz,1H),1.86-1.98(m,1H),1.79(td,J＝3.67,6.96Hz,1H),0.69-0.85(m,2H)。
步骤10：化合物2B的制备
将中间体2-9B(31mg,44.43μmol)溶于二氯甲烷(1.5mL)中，再加入三氟乙酸(42.31mg,371.05μmol)，20℃下反应1小时。反应液用二氯甲烷(20mL)稀释，加入饱和碳酸氢钠(10mL)，分相，水相用二氯甲烷(10mL)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液浓缩。粗产物经制备HPLC(色谱柱:O-Welch C18 150*30mm*5μm；流动相:[H2O(FA)-ACN]；B％:1％-41％,12min)纯化得化合物2B的甲酸盐。LCMS:MS(ESI)m/z:598.2[M+H]+。1H NMR(400MHz,CD3OD)δ9.06(s,1H),8.52(br s,1H),6.59(br d,J＝13.80Hz,1H),6.43(s,1H),6.33(s,2H),5.16(br d,J＝10.29Hz,2H),4.58-4.72(m,3H),4.48(d,J＝11.29Hz,1H),4.25(br s,2H),4.07(br d,J＝15.56Hz,1H),3.94(br d,J＝11.04Hz,2H),3.61(br d,J＝15.81Hz,1H),3.51(d,J＝10.54Hz,1H),2.99-3.12(m,2H),2.74(br d,J＝16.06Hz,1H),1.88-1.95(m,1H),1.72-1.85(m,1H),0.69-0.81(m,2H)。
实施例3


步骤1：中间体3-1的制备
将中间体1-1(25g,99.03mmol)溶于二氯甲烷(200mL)中，冷却至-30℃，随后依次加入N,N-二异丙基乙胺(38.40g,297.08mmol)和1-1B(21.02g,99.03mmol)，-30℃下反应2小时。直接减压浓缩得粗品3-1。MS:m/z＝428.1[M+1]+。
步骤2：中间体3-2A的制备
将中间体3-1(23g,53.70mmol)和中间体2-7(9.76g,59.07mmol)溶于无水甲苯(300mL)中，在0℃下缓慢加入叔丁醇钠(13.93g,145.00mmol)，0℃下反应0.5小时，再在20℃下反应0.5小时。反应液加乙酸乙酯200mL稀释，饱和食盐水洗(100mL)，无水硫酸钠干燥，过滤，滤液减压浓缩得粗产物3-2。粗产物经制备SFC(色谱柱:DAICEL CHIRALCEL OD(250mm*50mm,10μm)；流动相:[A(超临界二氧化碳),B(含有0.1％氨水的乙醇)]；B:40％)纯化得产物3-2A和3-2B。3-2A在分析SFC(色谱柱:Cellulose 2 100mm*4.6mm,3μm)流动相:[A(超临界二氧化碳),B(MeOH(0.05％DEA))]；B:40％)条件下的保留时间为4.964min，ee值为95.7％，1H NMR(400MHz,CD3OD)δ8.76(s,1H),4.98-4.87(m,2H),4.51–4.48(m,3H),4.34-4.29(m,1H),4.27(br s,2H),4.23-4.18(m,1H),3.61–3.58(m,3H),3.12(d,J＝9.5Hz,1H),2.78(br d,J＝16.8Hz,1H),2.71(dd,J＝4.0,9.6Hz,1H),2.45(br d,J＝16.8Hz,1H),1.83–1.72(m,2H),1.75-1.62(m,3H),1.55–1.51(m,1H),1.42(s,9H),0.60(q,J＝4.2Hz,1H),0.47-0.42(m,1H)。MS:m/z＝557.2[M+1]+。3-2B在同样条件下的保留时间为8.382min，ee值为96.5％。
步骤3：中间体3-4的合成
将原料3-3(2g,5.58mmol)溶于二氯甲烷(40mL)中，冰浴下0-10℃下加入N,N-二异丙基乙胺(4.33g,33.47mmol)，接着滴加三氟甲磺酸酐(6.30g,22.31mmol)，继续反应1.5小时。往反应液中加入水(20mL)充分搅拌，分去水相，有机相用无水硫酸钠干燥，浓缩。粗品通过柱层析(流动相：石油醚：乙酸乙酯＝10:1～2:1)分离得中间体3-4。
步骤4：中间体3-5的合成
将中间体3-4(3g,4.82mmol)，二苯甲酮亚胺(1.75g,9.64mmol)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(557.57mg,963.63μmol)，碳酸铯(4.71g,14.45mmol)溶于甲苯(60mL)中，接着加入三(二亚苄基茚丙酮)二钯(441.21mg,481.82μmol)，氮气保护下在100℃下反应2小时。将反应液冷却至25℃，滤去不溶物，浓缩去大部分甲苯，乙酸乙酯(30mL)稀释，水洗(20mL)，无水硫酸钠干燥，浓缩得到粗品。粗品通过柱层析(流动相：石油醚：乙酸乙酯＝10:1～1:1)分离得中间体3-5。
步骤5：中间体3-6的合成
将中间体3-5(3.1g,4.74mmol)，联硼酸频那醇酯(2.41g,9.48mmol)，[1,1'-双(二苯基膦)二茂铁]二氯化钯(693.88mg,948.30μmol)，醋酸钾(1.40g,14.22mmol)溶于甲苯(60mL)中，氮气保护下在110℃下反应18小时。将反应液冷却至25℃，滤去不溶物，乙酸乙酯(50mL)萃取，水洗(50mL)，无水硫酸钠干燥，浓缩。粗品通过柱层析(流动相：石油醚：乙酸乙酯＝10:1～2:1)分离得中间体3-6。MS(ESI)m/z:468.2[M+H2O-Ph2CO+1]+。
步骤6：中间体3-7的合成
将中间体3-2A(11.3g,20.29mmol)加入水(60mL)和无水二氧六环(240mL)中，再加入甲磺酸[正丁基二(1-金刚烷基)膦](2-氨基-1,1'-联苯-2-基)钯(II)(1.48g,2.03mmol)和中间体3-6(15.38g,24.34mmol)，碳酸铯(13.22g,40.57mmol)，87℃下反应2小时。反应液加乙酸乙酯(200mL)稀释，饱和食盐水(40mLx2)洗，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品经柱层析(流动相：二氯甲烷：甲醇＝50:1～20:1)分离得中间体3-7。MS(ESI)m/z:862.4[M+H2O-Ph2CO+1]+。
步骤7：中间体3-8的合成
将中间体3-7(8.4g,8.18mmol)加入到乙酸乙酯(30mL)和水(10mL)，再加入盐酸/乙酸乙酯(4M,62.37mL)，20℃下反应2小时。反应液加水(30mL)，有机相用水(30mLx2)洗，合并水相，用饱和碳酸氢钠调pH至7-8，用乙酸乙酯(80mLx3)萃取，合并有机相，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品3-8，直接用于下一步。MS(ESI)m/z:762.3[M+1]+。
步骤8：化合物3的合成
将中间体3-8(6.2g,8.14mmol)加入乙腈(70mL)中再加入四甲基氟化铵(2.29g,13.83mmol)，60℃下反应1小时。反应液加100mL乙酸乙酯稀释，30mL饱和碳酸氢钠洗，无水硫酸钠干燥，过滤，滤液减压浓缩得粗品。粗品经柱层析(流动相：二氯甲烷(10％氨气的甲醇溶液)：甲醇＝20:1～10:1)纯化得化合物3。SFC分析(色谱柱：Chiralcel OJ-3 100*4.6mm,3μm；流动相：A(超临界二氧化碳)和B(乙醇，含0.05％二乙胺)；梯度：B％:40％-40％)，保留时间3.761min，ee值为97.34％。手性HPLC分析(色谱柱：FLM Chiral NQ，150*4.6mm,3μm；流动相：A:(正己烷)和B：(乙醇，含有0.2％二乙胺，v/v)；梯度：B％:等度30％；洗脱时间：60min；柱温：35℃)显示保留时间17.387min。MS(ESI)m/z:606.3[M+1]+，1H NMR(400MHz, CD3OD)δ＝9.01(s,1H),7.76(dd,J＝5.6,9.2Hz,1H),7.28-7.19(m,2H),7.15(d,J＝2.3Hz,1H),5.08(br s,1H),5.01(br s,1H),4.70-4.54(m,3H),4.43(dd,J＝7.3,10.0Hz,1H),4.32(dd,J＝6.3,10.3Hz,1H),3.78-3.65(m,5H),3.39-3.34(m,1H),3.23(d,J＝9.5Hz,1H),2.93(br d,J＝17.3Hz,1H),2.82(dd,J＝4.0,9.3Hz,1H),2.56(br d,J＝17.1Hz,1H),1.91-1.76(m,5H),1.64(td,J＝3.6,6.9Hz,1H),0.72(q,J＝4.0Hz,1H),0.61-0.53(m,1H)。
实施例4
步骤1：中间体4-1的合成
将中间体3-2A(150mg,269.27μmol)、中间体1-3A(86.58mg,269.27μmol)、甲磺酸[正丁基二(1-金刚烷基)膦](2-氨基-1,1'-联苯-2-基)钯(II)(98.05mg,134.64μmol)、碳酸铯(263.20mg,807.82μmol)溶于二氧六环(8mL)中，氮气换气三次，随后加入水(0.5mL)，氮气保护下，80℃反应6小时。过滤除去不溶物，水洗(10mL)，乙酸乙酯萃取(30mL*2)，饱和食盐水洗(10mL)，无水硫酸钠干燥，减压浓缩除去有机溶剂得粗品，粗品经柱层析(流动相：二氯甲烷：甲醇＝100:1～50:1)纯化得中间体4-1。MS(ESI)m/z:716.3[M+1]+。
步骤2：化合物4的合成
将中间体4-1(22mg,30.1μmol)溶于二氯甲烷(5mL)，随后加入三氟乙酸(1.5mL)，25℃下反应3小时。体系加入饱和碳酸氢钠溶液至pH为8，随后加入二氯甲烷萃取(5mL*2)，无水硫酸钠干燥，减压浓缩除去有机溶剂得粗品产物。粗品经prep-HPLC(色谱柱:O-Welch C18 150*30mm*5μm；流动相:[A:水(0.5 ％甲酸)-B:乙腈]；B％:8％-48％,8min)分离得化合物4的甲酸盐。MS(ESI)m/z:616.2[M+1]+。1H NMR(400MHz,CD3OD)δ＝9.06(s,1H),8.51(br s,1H),6.94(s,1H),6.52(s,1H),5.16(br d,J＝10.79Hz,2H),4.71–4.80(m,2H),4.67(br d,J＝10.79Hz,1H),4.53(br d,J＝11.04Hz,1H),4.02–4.10(m,2H),3.84–3.90(m,2H),3.59–3.63(m,1H),3.50–3.53(m,1H),3.03-3.09(m,2H),2.73-2.78(m,1H),1.96-2.09(m,4H),1.90-1.93(m,1H),1.75-1.81(m,1H),0.87-0.97(m,1H),0.68-0.83(m,2H)。
实施例5

步骤1：化合物5-2的合成
氮气保护，将化合物5-1(21g,143.30mmol)溶于乙腈(210mL)中，加入N-碘代丁二酰亚胺(38.69g,171.96mmol)和一水合对甲苯磺酸(1.36g,7.16mmol)，75℃反应5小时。缓慢加入水(150mL)，水相用乙酸乙酯(150mL*3)萃取，分液。合并有机相，依次使用饱和亚硫酸钠溶液(15mL)和饱和食盐水(100mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗品5-2。MS(ESI)m/z:272.9[M+1]+。
步骤2：化合物5-3的合成
将化合物5-2(39.3g)溶于乙醇(435mL)中，加入三乙胺(43.79g,432.75mmol)和二氯二三苯基膦钯(10.12g,14.42mmol)，CO置换三次，然后CO氛围下(50Psi)，80℃反应47小时。减压浓缩，加入乙酸乙酯(300mL)和0.3M盐酸(300mL)，有固体析出，过滤，得到滤饼。滤液分液，水相使用乙酸乙酯(200mL*2)萃取，分液。合并有机相，使用饱和食盐水(200mL)清洗，无水硫酸钠干燥，过滤，将滤饼与有机相合并减压浓缩，得到粗品5-3。1H NMR(400MHz,CDCl3)δ8.54(s,1H),4.39(q,J＝7.1Hz,2H),1.41(t,J＝7.1Hz,3H)。
步骤3：化合物5-4的合成
氮气保护，将化合物5-3(41.3g,188.92mmol)溶于四氢呋喃(240mL)中，加入甲醇(80mL)和水(80mL)，分批次加入一水合氢氧化锂(23.78g,566.76mmol)，然后20℃反应5小时。将反应体系减压浓缩，向反应体系中加入水(100mL),水相使用4M盐酸调节pH至2，有固体析出，过滤。将滤饼溶于乙醇(200mL)，20℃搅拌16小时，过滤，滤饼减压干燥得到粗产品。将粗产品溶于石油醚(20mL)和乙酸乙酯(30mL)，20℃搅拌1小时，过滤，滤饼减压干燥,得到化合物5-4。1H NMR(400MHz,DMSO-d6)δ13.95-13.08(m,1H),8.35(s,1H),7.62(br s,2H)。
步骤4：化合物5-5的合成
氮气保护，将化合物5-4(17.8g,93.41mmol)溶于三氯氧磷(234.25g,1.53mol)，然后95℃反应3小时。将反应体系减压浓缩，加入四氢呋喃(360mL)，然后20℃分批次加入硫氰酸胺(21.33g,280.23mmol)，随后40℃反应16小时。向反应体系中加入水(300mL)，水相使用乙酸乙酯(300mL*3)萃取，分液。合并有机相，使用饱和食盐水(300mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。向粗产品中加入乙酸乙酯(40mL)和石油醚(20mL)，20℃搅拌0.5小时，过滤，滤饼减压干燥，得到化合物5-5。1H NMR(400MHz,DMSO-d6)δ13.33(br s,1H),12.91(s,1H),8.64(s,1H)。
步骤5：化合物5-6的合成
氮气保护，将化合物5-5(8.55g,36.91mmol)溶于N,N-二甲基甲酰胺(140mL)，20℃加入甲醇钠(2.09g,38.76mmol)，然后20℃缓慢滴加碘甲烷(4.72g,33.22mmol)，然后20℃反应5小时。向反应体系中加入冰水(300mL)，有固体析出，过滤，滤饼使用冰水(100mL)清洗，滤饼减压干燥，得到化合物5-6。1H NMR(400MHz,DMSO-d6)δ13.27(br s,1H),8.81(s,1H),2.61(s,3H)。
步骤6：化合物5-7的合成
氮气保护，将化合物5-6(3.5g,14.25mmol)和化合物5-6A(3.98g,14.67mmol)溶于二氧六环(70mL)、水(7mL)和乙醇(14mL)中，加入磷酸钾(9.07g,42.74mmol)和氯(2-二环己基膦基-2',4',6'-三异丙基-1,1'-联苯基)[2-(2'-氨基-1,1'-联苯)]钯(II)(1.68g,2.14mmol)，然后105℃反应1.5小时。向反应体系中加入水(200mL)，水相使用乙酸乙酯(200mL*2)萃取，分液。水相加入乙酸乙酯(200mL)变浑浊，通过硅藻土过滤，滤液分液。水相使用乙酸乙酯(200mL*2)萃取，分液。合并所有有机相，有机相使用饱和食盐水(100mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗产品通过柱层析(展开剂：石油醚：乙酸乙酯＝20:1～1:5,二氯甲烷：甲醇＝10:1)分离，得到化合物5-7。1H NMR(400MHz,DMSO-d6)δ13.19(br s,1H),9.03(s,1H),6.92(dd,J＝2.6,7.3Hz,1H),6.70(dd,J＝2.7,4.9Hz,1H),5.71(s,2H),2.62(s,3H)。
步骤7：化合物5-8的合成
氮气保护，将化合物5-7(2.1g,5.92mmol)溶于乙醇(63mL)中，0℃加入硫酸银(2.21g,7.10mmol)和碘单质(1.58g,6.22mmol)，然后缓慢升温至10℃反应2小时。向反应体系中加入饱和亚硫酸钠溶液(70mL)和乙酸乙酯(100mL)，过滤，分液。水相使用乙酸乙酯(100mL*3)萃取，分液。合并有机相，有机相使用无水硫酸钠干燥，过滤，减压浓缩得到粗品。粗产品通过柱层析(展开剂：石油醚：乙酸乙酯＝20:1～1:5)分离，得到化合物5-8。1H NMR(400MHz,CDCl3)δ10.47(br s,1H),9.34(s,1H),7.10(d,J＝8.1Hz,1H),2.78(s,3H)。
步骤8：化合物5-9的合成
冰水浴0℃氮气保护下，向化合物5-8(0.33g,686.56μmol)的N,N-二甲基甲酰胺(5mL)溶液中加入钠氢(96.11mg,2.40mmol)，搅拌30分钟后滴加4-甲氧基氯苄(236.55mg,1.51mmol)，得到的混合物自然升温至25℃搅拌14小时。向反应液中加入20mL饱和氯化铵水溶液和2*20mL乙酸乙酯搅拌10分钟，除去水相，有机相减压浓缩，得到粗品化合物5-9。
步骤9：化合物5-10的合成
氮气保护，将化合物5-9(0.11g)和N,N-二异丙基乙胺(59.16mg,457.73μmol)溶于四氢呋喃(2.7mL)，10℃加入1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐(95.28mg,183.09μmol)，10℃反应1小时。然后加入化合物1-1B(38.87mg,183.09μmol)，10℃反应17小时。向反应体系中加入水(10mL)，分液。水相使用二氯甲烷(10mL*3)萃取，分液，合并有机相，使用饱和食盐水(10mL)清洗，无水硫酸钠干燥，过滤，滤液减 压浓缩得到粗产品。粗产品通过柱层析(展开剂：乙酸乙酯/石油醚＝4.0％～20.0％)分离，得到化合物5-10。MS(ESI)m/z:915.0[M+1]+。
步骤10：化合物5-11的合成
氮气保护，将化合物5-10(0.15g,163.89μmol)和碘化亚铜(156.07mg,819.47μmol)溶于N,N-二甲基甲酰胺(3.75mL)，加入化合物5-10A(314.86mg,1.64mmol)，80℃反应2小时。缓慢加入水(10mL)和乙酸乙酯(20mL)，有固体析出，过滤。取滤液分液，水相使用乙酸乙酯(10mL*3)萃取，分液。合并有机相，使用饱和食盐水(5mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过柱层析(展开剂：乙酸乙酯/石油醚＝4.0％～20.0％)分离，得到化合物5-11。MS(ESI)m/z:857.1[M+1]+。
步骤11：化合物5-12的合成
氮气保护，将化合物5-11(0.06g,69.98μmol)溶于四氢呋喃(1.2mL)和水(0.4mL)中，0℃加入单过硫酸氢钾(82.38mg,489.89μmol)，缓慢升温至10℃反应4小时。补加单过硫酸氢钾(35.31mg,209.95μmol)，10℃反应2小时。缓慢加入饱和亚硫酸钠溶液(5mL)，水相使用乙酸乙酯(10mL*3)萃取，分液。合并有机相，使用饱和食盐水(5mL)清洗，无水硫酸钠干燥，过滤，减压浓缩，得到化合物5-12。MS(ESI)m/z:889.2[M+1]+；MS(ESI)m/z:873.2[M+1]+。
步骤12：化合物5-13的合成
氮气保护，将化合物5-12(0.05g,56.22μmol)和化合物2-7(13.93mg,84.33μmol)溶于四氢呋喃(1.5mL)，0℃加入叔丁醇钠(10.81mg,112.44μmol)，缓慢升温至10℃反应3小时。向反应体系中加入水(5mL)和乙酸乙酯(5mL)，过滤，滤液分液。水相使用乙酸乙酯(5mL*3)萃取，分液。合并有机相，使用饱和食盐水(5mL)清洗，无水硫酸钠干燥，过滤，减压浓缩，得到化合物5-13。MS(ESI)m/z:974.3[M+1]+。
步骤13：化合物5的合成
氮气保护，将化合物5-13(0.1g,102.62μmol)溶于二氯甲烷(2.5mL)，加入三氟乙酸(468.04mg,4.10mmol)，10℃反应6小时。将反应体系减压浓缩得到粗产品。粗产品通过两次高效液相制备色谱分离，得到化合物5。高效液相制备方法1：色谱柱：Phenomenex Luna 75*30mm*3μm；流动相A：水(0.04％盐酸)，流动相B：乙腈；运行梯度：B％:1％-43％，运行8min。高效液相制备方法2：色谱柱：Waters Xbridge BEH100*30mm*10μm；流动相A：水(10mM碳酸氢铵)，流动相B：乙腈；运行梯度：B％:30％-60％，运行8min。MS(ESI)m/z:634.2[M+1]+。
实施例6

步骤1：
氮气保护，将中间体Int-3A(0.2g,323.36μmol)和Int-4(181.67mg,323.36μmol)溶于四氢呋喃(8mL)和水(2mL)，加入磷酸钾(137.28mg,646.71μmol)和氯化(2-二环己基膦-2′,6′-二甲氧基-1,1′-联苯基)[2-(2′-氨基-1,1′-联苯基)]钯II)(23.30mg,32.34μmol)，然后50℃反应2小时。向反应体系中加入水(5mL)，水相使用乙酸乙酯(8mL*3)萃取，分液。有机相使用饱和食盐水(10mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Phenomenex luna C18 100*40mm*5μm；流动相:A(乙腈)和B(水，含0.04％盐酸)；梯度：B％:50％-80％，8min)分离，纯化得到化合物6-1A。MS m/z:973.1[M+1]+。
参考步骤1，使用中间体Int-3B为原料，得到化合物6-1B。MS m/z:973.1[M+1]+。
步骤2：
将化合物6-1A(0.2g,205.45μmol)通过制备SFC(色谱柱：ChiralPak IH,250*30mm,10μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％氨水)；梯度：B％:60％-60％，10min)分离，纯化分别得到化合物6-1A1和6-1A2。
化合物6-1A1：分析SFC(柱子：ChiralPak IH-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.463min，ee值97.49％。1H NMR(400MHz,CDCl3)δ＝7.30(s,1H),7.10(d,J＝8.8Hz,4H),6.94(d,J＝2.0Hz,1H),6.88(d,J＝8.8Hz,4H),6.52(d, J＝2.0Hz,1H),5.25(s,2H),5.04(d,J＝11.2Hz,1H),4.69(d,J＝10.8Hz,1H),4.55(s,4H),4.52-4.44(m,2H),4.44-4.32(m,3H),4.12-4.02(m,1H),3.81(s,6H),3.63(d,J＝15.6Hz,2H),3.56-3.43(m,1H),3.36(d,J＝17.6Hz,1H),3.19-3.08(m,1H),2.77(d,J＝17.6Hz,1H),2.07-1.87(m,4H),1.85-1.77(m,2H),1.51(s,9H),1.26(s,1H),0.91-0.84(m,1H).MS m/z:973.1[M+1]+。
化合物6-1A2：分析SFC(柱子：ChiralPak IH-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.983min，ee值95.66％。1H NMR(400MHz,CDCl3)δ＝7.30(s,1H),7.10(d,J＝8.8Hz,4H),6.94(d,J＝2.4Hz,1H),6.88(d,J＝8.4Hz,4H),6.51(d,J＝2.4Hz,1H),5.22(s,2H),5.06-4.83(m,1H),4.77-4.62(m,1H),4.54(s,4H),4.49-4.24(m,5H),4.13-3.94(m,1H),3.81(s,6H),3.70-3.46(m,3H),3.39-3.23(m,1H),3.18-3.02(m,1H),2.80-2.64(m,1H),2.09-1.89(m,4H),1.85-1.76(m,2H),1.52(s,9H),1.32-1.25(m,1H),0.90-0.75(m,1H).MS m/z:973.1[M+1]+.
步骤3：
将化合物6-1B(0.2g,205.45μmol)通过制备SFC(柱子：ChiralPak IH,250*30mm,10μm；流动相：A(超临界CO2)和B(甲醇，含0.1％氨水)；梯度：B％:40％-40％，11min)分离，纯化分别得到化合物6-1B1和化合物6-1B2。
化合物6-1B1:分析SFC(柱子：ChiralPak IH-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.330min，ee值94.45％。1H NMR(400MHz,CDCl3)δ＝7.26(s,1H),7.10(d,J＝8.4Hz,4H),6.94(d,J＝2.0Hz,1H),6.88(d,J＝8.4Hz,4H),6.51(d,J＝2.4Hz,1H),5.01(s,1H),4.93(s,1H),4.54(s,4H),4.42-4.16(m,6H),3.81(s,6H),3.65-3.50(m,3H),3.33-3.19(m,2H),2.94(d,J＝16.0Hz,1H),2.75(d,J＝6.4Hz,1H),2.46(dd,J＝15.6,2.0Hz,1H),2.00-1.92(m,2H),1.92-1.72(m,4H),1.52(s,9H),0.78-0.69(m,1H),0.54-0.42(m,1H).MS m/z:973.1[M+1]+。
化合物6-1B2:分析SFC(柱子：ChiralPak IH-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.495min，ee值98.01％。1H NMR(400MHz,CDCl3)δ＝7.25(s,1H),7.10(d,J＝8.4Hz,4H),6.93(s,1H),6.88(d,J＝8.4Hz,4H),6.51(s,1H),5.01(s,1H),4.92(s,1H),4.53(s,4H),4.39-4.24(m,5H),4.17(d,J＝8.8Hz,1H),3.81(s,6H),3.64-3.49(m,3H),3.33-3.19(m,2H),2.95(d,J＝18.8Hz,1H),2.75(d,J＝4.8Hz,1H),2.46(d,J＝16.4Hz,1H),1.99-1.89(m,2H),1.88-1.72(m,4H),1.52(s,9H),0.74(d,J＝1.6Hz,1H),0.53-0.43(m,1H).MS m/z:973.1[M+1]+.
步骤4：
化合物6A1:
氮气保护，将化合物6-1A1(0.07g,71.91μmol)溶于二氯甲烷(3.5mL)，加入三氟乙酸(1.07g,9.42mmol)，然后20℃反应3小时。减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Waters Xbridge BEH C18100*30mm*10μm；流动相:A(乙腈)和B(水，含10mM碳酸氢铵)；梯度：B％:35％-70％)分离，纯化得到化合物6A1。1H NMR(400MHz,CDCl3)δ＝7.31(d,J＝9.6Hz,1H),6.87(s,1H),6.42(s,1H),5.03(s,1H),4.94(s,1H),4.44-4.26(m,3H),4.26-4.11(m,3H),3.66(s,2H),3.53(t,J＝11.6Hz,2H),3.37-3.23(m,2H),2.98(d,J＝16.8Hz,1H),2.77(dd,J＝8.4,2.8Hz,1H),2.47(d,J＝16.4Hz,1H),2.23-2.04(m,2H),1.91-1.81(m,4H),1.56(s,1H),1.28(d,J＝10.8Hz,1H),0.89(t,J＝7.2Hz,1H),0.57-0.44(m,1H).MS m/z:633.1[M+1]+。
化合物6A2:
氮气保护，将化合物6-1A2(0.07g,71.91μmol)溶于二氯甲烷(3.5mL)，加入三氟乙酸(1.07g,9.42mmol)，然后20℃反应3小时。减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Waters Xbridge BEH C18100*30mm*10μm；流动相:A(乙腈)和B(水，含10mM碳酸氢铵)；梯度：B％:35％-70％)分离，纯化得到化合物6A2。1H NMR(400MHz,CDCl3)δ＝7.30(d,J＝10.0Hz,1H),6.87(s,1H),6.42(s,1H),5.02(s,1H),4.93(s,1H),4.42(d,J＝12.0Hz,1H),4.36-4.26(m,2H),4.21(d,J＝9.2Hz,3H),3.65(s,2H),3.61-3.54(m,1H),3.47(d,J＝12.4Hz,1H),3.35-3.23(m,2H),2.95(d,J＝16.8Hz,1H),2.84-2.72(m,1H),2.46(d,J＝16.4Hz,1H),2.11(br s,2H),1.92-1.80(m,4H),1.63-1.51(m,1H),1.40-1.19(m,1H),0.85-0.70(m,1H),0.60-0.42(m,1H).MS m/z:633.2[M+1]+。
步骤5：
化合物6B1:
氮气保护，将化合物6-1B1(0.06g,61.64μmol)溶于二氯甲烷(3mL)，加入三氟乙酸(921.00mg,8.08mmol)，然后20℃反应2小时。减压浓缩得到粗产品。粗产品通过高效液相色谱分离(色谱柱:Waters Xbridge Prep OBD C18 150*40mm*10μm；流动相:A(乙腈)和B(水，含0.05％氨水+10mM碳酸氢铵)；梯度：B％:30％-70％)，纯化得到化合物6B1。1H NMR(400MHz,CDCl3)δ＝7.31(d,J＝9.6Hz,1H),6.87(s,1H),6.42(s,1H),5.01(s,1H),4.92(s,1H),4.42(d,J＝12.8Hz,1H),4.33-4.24(m,2H),4.22-4.17(m,1H),4.14(s,2H),3.63(s,2H),3.58(s,1H),3.54(d,J＝12.4Hz,1H),3.45(d,J＝12.4Hz,1H),3.32-3.21(m,2H),2.95(d,J＝16.8Hz,1H),2.75(dd,J＝8.8,3.6Hz,1H),2.46(d,J＝17.2Hz,1H),1.92-1.86(m,2H),1.85-1.78(m,3H),1.58-1.52(m,1H),1.35-1.20(m,1H),0.74(q,J＝4.0Hz,1H),0.53-0.44(m,1H).MS m/z:633.2[M+1]+。
化合物6B2:
氮气保护，将化合物6-1B2(0.06g,61.64μmol)溶于二氯甲烷(3mL)，加入三氟乙酸(921.00mg,8.08mmol)，然后20℃反应2小时。减压浓缩得到粗产品。粗产品通过高效液相色谱分离(色谱柱:Waters Xbridge Prep OBD C18 150*40mm*10μm；流动相:A(乙腈)和B(水，含0.05％氨水+10mM碳酸氢铵)；梯度：B％:25％-65％)，纯化得到化合物6B2。1H NMR(400MHz,CDCl3)δ＝7.35-7.27(m,1H),6.88(s,1H),6.43(s,1H),5.03(s,1H),4.95(s,1H),4.46-4.26(m,3H),4.26-4.06(m,3H),3.66(s,3H),3.53(t,J＝10.8Hz,2H),3.29(d,J＝11.2Hz,2H),2.98(d,J＝15.6Hz,1H),2.78(s,1H),2.48(d,J＝14.8Hz,1H),1.85(s,5H),1.57(s,1H),1.38-1.22(m,1H),0.79(s,1H),0.51(s,1H).MS m/z:633.2[M+1]+。
实施例7

步骤1：
氮气保护，将中间体Int-3A(0.2g,323.36μmol)和中间体3-6(265.54mg,420.36μmol)溶于甲苯(2mL)和水(0.4mL)，加入磷酸钾(137.28mg,646.71μmol)和[(二(1-金刚烷基)-N-丁基膦)-2-(2-氨基联苯)氯化钯(II)(32.43mg,48.50μmol)，然后90℃反应6小时。向反应体系中加入水(7mL)，水相使用乙酸乙酯(7mL*3) 萃取，分液。有机相使用饱和食盐水(10mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Phenomenex luna C18 250*50mm*10μm；流动相:A(乙腈)和B(水，含0.04％盐酸)；梯度：B％:60％-80％，10min)分离，纯化得到化合物7-1A。MS m/z:879.3[M-C13H10O]+。
步骤2：
氮气保护，将中间体Int-3B(0.2g,323.36μmol)和中间体3-6(265.54mg,420.36μmol)溶于甲苯(2mL)和水(0.4mL)，加入磷酸钾(137.28mg,646.71μmol)和[(二(1-金刚烷基)-N-丁基膦)-2-(2-氨基联苯)氯化钯(II)(32.43mg,48.50μmol)，然后90℃反应4小时。向反应体系中加入水(7mL)，水相使用乙酸乙酯(7mL*3)萃取，分液。合并有机相，有机相使用饱和食盐水(10mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过柱层析(流动相：石油醚：乙酸乙酯＝50:1～1:1)分离，纯化得到化合物7-1B。MS m/z:522.4[M/2+1]+。
步骤3：
氮气保护，将7-1A(0.19g,216.12μmol)溶于二氯甲烷(7.5mL)，加入三氟乙酸(3.46g,30.36mmol)，然后20℃反应2小时。减压浓缩，得到粗品化合物7-2A。MS m/z:779.6[M+1]+。
参考步骤3，使用7-1B为原料，得到化合物7-2B。MS m/z:779.6[M+1]+。
步骤4：
氮气保护，将化合物7-2A(0.4g,143.58μmol)溶于N,N-二甲基甲酰胺(4mL)，加入氟化铯(2.18g,14.36mmol)和碳酸钠(91.31mg,861.50μmol)，20℃反应15小时。向反应体系中加入水(6mL)，水相使用乙酸乙酯(7mL*3)萃取，分液。合并有机相，使用饱和食盐水(5mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Phenomenex Luna C18 75*30mm*3μm；流动相:A(乙腈)和B(水，含0.04％甲酸)；梯度：B％:15％-55％)分离，再进一步通过制备SFC(柱子：DAICEL CHIRALPAK IG(250mm*30mm,10μm)；流动相：A(超临界CO2)和B(乙醇，含0.1％氨水)；梯度：B％:50％-50％，11min)分离，纯化分别得到化合物7A1和化合物7A2。
化合物7A1：分析SFC(柱子：ChiralPak IG-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.693min，ee值为97.92％。1H NMR(400MHz,CDCl3)δ＝7.67(dd,J＝8.8,5.6Hz,1H),7.28(s,1H),7.22(t,J＝8.8Hz,1H),7.12(d,J＝2.0Hz,1H),6.97(d,J＝2.0Hz,1H),5.03(s,1H),4.95(s,1H),4.41(d,J＝12.4Hz,1H),4.35(d,J＝10.4Hz,2H),4.23(d,J＝10.0Hz,1H),4.04-3.80(m,2H),3.66(s,3H),3.55(d,J＝12.4Hz,1H),3.51(d,J＝12.0Hz,1H),3.31(d,J＝4.4Hz,1H),3.28(s,1H),3.01(d,J＝16.8Hz,1H),2.82-2.74(m,2H),2.48(d,J＝16.8Hz,1H),1.90-1.86(m,5H),1.60-1.53(m,1H),1.32-1.19(m,1H),0.80(q,J＝4.4Hz,1H),0.56-0.48(m,1H).MS m/z:623.2[M+1]+。化合物7A2：分析SFC(柱子：ChiralPak IG-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为2.236min，ee值为98.26％。1H NMR(400MHz,CDCl3)δ＝7.67(dd,J＝8.8,6.0Hz,1H),7.28(s,1H),7.22(t,J＝8.8Hz,1H),7.25-7.19(m,1H),7.12(d,J＝2.0Hz,1H),5.03(s,1H),4.95(s,1H),4.44-4.30(m,3H),4.29-4.23(m,1H),4.07-3.83(m,2H),3.77-3.65(m,3H),3.57(t,J＝10.8Hz,2H),3.32(s,1H),3.29(d,J＝5.2Hz,1H),2.99(d,J＝16.4Hz,1H),2.83-2.73(m,2H),2.49(d,J＝16.8Hz,1H),1.94-1.86(m,5H),1.63-1.53(m,1H),1.30-1.11(m,1H),0.82(d,J＝4.0Hz,1H),0.53(q,J＝8.0Hz,1H).MS m/z:623.2[M+1]+。
步骤5：
氮气保护，将化合物7-2B(0.55g,198.24μmol)溶于N,N-二甲基甲酰胺(5mL)，加入氟化铯(3.01g,19.82mmol)和碳酸钠(126.07mg,1.19mmol)，20℃反应15小时。向反应体系中加入水(6mL)，水相使用乙酸乙酯(7mL*3)萃取，分液。合并有机相，使用饱和食盐水(5mL)清洗，无水硫酸钠干燥，过滤，减压浓缩得到粗产品。粗产品通过高效液相色谱(色谱柱:Phenomenex Luna C18 75*30mm*3μm；流动相:A(乙腈)和B(水，含0.04％甲酸)；梯度：B％:15％-55％)分离，再进一步通过制备SFC(柱子：DAICEL CHIRALPAK IG(250mm*30mm,10μm)；流动相：A(超临界CO2)和B(乙醇，含0.1％氨水)；梯度：B％:55％-55％，7min)分离，纯化分别得到化合物7B1和化合物7B2。
化合物7B1：分析SFC(柱子：ChiralPak IG-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为1.745min，ee值为98.56％。1H NMR(400MHz,CDCl3)δ＝7.67(dd,J＝9.2,6.0Hz,1H),7.30-7.25(m,1H),7.22(t,J＝8.8Hz,1H),7.11(d,J＝1.6Hz,1H),6.97(d,J＝1.6Hz,1H),5.03(s,1H),4.94(s,1H),4.38(t,J＝14.0Hz,2H),4.32-4.27(m,1H),4.27-4.19(m,1H),4.11-3.76(m,2H),3.73-3.61(m,3H),3.53(t,J＝11.6Hz,2H),3.34-3.23(m,2H),2.98(d,J＝16.8Hz,1H),2.81-2.73(m,2H),2.48(d,J＝16.8Hz,1H),1.92-1.79(m,5H),1.60-1.52(m,1H),1.26(s,1H),0.79(q,J＝3.6Hz,1H),0.51(q,J＝7.6Hz,1H).MS m/z:623.2[M+1]+。
化合物7B2：分析SFC(柱子：ChiralPak IG-3,50*4.6mm,3μm；流动相：A(超临界CO2)和B(异丙醇，含0.1％异丙胺)；梯度：B％:5％-5％，3min)条件下的保留时间为2.343min，ee值为98.62％。1H NMR(400MHz,CDCl3)δ＝7.67(dd,J＝8.8,5.6Hz,1H),7.29-7.25(m,1H),7.22(t,J＝8.8Hz,1H),7.11(d,J＝2.4Hz,1H),6.97(d,J＝2.0Hz,1H),5.03(s,1H),4.95(s,1H),4.41(d,J＝12.0Hz,1H),4.36(d,J＝10.4Hz,2H),4.23(d,J＝10.0Hz,1H),4.07-3.78(m,2H),3.75-3.63(m,3H),3.59(d,J＝12.0Hz,1H),3.54(d,J＝12.4Hz,1H),3.31(d,J＝3.2Hz,1H),3.28(s,1H),3.00(d,J＝16.8Hz,1H),2.82-2.73(m,2H),2.48(d,J＝16.8Hz,1H),1.93-1.80(m,5H),1.61-1.51(m,1H),1.26(s,1H),0.81(q,J＝4.0Hz,1H),0.55-0.47(m,1H).MS m/z:623.2[M+1]+。
实施例8
步骤1：
将化合物8-1(2g,6.05mmol)和1-1B(1.35g,6.36mmol)溶于二氯甲烷(20mL)，氮气置换三次，-40℃ 滴加三乙胺(1.84g,18.16mmol)。滴加完毕，升至25℃，反应3小时。倒入50mL水中，分液。水相用二氯甲烷萃取(10mL*3)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至3:1)，得到化合物8-2。MS m/z:505.0，507.0[M+1]+。
步骤2：
将8-2(646mg,1.28mmol)和氟化钾(1.48g,25.52mmol)溶于二甲基亚砜(12mL)，氮气置换三次，加热至120℃，搅拌1小时。反应液倒入60mL水中，乙酸乙酯萃取(10mL*3)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至5:1)，得到8-3。1H NMR(400MHz,CDCl3)δ7.80(d,J＝1.6Hz,1H),4.22-4.58(m,4H),3.51-3.89(m,2H),1.90-2.04(m,2H),1.66-1.84(m,2H),1.53(s,9H)。
步骤3：
将Int-5(333.98mg,1.23mmol)，8-3(502mg,1.03mmol)和磷酸钾(435.17mg,2.05mmol)溶于四氢呋喃(10mL)和水(2.5mL)，氮气置换三次，加入(2-二环己基膦-2,4,6-三异丙基-1,1-联苯)[2-(2-胺基-1,1-联苯]甲磺酸钯(86.76mg,102.50μmol,0.1eq)，氮气置换三次，35℃搅拌3小时。反应液冷却，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至4:1)，得到8-4。1H NMR(400MHz,CDCl3)δ7.81(d,J＝1.6Hz,1H),6.90(dd,J＝7.2,2.4Hz,1H),6.65(dd,J＝5.2,2.4Hz,1H),4.29-4.56(m,4H),3.51-3.87(m,2H),1.93-2.03(m,2H),1.77(br d,J＝8.0Hz,2H),1.54(s,9H)。
步骤4：
将8-4(330mg,595.25μmol),N-碘代丁二酰亚胺(200.88mg,892.87μmol)溶于N，N-二甲基甲酰胺(3.3mL),氮气置换三次，加热50℃，搅拌28小时。反应液倒入5mL饱和碳酸氢钠水溶液和5mL饱和硫代硫酸钠水溶液中，乙酸乙酯萃取(5mL*3)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至3:1)，得到化合物8-5。1H NMR(400MHz,CDCl3)δ7.85(d,J＝1.6Hz,1H),7.11(d,J＝8.4Hz,1H),4.30-4.63(m,4H),3.52-3.91(m,2H),1.94-2.05(m,2H),1.72-1.88(m,2H),1.54(s,9H)。
步骤5：
将氢化钠(36.69mg,917.26μmol,60％)溶于四氢呋喃(1mL)，氮气置换三次，冷却至0℃，加入2-7(151.56mg,917.26μmol)的四氢呋喃(1.5mL)溶液，升至20℃，搅拌10分钟。冷却至0℃，加入8-5(208mg,305.75μmol)的四氢呋喃(2.5mL)溶液，升至20℃，搅拌4小时。反应液倒入10mL饱和氯化铵水溶液中，乙酸乙酯萃取(2mL*3)。合并有机相，无水硫酸钠干燥，过滤，减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至1:2)，得到8-6。MS m/z:825.1[M+1]+。
步骤6：
将8-6(180mg,218.05μmol)，甲基硼酸(39.16mg,654.14μmol)和碳酸钾(60.27mg,436.09μmol)溶于二氧六环(2mL)和水(0.5mL)中，氮气置换三次，加入[1,1-双(二苯基磷)二茂铁]二氯化钯(15.95mg,21.80μmol,0.1eq)，氮气置换三次，升至80℃，搅拌60小时。反应液减压浓缩。粗品通过柱层析分离纯化(洗脱剂：石油醚:乙酸乙酯＝1:0至1:2)，得到粗品。粗品经过制备SFC分离纯化(色谱柱：DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相：A(超临界二氧化碳)和B(异丙醇，含0.1％氨水)；梯 度：B％＝45％-45％)，分别得到8-7A和8-7B的混合物、8-7C和8-7D。8-7C在SFC分析方法(色谱柱：Chiralpak AD-3,50×4.6mm,3μm；流动相：A(超临界二氧化碳)和B(异丙醇，含0.1％异丙胺)；梯度：B％＝5％-50％-5％，3.0min)中的保留时间为1.468min，手性异构体过量97.36％。8-7D在SFC分析方法(色谱柱：Chiralpak AD-3,50×4.6mm,3μm；流动相：A(超临界二氧化碳)和B(异丙醇，含0.1％异丙胺)；梯度：B％＝5％-50％-5％，3.0min)中的保留时间为1.603min，手性异构体过量100％。
步骤7：
将8-7A和8-7B的混合物(32mg,44.84μmol)溶于二氯甲烷(0.6mL)，加入三氟乙酸(0.2mL)，15℃搅拌0.5小时。反应液中滴加氨水至pH为9,减压浓缩。粗品经高效液相制备色谱分离纯化(色谱柱：Phenomenex Luna 100*30mm*3μm；流动相A：水(0.2％甲酸)，流动相B：乙腈；运行梯度：B％:1％-30％，运行8min)得到粗品。粗品经制备SFC分离纯化(色谱柱：DAICEL CHIRALPAK AD(250mm*30mm,10μm)；流动相：A(超临界二氧化碳)和B(乙醇，含0.1％氨水)；梯度：B％＝50％-50％，运行11min)，得到8A和8B。
8A在SFC分析方法(色谱柱：Chiralpak IG-3,50×4.6mm,3μm；流动相：A(超临界二氧化碳)和B(乙醇，含0.1％异丙胺)；梯度：B％＝5％-50％-5％，3.0min)中的保留时间为1.727min，手性异构体过量100％。1H NMR(400MHz,CD3OD)δ：7.91(s,1H),7.01(d,J＝8.4Hz,1H),4.95-5.10(m,2H),4.47(br d,J＝12.4Hz,2H),4.25-4.42(m,2H),3.56-3.75(m,5H),3.26-3.30(m,1H),3.20(br d,J＝9.6Hz,1H),2.91(br d,J＝16.4Hz,1H),2.79(br dd,J＝9.2,3.6Hz,1H),2.53(br d,J＝17.2Hz,1H),1.97(s,3H),1.81(br s,5H),1.56-1.70(m,1H),0.70(q,J＝4.0Hz,1H),0.49-0.59(m,1H)。MS m/z:613.2[M+1]+。
8B在SFC分析方法(色谱柱：Chiralpak IG-3,50×4.6mm,3μm；流动相：A(超临界二氧化碳)和B(乙醇，含0.1％异丙胺)；梯度：B％＝5％-50％-5％，3.0min)中的保留时间为2.216min，手性异构体过量99.22％。1H NMR(400MHz,CD3OD)δ：7.92(d,J＝2.0Hz,1H),7.01(d,J＝8.4Hz,1H),4.96-5.10(m,2H),4.47(br t,J＝13.2Hz,2H),4.25-4.41(m,2H),3.57-3.74(m,5H),3.26-3.30(m,1H),3.20(d,J＝9.6Hz,1H),2.91(br d,J＝16.8Hz,1H),2.79(dd,J＝9.6,4.0Hz,1H),2.53(br d,J＝16.4Hz,1H),1.76-1.91(m,8H),1.56-1.69(m,1H),0.70(q,J＝4.4Hz,1H),0.49-0.59(m,1H)。MS m/z:613.2[M+1]+。
步骤8：
将8-7C溶于二氯甲烷(0.6mL)，加入三氟乙酸(0.2mL)，15℃搅拌0.5小时。反应液中滴加氨水至pH为9，减压浓缩。粗品经高效液相制备色谱分离纯化(色谱柱：Phenomenex Luna 100*30mm*3μm；流动相A：水(0.2％甲酸)，流动相B：乙腈；运行梯度：B％:1％-30％，运行8min)得到8C的甲酸盐。1H NMR(400MHz,CD3OD)δ8.51(br s,1H),7.94(s,1H),7.02(d,J＝8.0Hz,1H),5.09(d,J＝16.4Hz,2H),4.50-4.63(m,3H),4.41(d,J＝10.8Hz,1H),3.86-4.08(m,3H),3.76(br dd,J＝12.8,8.8Hz,2H),3.48(br d,J＝15.6Hz,1H),3.34-3.41(m,1H),2.92-3.03(m,2H),2.66(br d,J＝16.4Hz,1H),1.93-2.07(m,7H),1.81-1.92(m,1H),1.66-1.76(m,1H),0.71-0.78(m,1H),0.60-0.69(m,1H)。MS m/z:613.2[M+1]+。
步骤9：
将8-7D(21mg,29.43μmol)溶于二氯甲烷(0.6mL)，加入三氟乙酸(0.2mL)，15℃搅拌0.5小时。反应液中滴加氨水至pH为9,减压浓缩。粗品经高效液相制备色谱分离纯化(色谱柱：Phenomenex Luna 100*30mm*3μm；流动相A：水(0.2％甲酸)，流动相B：乙腈；运行梯度：B％:1％-30％，运行8min)， 得到8D的甲酸盐。1H NMR(400MHz,CD3OD)δ8.53(br s,1H),7.93(s,1H),7.02(d,J＝8.0Hz,1H),5.07(br d,J＝18.8Hz,2H),4.42-4.65(m,3H),4.37(br d,J＝10.8Hz,1H),3.81-3.97(m,3H),3.73(br d,J＝12.4Hz,2H),3.43(br d,J＝15.6Hz,1H),3.34(br s,1H),2.84-3.03(m,2H),2.62(br d,J＝17.2Hz,1H),1.97(s,7H),1.81-1.89(m,1H),1.64-1.73(m,1H),0.69-0.81(m,1H),0.57-0.66(m,1H)。MS m/z:613.2[M+1]+。
实验例1.GP2D细胞p-ERK抑制测试
1.目的
通过HTRF的方法，筛选出能有效抑制GP2D细胞p-ERK的化合物。
2.实验流程
1).GP2D细胞种于透明96孔细胞培养板中，80μL细胞悬液每孔，每孔包含8000个细胞，细胞板放入二氧化碳培养箱，37度过夜孵育；
2).取2μL化合物加入78μL细胞培养基，混匀后，取20μL化合物溶液加入到对应细胞板孔中，细胞板放回二氧化碳培养箱继续孵育1小时；
3).结束孵育后，弃掉细胞上清加入50μL 1X细胞裂解液每孔，室温摇晃孵育30分钟；
4).使用detection buffer将Phospho-ERK1/2 Eu Cryptate antibody和Phospho-ERK1/2 d2 antibody稀释20倍；
5).取16μL细胞裂解物上清每孔到新的384白色微孔板中，再加入2μL Phospho-ERK1/2 Eu Cryptate antibody稀释液和2μL Phospho-ERK1/2 d2 antibody稀释液，常温孵育至少4小时；
6).孵育结束后使用多标记分析仪读取HTRF excitation:320nm,emission:615nm，665nm；
7).计算待测化合物IC50。
3.实验结果
结果见表2。
表2化合物对GP2D细胞p-ERK抑制的IC50值
实验结论：本发明化合物具有显著的GP2D p-ERK抑制作用。
实验例2.GP2D 3D CTG实验
1.实验目的：
本实验旨在验证本发明化合物对KRAS G12D突变的GP2D人结肠癌细胞的增殖抑制效果。
2.实验材料：
细胞株GP2D、DMEM培养基购自GIBCO，FBS购自Hyclone,L-glutamine购自Invitrogen。96-孔板购自Ultra Low Cluster。3D Cell Viability Assay(3D细胞活率化学发光检测试剂)试剂购自Promega，2104EnVision读板器购自PerkinElmer。
3.实验方法：
将GP2D细胞按DMEM+10％FBS+2mM L-glutamine培养条件在37℃，5％CO2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。将GP2D细胞种于96孔U底细胞培养板中，135μL细胞悬液每孔，其中包含6000个GP2D细胞。将培养板在37℃，5％CO2，及100％相对湿度的培养箱中培养过夜。将待测化合物用排枪进5倍稀释至第8个浓度，即从200μM稀释至2.56nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是1μM至0.0128nM。细胞板置于二氧化碳培养箱中培养5天。加入化合物的细胞板结束孵育后，向细胞板中加入每孔100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。
5.实验结果：
结果见表3。
表3化合物对GP2D细胞抗增殖的IC50值
实验结论：本发明化合物对KRAS G12D突变的GP2D细胞具有显著的抗增殖活性。
实验例3.AsPC-1 3D CTG实验
1.实验目的：
本实验旨在验证本发明化合物对KRAS G12D突变的AsPC-1人胰腺癌细胞的增殖抑制效果。
2.实验材料：
细胞株AsPC-1、RPMI-1640培养基购自GIBCO，FBS购自Hyclone。96-孔板购自Ultra Low Cluster，3D Cell Viability Assay(3D细胞活率化学发光检测试剂)试剂购自Promega，2104EnVision读板器购自PerkinElmer。
3.实验方法：
将AsPC-1细胞按RPMI-1640+10％FBS培养条件在37℃，5％CO2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。将AsPC-1细胞种于96孔U底细胞培养板中，135μL细胞悬液每孔，其中包含500个AsPC-1细胞。将培养板在37℃，5％CO2，及100％相对湿度的培养箱中培养过夜。将待测化合物用排枪进5倍稀释至第8个浓度，即从200μM稀释至2.56nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是1μM至0.0128nM。细胞板置于二氧化碳培养箱中培养7天。加入化合物的细胞板结束孵育后，向细胞板中加入每孔100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。
5.实验结果：
结果见表4。
表4化合物对AsPC-1细胞抗增殖的IC50值
实验结论：本发明化合物对KRAS G12D突变的AsPC-1细胞具有显著的抗增殖活性。
实验例4.PANC04.03 3D CTG实验
1.实验目的：
本实验旨在验证本发明化合物对KRAS G12D突变的PANC04.03人胰腺癌细胞的增殖抑制效果。
2.实验材料：
细胞株PANC04.03、RPMI-1640培养基购自GIBCO，FBS购自Hyclone，human insulin购自Yeasen。96-孔板购自Ultra Low Cluster，3D Cell Viability Assay(3D细胞活率化学发光检测试剂)试剂购自Promega，2104 EnVision读板器购自PerkinElmer。
3.实验方法：
将PANC04.03细胞按RPMI-1640+15％FBS+5ug/ml human insulin培养条件在37℃，5％CO2的培养箱中进行培养。定期传代，取处于对数生长期的细胞用于铺板。将PANC04.03细胞种于96孔U底细胞培养板中，135μL细胞悬液每孔，其中包含2000个PANC04.03细胞。将培养板在37℃，5％CO2，及100％相对湿度的培养箱中培养过夜。将待测化合物用排枪进5倍稀释至第8个浓度，即从200μM稀释至2.56 nM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移20μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是1μM至0.0128nM。细胞板置于二氧化碳培养箱中培养7天。加入化合物的细胞板结束孵育后，向细胞板中加入每孔100μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
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，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。
5.实验结果：
结果见表5。
表5化合物对PANC04.03细胞抗增殖的IC50值
实验结论：本发明化合物对KRAS G12D突变的PANC04.03细胞具有显著的抗增殖活性。
实验例5.体内药代动力学实验
1.实验目的：
本实验旨在考察本发明化合物在CD-1小鼠口服及静脉注射下的药代动力学特征。
2.实验方法：
受试化合物与10％二甲基亚砜+90％(10％羟丙基-β-环糊精(HP-β-CD)水溶液)混合，涡旋并超声，分别制备得到0.6、3.0和10.0mg/mL澄清溶液。选取7至10周龄的雄性CD-1小鼠，静脉注射(i.v.)给予候选化合物溶液，剂量为3mg/kg(给药浓度0.6mg/mL)。口服(p.o.)给予候选化合物溶液，剂量为30mg/kg(给药浓度3.0mg/mL)或100mg/kg(给药浓度10.0mg/mL)。收集一定时间的全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件(美国Pharsight公司)计算药代参数。
3.实验结果：
结果见表6。
表6化合物3在CD-1小鼠体内的PK性质

实验结论：本发明化合物在小鼠体内具有较好的药代动力学特征。
实验例6.GP2D肿瘤模型体内药效学实验
1.实验目的：
人结肠癌GP2D细胞裸小鼠皮下移植肿瘤Balb/c Nude小鼠模型的体内药效学研究
2.实验方法：
细胞培养：人结肠癌GP2D细胞体外单层培养，培养条件为DMEM/F12培养基中加20％胎牛血清，37℃5％二氧化碳孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，重悬于适量PBS中，1:1加入基质胶，获取细胞密度为25x 106cells/mL的细胞悬液。
细胞接种：将0.2mL(5×106cells/mouse个)GP2D细胞(加基质胶，体积比为1:1)皮下接种于每只小鼠的右后背。
实验操作：肿瘤平均体积达到140mm3时，根据肿瘤体积进行随机分组，每组6只，空白组给药剂量为0，测试组给药剂量分别为30mg/kg、100mg/kg，给药体积10μL/g，口服给药，给药28天，每天两次。
3.肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即D0)测量所得 平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。
TGI(％)，反映肿瘤生长抑制率。TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
4.实验结果：
实验结果见表7。
表7化合物3在GP2D肿瘤模型体内药效模型中的药效结果
实验结论：本发明化合物具有优异的肿瘤抑制效果。
实验例7.PANC04.03肿瘤模型体内药效学实验
1.实验目的：
人胰腺癌PANC04.03细胞裸小鼠皮下移植肿瘤Balb/c Nude小鼠模型的体内药效学研究
2.实验方法：
细胞培养：人胰腺癌PANC04.03细胞体外单层培养，培养条件为RPMI-1640+15％FBS+10units/mL insulin,37℃,5％二氧化碳孵箱培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％，数量到达要求时，收取细胞，计数，重悬于适量PBS中，获取细胞密度为25x 106cells/mL的细胞悬液。
细胞接种：将0.2mL(5×106cells/mouse个)PANC04.03细胞皮下接种于每只小鼠的右后背。
实验操作：肿瘤平均体积达到190mm3时，根据肿瘤体积进行随机分组，每组6只，空白组给药剂量为0，测试组给药剂量分别为30mg/kg、100mg/kg,150mg/kg，给药体积10μL/g，口服给药，给药28天，每天两次。
3.肿瘤测量和实验指标：
每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。相对肿瘤增殖率T/C(％)＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即D0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。
TGI(％)，反映肿瘤生长抑制率。TGI(％)＝[1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积)/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)]×100％。
4.实验结果：
实验结果见表8。
表8化合物3在小鼠PACN04.03体内药效模型中的药效结果
实验结论：本发明化合物具有优异的肿瘤抑制效果。

Claims (19)

  1. 本发明提供了式(III-1)所示化合物或其药学上可接受的盐，
    其中，
    X选自CH、C-Rx、N和N+-O-；优选的，X选自N和N+-O-；Rx选自F、Cl、Br；
    R1选自所述分别独立地任选被1、2、3或4个Ra取代；
    R3选自H和D；
    各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C1-3卤代烷基、C2-4烯基、C2- 4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
    各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
  2. 式(III-1)所示化合物或其药学上可接受的盐，
    其中，
    X选自CH、N和N+-O-；优选的，X选自N和N+-O-；
    R1选自所述分别独立地任选被1、2、3或4个Ra取代；
    R3选自H和D；
    各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
    各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
  3. 式(II)和(III-2)所示化合物或其药学上可接受的盐，
    其中，
    R1选自苯基、吡啶基和萘基，所述苯基、吡啶基和萘基分别独立地任选被1、2、3或4个Ra取代；
    R2选自所述分别独立地任选被1、2或3个Rc取代；
    R3选自H和D；
    各Ra分别独立地选自F、Cl、Br、I、OH、NH2、C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、-C1-3烷基-环丙基和环丙基，所述C1-3烷基、C1-3烷氧基、C2-4烯基、C2-4炔基、环丙基和-C1-3烷基-环丙基分别独立地任选被1、2或3个R取代；
    Rb选自H、CN、CH3和OCH3；
    各Rc分别独立地选自F、Cl、Br、I、CH2F、CHF2、CF3和CH2CF3；
    各R分别独立地选自F、Cl、Br、I、CH2F、CHF2和CF3。
  4. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，各Ra分别独立地选自F、Cl、OH、NH2、CH3、CH2CH3、CH(CH3)2、OCH3、OCH2CH3、OCH(CH3)2、-C≡CH、-C≡CCH3、和环丙基，所述CH3、CH2CH3、CH(CH3)2、OCH3、OCH2CH3、OCH(CH3)2、-C≡CH、-C≡CCH3、和环丙基分别独立地任选被1、2或3个R取代。
  5. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，各Ra分别独立地选自F、Cl、OH、NH2、CH3、CHF2、CF3、CH2CF3、CH(CH3)CF3、OCH3、OCF3、-C≡CH、-C≡CCH3、
  6. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，R1选自
  7. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，R1选自 所述分别独立地任选被1、2、3或4个Ra取代。
  8. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，R1选自
  9. 根据权利要求1～3任意一项所述化合物或其药学上可接受的盐，其中，R1选自
  10. 根据权利要求3所述化合物或其药学上可接受的盐，其中，R2选自
  11. 根据权利要求3所述化合物或其药学上可接受的盐，其中，R2选自
  12. 根据权利要求1或2所述化合物或其药学上可接受的盐，其中，X选自N。
  13. 根据权利要求1或2所述化合物或其药学上可接受的盐，其化合物选自，
    其中，R1和R3如权利要求1所定义。
  14. 根据权利要求1或2所述化合物或其药学上可接受的盐，其化合物选自，
    其中，X、R1和R3如权利要求1所定义。
  15. 根据权利要求3所述化合物或其药学上可接受的盐，其化合物选自，
    其中，R1和R3如权利要求3所定义。
  16. 下列所示化合物或其药学上可接受的盐，



  17. 根据权利要求16所述的化合物或其药学上可接受的盐，其选自，
















  18. 根据权利要求17所述化合物或其药学上可接受的盐，其化合物选自，
  19. 根据权利要求1～18任意一项所述化合物或其药学上可接受的盐，其在制备治疗KRASG12D突变的实体瘤化合物中的应用。
PCT/CN2023/106806 2022-07-12 2023-07-11 哌嗪桥取代的杂环并嘧啶类化合物 Ceased WO2024012456A1 (zh)

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