WO2021238999A1 - 氟代吡咯并吡啶类化合物及其应用 - Google Patents

氟代吡咯并吡啶类化合物及其应用 Download PDF

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Publication number
WO2021238999A1
WO2021238999A1 PCT/CN2021/096143 CN2021096143W WO2021238999A1 WO 2021238999 A1 WO2021238999 A1 WO 2021238999A1 CN 2021096143 W CN2021096143 W CN 2021096143W WO 2021238999 A1 WO2021238999 A1 WO 2021238999A1
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Prior art keywords
compound
reaction
ethyl acetate
crude product
synthesis
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PCT/CN2021/096143
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English (en)
French (fr)
Inventor
方堃
陈曙辉
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深圳市瓴方生物医药科技有限公司
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Priority to KR1020227044273A priority Critical patent/KR20230012586A/ko
Priority to EP21813936.8A priority patent/EP4159734B1/en
Priority to CN202180037895.4A priority patent/CN115667258B/zh
Priority to US17/928,295 priority patent/US20230212165A1/en
Priority to AU2021282122A priority patent/AU2021282122B2/en
Priority to JP2022570742A priority patent/JP7418051B2/ja
Publication of WO2021238999A1 publication Critical patent/WO2021238999A1/zh

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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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a class of fluoropyrrolopyridine compounds. Specifically, it relates to the compound represented by formula (II), its isomers or pharmaceutically acceptable salts thereof.
  • DDR DNA damage response
  • the protein in the cell directly recognizes the abnormal DNA structure and activates the related kinases of the DDR pathway to deal with extensive DNA damage and increased replication pressure in cancer cells.
  • the DDR pathway can enable cells to survive in the face of genome instability and replication pressure, or mediate irreparable damaged cell senescence or programmed death.
  • the defect of DDR gene promotes the mutation of the driver gene, the heterogeneity of the tumor and the escape of apoptosis through a variety of ways, so as to promote the growth of the tumor.
  • ATR capillary dilatation ataxia mutations and RAD-3 related protein kinases
  • PIKKs phosphatidylinositol-3-kinase-related kinases
  • ATR protein kinase produces a coordinated response to DNA damage, replication stress and cell cycle interference.
  • ATR and ATM belong to the PIKK family of serine/threonine protein kinases. They are a common component of cell cycle and DNA damage repair. Others include Chkl, BRCA1, and p53.
  • ATR is mainly responsible for DNA replication stress (replication fork stagnation) and repair of single-strand breaks.
  • ATR is activated by the DNA single-stranded structure.
  • the DNA polymerase stays in the DNA replication process, and the replication helicase continues to unwind at the front end of the DNA replication fork, resulting in the production of long single-stranded DNA (ssDNA), which is then combined by the single-stranded DNA and RPA (replication protein A).
  • the ATR/ATR-acting protein complex recruited by RPA to the damage site activates the RAD17/rfc2-5 complex to bind to the damage site, and the DNA-ssDNA junction Activating Rad9-HUS1-RAD1(9-1-1) heterotrimer, 9-1-1 in turn recruits TopBP1 to activate ATR.
  • ATR promotes DNA repair through downstream targets, stabilizes and restarts stalled replication forks and transient cell cycle arrest. These functions are achieved by ATR by mediating the downstream target Chk1.
  • ATR functions as a checkpoint for the cell cycle of DNA damage in the S phase.
  • ATR can mediate the degradation of CDC25A through Chk1, thereby delaying the process of DNA replication and giving time to repair the replication fork.
  • ATR is also the main regulator of G2/M cell cycle checkpoints, preventing cells from entering mitosis prematurely before DNA replication is complete or DNA damage.
  • This ATR-dependent G2/M cell cycle arrest is mainly mediated by two mechanisms: 1. Degradation of CDC25A. 2. Phosphorylate Cdc25C by Chk1 to bind it to 14-3-protein. The binding of Cdc25C to 14-3-3 protein promotes its export from the nucleus and cytoplasmic isolation, thereby inhibiting its ability to dephosphorylate and activate nuclear Cdc2, which in turn prevents entry into mitosis.
  • ATM gene mutations often occur in tumor cells, indicating that the loss of ATM activity is beneficial to the survival of cancer cells.
  • ATM kinase inactivation will make cells more dependent on ATR-mediated signaling pathways, and the combined inactivation of ATR and ATM can induce the synthetic lethality of cancer cells. Therefore, inhibiting ATR may be an effective method for cancer treatment in the future.
  • the present invention provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • T 1 , T 2 , T 3 , T 4 and T 5 are independently C, CH or N respectively;
  • E 1 is O or S
  • R 1 each independently H or C 1-3 alkyl, wherein said C 1-3 alkyl optionally substituted with 1, 2 or 3 R a;
  • R 2 is each independently H or C 1-3 alkyl, wherein said C 1-3 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • R 3 is H or C 1-3 alkyl
  • R 4 is each independently H, C 1-3 alkyl, -OC 1-3 alkyl or -S(O) 2 -C 1-3 alkyl, wherein said C 1-3 alkyl, -OC 1 -3 alkyl and -S(O) 2 -C 1-3 alkyl are each independently optionally substituted with 1, 2 or 3 R c ;
  • R a , R b and R c are each independently F, Cl, Br or I;
  • n 1, 2 or 3.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 each independently H or C 1-3 alkyl, wherein said C 1-3 alkyl optionally substituted with 1, 2 or 3 R a;
  • R 2 is each independently H or C 1-3 alkyl, wherein said C 1-3 alkyl is optionally substituted with 1, 2 or 3 R b ;
  • R a and R b are F, Cl, Br, or I, respectively.
  • R 1 is H or CH 3 , wherein CH 3 is optionally substituted with 1, 2 or 3 Ra, and other variables are as defined in the present invention.
  • R 1 is CH 3 , and other variables are as defined in the present invention.
  • R 1 is H or CH 3 , and other variables are as defined in the present invention.
  • R 2 is H or CH 3 , wherein CH 3 is optionally substituted with 1, 2 or 3 R b , and other variables are as defined in the present invention.
  • R 2 is CH 3 , and other variables are as defined in the present invention.
  • R 2 is H or CH 3 , and other variables are as defined in the present invention.
  • R 3 is independently H or CH 3 , and other variables are as defined in the present invention.
  • R 4 is independently H, CH 3 , -O-CH 3 or -S(O) 2 -CH 3 , and other variables are as defined in the present invention.
  • the above-mentioned ring A is Other variables are as defined in the present invention.
  • the above-mentioned ring A is Other variables are as defined in the present invention.
  • the above-mentioned compound has a structure of formula (I-1) or (I-2)
  • R 1 and R 2 are as defined in the present invention.
  • the present invention also provides a compound of the following formula or a pharmaceutically acceptable salt thereof,
  • the present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of a medicine for the treatment of ATR-related diseases.
  • the compound of the present invention has strong inhibitory activity against ATR enzyme; at the same time, it has a better inhibitory effect against LoVo tumor cells lacking ATM signaling pathway; at the same time, the compound of the present invention has good PK parameters such as exposure and bioavailability, and is suitable for medication;
  • the compound of the present invention can significantly inhibit the growth of human gastric cancer SNU-601 xenograft tumors, and is relatively tolerant to mice.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are within the scope of reliable medical judgment and are suitable for use in contact with human and animal tissues. , Without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to a salt of the compound of the present invention, which is prepared from a compound with specific substituents discovered in the present invention and a relatively non-toxic acid or base.
  • a base addition salt can be obtained by contacting the compound with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salt or similar salts.
  • the acid addition salt can be obtained by contacting the compound with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, the organic acid includes, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , And salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and
  • the pharmaceutically acceptable salt of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods.
  • such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of both.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers Isomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomers or diastereomer-enriched mixtures, all of these mixtures belong to this Within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All these isomers and their mixtures are included in the scope of the present invention.
  • enantiomers or “optical isomers” refer to stereoisomers that are mirror images of each other.
  • cis-trans isomer or “geometric isomer” is caused by the inability to rotate freely because of double bonds or single bonds of ring-forming carbon atoms.
  • diastereomer refers to a stereoisomer in which a molecule has two or more chiral centers and the relationship between the molecules is non-mirror mirror image.
  • wedge-shaped solid line keys And wedge-shaped dashed key Represents the absolute configuration of a three-dimensional center, with a straight solid line key And straight dashed key Indicates the relative configuration of the three-dimensional center, using wavy lines Represents a wedge-shaped solid line key Or wedge-shaped dashed key Or use wavy lines Represents a straight solid line key And straight dashed key
  • the following formula (A) means that the compound exists as a single isomer of formula (A-1) or formula (A-2) or as two isomers of formula (A-1) and formula (A-2)
  • the following formula (B) means that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of two of formula (B-1) and formula (B-2) A mixture of isomers exists.
  • the following formula (C) means that the compound exists as a single isomer of formula (C-1) or formula (C-2) or as two isomers of formula (C-1) and formula (C-2) Exist as a mixture.
  • tautomer or “tautomeric form” means that at room temperature, the isomers of different functional groups are in dynamic equilibrium and can be transformed into each other quickly. If tautomers are possible (such as in solution), the chemical equilibrium of tautomers can be reached.
  • proton tautomer also called prototropic tautomer
  • Valence isomers include some recombination of bond electrons to carry out mutual transformations.
  • keto-enol tautomerization is the tautomerization between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in one isomer”, “enriched in isomers”, “enriched in one enantiomer” or “enriched in enantiomers” refer to one of the isomers or pairs of
  • the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater, or 99.5% or greater, or 99.6% or greater, or 99.7% or greater, or 99.8% or greater, or greater than or equal 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90%, and the content of the other isomer or enantiomer is 10%, the isomer or enantiomer excess (ee value) is 80% .
  • optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one wants to obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure The desired enantiomer.
  • the molecule when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), it forms a diastereomeric salt with a suitable optically active acid or base, and then passes through a conventional method known in the art The diastereoisomers are resolved, and then the pure enantiomers are recovered.
  • the separation of enantiomers and diastereomers is usually accomplished through the use of chromatography, which employs a chiral stationary phase and is optionally combined with chemical derivatization (for example, the formation of amino groups from amines). Formate).
  • the compound of the present invention may contain unnatural proportions of atomic isotopes on one or more of the atoms constituting the compound.
  • compounds can be labeled with radioisotopes, such as tritium ( 3 H), iodine-125 ( 125 I), or C-14 ( 14 C).
  • deuterium can be substituted for hydrogen to form deuterated drugs.
  • the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have reduced toxic side effects and increased drug stability. , Enhance the efficacy, prolong the biological half-life of drugs and other advantages.
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by substituents, and can include deuterium and hydrogen variants, as long as the valence of the specific atom is normal and the substituted compound is stable of.
  • oxygen it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups.
  • optionally substituted means that it can be substituted or unsubstituted. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis that they can be chemically realized.
  • any variable such as R
  • its definition in each case is independent.
  • the group can optionally be substituted with up to two Rs, and R has independent options in each case.
  • combinations of substituents and/or variants thereof are only permitted 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.
  • substituents When a substituent is vacant, it means that the substituent is absent. For example, when X in A-X is vacant, it means that the structure is actually A.
  • substituents do not indicate which atom is connected to the substituted group, such substituents can be bonded via any atom.
  • a pyridyl group can pass through any one of the pyridine ring as a substituent. The carbon atom is attached to the substituted group.
  • the middle linking group L is -MW-, at this time -MW- can be formed by connecting ring A and ring B in the same direction as the reading order from left to right It can also be formed by connecting ring A and ring B in the direction opposite to the reading order from left to right Combinations of the linking groups, substituents, and/or variants thereof are only permitted if such combinations result in stable compounds.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • connection mode of the chemical bond is not positioned, and there is a H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will correspondingly decrease with the number of chemical bonds connected to become the corresponding valence number ⁇ The group.
  • the chemical bond between the site and other groups can be a straight solid bond Straight dashed key Or wavy line Express.
  • the straight solid bond in -OCH 3 means that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy line in indicates that the phenyl group is connected to other groups through the 1 and 2 carbon atoms;
  • C 1-3 alkyl is used to indicate a linear or branched saturated hydrocarbon group composed of 1 to 3 carbon atoms.
  • the C 1-3 alkyl group includes C 1-2 and C 2-3 alkyl groups, etc.; it 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.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , including any range from n to n+m, for example, C 1-12 includes C 1-3 , C 1-6 , C 1-9 , C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12, etc.; similarly, from n to n +m member means that the number of atoms in the ring is n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, and 9-membered ring, and 9
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (for example, a nucleophilic substitution reaction).
  • representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, and iodine; sulfonate groups such as mesylate, tosylate, p-bromobenzenesulfonate, p-toluenesulfonic acid Esters, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy and the like.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc) ; Arylmethyloxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethyloxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups, such as trimethylsilyl (TMS) and tert-butyldi
  • hydroxyl protecting group refers to a protecting group suitable for preventing side reactions of the hydroxyl group.
  • Representative hydroxy protecting groups include but are not limited to: alkyl, such as methyl, ethyl and tert-butyl; acyl, such as alkanoyl (such as acetyl); arylmethyl, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl such as methyl, ethyl and tert-butyl
  • acyl such as alkanoyl (such as acetyl)
  • arylmethyl such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenyl
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent alternatives, preferred implementations include but are not limited to the embodiments of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • SXRD single crystal X-ray diffraction
  • the cultivated single crystal is collected with the Bruker D8 venture diffractometer to collect the diffraction intensity data
  • the light source is CuK ⁇ radiation
  • the scanning method After scanning and collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure to confirm the absolute configuration.
  • the solvent used in the present invention is commercially available.
  • the present invention uses the following abbreviations: aq stands for water; eq stands for equivalent or equivalent.
  • the compound is used according to the conventional naming principle in the field or The software is named, and the commercially available compounds use the supplier catalog name.
  • Place 1-C (160mg, 519.86 ⁇ mol) in a microwave reactor, add ethylene glycol dimethyl ether (5mL), add Intermediate I (174.82mg, 519.86 ⁇ mol), sodium carbonate aqueous solution (2M, 1.82mL) And [1,1-bis(diphenylphosphine)ferrocene]dichloropalladium dichloromethane complex (42.45mg, 51.99 ⁇ mol), bubbling with nitrogen for 2 minutes, heating to 110°C in microwave and stirring for 0.5 hours.
  • ethylene glycol dimethyl ether 5mL
  • Intermediate I 174.82mg, 519.86 ⁇ mol
  • sodium carbonate aqueous solution (2M, 1.82mL
  • [1,1-bis(diphenylphosphine)ferrocene]dichloropalladium dichloromethane complex 42.45mg, 51.99 ⁇ mol
  • Glacial acetic acid (15 mL) was added to compound 2-C (1.3 g, 4.38 mmol) and acetylhydrazine (1.62 g, 21.90 mmol), and the reaction was heated to 100° C. and stirred for 1.5 hours.
  • the crude product was subjected to column chromatography (petroleum ether/ethyl acetate: 30% to 100%). ) Purification to obtain compound 2-D.
  • reaction solution was washed once with saturated sodium bicarbonate, and the organic phase was extracted with 150 mL of ethyl acetate (50*3), washed once with saturated brine, dried over anhydrous sodium sulfate, filtered to obtain the filtrate, and depressurized dry.
  • the crude product was separated by column chromatography (methanol/dichloromethane: 0-20%) to obtain compound 2, and then passed through a preparative chromatography column (Phenomenex Gemini-NX 80*30mm*3 ⁇ m; mobile phase: [water (10mM NH 4 HCO 3 )-ACN]; ACN%: 20%-50%, 9 minutes) to obtain compound 2.
  • MS m/z 408.1 [M+H] + .
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (30 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 70% to 90%) to obtain compound 6-A.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (15 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (tetrahydrofuran/petroleum ether: 50-90%) to obtain compound 6.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (20 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 50%-60%) to obtain compound 9-A.
  • Place 7-A (200mg, 651.91 ⁇ mol) in a microwave reactor, add 1,4-dioxane (3mL) and water (0.3mL), add I (438.45mg, 1.30mmol), sodium carbonate (172.74) mg,1.63mmol) and [1,1-bis(diphenylphosphine)ferrocene]dichloride palladium dichloromethane complex (53.24mg, 65.19 ⁇ mol), bubbling with nitrogen for 2 minutes, microwave heating to 110 Stir at °C for 1 hour.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (15 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (tetrahydrofuran/petroleum ether: 70-90%) to obtain compound 9.
  • Place 1-B 500 mg, 1.48 mmol in a microwave reactor, add 1,4-dioxane (6 mL) and water (0.6 mL).
  • 11-A 315.21mg, 1.62mmol
  • sodium carbonate 469.58mg, 4.43mmol
  • [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride 75.64mg, 103.38umol
  • Place 1-B 500 mg, 1.48 mmol in a microwave reactor, and add 1,4-dioxane (5 mL) and water (0.5 mL).
  • 1,4-dioxane 5 mL
  • water 0.5 mL
  • 12-A 458.37mg, 1.62mmol
  • sodium carbonate 469.58mg, 4.43mmol
  • [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride 75.64mg, 103.38 ⁇ mol
  • nitrogen Purge for 2 minutes, microwave heating to 110°C and stirring for 0.5 hour.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (20 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 50% to 70%) to obtain compound 13-A.
  • reaction liquid was cooled, tetraethylammonium fluoride hydrate (139.03 mg, 831.19umol) was added to the reaction liquid, and stirred at 25°C for 2 hours.
  • the reaction system was diluted with water (20 mL), washed with ethyl acetate (15 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (tetrahydrofuran/petroleum ether: 50-70%) to obtain compound 13.
  • 1,4-dimethylpyrazole-5-manalol borate 216.47mg, 974.68umol, 1.1eq
  • sodium carbonate 140.87mg, 1.33 mmol, 664.56 ⁇ L, 1.5eq
  • 1,4-dioxane (3mL) solution was added [1,1-bis(diphenylphosphine)ferrocene]palladium dichloride (64.83mg, 88.61 ⁇ mol, 0.1 eq), bubbling with nitrogen, and the reaction was stirred at 100°C for 1 hour in the microwave.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (20 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 40% to 70%) to obtain compound 14-A.
  • reaction solution was cooled, the reaction system was diluted with water (20 mL), washed with ethyl acetate (20 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 40%-50%) to obtain compound 15-A.
  • reaction solution was cooled, tetraethylammonium fluoride hydrate (128.55mg, 768.54umol) was added to the reaction solution, and stirred at 25°C for 2 hours.
  • the reaction system was diluted with water (20 mL), washed with ethyl acetate (15 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (ethyl acetate/dichloromethane: 50-60%) to obtain compound 15.
  • reaction solution was cooled, the reaction system was diluted with water (40 mL), washed with ethyl acetate (20 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether/ethyl acetate: 0%-20%) to obtain compound 16-A.
  • the reaction system was diluted with water (40 mL), washed with ethyl acetate (15 mL ⁇ 3), washed with saturated brine (10 mL), and dried with anhydrous sodium sulfate. After filtering off the desiccant, the solvent was removed under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (ethyl acetate/petroleum ether: 50-75%) to obtain compound 16.
  • the inhibitory activity of the test compound on human ATR kinase was evaluated by measuring the IC 50 value.
  • ATR/ATRIP(h) was incubated in assay buffer containing 50nM GST-cMyc-p53 and Mg/ATP (10uM). The reaction is initiated by adding a Mg/ATP mixture. After incubating for 30 minutes at room temperature, a stop solution containing EDTA was added to terminate the reaction. Finally, the assay buffer solution containing d 2 labeled anti-GST monoclonal antibody and anti-phospho-Ser15 antibody against p53 phosphorylation europium labeled. Then read the plate in time-resolved fluorescence mode and perform homogeneous time resolution.
  • HTRF fluorescence
  • the culture plate is placed at room temperature for 10 minutes to stabilize the luminescence signal.
  • IR(%) (1—(RLU compound—RLU blank control)/(RLU vehicle control—RLU blank control))*100%.
  • the inhibition rate of different concentration of compound in Excel and GraphPad Prism software used for calculating inhibition curves and associated parameters, including the minimum inhibitory rate, the maximum inhibition rate and IC 50.
  • the experimental results show that the compound of the present invention has a good inhibitory effect on LoVo tumor cells lacking ATM signaling pathway.
  • Test sample On the basis of the above experiment, select compound 1 to carry out further experiments.
  • mice The purpose of this study is to determine the pharmacokinetic parameters of compound 1 and calculate its oral bioavailability in female Balb/c Nude mice.
  • This project uses 4 female Balb/c Nude mice, two mice are injected intravenously, the dosage is 1mg/kg, collected 0h (before administration) and 0.0833, 0.25, 0.5, 1, after administration, Plasma samples at 2, 4, 6, 8, and 24 hours, and the other two mice were orally administered by gavage at a dose of 10 mg/kg or 25 mg/kg, collected at 0 hours (before administration) and 0.25, 0.5 after administration , 1, 2, 4, 6, 8, 24h plasma samples, and then perform LC/MS/MS analysis on the collected samples and collect the data.
  • the collected analysis data is used to calculate the relevant pharmacokinetic parameters with Phoenix WinNonlin 6.2.1 software .
  • C 0 (nM) is the concentration of the drug in the body at 0 minutes
  • Cl (mL/min/kg) is the clearance rate of the drug in the body
  • V dss (L/kg) is the volume of distribution of the drug in the body
  • T 1/2 (h) is Half-life
  • AUC 0-t (nM.h) is the amount of drug exposure in the body
  • Cmax (nM) is the highest concentration of the drug in the body.
  • the compound of the present invention has good in vivo pharmacokinetic properties such as exposure and bioavailability.
  • the main purpose of this study is to study the anti-tumor efficacy of the test substance on the human gastric cancer cell SNU-601 xenograft model.
  • Human gastric cancer SNU-601 cells sourced from KCLB (Cat. No. 00601), are protected and maintained by Huiyuan Biotechnology (Shanghai) Co., Ltd.
  • the in vitro culture conditions are RPMI 1640 medium (containing 300mg/L L-glutamine) with 10% fetal calf serum, 25mM HEPES and 25mM sodium bicarbonate, 37°C 5% CO2 incubator culture, passage two to three times a week. When the number of cells reaches the requirement, the cells are collected and counted.
  • Dosage Compound 1 was administered orally in three doses of 15 mg/kg (3 days off for 4 days), 10 mg/kg (3 days off for 4 days), and 5 mg/kg (continuous administration).
  • the tumor diameter was measured with a vernier caliper three times a week.
  • V1 and BW1 refer to the tumor volume and weight of a certain animal on the day of group administration
  • Vt and BWt refer to the tumor volume and weight of a certain animal at a certain time.
  • TGI percent or the relative tumor proliferation rate T/C (%).
  • Relative tumor proliferation rate T/C(%) TRTV/CRTV ⁇ 100 (TRTV: average RTV of the treatment group; CRTV: average RTV of the negative control group).
  • RTV relative tumor volume
  • TGI (%) reflects the tumor growth inhibition rate.
  • TGI(%) [(1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the beginning of the treatment group))/(Average tumor volume at the end of treatment in the solvent control group-start treatment in the solvent control group Time average tumor volume)] ⁇ 100.
  • Tweight and Cweight represent the tumor weight of the administration group and the vehicle control group, respectively.
  • the human gastric cancer cell SNU601 xenograft tumor model was used to evaluate the in vivo efficacy of the test compound. During the entire administration period, no animal stopped the drug due to a weight loss of more than 10%. On the 21st day after the administration, the efficacy test was ended. On the 21st day after administration, the tumor volume in the vehicle group reached 890.01 ⁇ 184.62mm 3 . Compared with the vehicle control group, compound 1 showed certain anti-tumor effects at the doses of 15 mg/kg, 10 mg/kg and 5 mg/kg. Their corresponding tumor volumes were 108.74 ⁇ 9.67mm 3 , 136.74 ⁇ 14.46mm3 and 229.99 ⁇ 24.42mm 3 , respectively. The tumor inhibition rate TGI was 104.81% (p ⁇ 0.01), 101.08% (p ⁇ 0.01)) and 88.61% ( p ⁇ 0.01)).
  • the compounds of the present invention can significantly inhibit the growth of human gastric cancer SNU-601 xenograft tumors, and are relatively tolerant to mice.

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Abstract

本发明公开了一类氟代吡咯并吡啶类化合物。具体公开了式(Ⅱ)所示化合物、其异构体或其药学上可接受的盐。

Description

氟代吡咯并吡啶类化合物及其应用
相关申请的交叉引用
本申请要求申请日为2020.05.29的中国专利申请CN202010478432.9、和申请日为2021.02.01的中国专利申请CN202110139687.7的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一类氟代吡咯并吡啶类化合物。具体涉及式(II)所示化合物、其异构体或其药学上可接受的盐。
背景技术
DNA损伤应答(DDR)由多种多样的信号通路确保活细胞基因组的完整性。细胞内的蛋白质会直接识别异常的DNA结构并激活DDR通路的相关激酶，以应对广泛的DNA损伤和癌细胞内增加的复制压力。DDR通路能够使细胞在面对基因组不稳定和复制压力时存活，或者介导不可修复的损伤细胞衰老或程序性死亡。DDR基因的缺陷通过多种途径促进驱动基因变异、肿瘤异质性以及逃避凋亡，达到促进肿瘤生长作用。
ATR(毛细管扩张共济失调突变和RAD-3相关蛋白激酶)属于PIKKs(磷脂酰肌醇-3-激酶-相关激酶)家族，参与DNA的损伤修复以维护基因的稳定。ATR蛋白激酶对DNA的损伤，复制压力应激和细胞周期的干扰产生协同应答。ATR和ATM同属于丝氨酸/苏氨酸蛋白激酶的PIKK家族，他们是细胞周期和DNA损伤修复的共同组成部分，其他还包括，Chkl,BRCAl,p53。ATR主要负责DNA复制应激(复制叉停滞)、单链断裂的修复工作。
当DNA双链断裂出现切除或复制叉停滞时，ATR被DNA单链结构所激活。DNA聚合酶停留在DNA复制过程中，复制解旋酶继续在DNA复制叉前端解旋，导致长的单链DNA(ssDNA)的产生，然后由单链DNA和RPA(复制蛋白A)结合。复制应激或DNA损伤时由RPA招募的ATR/ATR作用蛋白的复合物到损伤位点，RPA-单链DNA复合物激活RAD17/rfc2-5复合物结合到损伤位点，DNA-ssDNA连接处活化Rad9-HUS1-RAD1(9-1-1)异源三聚体，9-1-1反过来招募TopBP1激活ATR。一旦ATR被激活，ATR通过下游目标促进DNA修复、稳定和重新启动停滞的复制叉和短暂的细胞周期阻滞。这些功能是ATR通过介导下游靶Chk1来得以实现。ATR在S期起着DNA损伤细胞周期检查点的作用。它能通过Chk1介导CDC25A的降解，从而延缓DNA的复制进程，给修复复制叉提供了时间。ATR也是G2/M细胞周期检查点的主要调控者，在DNA复制完成或DNA 损伤之前，阻止细胞过早进入有丝分裂。这种依赖ATR的G2/M细胞周期阻滞主要是通过两种机制介导：1。CDC25A的降解。2.通过Chk1磷酸化Cdc25C使之与14-3-蛋白结合。Cdc25C与14-3-3蛋白的结合促进其从细胞核的输出和细胞质隔离，从而抑制其去磷酸化和激活核Cdc2的能力，这进而阻止进入有丝分裂。
ATM基因在肿瘤细胞中常发生突变，表明ATM活性丧失有利于癌细胞的存活。ATM激酶失活会使细胞更依赖ATR介导的信号通路，ATR和ATM的联合失活可以诱导癌细胞的合成致死性。因此，抑制ATR可能是未来的癌症治疗中一种有效的方法。
发明内容
本发明提供了式(Ⅱ)所示化合物或其药学上可接受的盐，
Figure PCTCN2021096143-appb-000001
其中，
环A为
Figure PCTCN2021096143-appb-000002
T 1、T 2、T 3、T 4和T 5分别独立地为C、CH或N；
E 1为O或S；
R 1分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R a取代；
R 2分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
R 3为H或C 1-3烷基；
R 4分别独立地为H、C 1-3烷基、-O-C 1-3烷基或-S(O) 2-C 1-3烷基，其中所述C 1-3烷基、-O-C 1-3烷基和-S(O) 2-C 1-3烷基分别独立地任选被1、2或3个R c取代；
R a、R b和R c分别独立地为F、Cl、Br或I；
m为1、2或3。
本发明提供了式(Ⅰ)所示化合物或其药学上可接受的盐，
Figure PCTCN2021096143-appb-000003
结构单元
Figure PCTCN2021096143-appb-000004
为
Figure PCTCN2021096143-appb-000005
R 1分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R a取代；
R 2分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
R a和R b分别独立地为F、Cl、Br或I。
本发明的一些方案中，上述R 1为H或CH 3，其中CH 3任选地被1、2或3个Ra取代，其他变量如本发明所定义。
本发明的一些方案中，上述R 1为CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述R 1为H或CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述R 2为H或CH 3，其中CH 3任选地被1、2或3个R b取代，其他变量如本发明所定义。
本发明的一些方案中，上述R 2为CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述R 2为H或CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述R 3分别独立地为H或CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述R 4分别独立地为H、CH 3、-O-CH 3或-S(O) 2-CH 3，其他变量如本发明所定义。
本发明的一些方案中，上述环A为
Figure PCTCN2021096143-appb-000006
Figure PCTCN2021096143-appb-000007
其他变量如本发明所定义。
本发明的一些方案中，上述环A为
Figure PCTCN2021096143-appb-000008
Figure PCTCN2021096143-appb-000009
Figure PCTCN2021096143-appb-000010
其他变量如本发明所定义。
本发明的一些方案中，上述化合物具有式(Ⅰ-1)或(Ⅰ-2)结构
Figure PCTCN2021096143-appb-000011
其中，R 1和R 2如本发明所定义。
本发明的一些方案中，上述结构单元
Figure PCTCN2021096143-appb-000012
为
Figure PCTCN2021096143-appb-000013
其他变量如本发明所定义。
本发明还有一些方案是由上述各变量任意组合而来。
本发明还提供了下式化合物或其药学上可接受的盐，
Figure PCTCN2021096143-appb-000014
Figure PCTCN2021096143-appb-000015
本发明还提供了上述化合物或其药学上可接受的盐在制备治疗ATR相关疾病的药物中的应用。
技术效果
本发明化合物针对ATR酶有较强的抑制活性；同时针对ATM信号通路缺失的LoVo肿瘤细胞有较好的抑制作用；同时，本发明化合物暴露量、生物利用度等PK参数良好，适于用药；另外，本发明化合物可以明显抑制人胃癌SNU-601异种移植瘤的生长，且对小鼠相对耐受。
定义和说明
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、 邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键
Figure PCTCN2021096143-appb-000016
和楔形虚线键
Figure PCTCN2021096143-appb-000017
表示一个立体中心的绝对构型，用直形实线键
Figure PCTCN2021096143-appb-000018
和直形虚线键
Figure PCTCN2021096143-appb-000019
表示立体中心的相对构型，用波浪线
Figure PCTCN2021096143-appb-000020
表示楔形实线键
Figure PCTCN2021096143-appb-000021
或楔形虚线键
Figure PCTCN2021096143-appb-000022
或用波浪线
Figure PCTCN2021096143-appb-000023
表示直形实线键
Figure PCTCN2021096143-appb-000024
和直形虚线键
Figure PCTCN2021096143-appb-000025
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线
Figure PCTCN2021096143-appb-000026
连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
Figure PCTCN2021096143-appb-000027
除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同 位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
Figure PCTCN2021096143-appb-000028
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
Figure PCTCN2021096143-appb-000029
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
Figure PCTCN2021096143-appb-000030
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点 存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
Figure PCTCN2021096143-appb-000031
直形虚线键
Figure PCTCN2021096143-appb-000032
或波浪线
Figure PCTCN2021096143-appb-000033
表示。例如-OCH 3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
Figure PCTCN2021096143-appb-000034
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
Figure PCTCN2021096143-appb-000035
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；
Figure PCTCN2021096143-appb-000036
表示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括
Figure PCTCN2021096143-appb-000037
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2021096143-appb-000038
仍包括
Figure PCTCN2021096143-appb-000039
这种连接方式的基团，只是在连接1个化学键时，该位点的H会对应减少1个变成相应的一价哌啶基。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，C n-n+m或C n-C n+m包括n至n+m个碳的任何一种具体情况，例如C 1-12包括C 1、C 2、C 3、C 4、C 5、C 6、C 7、C 8、C 9、C 10、C 11、和C 12，也包括n至n+m中的任何一个范围，例如C 1-12包括C 1-3、C 1-6、C 1-9、C 3-6、C 3-9、C 3-12、C 6-9、C 6-12、和C 9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲核取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰 基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
Figure PCTCN2021096143-appb-000040
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：aq代表水；eq代表当量、等量。
化合物依据本领域常规命名原则或者使用
Figure PCTCN2021096143-appb-000041
软件命名，市售化合物采用供应商目录名称。
具体实施方式
下面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。
中间体I
Figure PCTCN2021096143-appb-000042
合成路线：
Figure PCTCN2021096143-appb-000043
步骤1：化合物I-2的合成
向化合物I-1(500mg,3.67mmol)的四氢呋喃(30mL)溶液中加入钠氢(220.36mg,5.51mmol,60％)，在室温20℃下反应1小时。加入三异丙基硅烷(849.80mg,4.41mmol)，室温20℃反应2.5小时。反应结束后，向反应液中加入10mL饱和氯化铵水溶液淬灭反应。加入60mL水，用乙酸乙酯(70mL*3)萃取。有机相经饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-20％)得到化合物I-2。
MS m/z:293.0[M+H] +
步骤2：化合物I的合成
将化合物I-2(200mg,683.84μmol)置于三口瓶中，加入无水四氢呋喃(12mL)，氮气置换。将反应液冷却至-78℃，加入二异丙基氨基锂(2M,683.84μL,2eq)，并搅拌30分钟，加入硼酸三甲脂(99.48mg,957.38μmol,108.13μL,1.4eq)，升至室温18℃搅拌1小时。反应结束后，向反应液中加入10mL饱和氯化铵水溶液，搅拌15分钟。用乙酸乙酯(30mL*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。得中间体I。
MS m/z:337.1[M+H] +。
实施例1
Figure PCTCN2021096143-appb-000044
Figure PCTCN2021096143-appb-000045
步骤1：化合物1-B的合成
在室温下向化合物1-A(3.22g,11.76mmol,1eq)的N,N-二甲基甲酰胺(20.00mL)溶液中加入(R)-3-甲基吗啡啉(2.38g,23.51mmol,2eq)，碳酸钾(3.25g,23.51mmol,63.60μL,2eq)，然后在130℃，氮气氛围搅拌18小时。反应体系用水(60mL)稀释，乙酸乙酯(50mL×3)洗涤，饱和食盐水(50mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品用柱层析(石油醚/乙酸乙酯:0％～20％)纯化得到化合物1-B.
MS-ESI m/z:339.0[M+H] +。
步骤2：化合物1-C的合成
向化合物1-B(0.75g,2.22mmol,1eq)，1,4-二甲基-1H-1,2,3-三氮唑(258.16mg,2.66mmol,1.2eq)和碳酸钾(918.48mg,6.65mmol,3eq)的N,N-二甲基乙酰胺(2mL)溶液中加入醋酸钯(34.81mg,155.06μmol,0.07eq)和三环己基膦(93.18mg,332.28μmol,107.72μL,0.15eq)，用氮气鼓泡，反应在微波110℃搅拌1小时。待反应液冷却后，过滤，减压浓缩得到粗品，粗品经柱层析(石油醚/乙酸乙酯:8％～50％)纯化得到化合物1-C。
MS-ESI m/z:308.1[M+H] +。
步骤3：化合物1的合成
将1-C(160mg,519.86μmol)置于微波反应器中，加入乙二醇二甲醚(5mL)，加入中间体I(174.82mg,519.86μmol)，碳酸钠的水溶液(2M,1.82mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(42.45mg,51.99μmol)，氮气鼓泡2分钟，微波加热至110℃搅拌0.5小时。反应结束后，向反应液中加入60mL水，用210mL(70*3)乙酸乙酯萃取反应液，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液，并减压干燥。粗品经柱层析分离(四氢呋喃/石油醚：20-80％)得到化合物1。
MS m/z:408.2[M+H] +
1H NMR(400MHz,CDCl 3)δppm 1.37(br d,J＝6.27Hz,3H)2.42(s,3H)3.27-3.40(m,1H)3.67(br t,J＝10.92Hz,1H)3.84(br s,2H)4.00-4.13(m,5H)4.38(br d,J＝4.02Hz,1H)6.46(br s,1H)7.08(br s,1H)7.14(br s,1H)7.54(br s,1H)8.40(br s,1H)8.85(br s,1H)
实施例2
Figure PCTCN2021096143-appb-000046
步骤1：化合物2-B的合成
向化合物2-A(1.5g,9.20mmol)、(R)-3-甲基吗啡啉(977.31mg,9.66mmol)的1-甲基-2-吡咯烷酮(10mL)的溶液中加入碳酸钠(1.27g,11.96mmol)，反应用微波在210℃搅拌2小时后再升温到220度搅拌3*2小时后，(分2批次投反应)，反应液加水(100mL)稀释，水相用乙酸乙酯(70mL×3)萃取，合并有机相，有机相分别用水(40mL×3)洗和饱和食盐水(80mL)洗，有机相用无水硫酸钠干燥，过滤，有机相缩得到粗品，粗品经柱层析(石油醚/乙酸乙酯:10％～35％)纯化得到化合物2-B。
MS-ESI m/z:227.9[M+H] +。
步骤2：化合物2-C的合成
向化合物2-B(0.95g,4.17mmol)的N,N-二甲基甲酰胺(10mL)的溶液中加N,N-二甲基乙酰胺二甲基缩醛(2.22g,16.69mmol)，反应在100℃搅拌1小时后，减压浓缩得到粗品化合物2-C。
MS-ESI m/z:297.1[M+H] +。
步骤3：化合物2-D的合成
向化合物2-C(1.3g,4.38mmol)、乙酰肼(1.62g,21.90mmol)加入冰醋酸(15mL)的，反应升温到100℃搅拌1.5小时。反应液用饱和碳酸钠调pH＝7-8，用乙酸乙酯(100mL×3)萃取，有机相减压浓缩得到粗品，粗品经柱层析(石油醚/乙酸乙酯:30％～100％)纯化得到化合物2-D。
MS-ESI m/z:307.9[M+H] +。
步骤4：化合物2的合成
将2-D(210mg,682.31μmol)置于微波反应器中，加入乙二醇二甲醚(2mL)，加入中间体I(269.94mg,682.31μmol)，碳酸钠的水溶液(2M,2.39mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(55.72mg,68.23μmol)，氮气鼓泡2分钟，微波加热至110℃搅拌0.5小时。反应结束后，将反应液用饱和碳酸氢钠洗涤一次，有机相用乙酸乙酯150mL(50*3)萃取，用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液，并减压干燥。粗品经柱层析分离(甲醇/二氯甲烷：0-20％)得到化合物2，又经通过制备色谱柱(Phenomenex Gemini-NX 80*30mm*3μm；流动相：[水(10mM NH 4HCO 3)-ACN]；ACN％:20％-50％,9分钟)纯化，得到化合物2。MS m/z:408.1[M+H] +。
1H NMR(400MHz,CDCl 3)δppm 1.37(d,J＝6.78Hz,3H)2.42(s,6H)3.35(td,J＝12.61,3.64Hz,1H)3.67(td,J＝11.80,3.01Hz,1H)3.78-3.89(m,2H)4.02-4.11(m,2H)4.33(br d,J＝7.03Hz,1H)6.32(s,1H)7.02-7.09(m,2H)7.58(t,J＝2.76Hz,1H)8.42(s,1H)9.25(br s,1H)。
实施例3
Figure PCTCN2021096143-appb-000047
步骤1：化合物3-C的合成
将3-A(500mg,3.16mmol)置于微波反应器中，加入1,4-二氧六环(10mL)和水(1mL)。加入化合物3-B(866.96mg,3.16mmol)，碳酸钠(838.54mg,7.91mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(155.49mg,221.52μmol)，氮气鼓泡2分钟，微波加热至110℃搅拌0.5小时。反应结束后向反应液中加入60mL水，用乙酸乙酯210mL(70*3)萃取。有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙 酸乙酯/石油醚：0-10％)得到化合物3-C。MS m/z:224.8/226.7[M+H] +。
步骤2：化合物3-D的合成
将化合物3-C(370mg,1.64mmol)置于微波反应器中，加入(R)-3-甲基吗啡啉(498.83mg)，微波加热至200℃搅拌1小时。反应结束后将反应液减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-30％)得到化合物3-D。MS m/z:289.9[M+H] +。
步骤3：化合物3的合成
将化合物3-D(414.1mg,1.43mmol)溶于1,4-二氧六环(10mL)中，加入中间体I(624.77mg,1.86mmol),碳酸钠的水溶液(2M,2.14mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(58.35mg,71.46μmol)。加热至80℃反应16小时后将温度降至室温25℃，加入四乙基氟化铵水合物(239.04mg,1.43mmol)搅拌2小时。点板监测反应完全。反应结束后向反应液中加入60mL水。将反应液通过硅藻土过滤，滤液用乙酸乙酯210mL(70*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(四氢呋喃/石油醚：10-50％),又经通过制备色谱柱(Phenomenex Gemini-NX 80*30mm*3μm；流动相：[水(10mM NH4HCO3)-ACN]；ACN％:37％-67％,9分钟)纯化，得到化合物3。MS m/z:390.0[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.74(br d,J＝4.6Hz,1H),8.65(br s,1H),8.37(s,1H),7.86-7.78(m,2H),7.69(d,J＝2.3Hz,1H),7.49(t,J＝2.8Hz,1H),7.37-7.30(m,2H),7.07(br s,1H),4.57(br d,J＝6.6Hz,1H),4.07(br d,J＝2.5Hz,1H),4.04(d,J＝3.0Hz,1H),3.83(d,J＝1.4Hz,2H),3.67(dt,J＝2.6,11.6Hz,1H),3.38(dt,J＝3.8,12.6Hz,1H),1.36(d,J＝6.8Hz,3H)
实施例4
Figure PCTCN2021096143-appb-000048
步骤1：化合物4-C的合成
将4-A(500mg,2.91mmol)置于微波反应器中，加入1,4-二氧六环(10mL)和水(1mL)。加入3-B(875.89mg,3.20mmol)，碳酸钠(924.21mg,8.72mmol)和[1,1-双(二苯 基膦)二茂铁]二氯化钯(142.81mg,203.46μmol)，氮气吹扫2分钟，微波加热至110℃搅拌0.5小时。反应结束后向反应液中加入60mL水，用乙酸乙酯210mL(70*3)萃取。有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-5％)得到化合物4-B。MS m/z:238.87[M+H] +。
步骤2：化合物4-C的合成
将化合物4-B(600mg,2.51mmol)置于微波反应器中，加入(R)-3-甲基吗啡啉(761.45mg,7.53mmol)，微波加热至200℃搅拌1小时。反应结束后将反应液减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-25％)得到化合物4-C。MS m/z:303.9[M+H] +。
步骤3：化合物4的合成
将化合物4-C(682.1mg,2.25mmol)溶于1,4-二氧六环(10mL)中，加入中间体I(981.59mg,2.92mmol),碳酸钠的水溶液(2M,3.37mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(91.68mg,112.27μmol)。加热至80℃反应16小时后保护基未完全脱掉，降至室温20℃左右加入四乙基氟化铵水合物(375.57mg,2.25mmol,)搅拌2小时。点板监测反应完成。反应结束后将反应液通过硅藻土过滤，滤液直接减压干燥。粗品经柱层析分离(四氢呋喃/石油醚：10-60％)得到化合物4。MS m/z:404.1[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.79(br s,1H),8.55(d,J＝4.1Hz,1H),8.33(s,1H),7.63(d,J＝7.6Hz,1H),7.46(t,J＝2.8Hz,1H),7.31(d,J＝2.6Hz,1H),7.24(d,J＝4.9Hz,1H),7.04(br s,1H),6.74(s,1H),4.50(br d,J＝6.6Hz,1H),4.08-3.92(m,2H),3.81(s,2H),3.65(dt,J＝2.9,11.7Hz,1H),3.35(dt,J＝3.6,12.5Hz,1H),2.45(s,3H),1.34(d,J＝6.6Hz,3H)
实施例5
Figure PCTCN2021096143-appb-000049
步骤1：化合物5-B的合成
将1-B(300mg,886.08μmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3 mL)。加入5-A(201.83mg,974.68μmol)，碳酸钠(281.74mg,2.66mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(43.54mg,62.03μmol)，氮气鼓泡2分钟，微波加热至100℃搅拌1小时。反应结束后将反应液减压干燥，然后拌样纯化。粗品经柱层析分离(乙酸乙酯/石油醚：0-25％)得到化合物5-B。MS m/z:291.9[M+H] +。
步骤2：化合物5的合成
将化合物5-B(158.9mg,544.60μmol)溶于1,4-二氧六环(10mL)中，加入中间体I(238.08mg,707.98μmol)，碳酸钠的水溶液(2M,816.90μL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(31.13mg,38.12μmol)。加热至100℃反应16小时。反应结束后将反应液通过硅藻土过滤，滤液直接减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：10-60％)得到化合物5。MS m/z:392.0[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.48(br s,1H),8.36(s,1H),7.49(t,J＝2.7Hz,1H),7.08(br s,1H),6.78(s,1H),6.60(s,1H),6.40(br d,J＝1.8Hz,1H),6.23(t,J＝3.1Hz,1H),4.41(br d,J＝7.0Hz,1H),4.09-3.97(m,2H),3.83(s,2H),3.79(s,3H),3.72-3.61(m,1H),3.32(dt,J＝3.6,12.6Hz,1H),1.34(d,J＝6.6Hz,3H)
实施例6
Figure PCTCN2021096143-appb-000050
步骤1：化合物6-A的合成
向化合物1-B(800mg,2.36mmol,1eq)，1-甲基-1,2,3-三氮唑(294.50mg,3.54mmol,1.5eq)和乙酸钾(436.79mg,4.73mmol)的N,N-二甲基甲酰胺(10mL)溶液中加入醋酸钯(53.05mg,236.29μmol,0.1eq)和三环己基膦(132.52mg,472.57μmol,0.2eq)，用氮气鼓泡，反应在微波120℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(30mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。 粗品经柱层析(石油醚/乙酸乙酯:70％～90％)纯化得到化合物6-A。
MS-ESI m/z:295.1[M+H] +。
步骤2：化合物6的合成
将6-A(500mg,1.70mmol)置于微波反应器中，加入1,4-二氧六环(5mL)和水(0.5mL)，加入中间体I(572.40mg,1.70mmol)，碳酸钠(451.02mg,4.26mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(139.00mg,170.21μmol)，氮气置换3次，在80℃搅拌12小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(四氢呋喃/石油醚：50-90％)得到化合物6。
MS m/z:394.1[M+H] +
1H NMR(400MHz,CDCl 3)δ8.69(br s,1H),8.40(s,1H),7.84(s,1H),7.54(t,J＝2.9Hz,1H),7.23(dd,J＝0.9,3.1Hz,1H),7.06(t,J＝2.1Hz,1H),6.55(d,J＝1.0Hz,1H),4.43(br d,J＝7.3Hz,1H),4.19(s,3H),4.11-3.97(m,2H),3.88-3.78(m,2H),3.67(dt,J＝3.0,11.8Hz,1H),3.35(dt,J＝3.9,12.6Hz,1H),1.37(d,J＝6.8Hz,3H)
实施例7
Figure PCTCN2021096143-appb-000051
步骤1：化合物7-B的合成
将1-B(300mg,886.08μmol)置于微波反应器中，加入1,4-二氧六环(2mL)和水(0.2mL)。加入7-A(213.54mg,974.68μmol)，碳酸钠(281.74mg,2.66mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(64.83mg,88.61μmol)，氮气吹扫2分钟，微波加热至100℃搅拌1.5小时。反应结束后将反应液减压干燥，然后拌样纯化。粗品经柱层析分离(乙酸乙酯/石油醚：10-40％)得到化合物7-B。MS m/z:303.9[M+H] +。
步骤2：化合物7的合成
将化合物7-B(274mg,901.95μmol)溶于1,4-二氧六环(10mL)中，加入中间体I(454.97mg,1.35mmol),碳酸钠的水溶液(2M,1.35mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(51.56mg,63.14umol)。氮气置换3次，加热至80℃反应16小时。反应结束后将反应液降至室温，向反应液中加入40mL水，用乙酸乙酯150mL(50*3)萃取。有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：10-50％)得到化合物7。MS m/z:404.1[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.70-8.55(m,2H),8.38(s,1H),7.72(br d,J＝6.9Hz,1H),7.52(br s,1H),7.33(br s,1H),7.18(br d,J＝2.3Hz,1H),7.09(br s,1H),6.49(s,1H),4.40(br d,J＝6.6Hz,1H),4.09-3.98(m,2H),3.83(s,2H),3.73-3.62(m,1H),3.39-3.28(m,1H),2.68(s,3H),1.36(br d,J＝6.8Hz,3H)
实施例8
Figure PCTCN2021096143-appb-000052
步骤1：化合物1-B的合成
将1-B(300mg,886.08μmol)置于微波反应器中，加入1,4-二氧六环(2mL)和水(0.2mL)。加入8-A(212.58mg,974.68μmol)，碳酸钠(281.74mg,2.66mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(64.83mg,88.61μmol)，氮气鼓泡2分钟，微波加热至100℃搅拌1.5小时。反应结束后将反应液减压干燥，然后拌样纯化。粗品经柱层析分离(乙酸乙酯/石油醚：5-20％)得到化合物8-B。MS m/z:302.9[M+H] +。
步骤2：化合物8的合成
将化合物8-B(285mg,941.22μmol)溶于1,4-二氧六环(10mL)中，加入I(474.78mg,1.41mmol),碳酸钠的水溶液(2M,1.41mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(53.80mg,65.89umol)。氮气置换3次，加热至80℃反应16小时。反应结束 后向反应液中加入40mL水，用乙酸乙酯150mL(50*3)萃取。有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：10-50％)得到化合物8。MS m/z:403.1[M+H] +。
1H NMR(400MHz,CDCl 3)δ7.49(br s,1H),7.30(br d,J＝7.9Hz,6H),7.20(br s,1H),7.06-6.87(m,1H),6.58(br s,1H),4.42(br s,1H),4.12-4.02(m,2H),3.85(br s,2H),3.72(br s,1H),3.39(br s,1H),2.38(s,3H),1.38(br d,J＝5.5Hz,3H)
实施例9
Figure PCTCN2021096143-appb-000053
步骤1：化合物9-A的合成
向化合物1-B(300mg,886.08μmol,1eq)，3,5-二甲基吡唑-4-嚬哪醇硼酸酯(216.47mg,974.68μmol,1.1eq)，碳酸钠(140.87mg,1.33mmol,664.56μL,1.5eq)和1,4-二氧六环(3mL)溶液中加入[1,1-双(二苯基膦)二茂铁]二氯化钯(64.83mg,88.61μmol,0.1eq)，用氮气鼓泡，反应在微波100℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(20mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析(石油醚/乙酸乙酯:50％～60％)纯化得到化合物9-A。
MS-ESI m/z:306.9[M+H] +。
步骤2：化合物9的合成
将7-A(200mg,651.91μmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3mL)，加入I(438.45mg,1.30mmol)，碳酸钠(172.74mg,1.63mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(53.24mg,65.19μmol)，氮气鼓泡2分钟，微波加热至110℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(四氢呋喃/石油醚：70-90％)得到化合物9。
MS m/z:407.1[M+H] +
1H NMR(400MHz,DMSO-d 6)δ12.45(br s,1H),11.74(br s,1H),8.35(s,1H),7.93-7.57(m,1H),6.99(d,J＝2.0Hz,1H),6.82(br s,1H),6.61(s,1H),4.38(br s,1H),4.18-3.90(m,2H),4.17-3.89(m,1H),3.79-3.70(m,1H),3.69-3.63(m,1H),3.57-3.47(m,1H),2.29(br d,J＝19.6Hz,6H),1.19(br d,J＝6.5Hz,3H)
实施例10
Figure PCTCN2021096143-appb-000054
步骤1：化合物10-B的合成
将10-A(300mg,1.48mmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3mL)。加入3-B(445.99mg,1.63mmol)，碳酸钠(470.60mg,4.44mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(72.72mg,103.60umol)，氮气吹扫2分钟，微波加热至110℃搅拌0.5小时。反应结束后向反应液中加入60mL水，用乙酸乙酯210mL(70*3)萃取。有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-20％)得到化合物10-B。MS m/z:268.8[M+H] +。
步骤2：化合物10-C的合成
将化合物10-B(300mg,1.11mmol)置于微波反应器中，加入(R)-3-甲基吗啡啉(112.75mg,1.11mmol)，微波加热至200℃搅拌2小时。反应结束后将反应液减压干燥，将大部分(R)-3-甲基吗啡啉减压除去。粗品经柱层析分离(乙酸乙酯/石油醚：0-30％)得到化合物10-C。MS m/z:333.9[M+H] +。
步骤3：化合物10的合成
将化合物10-C(300mg,898.71μmol)溶于1,4-二氧六环(3mL)中，加入中间体I(453.33mg,1.35mmol),碳酸钠的水溶液(2M,1.35mL)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(73.39mg,89.87μmol)。氮气置换3次，加热至100℃反应16小时后将温 度降至室温40℃左右，加入四乙基氟化铵水合物(150.32mg,898.71μmol)搅拌2小时。反应结束后向反应液中加入60mL水。用乙酸乙酯210mL(70*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：0-40％)得到化合物10。MS m/z:434.1[M+H] +。
1H NMR(400MHz,CDCl 3)δ8.80(br s,1H),8.33(br s,1H),8.08(s,1H),7.51(br s,1H),7.16(d,J＝2.3Hz,1H),7.05(br s,1H),6.69(s,1H),6.50(s,1H),4.40(br d,J＝6.0Hz,1H),4.10-4.03(m,2H),3.97(s,3H),3.84(s,2H),3.74-3.63(m,1H),3.41-3.27(m,1H),2.35(s,3H),1.36(d,J＝6.5Hz,3H)
实施例11
Figure PCTCN2021096143-appb-000055
步骤1：化合物1-C的合成
将1-B(500mg,1.48mmol)置于微波反应器中，加入1,4-二氧六环(6mL)和水(0.6mL)。加入11-A(315.21mg,1.62mmol)，碳酸钠(469.58mg,4.43mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(75.64mg,103.38umol)，氮气吹扫2分钟，微波加热至110℃搅拌0.5小时。反应结束后向反应液中加入40mL水，用乙酸乙酯150mL(50*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：0-30％)得到化合物11-B。MS m/z:278.9[M+H] +。
步骤2：化合物11-C的合成
将化合物11-B(615mg,2.21mmol)置于微波反应器中，加入1,4-二氧六环(10mL)和水(1mL)，加入中间体I(964.56mg,2.87mmol),碳酸钠(701.56mg,6.62mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(126.13mg,154.45μmol)。氮气鼓泡2分钟，加热至110℃反应1小时。反应结束后将反应液通过硅藻土过滤得滤液，减压干燥得粗品化合物11-C。MS m/z:535.2[M+H] +。
步骤3：化合物11的合成
将化合物11-C(1.02g,1.91mmol)溶于1,4-二氧六环(20mL)中，加入四乙基氟化铵水合物(319.05mg,1.91mmol)，加热至40℃搅拌2小时。反应结束后向反应液中加入40mL水，用乙酸乙酯150mL(50*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：10-50％)得到化合物11。MS m/z:379.0[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ13.09(br s,1H),11.76(br s,1H),8.37(s,1H),7.90-7.79(m,1H),7.75(t,J＝2.8Hz,1H),7.53(br s,1H),7.14(s,1H),6.93(d,J＝2.3Hz,1H),6.79(br s,1H),4.48(br d,J＝6.5Hz,1H),4.02-3.91(m,2H),3.79-3.72(m,1H),3.72-3.65(m,1H),3.53(dt,J＝2.8,11.7Hz,1H),3.17-3.16(m,1H),1.20(d,J＝6.5Hz,3H)
实施例12
Figure PCTCN2021096143-appb-000056
步骤1：化合物12-B的合成
将1-B(500mg,1.48mmol)置于微波反应器中，加入1,4-二氧六环(5mL)和水(0.5mL)。加入12-A 458.37mg,1.62mmol)，碳酸钠(469.58mg,4.43mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯(75.64mg,103.38μmol)，氮气吹扫2分钟，微波加热至110℃搅拌0.5小时。反应结束后向反应液中加入40mL水，用乙酸乙酯150mL(50*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/石油醚：5-30％)得到化合物12-B。MS m/z:366.9[M+H] +。
步骤2：化合物12-C的合成
将化合物12-B(1.36mmol)置于微波反应器中，加入1,4-二氧六环(5mL)和水(0.5mL)，加入I(595.82mg,1.77mmol),碳酸钠(433.36mg,4.09mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(111.30mg,136.29μmol)。氮气吹扫2分钟，加热至110℃反应1小时。反应结束后将反应液通过硅藻土过滤得滤液，减压干燥得粗品化合物12-C。MS m/z:623.2[M+H] +。
步骤3：化合物12的合成
将化合物12-C(1.19g,1.91mmol)溶于1,4-二氧六环(20mL)中，加入四乙基氟化铵水合物(319.05mg,1.91mmol)，加热至40℃搅拌2小时。反应结束后向反应液中加入50mL水，用乙酸乙酯150mL(50*3)萃取，有机相用饱和食盐水洗涤一次，用无水硫酸钠干燥，过滤得滤液并减压干燥。粗品经柱层析分离(乙酸乙酯/二氯甲烷：10-60％)得到化合12。MS m/z:467.1[M+H] +。
1H NMR(400MHz,DMSO-d 6)δ11.79(br s,1H),8.39(s,1H),8.10-8.01(m,4H),7.77(br s,1H),7.36(br s,1H),7.07(s,1H),6.82(br s,1H),4.56(br d,J＝6.1Hz,1H),4.08(br d,J＝12.3Hz,1H),3.98(br d,J＝8.9Hz,1H),3.81-3.73(m,1H),3.72-3.65(m,1H),3.54(br t,J＝10.8Hz,1H),3.28(s,3H),3.24-3.15(m,1H),1.23(br d,J＝6.5Hz,3H)
实施例13
Figure PCTCN2021096143-appb-000057
步骤1：化合物13-A的合成
向化合物1-B(300mg,886.08μmol,1eq)，1,3-二甲基吡唑-4-嚬哪醇硼酸酯(216.47mg,974.68μmol,1.1eq)和碳酸钠(140.87mg,1.33mmol,664.56μL,1.5eq)1,4-二氧六环(3mL)溶液中加入[1,1-双(二苯基膦)二茂铁]二氯化钯(64.84mg,88.61μmol,0.1eq)，用氮气 鼓泡，反应在微波100℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(20mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析(石油醚/乙酸乙酯:50％～70％)纯化得到化合物13-A。
MS-ESI m/z:306.9[M+H] +。
步骤2：化合物13的合成
将13-A(170mg,554.13μmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3mL)，加入中间体I(372.69mg,1.11mmol)，碳酸钠(146.83mg,1.39mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(45.25mg,55.41μmol)，氮气鼓吹2分钟，微波加热至110℃搅拌1小时。待反应液冷却后，向反应液中加入四乙基氟化铵水合物(139.03mg,831.19umol)，在25℃搅拌2小时。反应体系用水(20mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(四氢呋喃/石油醚：50-70％)，得到化合物13。
MS m/z:407.1[M+H] +
1H NMR(400MHz,DMSO-d 6)δ11.76(br s,1H),8.37(s,1H),8.15(s,1H),7.76(br s,1H),7.15(d,J＝2.0Hz,1H),6.81(br s,1H),6.76(s,1H),4.44(br d,J＝6.0Hz,1H),4.02-3.92(m,2H),3.81(s,3H),3.78-3.72(m,1H),3.71-3.65(m,1H),3.58-3.48(m,1H),3.15(dt,J＝3.5,12.7Hz,1H),2.38(s,3H),1.20(d,J＝6.5Hz,3H)
实施例14
Figure PCTCN2021096143-appb-000058
步骤1：化合物14-A的合成
向化合物1-B(300mg,886.08μmol,1eq)，1,4-二甲基吡唑-5-嚬哪醇硼酸酯(216.47mg,974.68umol,1.1eq)和碳酸钠(140.87mg,1.33mmol,664.56μL,1.5eq)1,4-二氧六环(3mL) 溶液中加入[1,1-双(二苯基膦)二茂铁]二氯化钯(64.83mg,88.61μmol,0.1eq)，用氮气鼓泡，反应在微波100℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(20mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析(石油醚/乙酸乙酯:40％～70％)纯化得到化合物14-A。
MS-ESI m/z:306.9[M+H] +。
步骤2：化合物14的合成
将1-B(230mg,749.70μmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3mL)，加入中间体I(504.22mg,1.5mmol)，碳酸钠(198.65mg,1.87mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(61.22mg,74.97μmol)，氮气鼓吹2分钟，微波加热至110℃搅拌1小时。待反应液冷却后，向反应液中加入四乙基氟化铵水合物(188.10mg,1.12mmol)，在25℃搅拌2小时。反应体系用水(20mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(四氢呋喃/石油醚：50-70％)得到化合物14。
MS m/z:407.1[M+H] +
1H NMR(400MHz,DMSO-d 6)δ11.81(br s,1H),8.39(s,1H),7.78(t,J＝2.8Hz,1H),7.38(s,1H),7.05(d,J＝2.3Hz,1H),6.84(br s,1H),6.78(s,1H),4.44(br d,J＝6.5Hz,1H),4.06(br d,J＝12.0Hz,1H),3.97(br dd,J＝3.0,11.0Hz,1H),3.83(s,3H),3.78-3.72(m,1H),3.71-3.65(m,1H),3.53(dt,J＝2.8,11.8Hz,1H),3.17(dt,J＝3.6,12.7Hz,1H),2.07(s,3H),1.22(d,J＝6.5Hz,3H)
实施例15
Figure PCTCN2021096143-appb-000059
步骤1：化合物15-A的合成
向化合物1-B(300mg,886.08μmol,1eq)，I(202.80mg,974.68μmol,1.1eq)和碳酸钠(140.87mg,1.33mmol,664.56μL,1.5eq)1,4-二氧六环(3mL)溶液中加入[1,1-双(二苯基膦)二茂铁]二氯化钯(64.83mg,88.61μmol,0.1eq)，用氮气鼓泡，反应在微波100℃搅拌1小时。待反应液冷却后，反应体系用水(20mL)稀释，乙酸乙酯(20mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析(石油醚/乙酸乙酯:40％～50％)纯化得到化合物15-A。
MS-ESI m/z:292.9[M+H] +。
步骤2：化合物15的合成
将15-A(150mg,512.36μmol)置于微波反应器中，加入1,4-二氧六环(3mL)和水(0.3mL)，加入中间体I(172.30mg,512.36μmol)，碳酸钠(135.76mg,1.28mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(41.84mg,51.24μmol)，氮气鼓吹2分钟，微波加热至110℃搅拌1小时。待反应液冷却后，向反应液中加入四乙基氟化铵水合物(128.55mg,768.54umol)，在25℃搅拌2小时。反应体系用水(20mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(乙酸乙酯/二氯甲烷：50-60％)得到化合物15。
MS m/z:393.1[M+H] +
1H NMR(400MHz,DMSO-d 6)δ11.80(br s,1H),8.39(s,1H),7.78(t,J＝2.8Hz,1H),7.53(d,J＝2.0Hz,1H),7.17(d,J＝2.0Hz,1H),6.87(s,1H),6.82(br s,1H),6.60(d,J＝1.8Hz,1H),4.49(br d,J＝6.5Hz,1H),4.08-3.99(m,2H),3.97(s,3H),3.80-3.73(m,1H),3.72-3.64(m,1H),3.53(dt,J＝2.6,11.6Hz,1H),3.18(dt,J＝3.5,12.7Hz,1H),1.22(d,J＝6.5Hz,3H)
实施例16
Figure PCTCN2021096143-appb-000060
步骤1：化合物1-B的合成
向化合物1-B(500mg,1.48mmol,1eq)，3,5-二甲基异噁唑-4-嚬哪醇硼酸酯(362.38mg,1.62mmol,1.1eq)和碳酸钠(234.79mg,2.22mmol,1.11mL,1.5eq)1,4-二氧六环(8mL)溶液中加入[1,1-双(二苯基膦)二茂铁]二氯化钯(108.06mg,147.68μmol,0.1eq)，氮气置换3次，反应在90℃搅拌12小时。待反应液冷却后，反应体系用水(40mL)稀释，乙酸乙酯(20mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析(石油醚/乙酸乙酯:0％～20％)纯化得到化合物16-A。
MS-ESI m/z:307.9[M+H] +。
步骤2：化合物16的合成
将16-A(370mg,1.2mmol)置于微波反应器中，加入1,4-二氧六环(5mL)和水(0.5mL)，加入I(727.69mg,2.16mmol)，碳酸钠(318.54mg,3.01mmol)和[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷络合物(98.17mg,120.22μmol)，氮气置换3次，在80℃搅拌12小时。待反应液冷却后，向反应液中加入四乙基氟化铵水合物(301.62mg,1.80mmol)，在25℃搅拌2小时。反应体系用水(40mL)稀释，乙酸乙酯(15mL×3)洗涤，饱和食盐水(10mL)洗涤，无水硫酸钠干燥。滤去干燥剂后，减压除去溶剂得到粗品。粗品经柱层析分离(乙酸乙酯/石油醚：50-75％)得到化合物16。
MS m/z:408.1[M+H] +
1H NMR(400MHz,CHLOROFORM-d)δ9.18(br s,1H),8.37(s,1H),7.53(t,J＝2.6Hz,1H),7.11(d,J＝2.5Hz,1H),7.07(br s,1H),6.43(s,1H),4.38(br d,J＝6.8Hz,1H),4.10-3.97(m,2H),3.88-3.74(m,2H),3.67(dt,J＝3.0,11.8Hz,1H),3.32(dt,J＝3.6,12.6Hz,1H),2.51(s,3H),2.37(s,3H),1.35(d,J＝6.8Hz,3H)
实验例1：体外细胞活性实验
通过测定IC 50值来评价受试化合物对人的ATR激酶的抑制活性.
将ATR/ATRIP(h)在含有50nM GST-cMyc-p53和Mg/ATP(10uM)的测定缓冲液中培育。反应通过添加Mg/ATP混合物来引发。在室温下培育30分钟后，加入含有EDTA的终止溶液终止反应。最后，加入含有d 2标记的抗GST单克隆抗体的检测缓冲液和抗磷酸化p53的铕标记的抗磷酸Ser15抗体。然后以时间分辨荧光模式读取平板并进行均相时间分辨。
根据公式HTRF＝10000×(Em665nm/Em620nm)确定荧光(HTRF)信号。
表1 体外ATR酶活性实验结果
化合物编号 IC 50(nM)
化合物1 33
化合物2 69
化合物5 336
化合物6 211
化合物7 78
化合物8 425
化合物9 31
化合物10 182
化合物13 500
化合物14 57
化合物15 204
化合物16 78
实验结果表明，本发明化合物针对ATR酶有较强的抑制活性。
实验例2：体外细胞活性实验
本实验通过检测化合物在肿瘤细胞系LoVo中对体外细胞活性的影响而研究化合物抑制细
胞增殖的作用。
CellTiter-Glo发光法细胞活性检测
以下步骤按照PromegaCellTiter-Glo发光法细胞活性检测试剂盒(Promega-G7573)的说明书来进行。
(1).将CellTiter-Glo缓冲液融化并放置至室温。
(2).将CellTiter-Glo底物放置至室温。
(3).在一瓶CellTiter-Glo底物中加入CellTiter-Glo缓冲液以溶解底物，从而配制CellTiter-Glo工作液。
(4).缓慢涡旋震荡使充分溶解。
(5).取出细胞培养板放置30分钟使其平衡至室温。
(6).在每孔中加入50μL(等于每孔中细胞培养液一半体积)的CellTiter-Glo工作液。用铝箔纸包裹细胞板以避光。
(7).将培养板在轨道摇床上振摇2分钟以诱导细胞裂解。
(8).培养板在室温放置10分钟以稳定发光信号。
(9).在SpectraMax i3x of Molecular Devices读板器上检测发光信号。
数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(1–(RLU化合物–RLU空白对照)/(RLU溶媒对照–RLU空白对照))*100％。在Excel中计算不同浓度化合物的抑 制率，然后用GraphPad Prism软件作抑制曲线图和计算相关参数，包括最小抑制率，最大抑制率及IC 50。
实验结果见表2：
表2 体外LoVo细胞抑制增殖实验结果
化合物编号 LoVo细胞增殖IC 50(μM)
化合物1 0.102
化合物2 0.234
实验结果表明，本发明化合物针对ATM信号通路缺失的LoVo肿瘤细胞有较好的抑制作用。
实验例3：体内药代动力学性质研究
供试样品：在上述实验的基础上，选择化合物1开展进一步实验。
实验方法：该研究的目的是为了测定该化合物1药代动力学参数，并计算其在雌性Balb/c Nude小鼠中的口服生物利用度。该项目使用4只雌性Balb/c Nude小鼠，两只小鼠进行静脉注射给药，给药剂量为1mg/kg，收集0h(给药前)和给药后0.0833，0.25，0.5，1，2，4，6，8，24h的血浆样品，另外两只小鼠口服灌胃给药，给药剂量为10mg/kg或者25mg/kg，收集0h(给药前)和给药后0.25，0.5，1，2，4，6，8，24h的血浆样品，然后对收集的样品进行LC/MS/MS分析并采集数据，采集的分析数据用Phoenix WinNonlin 6.2.1软件计算相关药代动力学参数。
实验结果见表3：
表3 体内药代动力学实验结果
3.1静脉注射给药结果
Figure PCTCN2021096143-appb-000061
3.2口服给药结果
给药剂量(mg/kg) C max(nM) T 1/2(h) AUC 0-t(nM.h) F(％)
10 9565 1.4 22992 91.6
25 17396 3.8 82019 -
注：C 0(nM)为0分钟时体内药物浓度；Cl(mL/min/kg)为药物体内清除率；V dss(L/kg)为药物体内分布容积；T 1/2(h)为半衰期；AUC 0-t(nM.h)为体内药物暴露量；Cmax(nM)为体内药物最高浓度。
实验结论：本发明化合物有良好的暴露量和生物利用度等体内药代动力学性质。
实验例4：人胃癌细胞SNU-601 CDX体内药效研究
实验目的：
本研究主要目的是在人胃癌细胞SNU-601异种移植瘤模型上研究受试物的抗肿瘤药效。
实验方法：
1.实验动物
种属：小鼠
品系：CB17 SCID小鼠
供应商：华阜康实验动物技术有限公司
周龄：6－8周龄
性别：雌性
2.细胞培养
人胃癌SNU-601细胞，来源KCLB(货号：00601)，由辉源生物科技(上海)有限公司保种维持传代。体外培养条件为RPMI 1640培养基(含有300mg/L L-谷氨酰胺)中加入10％胎牛血清、25mM HEPES和25mM碳酸氢钠，37℃ 5％CO2孵箱培养，一周两到三次传代。当细胞数量到达要求时，收取细胞，计数。将0.2mL(5×106个)SNU-601细胞(重悬于DPBS:Matrigel＝1:1)皮下接种于每只小鼠的右后背，肿瘤平均体积达到147.61mm3时开始分组给药。
3.受试物的给药剂量
给药剂量：化合物1分别采用15mg/kg(给3天休4天)，10mg/kg(给3天休4天)和5mg/kg(连续给药)三个剂量口服给药。
4.肿瘤测量和实验指标
每周三次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5×a×b 2，a和b分别表示肿瘤的长径和短径。相对肿瘤体积(RTV)的计算公式为：RTV(％)＝(Vt/V1)×100；动物体重变化(BWC)的计算公式为：BWC(％)＝(BWt–BW1)/BW1×100，其中，V1和BW1指某只动物分组给药当天的肿瘤体积和体重，Vt和BWt指某只动物某一次测量的肿瘤体积和体重。
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：治疗组平均RTV；CRTV：阴性对照组平均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/ V1，其中V1是分组给药时(即D1)测量所得肿瘤体积，Vt为某一次测量时的肿瘤体积，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。
在实验结束后将检测肿瘤重量，并计算Tweight/Cweight百分比，Tweight和Cweight分别表示给药组和溶媒对照组的瘤重。
5.实验结果
本次实验，采用人胃癌细胞SNU601异种移植瘤模型，评价受试物化合物的体内药效。在整个给药期间，并无动物因体重下降超过10％而停药，给药后第21天，结束药效试验。在给药后第21天，溶媒组的肿瘤体积达到890.01±184.62mm 3。与溶媒对照组相比，化合物1在15mg/kg、10mg/kg及5mg/kg给药剂量下均表现出一定的抗肿瘤效果。它们对应的肿瘤体积分别为108.74±9.67mm 3、136.74±14.46mm3及229.99±24.42mm 3，抑瘤率TGI分别为104.81％(p＜0.01)、101.08％(p＜0.01))及88.61％(p＜0.01))。
在整个实验期间，化合物1给药组动物体重均无明显下降，动物状态无异常。
综上，本发明化合物可以明显抑制人胃癌SNU-601异种移植瘤的生长，且对小鼠相对耐受。

Claims (12)

  1. 式(Ⅱ)所示化合物或其药学上可接受的盐，
    Figure PCTCN2021096143-appb-100001
    其中，
    环A为
    Figure PCTCN2021096143-appb-100002
    T 1、T 2、T 3、T 4和T 5分别独立地为C、CH或N；
    E 1为O或S；
    R 1分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R a取代；
    R 2分别独立地为H或C 1-3烷基，其中所述C 1-3烷基任选被1、2或3个R b取代；
    R 3为H或C 1-3烷基；
    R 4分别独立地为H、C 1-3烷基、-O-C 1-3烷基或-S(O) 2-C 1-3烷基，其中所述C 1-3烷基、-O-C 1-3烷基和-S(O) 2-C 1-3烷基分别独立地任选被1、2或3个R c取代；
    R a、R b和R c分别独立地为F、Cl、Br或I；
    m为1、2或3。
  2. 根据权利要求1所述化合物或其药学上可接受的盐，其中，化合物具有式(Ⅰ-1)或(Ⅰ-2)结构
    Figure PCTCN2021096143-appb-100003
    其中，
    R 1和R 2如权利要求1所定义。
  3. 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，R 1分别独立地为H或 CH 3，其中所述CH 3任选地被1、2或3个R a取代。
  4. 根据权利要求3所述化合物或其药学上可接受的盐，其中，R 1分别独立地为H或CH 3。
  5. 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，R 2分别独立地为H或CH 3，其中CH 3任选地被1、2或3个R b取代。
  6. 根据权利要求5所述化合物或其药学上可接受的盐，其中，R 2分别独立地为H或CH 3。
  7. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 3分别独立地为H或CH 3。
  8. 根据权利要求1所述化合物或其药学上可接受的盐，其中，R 4分别独立地为H、CH 3、-O-CH 3或-S(O) 2-CH 3。
  9. 根据权利要求1所述化合物或其药学上可接受的盐，其中，环A为
    Figure PCTCN2021096143-appb-100004
    Figure PCTCN2021096143-appb-100005
  10. 根据权利要求9所述化合物或其药学上可接受的盐，其中，环A为
    Figure PCTCN2021096143-appb-100006
    Figure PCTCN2021096143-appb-100007
  11. 下式化合物或其药学上可接受的盐，
    Figure PCTCN2021096143-appb-100008
    Figure PCTCN2021096143-appb-100009
  12. 根据权利要求1～11任意一项所述化合物或其药学上可接受的盐在制备治疗ATR相关疾病的药物中的应用。
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