WO2021104461A1 - 二氮杂吲哚类衍生物及其作为Chk1抑制剂的应用 - Google Patents

二氮杂吲哚类衍生物及其作为Chk1抑制剂的应用 Download PDF

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WO2021104461A1
WO2021104461A1 PCT/CN2020/132306 CN2020132306W WO2021104461A1 WO 2021104461 A1 WO2021104461 A1 WO 2021104461A1 CN 2020132306 W CN2020132306 W CN 2020132306W WO 2021104461 A1 WO2021104461 A1 WO 2021104461A1
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Prior art keywords
compound
reaction
pharmaceutically acceptable
add
acid
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English (en)
French (fr)
Inventor
廖勇刚
钱文远
韦昌青
肖瑶
奚正英
张臣
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 EP20894671.5A priority Critical patent/EP4067351B1/en
Priority to US17/779,310 priority patent/US11634424B2/en
Priority to CN202080081812.7A priority patent/CN114746413B/zh
Priority to JP2022531590A priority patent/JP7260718B2/ja
Priority to ES20894671T priority patent/ES3014705T3/es
Publication of WO2021104461A1 publication Critical patent/WO2021104461A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • 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 quotes the following priorities:
  • the present invention relates to a series of diazaindole derivatives and their application in preparing medicines for diseases related to Chk1. Specifically, it relates to a compound represented by formula (I) and a pharmaceutically acceptable salt thereof.
  • DNA Deoxyribonucleic acid
  • endogenous such as reactive oxygen species, free radicals and other active compounds generated during metabolism in the body, replication stress, etc.
  • exogenous such as ionizing radiation, ultraviolet rays, alkylating agents, Carcinogens, virus infections, etc.
  • DNA double-strand break which is considered to be the most serious DNA damage, and is repaired in two different ways, namely non-homologous ends Connection (Non-homologous End Joining, NHEJ) and homologous recombination (Homologous Recombination, HR);
  • DNA single strand break which is a specific type that occurs on a stalled replication fork
  • DSB DNA double-strand break
  • SSB DNA single strand break
  • DDR DNA damage response
  • the cell cycle checkpoint has a conservative signal regulation system.
  • the ATR/Chk1 kinase pathway and ATM/Chk2 kinase pathway play an important role in the molecular mechanisms of cell cycle checkpoints involved in DNA damage response.
  • the ATM/Chk2 pathway is usually activated; and in the case of structural damage such as DNA single-strand breaks or DNA alkylation, the ATR/Chk1 pathway is activated. After the two are activated, they activate a series of downstream substrates, such as Cdc25, cyclin, and Wee1, which makes the cell cycle arrest.
  • the ATR-Chk1-Cdc25 reaction pathway is the most important pathway for cells to deal with ionizing radiation and replication pressure.
  • the principle is roughly that the cell activates ATR after recognizing the signal of DNA damage, thereby phosphorylating multiple serines on Chk1
  • activated Chk1 then phosphorylates Cdc25, which accelerates the ubiquitination and degradation of Cdc25; at the same time, activated Chk1 activates Wee1, which in turn phosphorylates and inactivates the CDK1/Cyclin B complex.
  • the final result is cell cycle arrest.
  • the cells are prevented from entering the mitotic phase in the presence of DNA damage or unreplicated DNA.
  • Chk1 is highly expressed in certain malignant tumors, and tumor cells lacking Chk1 expression often show multiple defects, such as slow cell proliferation, disappearance of cell cycle checkpoint arrest response, and increased sensitivity to DNA damaging agents. Releasing the blocking effect of Chk1 on cell cycle checkpoints to promote tumor cell apoptosis, this mechanism is the research basis of many Chk1 inhibitors and is also a new hot spot in current anti-tumor research.
  • Chk1 is the main kinase that controls S and G2/M checkpoints Therefore, inhibiting Chk1 when tumor cell DNA is damaged can hinder the self-repair of tumor cells, so as to achieve the purpose of enhancing tumor cell killing.
  • Normal cells have a perfect cell cycle checkpoint regulation function, which can repair and protect themselves, so they are relatively insensitive to Chk1 inhibitors. This makes Chk1 inhibitors very selective in targeting tumor cells in tumor therapy. .
  • Chk1 inhibitors can not only be used alone to stop or delay the proliferation of tumor cells, but also can be combined with chemotherapy or radiotherapy to kill tumor cells through a combined lethal (syntheticlethal) mechanism of action to treat tumors.
  • lethal syntheticlethal
  • Chk1 inhibition There are many kinds of Chk1 inhibition in WO05/066163, WO04/063198, WO03/093297 and WO02/070494. It was reported in WO 05/009435.
  • LY2606368 (Prexasertib) developed by Lilly Company is carrying out a variety of anti-tumor clinical research.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • T 1 is selected from CH and N;
  • L is selected from a single bond and -CH 2 -;
  • n is selected from 0, 1, 2, 3 and 4;
  • n and q are independently selected from 1 and 2 respectively;
  • R 1 is selected from H, F, Cl, Br and I;
  • R 2 is selected from H, D, F, Cl, Br and I;
  • R 3 is selected from H, F, Cl, Br, I, CN and C 1-3 alkyl, said C 1-3 alkyl optionally substituted with 1, 2 or 3 R a;
  • Each R 4 is independently selected from H, F, Cl, Br, I, C 1-3 alkyl and C 1-3 alkoxy, and the C 1-3 alkyl and C 1-3 alkoxy are either Choose to be replaced by 1, 2 or 3 R b ;
  • R 5 is selected from H and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R c ;
  • R a, R b and R c are each independently selected from H, D, F, Cl, Br and I.
  • the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • T 1 is selected from CH and N;
  • L is selected from a single bond and -CH 2 -;
  • n is selected from 0, 1, 2, 3 and 4;
  • n and q are independently selected from 1 and 2 respectively;
  • R 1 is selected from H, F, Cl, Br, I;
  • R 2 is selected from H, D, F, Cl, Br and I;
  • R 3 is selected from H, F, Cl, Br, I, CN and C 1-3 alkyl, said C 1-3 alkyl optionally substituted with 1, 2 or 3 R a;
  • Each R 4 is independently selected from H, F, Cl, Br, I, C 1-3 alkyl and C 1-3 alkoxy, and the C 1-3 alkyl and C 1-3 alkoxy are either Choose to be replaced by 1, 2 or 3 R b ;
  • R 5 is selected from H and C 1-3 alkyl, the C 1-3 alkyl is optionally substituted with 1, 2 or 3 R c ;
  • R a, R b and R c are each independently selected from H, F, Cl, Br and I.
  • R 1 is selected from H and F, and other variables are as defined in the present invention.
  • R 3 is selected from H and CH 3 , and other variables are as defined in the present invention.
  • R 4 is independently selected from H, F, Cl, CH 3 and OCH 3 , and the CH 3 and OCH 3 are optionally substituted by 1, 2 or 3 R b , and other variables As defined in the present invention.
  • R 4 is independently selected from H, F, Cl, CH 3 , CF 3 , OCH 3 , OCD 3 and OCF 3 , and other variables are as defined in the present invention.
  • R 4 is independently selected from H, F, Cl, CH 3 , CF 3 , OCH 3 and OCF 3 , and other variables are as defined in the present invention.
  • R 5 is selected from H, CH 3 and CD 3 , and other variables are as defined in the present invention.
  • R 5 is selected from H and CH 3 , and other variables are as defined in the present invention.
  • the above-mentioned compound or a pharmaceutically acceptable salt thereof is selected from,
  • T 1 , R 1 , R 2 , R 3 , R 4 , R 5 , L and m 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 use of the above-mentioned compound or its pharmaceutically acceptable salt in the preparation of medicines for diseases related to Chk1.
  • the above application is characterized in that the drug is a drug used to treat solid tumors such as pancreatic cancer.
  • the compound designed in the present invention exhibits good Chk1 inhibitory activity, has good oral exposure and bioavailability, and is suitable for oral administration.
  • it is treated with the representative compound of the present invention and has significant inhibition The effect of tumor growth. Therefore, the compounds of the present invention have potential application value for treating diseases related to Chk1 (such as diseases related to cell proliferation).
  • 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, such as 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 acidic functional groups
  • 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 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.
  • 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.
  • deuterated drugs have reduced toxic side effects and increased drug stability. , Enhance the efficacy, extend the biological half-life of drugs and other advantages. All changes in the isotopic composition of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • 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.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • the connection method 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 Said.
  • 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;
  • D in the present invention represents deuterium ( 3 H).
  • 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 include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.
  • C 1-3 alkoxy refers to those alkyl groups containing 1 to 3 carbon atoms that are attached to the rest 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 and the like.
  • Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C n-n+m or C n -C n+m includes any specific case of n to n+m carbons, for example, C 1-12 includes C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , and C 12 , 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 triflate; chlorine, bromine, iodine; sulfonate groups, such as methanesulfonate, 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
  • hydroxy 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 groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanoyl groups (such as acetyl); arylmethyl groups, such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and so on.
  • alkyl groups such as methyl, ethyl, and tert-butyl
  • acyl groups such as alkanoyl groups (such as acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (P
  • 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 combining them 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 field.
  • SXRD single crystal X-ray diffraction
  • the cultured 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.
  • HCl hydrochloric acid
  • MeOH stands for methanol
  • EtOH stands for ethanol
  • THF tetrahydrofuran
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • n-BuLi stands for n-butyl Lithium
  • NaH stands for sodium hydrogen
  • TFA trifluoroacetic acid
  • min stands for minutes.
  • the hydrochloride or trifluoroacetate of the compound of the present invention is added with saturated sodium bicarbonate solution to adjust the pH to neutral, and then separated by high performance liquid chromatography (neutral, ammonium bicarbonate system) to obtain the free base of the compound.
  • Step 5 Dissolve compound 1-7 (9.5g, 46.5mmol) in THF (50mL), add NaH (2.1g, 51.2mmol, purity 60%) at 0°C and stir for 0.5 hours, then add dicarbonate Tert-butyl ester (11.2 g, 51.2 mmol) was kept at 0°C for 0.5 hours and then the reaction solution was concentrated.
  • MS ESI calculated value C 15 H 20 N4O 3 [M+H] + 305, measured value 305.
  • Step 6 Dissolve compound 1-3 (130.0mg, 0.4mmol) and compound 1-8 (117.4mg, 0.4mmol) in 1,4-dioxane (10mL), add cesium carbonate (188.6mg, 0.6 mmol) and methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'- Amino-1,1'-biphenyl-2-yl)palladium(II) (35.0mg, 0.4mmol) was reacted at 95°C and stirred for 3 hours, filtered, and the filtrate was concentrated to obtain compound 1-9.
  • Step 7 Dissolve compound 1-9 (0.1g, 0.16mmol) in dichloromethane (3mL), add trifluoroacetic acid (3mL), and stir the reaction at 25°C for 0.5 hours. After the reaction is complete, it is concentrated.
  • the crude product is prepared efficiently Liquid chromatography (chromatographic column: Boston Prime C18 150*30mm 5 ⁇ m; mobile phase: [water (0.05% ammonia)-acetonitrile]; acetonitrile%: 36%-46%, 9min.) to obtain compound 1.
  • Step 1 Dissolve compound 1-1 (0.2g, 1.3mmol) in acetonitrile (5mL), add 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis (Tetrafluoroborate) (551.7mg, 1.6mmol) and pyridine (754.4mg, 9.5mmol) were reacted at 25°C and stirred for 12 hours.
  • MS ESI calculated C 6 H 3 ClFN 3 [M+H] + 172, found 172.
  • Step 2 Dissolve compound 2-1 (100.0mg, 582.9 ⁇ mol) in DMF (5mL), add NaH (34.9mg, 874.3 ⁇ mol, purity 60%) at 25°C and stir for 1 hour, then add 1-2 (162.8 mg,582.9 ⁇ mol), the reaction was stirred at 80°C for 2 hours, water (10mL) and ethyl acetate (50mL) were added for extraction and then concentrated.
  • Step 3 Dissolve compound 2-2 (60.0mg, 169.1 ⁇ mol) and 1-8 (40.0mg, 131.4 ⁇ mol) in 1,4-dioxane (5mL), and add methanesulfonic acid (2-dioxane) in sequence.
  • the reaction was stirred at 90°C for 2 hours under nitrogen protection. After the reaction was completed, it was cooled and filtered. After the filtrate was concentrated, the crude compound 2-3 was obtained directly.
  • MS ESI calculated value C 31 H 39 FN 8 O 5 [M+H] + 623, measured value 623.
  • Step 4 Dissolve compound 2-3 (30.5mg, 48.5 ⁇ mol) in dichloromethane (2mL), add trifluoroacetic acid (2mL), stir at 25°C for 0.5 hours, after the reaction is completed, the reaction solution is concentrated, the crude product is prepared efficiently Liquid chromatography (column: Xtimate C18 150*25mm*5 ⁇ m; mobile phase: [water (0.075%TFA)-acetonitrile]; acetonitrile%: 24%-44%, 7min.) to obtain compound 2 trifluoroacetic acid salt.
  • Step 3 Dissolve compound 3-3 (115.3mg, 328.6 ⁇ mol) and 1-8 (100.0mg, 328.6 ⁇ mol) in 1,4-dioxane (5mL), methanesulfonic acid (2-dicyclohexylphosphine) -3,6-Dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2- Base) palladium(II) (29.8mg, 32.9 ⁇ mol) and cesium carbonate (214.1mg, 657.2 ⁇ mol) were added to the reaction solution, and reacted at 90°C for 0.5 hours under nitrogen protection. After the reaction, the reaction solution was concentrated to obtain compound 3. -4 The crude product was directly used in the next reaction. MS ESI calculated value C 32 H 42 N8O 5 [M+H] + 619, measured value 619.
  • Step 4 Dissolve compound 3-4 (150.2mg, 242.8 ⁇ mol) in dichloromethane (2mL), add trifluoroacetic acid (2mL), stir at 25°C for 0.5 hours, concentrate after the reaction, and prepare the crude product for high performance liquid phase Chromatography (chromatographic column: Xtimate C18 150*25mm*5 ⁇ m; mobile phase: [water (0.075%TFA)-acetonitrile]; acetonitrile%: 15%-45%, 7min.) to obtain the trifluoroacetate of compound 3.
  • Step 1 Dissolve compound 1-1 (1.0g, 6.5mmol) in DMF (20mL), add KOH (1.5g, 26.1mmol) and iodine granules (3.3g, 13.0mmol) at 0°C, and react at 0°C After stirring for 1 hour, the temperature was raised to 25°C and stirring was continued for 2 hours. After the reaction was completed, ice water (100 mL) was added and then filtered. After the filter cake was dried, the crude product of compound 6-1 was obtained, which was directly used in the next reaction. MS ESI calculated value C 6 H 3 ClIN 3 [M+H] + 280, found value 280.
  • Step 3 Dissolve compound 6-2 (0.9g, 1.9mmol) in THF (10mL), add n-BuLi (2.5M, 394.20 ⁇ L) dropwise at -78°C under nitrogen protection, stir at -78°C for 0.5 hours and add Deuterium water (32.9mg, 1.6mmol) was stirred for half an hour. After the reaction was completed, saturated brine (2mL) was added to quench the reaction, extracted with ethyl acetate (50mL), and the organic phase was dried and concentrated to obtain compound 6-3. Used in the next reaction. MS ESI calculated value C 16 H 20 DClN 4 O 2 [M+H] + 338, measured value 338.
  • Step 4 Dissolve compound 6-3 (270.0 mg, 799.24 ⁇ mol) and compound 1-8 (342.0 mg, 1.9 mmol) in 1,4-dioxane (10 mL), and add methanesulfonic acid (2-dioxane) in sequence.
  • the reaction was stirred at 100°C for 1.5 hours. After the reaction is completed, it is cooled and filtered, and the filtrate is concentrated to obtain compound 6-4 directly for the next reaction.
  • Step 5 Dissolve compound 6-4 (0.1g, 0.2mmol) in dichloromethane (3mL), add trifluoroacetic acid (3mL), stir at 25°C for 0.5 hours, after the reaction is completed, concentrate, the crude product is prepared by HPLC Chromatography (chromatographic column: Waters Xbridge 150*25 5 ⁇ ; mobile phase: [water (10mM NH 4 HCO 3 )-acetonitrile]; acetonitrile%: 0%-60%, 10 min.) to obtain compound 6.
  • Step 1 Add n-BuLi (4.3mL, 2.5M) dropwise to a THF (10mL) solution of acetonitrile (445.8mg, 10.8mmol) at -78°C, stir at -78°C for 0.5 hours, and then add compound 7- A solution of 1 (1.0 g, 5.4 mmol) in THF (5 mL) was stirred at -78°C for 0.5 hour. After the reaction was completed, it was quenched by adding 10% citric acid aqueous solution (5 mL), extracted with ethyl acetate (50 mL), dried and concentrated to obtain the crude compound 7-2, which was directly used in the next step.
  • MS ESI calculated value C 10 H 8 FNO 2 [M+H] + 194, measured value 194.
  • Step 2 Add hydrazine hydrate (0.8mL, 15.6mmol) dropwise to the solution of compound 7-2 (117.5mg, 0.4mmol) in ethanol (10mL) and acetic acid (1mL), stir at 100°C for 2 hours, and concentrate after the reaction is complete
  • MS ESI calculated value C 10 H 10 FN 3 O[M+H] + 208, measured value 208.
  • Step 4 Dissolve compound 7-4 (60.0 mg, 0.2 mmol) and compound 1-3 (74.4 mg, 0.2 mmol) in 1,4-dioxane (10 mL), and add cesium carbonate (95.4 mg, 0.3mmol) and methanesulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-Amino-1,1'-biphenyl-2-yl)palladium(II) (17.7mg, 0.02mmol), stirred at 95°C for 3 hours, after the completion of the reaction, the reaction solution was filtered and concentrated to obtain crude compound 7-5 for direct use To the next step. MS ESI calculated value C 31 H 38 FN 7 O 5 [M+H] + 608, actual value 608.
  • Step 5 Dissolve compound 7-5 (110.0mg, 0.2mmol) in dichloromethane (2mL), add trifluoroacetic acid (2mL), stir at 25°C for 0.5 hours, concentrate after the reaction, and prepare the crude product for high performance liquid phase Chromatography (chromatographic column: Phenomenex Ge min.i-NX150*30mm*5 ⁇ m; mobile phase: [water (10mM NH 4 HCO 3 )-acetonitrile]; acetonitrile%: 27%-49.5%, 6min.) to obtain compound 7.
  • Step 1 Dissolve 2-fluoroacetonitrile (353.8mg, 6.0mmol) and compound 15-1 (1.0g, 5.3mmol) in THF (20mL), reduce the temperature to -78°C, and add bis( Lithium trimethylsilyl)amide (1M, 11.0 mL) and stirred at this temperature for 0.5 hours. After the reaction was completed, water (30 mL) was added, extracted with ethyl acetate (50 mL), and the organic phase was dried and concentrated to obtain the crude compound 15-2, which was directly used in the next reaction.
  • Step 2 Compound 15-2 (1.0 g, 4.7 mmol) and hydrazine hydrate (483.8 mg, 9.5 mmol) were dissolved in EtOH (10 mL) and AcOH (0.1 mL), and the reaction was stirred at 100° C. for 2 hours. After the reaction is completed, the crude product of compound 15-3 is obtained after concentration, which is directly used in the next reaction. MS ESI calculated value C 10 H 9 F 2 N 3 O [M+H] + 226, measured value 226.
  • Step 4 Dissolve compound 15-4 (135.2 mg, 415.7 ⁇ mol) and compound 1-3 (140.0 mg, 415.7 ⁇ mol) in 1,4-dioxane (10 mL), and add methanesulfonic acid (2-dioxane) in sequence.
  • Cyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl) -2-yl)palladium(II) 37.6mg, 41.5 ⁇ mol
  • cesium carbonate 270.8mg, 831.3 ⁇ mol
  • Step 5 Dissolve compound 15-5 (100.0mg, 159.8 ⁇ mol) in dichloromethane (3mL), add trifluoroacetic acid (3mL), and stir at 25°C for 0.5 hours. After the reaction is completed, the reaction solution is concentrated, and the crude product is prepared efficiently Liquid chromatography (chromatographic column: Venusil ASB Phenyl250*50mm 10 ⁇ m; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 20%-50%, 10min.) to obtain the hydrochloride of compound 15.
  • Step 1 Dissolve compound 2-2 (110.0 mg, 310.0 ⁇ mol) and compound 7-4 (95.3 mg, 310.0 ⁇ mol) in 1,4-dioxane (10 mL), and add methanesulfonic acid (2-dioxane) in sequence.
  • the reaction was stirred at 100°C for 1.5 hours. After the reaction is completed, it is cooled and filtered, and the filtrate is concentrated to obtain compound 16-1.
  • Step 4 Dissolve compound 16-1 (50.0mg, 79.9 ⁇ mol) in dichloromethane (2mL), add trifluoroacetic acid (2mL), stir at 25°C for 0.5 hours, after the reaction is complete, the reaction solution is concentrated, the crude product is prepared efficiently Liquid chromatography (chromatographic column: Venusil ASB Phenyl 250*50mm10 ⁇ m; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 25%-55%, 10min.) to obtain the hydrochloride of compound 16.
  • 1 H NMR 400MHz, CD 3 OD
  • MS ESI calculated value C 7 H 6 ClN 3 [M+H] + 168, found value 168.
  • Step 4 Add compound 17-5 (100.0 mg, 370.5 mg) and compound 7-4 (113.8 mg, 370.5 ⁇ mol) to anhydrous 1,4-dioxane (2 mL), and then add methanesulfonic acid in sequence (2-Dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1' -Biphenyl-2-yl)palladium(II) (33.6mg, 37.1 ⁇ mol) and cesium carbonate (241.4mg, 741.0 ⁇ mol), the reaction was stirred at 100°C for 1 hour.
  • Step 5 Dissolve compound 17-6 (0.1g, 160.9 ⁇ mol) in dichloromethane (2ml), add trifluoroacetic acid (2mL), stir for 5 minutes at 25°C, concentrate after the reaction is complete, and prepare the crude product for HPLC Chromatography (chromatographic column: Venusil ASB Phenyl 250*50mm 10 ⁇ m; mobile phase: [water (0.05% HCl)-acetonitrile]; acetonitrile%: 20%-50%, 10 min.) to obtain the hydrochloride of compound 17.
  • chromatographic column Venusil ASB Phenyl 250*50mm 10 ⁇ m
  • mobile phase [water (0.05% HCl)-acetonitrile]
  • acetonitrile% 20%-50%, 10 min.
  • Step 1 Dissolve compound 1-3 (150 mg, 0.445 mmol) in DCM (20 mL), add HCl/MeOH (4M, 20 mL), stir the reaction at 25°C for 0.5 hours and then concentrate to obtain crude compound 18-1, which is used directly To the next step.
  • MS ESI calculated C 11 H 13 ClN 4 [M+H] + 237, found 237.
  • Step 3 Dissolve compound 18-2 (85.0mg, 0.339mmol) and compound 1-8 (108.2mg, 0.339mmol) in dioxane (10mL), add cesium carbonate (165.69mg, 0.508mmol) and methyl Sulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1, 1'-Biphenyl-2-yl)palladium(II) (30.73mg, 0.034mmol) was stirred at 95°C for 1 hour. After the reaction, the reaction solution was filtered and concentrated to obtain the crude compound 18-3, which was directly used in the next reaction. MS ESI calculated value C 27 H 34 N 8 O 3 [M+H] + 519, measured value 519.
  • Step 4 Dissolve compound 18-3 (80.0mg, 0.154mmol) in DCM (5mL), add trifluoroacetic acid (2.0mL), and stir at 25°C for 0.5 hours. After the reaction is completed, the reaction solution is concentrated, and the crude product is prepared as a high-efficiency solution Phase chromatography (column: Boston Green ODS150*30mm*5 ⁇ m; mobile phase: [water (0.075%TFA)-acetonitrile]; acetonitrile%: 18%-48%, 8min.) to obtain the trifluoroacetate of compound 18 .
  • Phase chromatography columnumn: Boston Green ODS150*30mm*5 ⁇ m; mobile phase: [water (0.075%TFA)-acetonitrile]; acetonitrile%: 18%-48%, 8min.
  • Step 2 Add acetonitrile (3.0mL, 57.6mmol) to tetrahydrofuran (100mL), slowly dropwise add n-butyllithium (2.5M, 24.6mL, 61.5mmol) to the reaction solution at -78°C, and then keep it After the temperature continued to react for 30 minutes, compound 20-1 (7.2g, 38.46mmol) was slowly added dropwise to the reaction solution. The reaction was continued for 30 minutes, and then warmed to room temperature.
  • n-butyllithium 2.5M, 24.6mL, 61.5mmol
  • Step 4 Dissolve compound 20-3 (7.0g, 33.7mmol) in tetrahydrofuran (50mL), add sodium hydride (1.3g, 33.7mmol, purity 60%) at 0°C and stir for 0.5 hours, then add dicarbonic acid Di-tert-butyl ester (7.3g, 33.7mmol) was kept at 0°C for 0.5 hours and then the reaction solution was concentrated.
  • MS ESI calculated value C 15 H 17 D 3 N 4 O 3 [M+H] + 308, actual value 308.
  • Step 2 Dissolve compound 1-8 (100.0mg, 0.3mmol) and compound 21-1 (100.0mg, 0.4mmol) in dioxane (10mL), add cesium carbonate (214.1mg, 0.7mmol) and methyl Sulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1, 1'-Biphenyl-2-yl)palladium(II) (30.0 mg, 32.0 ⁇ mol) was stirred at 95°C for 1 hour, and the reaction solution was filtered and concentrated to obtain the crude compound 21-2, which was directly used in the next reaction.
  • Step 3 Dissolve compound 21-2 (171.0mg, 0.3mmol) in DCM (10mL), add trifluoroacetic acid (3.0mL), stir at 25°C for 0.5 hours, after the reaction is completed, the reaction solution is concentrated, and the crude product is prepared as a high-efficiency solution Phase chromatography (column: Phenomenex Gemini-NX80*40mm*3 ⁇ m; mobile phase: [water (0.05% ammonia + 10mM ammonium bicarbonate)-acetonitrile]; acetonitrile%: 27%-57%, 8min.) to obtain compound 21 .
  • Phase chromatography (column: Phenomenex Gemini-NX80*40mm*3 ⁇ m; mobile phase: [water (0.05% ammonia + 10mM ammonium bicarbonate)-acetonitrile]; acetonitrile%: 27%-57%, 8min.) to obtain compound 21 .
  • Step 1 Dissolve compound 21-1 (100.0mg, 0.3mmol) and compound 20-4 (99.0mg, 0.4mmol) in dioxane (10mL), add cesium carbonate (214.1mg, 0.6mmol) and methyl Sulfonic acid (2-dicyclohexylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl) (2'-amino-1, 1'-Biphenyl-2-yl)palladium(II) (30.0 mg, 32.0 ⁇ mol) was stirred at 95°C for 1 hour, and the reaction solution was filtered and concentrated to obtain the crude compound 22-1, which was directly used in the next reaction.
  • the calculated value of MS ESI is C 27 H 28 D 6 N 8 O 3 [M+H] + 525, and the measured value is 525.
  • Step 2 Dissolve compound 22-1 (170.0mg, 0.3mmol) in DCM (10mL), add trifluoroacetic acid (3.0mL), stir at 25°C for 0.5 hours, after the reaction is completed, the reaction solution is concentrated, and the crude product is prepared as a high-efficiency solution Phase chromatography (column: Phenomenex Gemini-NX80*40mm*3 ⁇ m; mobile phase: [water (0.05% ammonia + 10mM sodium bicarbonate)-acetonitrile]; acetonitrile%: 27%-57%, 8min.) to obtain compound 22 .
  • Phase chromatography columnumn: Phenomenex Gemini-NX80*40mm*3 ⁇ m; mobile phase: [water (0.05% ammonia + 10mM sodium bicarbonate)-acetonitrile]; acetonitrile%: 27%-57%, 8min.
  • the compound of the present invention for experiment is self-made, and its chemical name and structural formula are shown in the preparation examples of each compound.
  • the assay mixture containing the compound of the present invention and Chk1 and Chk2 kinases was mixed together and incubated in a microtiter plate, and the compounds were tested by monitoring the phosphorylation of Chk1 and Chk2 kinase on a synthetic peptide substrate with a specific amino acid sequence (KKKVSRSGLYRSPSMPENLNRPR) CHK1 inhibitor activity.
  • the experimental test was carried out on the KinaseProfiler TM protein kinase activity detection platform of Eurofins, and the experimental results were provided by the company.
  • Chk1 and Chk2 kinase use 20mM MOPS (morpholine propane sulfonic acid), 1mM EDTA (ethylenediaminetetraacetic acid), 0.04% Brij-35, 5% glycerol, 0.1% 2-mercaptoethanol, 1mg/mL BSA (bovine serum albumin) was diluted and added to the reaction system.
  • the reaction system contained the compound of the example, 8mM MOPS pH 7.0, 0.2mM EDTA, 200 ⁇ M polypeptide substrate (KKKVSRSGLYRSPSMPENLNRPR), 10mM magnesium acetate and a certain concentration of [ ⁇ -33P] -ATP (intensity approximately 500cpm/pmol).
  • the compound of the present invention exhibits good Chk1 inhibitory activity and has potential application value for treating diseases related to Chk1 (such as diseases related to cell proliferation).
  • mice the clear solution obtained by dissolving the test compound was injected via tail vein and intragastrically administered to female C57BL/6 mice (overnight fasting, 7-9 weeks old).
  • the intravenous injection group was administered at a dose of 1 mg/kg, at 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours
  • the intragastric group was administered at a dose of 10 mg/kg
  • blood was collected from the mandibular vein and centrifuged to obtain plasma.
  • PK parameters Compound 18 Compound 19 Compound 20 Intravenous injection T 1/2 (h) 0.9 1.1 1.4 Vd(L/kg) 1.9 2.4 2.7 Cl(ml/min/kg) 35.3 33.1 30.9 Intravenous AUC 0-last (nM.hr) 1138 1148 1212 C max (nM) 1223 1215 1255 Oral AUC 0-last (nM.hr) 3629 3912 5135 F(%) 33.2 39.1 41.9
  • T 1/2 half-life
  • C max peak concentration
  • Vd (L/kg) apparent volume of distribution
  • AUC 0-last the area under the plasma concentration-time curve from time 0 to 24h;
  • the compound of the present invention has good oral bioavailability and higher exposure, which is beneficial to produce good in vivo efficacy.
  • mice Female BALB/c Nude nude mice, 6-8 weeks old, weighing 18-22 grams; Supplier: Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Experimental methods and procedures:
  • Human ovarian cancer OVCAR-3 (ATCC-HTB-161) cells are cultured in a monolayer in vitro, and the culture conditions are RPMI1640 medium with 20% fetal bovine serum, 0.01 mg/mL bovine insulin, and 1% double antibody at 37°C. Culture in a 5% CO 2 incubator. Use pancreatin for routine digestion and passage twice a week. When the cell saturation is 80%-90% and the number reaches the requirement, the cells are collected, counted, and inoculated.
  • OVCAR-3 cells 0.1 mL (10 ⁇ 10 6 cells) of OVCAR-3 cells were subcutaneously inoculated on the right back of each mouse, and group administration was started when the average tumor volume reached 122 mm 3.
  • test compound 18 and compound 19 were prepared as a clear solution of 2 mg/mL, and the solvent was 10% dimethyl sulfoxide-20% polyethylene glycol 400-5% Tween 80-65% water.
  • the tumor diameter was measured with vernier calipers twice a week.
  • TGI tumor growth inhibition rate
  • TGI(%) [(1-(Average tumor volume at the end of a certain treatment group-average tumor volume at the start of the treatment group))/(Average tumor volume at the end of treatment in the solvent control group-start treatment in the solvent control group Average tumor volume at time)] ⁇ 100%.
  • RTV relative tumor volume
  • the statistical analysis is based on the RTV data at the end of the experiment using SPSS software.
  • the comparison between the two groups is analyzed by T test , and the comparison between three or more groups is analyzed by one-way ANOVA. If the variance is uniform (the F value is not significantly different), the Tukey's method is used for analysis. If the variance is not uniform (F value) Significant difference), using Games-Howell method to test. p ⁇ 0.05 considered a significant difference.
  • Example 18 Administration of human ovarian cancer OVCAR-3 subcutaneous xenograft tumor BALB/c nude nude mice Example 18, and the changes in tumor volume of each group after treatment in Example 19 are shown in Table 5.
  • the p value of the relative tumor volume (RTV) between the drug-administered group and the vehicle control group is obtained by analyzing the tumor volume by one-way ANOVA. Due to uneven variance (the F value is significantly different), the Games-Howell method is used Perform post-hoc inspection. p ⁇ 0.05 considered a significant difference.

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Abstract

式I所示的二氮杂吲哚类衍生物及其在制备与Chk1相关疾病的药物中的应用。

Description

二氮杂吲哚类衍生物及其作为Chk1抑制剂的应用
本发明引用如下优先权：
CN201911206315.0，申请日2019年11月29日；
CN202010790385.1，申请日2020年08月07日。
技术领域
本发明涉及了一系列二氮杂吲哚类衍生物，及其在制备与Chk1相关疾病的药物中的应用。具体涉及了式(Ι)所示化合物及其药学上可接受的盐。
背景技术
脱氧核醣核酸(deoxyribonucleic acid，DNA)作为机体遗传物质，其分子结构保持相对稳定对细胞的存活与功能具有重要意义。细胞中的DNA由于受到各种内源性(如活性氧、体内代谢过程中产生的自由基和其它活性化合物、复制应激反应等)和外源性(如离子辐射、紫外线、烷化剂、致癌物、病毒侵染等)因素的干扰，常常持续遭受着各种损伤。这些损伤将导致基因组发生不同程度的改变，可能演变成转录和复制错误，如果不修复或者错误修复，最终导致细胞死亡或基因突变。最常见的DNA损伤有两种：(1)DNA双链断裂(double-strand break，DSB)，这被认为是最严重的DNA损伤，通过两种不同的方式进行途径修复，即非同源末端连接(Non-homologous End Joining，NHEJ)和同源重组(Homologous Recombination，HR)；(2)DNA单股断裂(Single strand break，SSB)，这是一种在停滞的复制叉上发生的特定类型的病变，但也是DSB修复期间形成的常见中间体。
细胞在分裂过程中，其遗传物质复制并均等地分配给两个子细胞，经历了一系列细胞周期。在细胞进入有丝分裂期(mitotic period，M期)之前需要完成DNA的复制，损伤的DNA必须完成修复，否则会引发有丝分裂灾难，造成细胞死亡。为了保持基因组的完整性，细胞在整个进化过程中形成了一种称为DNA损伤反应(DDR)的复杂机制介导受损DNA的检测和修复。简单来讲，细胞DNA受到损伤时会激活细胞周期检查点，造成细胞周期停滞，从而有利于细胞进行DNA修复。
细胞周期检查点有一套保守的信号调控体系。在参与DNA损伤反应的细胞周期检查点分子机制中，ATR/Chk1激酶通路和ATM/Chk2激酶通路起着重要的作用。在DNA双链断裂时，通常是ATM/Chk2通路被激活；而在DNA单链断裂或者DNA烷基化等结构性损伤时，则是ATR/Chk1通路被激活。二者被活化后激活一系列的下游底物，如Cdc25、cyclin和Wee1等，使得细胞周期停滞。在信号的传递过程中ATR-Chk1-Cdc25反应通路是细胞应对离子辐射和复制压力的最主要通路，其原理大致为细胞在识别DNA损伤的信号后激活ATR，从而磷酸化Chk1上的多个丝氨酸位点，激活的Chk1继而磷酸化Cdc25，加速Cdc25的泛素化降解；同时，活化的Chk1激活Wee1，进而使CDK1/Cyclin B的复合物磷酸化而失活，最终的结果是使细胞周期停滞在S期和G2/M期，从而阻止细胞在DNA损伤或未复制的DNA存在下进入有丝分裂期。
一些研究发现在某些恶性肿瘤中Chk1高表达，Chk1表达缺失的肿瘤细胞往往会表现出多重缺陷，如细胞增殖缓慢、细胞周期检测点的停滞反应消失和对DNA损伤剂的敏感性增加等。通过解除Chk1对细胞周期检测点的阻滞作用进而促使肿瘤细胞凋亡，这一机制是众多Chk1抑制剂的研究基础，也是目前抗肿瘤研究的新热点。由于近50％的肿瘤细胞存在抑癌基因p53缺陷导致其G1检查点缺失，因此大多数肿瘤细胞主要依赖于S和G2/M检查点，而Chk1是控制S和G2/M检查点的主要激酶，因此在肿瘤细胞DNA损伤时抑制Chk1能够阻碍肿瘤细胞的自我修复，从而达到增强杀伤肿瘤细胞的目的。而正常细胞具有完善的细胞周期检查点调控功能，可以进行自我修复和保护，因而对Chk1抑制剂相对不敏感，这就使得Chk1抑制剂在肿瘤治疗中有很好的靶向肿瘤细胞的选择性。
Chk1抑制剂不仅可单独使用来终止或者延缓肿瘤细胞的增殖，还可以联合化疗或放疗通过联合致死(syntheticlethal)作用机制来杀伤肿瘤细胞从而治疗肿瘤。有多种Chk1抑制在WO 05/066163、W004/063198、WO 03/093297和WO 02/070494。WO 05/009435中被报道。Lilly公司开发的LY2606368(Prexasertib)正在开展多种抗肿瘤的临床研究。
Figure PCTCN2020132306-appb-000001
目前，仍然存在开发新的Chk1抑制剂用于治疗与细胞增殖相关的疾病的需求。
发明内容
本发明提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2020132306-appb-000002
其中，
T 1选自CH和N；
L选自单键和-CH 2-；
m选自0、1、2、3和4；
n和q分别独立地选自1和2；
R 1选自H、F、Cl、Br和I；
R 2选自H、D、F、Cl、Br和I；
R 3选自H、F、Cl、Br、I、CN和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
各R 4分别独立地选自H、F、Cl、Br、I、C 1-3烷基和C 1-3烷氧基，所述C 1-3烷基和C 1-3烷氧基任选被1、2或3个R b取代；
R 5选自H和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R c取代；
R a、R b和R c分别独立地选自H、D、F、Cl、Br和I。
本发明提供了式(I)所示化合物或其药学上可接受的盐，
Figure PCTCN2020132306-appb-000003
其中，
T 1选自CH和N；
L选自单键和-CH 2-；
m选自0、1、2、3和4；
n和q分别独立地选自1和2；
R 1选自H、F、Cl、Br、I；
R 2选自H、D、F、Cl、Br和I；
R 3选自H、F、Cl、Br、I、CN和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
各R 4分别独立地选自H、F、Cl、Br、I、C 1-3烷基和C 1-3烷氧基，所述C 1-3烷基和C 1-3烷氧基任选被1、2或3个R b取代；
R 5选自H和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R c取代；
R a、R b和R c分别独立地选自H、F、Cl、Br和I。
在本发明的一些方案中，上述R 1选自H和F，其他变量如本发明所定义。
在本发明的一些方案中，上述R 3选自H和CH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4分别独立地选自H、F、Cl、CH 3和OCH 3，所述CH 3和OCH 3任选被1、2或3个R b取代，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4分别独立地选自H、F、Cl、CH 3、CF 3、OCH 3、OCD 3和OCF 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 4分别独立地选自H、F、Cl、CH 3、CF 3、OCH 3和OCF 3，其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000004
选自
Figure PCTCN2020132306-appb-000005
其他变量 如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000006
选自
Figure PCTCN2020132306-appb-000007
其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000008
选自
Figure PCTCN2020132306-appb-000009
Figure PCTCN2020132306-appb-000010
Figure PCTCN2020132306-appb-000011
其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000012
选自
Figure PCTCN2020132306-appb-000013
Figure PCTCN2020132306-appb-000014
其他变量如本发明所定义。
在本发明的一些方案中，上述R 5选自H、CH 3和CD 3，其他变量如本发明所定义。
在本发明的一些方案中，上述R 5选自H和CH 3，其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000015
选自
Figure PCTCN2020132306-appb-000016
Figure PCTCN2020132306-appb-000017
其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000018
选自
Figure PCTCN2020132306-appb-000019
其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000020
选自
Figure PCTCN2020132306-appb-000021
Figure PCTCN2020132306-appb-000022
其他变量如本发明所定义。
在本发明的一些方案中，上述结构片段
Figure PCTCN2020132306-appb-000023
选自
Figure PCTCN2020132306-appb-000024
Figure PCTCN2020132306-appb-000025
其他变量如本发明所定义。
在本发明的一些方案中，上述化合物或其药学上可接受的盐，其选自，
Figure PCTCN2020132306-appb-000026
其中，T 1、R 1、R 2、R 3、R 4、R 5、L和m如本发明所定义。
本发明还有一些方案是由上述变量任意组合而来。
本发明还提供了下式化合物或其药学上可接受的盐，
Figure PCTCN2020132306-appb-000027
Figure PCTCN2020132306-appb-000028
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与Chk1相关疾病的药物中的应用。
在本发明的一些方案中，上述应用，其特征在于，所述药物是用于治疗胰腺癌等实体瘤的药物。
技术效果
本发明所设计的化合物展示了良好的Chk1抑制活性，有良好的口服暴露量和生物利用度，适合口服给药，在小鼠卵巢癌模型中给予本发明中代表性化合物治疗，具有显著的抑制肿瘤生长的效果。因此，本发明化合物具有用于治疗与Chk1相关的疾病(如细胞增殖相关疾病)的潜在应用价值。
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。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。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚( 3H)，碘-125( 125I)或C-14( 14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
术语“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“被取代的”是指特定原子上的任意一个或多个氢原子被取代基取代，可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧(即＝O)时，意味着两个氢原子被取代。氧取代不会发生在芳香基上。术语“任选被取代的”是指可以被取代，也可以不被取代，除非另有规定，取代基的种类和数目在化学上可以实现的基础上可以是任意的。
当任何变量(例如R)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R所取代，则所述基团可以任选地至多被两个R所取代，并且每种情况下的R都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR) 0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
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 PCTCN2020132306-appb-000029
直形虚线键
Figure PCTCN2020132306-appb-000030
或波浪线
Figure PCTCN2020132306-appb-000031
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 PCTCN2020132306-appb-000032
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 PCTCN2020132306-appb-000033
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 PCTCN2020132306-appb-000034
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 PCTCN2020132306-appb-000035
这4种连接方式，即使-N-上画出了H原子，但是
Figure PCTCN2020132306-appb-000036
仍包括
Figure PCTCN2020132306-appb-000037
这种连接方式的基团，只是在连接1个化学键时，该位点的的H会对应减少1个变成相应的一价哌啶基。
除非另有规定，本发明所述D代表氘( 3H)。
除非另有规定，术语“C 1-3烷基”用于表示直链或支链的由1至3个碳原子组成的饱和碳氢基团。所述C 1-3烷基包括C 1-2和C 2-3烷基等；其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C 1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)等。
除非另有规定，术语“C 1-3烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至3个碳原子的烷基基团。所述C 1-3烷氧基包括C 1-2、C 2-3、C 3和C 2烷氧基等。C 1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，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 PCTCN2020132306-appb-000038
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：HCl代表盐酸；MeOH代表甲醇；EtOH代表乙醇；THF代表四氢呋喃；DCM代表二氯甲烷；DMF代表N，N-二甲基甲酰胺；n-BuLi代表正丁基锂；NaH代表钠氢；TFA代表三氟乙酸；min代表分钟。
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。本发明化合物的盐酸盐或三氟乙酸盐加入碳酸氢钠溶液中，乙酸乙酯萃取，有机相用无水硫酸钠干燥，减压下浓缩可得到相应的化合物。或者，本发明化合物的盐酸盐或三氟乙酸盐，加入饱和碳酸氢钠溶液调节pH到中性，经过高效液相色谱法分离(中性,碳酸氢铵体系)得到化合物的游离碱。
实施例1
Figure PCTCN2020132306-appb-000039
步骤1:将化合物1-1(200.0mg，1.3mmol)加入到NaH(57.3mg，1.4mmol，纯度60％)的DMF(10 mL)悬浊液中，25℃搅拌0.5小时后加入化合物1-2(363.8mg，1.3mmol)，反应于90℃搅拌16小时。反应完毕后加水(10ml)，乙酸乙酯萃取(10ml*3)，有机相浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物1-3。MS ESI计算值C 16H 21ClN 4O 2[M+H] +337，实测值337。
步骤2:将化合物1-4(30.0g,195.9mmol)，碘甲烷(78.3g,551.6mmol)溶于氯仿(500mL)中，加入碳酸银(59.4g,215.5mmol)后反应于60℃搅拌4小时，反应完毕后冷却、过滤，滤液浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物1-5。MS ESI计算值C 9H 11NO 3[M+H] +182，实测值182。
步骤3:将乙腈(3.9mL,74.5mmol)加入到THF(100mL)中，在-78℃下缓慢滴加n-BuLi(2.5M,29.8mL,74.5mmol)到反应液中，然后保持该温度继续反应30分钟后缓慢滴加化合物1-5(9.0g,49.7mmol)到反应液中，继续反应30分钟，然后升到室温，反应液倒入水(100mL)中，乙酸乙酯萃取(200mL)后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝2:1)得化合物1-6。MS ESI计算值C 10H 10N 2O 2[M+H] +191，实测值191。
步骤4:将化合物1-6(8.0g,42.1mmol)溶于乙醇(50mL)，依次加入水合肼(12.6g,252.4mmol,12.27mL)和醋酸(8.0mL,139.8mmol)，反应于100℃下搅拌2小时，反应完毕后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物1-7。MS ESI计算值C 10H 12N 4O[M+H] +205，实测值205。
步骤5:将化合物1-7(9.5g,46.5mmol)溶于THF(50mL)，在0℃下加入NaH(2.1g,51.2mmol,纯度60％)并搅拌0.5小时，然后将加入二碳酸二叔丁酯(11.2g,51.2mmol)，保持0℃下反应0.5小时后浓缩反应液，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物1-8。MS ESI计算值C 15H 20N4O 3[M+H] +305，实测值305。
步骤6:将化合物1-3(130.0mg，0.4mmol)和化合物1-8(117.4mg，0.4mmol)溶于1,4-二氧六环(10mL)，依次加入碳酸铯(188.6mg，0.6mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(35.0mg，0.4mmol)，反应于95℃搅拌3小时后过滤、滤液浓缩后得化合物1-9。MS ESI计算值C 31H 40N 8O 5[M+H] +605，实测值605。
步骤7:将化合物1-9(0.1g，0.16mmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(3mL)，反应于25℃搅拌0.5小时，反应完毕后浓缩，粗品经制备高效液相色谱(色谱柱:Boston Prime C18 150*30mm 5μm；流动相:[水(0.05％氨水)-乙腈]；乙腈％:36％-46％,9min.)分离得化合物1。 1H NMR(400MHz,CD 3OD)δ8.21(s,1H),7.97(d,J＝7.53Hz,1H),7.49(d,J＝3.76Hz,1H),7.03-7.05(brs,1H),6.95(d,J＝7.53Hz,1H),6.52(d,J＝4.02Hz,1H),4.69-4.81(m,1H),4.11(s,3H),3.26(br d,J＝14.05Hz,2H),2.86(br t,J＝11.92Hz,2H),2.50(s,3H),1.95-2.18(m,4H)。MS ESI计算值C 21H 24N 8O[M+H] +405，实测值405。
实施例2
Figure PCTCN2020132306-appb-000040
Figure PCTCN2020132306-appb-000041
步骤1:将化合物1-1(0.2g,1.3mmol)溶于乙腈(5mL)，依次加入1-氯甲基-4-氟-1,4-二氮杂双环[2.2.2]辛烷双(四氟硼酸盐)(551.7mg,1.6mmol)和吡啶(754.4mg,9.5mmol)，反应于25℃搅拌12小时后浓缩反应液，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物2-1。MS ESI计算值C 6H 3ClFN 3[M+H] +172，实测值172。
步骤2:将化合物2-1(100.0mg,582.9μmol)溶于DMF(5mL)，25℃下加入NaH(34.9mg,874.3μmol,纯度60％)并搅拌1小时，随后加入1-2(162.8mg,582.9μmol)，反应于80℃搅拌2小时，加入水(10mL)和乙酸乙酯(50mL)萃取后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物2-2。MS ESI计算值C 16H 20ClFN 4O 2[M+H] +355，实测值355。
步骤3:将化合物2-2(60.0mg,169.1μmol)和1-8(40.0mg,131.4μmol)溶于1,4-二氧六环(5mL)中，依次加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)和碳酸铯(85.6mg,262.8μmol)，反应在氮气保护下90℃搅拌2小时，反应完毕后冷却过滤，滤液浓缩后得化合物2-3粗品直接用于下一步反应。MS ESI计算值C 31H 39FN 8O 5[M+H] +623，实测值623。
步骤4:将化合物2-3(30.5mg,48.5μmol)溶于二氯甲烷(2mL)中，加入三氟乙酸(2mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Xtimate C18 150*25mm*5μm；流动相:[水(0.075％TFA)-乙腈]；乙腈％:24％-44％,7min.)分离得化合物2的三氟乙酸盐。 1H NMR(400MHz,CD 3OD)δ8.25(s,1H),8.00(d,J＝7.53Hz,1H),7.36-7.39(m,1H),6.96(d,J＝7.53Hz,1H),6.84(s,1H),5.14(br s,1H),4.94-5.03(m,2H),4.79-4.87(m,1H),4.10(s,3H),3.62(br d,J＝12.80Hz,2H),3.36-3.39(m,2H),2.50(s,3H),2.18-2.43(m,4H)。MS ESI计算值C 21H 23FN 8O[M+H] +423，实测值423。
实施例3
Figure PCTCN2020132306-appb-000042
步骤1:将化合物3-1(0.5g,2.3mmol)和三乙胺(352.5mg,3.5mmol)溶于二氯甲烷(30mL)中，甲烷磺酰氯(399.1mg,3.5mmol)加入到反应液中并在25℃下反应0.5小时，反应完毕后反应液浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝5:1)得化合物3-2。MS ESI计算值C 12H 23NO 5S[M+H] +294，实测值294。
步骤2:将化合物1-1(156.6mg,1.0mmol)溶于DMF(10mL)，加入NaH(61.20mg,1.53mmol,纯度60％)后于25℃搅拌1小时，随后加入3-2(0.3g,1.0mmol)，90℃继续反应2小时后加入水(10mL)，乙酸乙酯萃取后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物3-3。MS ESI计算值C 17H 23ClN 4O 2[M+H] +351，实测值351。
步骤3:将化合物3-3(115.3mg,328.6μmol)和1-8(100.0mg,328.6μmol)溶于1,4-二氧六环(5mL)，甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(29.8mg,32.9μmol)和碳酸铯(214.1mg,657.2μmol)分别加入到反应液中，在氮气保护下90℃反应0.5小时，反应完毕后反应液浓缩，得化合物3-4粗品直接用于下一步反应。MS ESI计算值C 32H 42N8O 5[M+H] +619，实测值619。
步骤4:将化合物3-4(150.2mg,242.8μmol)溶于二氯甲烷(2mL)中，加入三氟乙酸(2mL)，25℃搅拌0.5小时，反应完毕后浓缩，粗品经制备高效液相色谱(色谱柱:Xtimate C18 150*25mm*5μm；流动相:[水(0.075％TFA)-乙腈]；乙腈％:15％-45％,7min.)分离得化合物3的三氟乙酸盐。 1H NMR(400MHz,CD 3OD)δ8.25(s,1H),7.99(d,J＝7.78Hz,1H),7.56(d,J＝3.76Hz,1H),6.95(d,J＝7.78Hz,1H),6.81(s,1H),6.60(d,J＝3.76Hz,1H),4.71-4.96(m,1H),4.42(d,J＝7.03Hz,2H),4.11(s,3H),3.34-3.41(m,2H),2.93(dt,J＝2.76,12.92Hz,2H),2.48(s,3H),2.14-2.40(m,1H),1.96(s,1H),1.83(br d,J＝12.55Hz,2H),1.41-1.63(m,2H)。MS ESI计算值C 22H 26N 8O[M+H] +419，实测值419。 表1中所示化合物参考化合物3的方法用对应的醇作为原料而制备：
表1
Figure PCTCN2020132306-appb-000043
Figure PCTCN2020132306-appb-000044
实施例6
Figure PCTCN2020132306-appb-000045
步骤1:将化合物1-1(1.0g,6.5mmol)溶于DMF(20mL)中，0℃下加入KOH(1.5g,26.1mmol)和碘粒(3.3g,13.0mmol)，反应于0℃搅拌1小时后升温到25℃继续搅拌2小时。反应完毕后加入冰水(100mL)后过滤，滤饼干燥后得化合物6-1粗品，直接用于下一步反应。MS ESI计算值C 6H 3ClIN 3[M+H] +280，实测值280。
步骤2:将化合物6-1(1.6g,5.7mmol)溶于DMF(50mL)中，0℃下加入NaH(343.5mg,8.6mmol,纯度60％)，自然升温到25℃并搅拌半小时。然后加入化合物1-2(4.8g,17.2mmol)并加热至80℃搅拌12小时。反应完毕后浓缩，浓缩液用乙酸乙酯(80mL)稀释后分别用水(50mL)和盐水(50mL)洗涤，有机相干燥浓缩后经柱层析纯化(石油醚：乙酸乙酯＝2:1)得化合物6-2。MS ESI计算值C 16H 20ClIN 4O 2[M+H] +463，实测值463。
步骤3:将化合物6-2(0.9g，1.9mmol)溶于THF(10mL)中，氮气保护下于-78℃滴加n-BuLi(2.5M,394.20μL)，-78℃搅拌0.5小时加入氘水(32.9mg,1.6mmol)并搅拌半小时，反应完毕后加饱和食盐水(2mL)液淬灭反应，乙酸乙酯(50mL)萃取，有机相经干燥后浓缩得化合物6-3，直接用于下一步反应。MS ESI计算值C 16H 20DClN 4O 2[M+H] +338，实测值338。
步骤4:将化合物6-3(270.0mg,799.24μmol)和化合物1-8(342.0mg，1.9mmol)溶于1,4-二氧六环(10mL)，依次加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基 -2-基)钯(II)和碳酸铯(520.8mg,1.6mmol)，反应于100℃搅拌1.5小时。反应完毕后冷却过滤，滤液浓缩后得化合物6-4直接用于下一步反应。MS ESI计算值C 31H 39DN 8O 5[M+H] +606，实测值606。
步骤5:将化合物6-4(0.1g，0.2mmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(3mL)，25℃搅拌0.5小时，反应完毕后浓缩，粗品经制备高效液相色谱(色谱柱:Waters Xbridge 150*25 5μ；流动相:[水(10mM NH 4HCO 3)-乙腈]；乙腈％:0％-60％,10min.)分离得化合物6。 1H NMR(400MHz,CD 3OD)δ8.21(s,1H),7.96(d,J＝7.5Hz,1H),7.48(s,1H),7.09-6.92(m,2H),4.10(s,3H),3.28(br d,J＝13.1Hz,3H),2.96-2.84(m,3H),2.50(s,3H),2.19-2.02(m,4H)。MS ESI计算值C 21H 23DN 8O[M+H] +406，实测值406。
实施例7
Figure PCTCN2020132306-appb-000046
步骤1:在-78℃下往乙腈(445.8mg，10.8mmol)的THF(10mL)溶液中滴加n-BuLi(4.3mL，2.5M)，-78℃搅拌0.5小时，然后滴加化合物7-1(1.0g，5.4mmol)的THF(5mL)溶液，-78℃搅拌0.5小时。反应完毕后加10％柠檬酸水溶液(5mL)淬灭，乙酸乙酯(50mL)萃取，干燥浓缩得化合物7-2粗品，直接用于下一步。MS ESI计算值C 10H 8FNO 2[M+H] +194，实测值194。
步骤2:将水合肼(0.8mL，15.6mmol)滴加到化合物7-2(117.5mg，0.4mmol)的乙醇(10mL)和乙酸(1mL)溶液中，100℃搅拌2小时，反应完毕后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物7-3。MS ESI计算值C 10H 10FN 3O[M+H] +208，实测值208。
步骤3:在0℃下将化合物7-3(1.0g，4.8mmol)加入到NaH(231.6mg，5.8mmol，纯度60％)的THF(10mL)悬浊液中，0℃搅拌0.5小时，然后将二碳酸二叔丁酯(1.7g，5.8mmol)加入到反应中，25℃搅拌0.5小时。反应完毕后加水，乙酸乙酯萃取，浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物7-4。MS ESI计算值C 15H 18FN 3O 3[M+H] +308，实测值308。
步骤4:将化合物7-4(60.0mg，0.2mmol)，化合物1-3(74.4mg，0.2mmol)溶于1,4-二氧六环(10mL)中，依次加入碳酸铯(95.4mg，0.3mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(17.7mg，0.02mmol)，95℃搅拌3小时，反应完毕后反应液过滤浓缩，得化合物7-5粗品直接用于下一步。MS ESI计算值C 31H 38FN 7O 5[M+H] +608，实测值608。
步骤5:将化合物7-5(110.0mg，0.2mmol)溶于二氯甲烷(2mL)中，加入三氟乙酸(2mL)，25℃搅拌0.5小时，反应完毕后浓缩，粗品经制备高效液相色谱(色谱柱:Phenomenex Ge min.i-NX150*30mm*5μm；流动相:[水(10mM NH 4HCO 3)-乙腈]；乙腈％:27％-49.5％,6min.)分离得化合物7。 1H NMR(400MHz,CD 3OD)δ8.18(s,1H),7.47(d,J＝3.50Hz,1H),7.35-7.43(m,1H),7.29(br s,1H),7.03(d,J＝8.50Hz,1H),6.87-6.95(m,1H),6.52(d,J＝3.63Hz,1H),4.63(br s,1H),4.02(s,3H),3.27(br s,2H),2.90(br t,J＝11.88Hz,2H),1.98-2.20(m,4H)。MS ESI计算值C 21H 22FN 7O[M+H] +408，实测值408。表2中所示化合物参考化合物7的方法用对应的酯为原料而制备：
表2
Figure PCTCN2020132306-appb-000047
Figure PCTCN2020132306-appb-000048
Figure PCTCN2020132306-appb-000049
Figure PCTCN2020132306-appb-000050
实施例15
Figure PCTCN2020132306-appb-000051
步骤1:将2-氟乙腈(353.8mg,6.0mmol)和化合物15-1(1.0g,5.3mmol)溶于THF(20mL)中，将温度降至-78℃，氮气保护下滴加二(三甲基硅基)氨基锂(1M,11.0mL)并在该温度下搅拌0.5小时。反应 完毕后加入水(30mL)，用乙酸乙酯(50mL)萃取，有机相干燥浓缩后得化合物15-2粗品，直接用于下步反应。
步骤2:将化合物15-2(1.0g,4.7mmol)和水合肼(483.8mg,9.5mmol)溶于EtOH(10mL)和AcOH(0.1mL)中，反应于100℃搅拌2小时。反应完毕后浓缩后得化合物15-3粗品，直接用于下一步反应。MS ESI计算值C 10H 9F 2N 3O[M+H] +226，实测值226。
步骤3:将化合物15-3(200.0mg,888.1μmol)溶于THF(10mL)，0℃下加入NaH(42.6mg,1.07mmol,纯度60％)并在该温度下搅拌0.5小时后加入二碳酸二叔丁酯(232.60mg,1.07mmol)，反应于25℃搅拌0.5小时。反应完毕后加入水(30mL)，然后用乙酸乙酯(50mL)萃取，滤液浓缩后经经柱层析纯化(石油醚：乙酸乙酯＝2:1)得化合物15-4。MS ESI计算值C 15H 17F 2N 3O 3[M+H] +326，实测值326。
步骤4:将化合物15-4(135.2mg,415.7μmol)和化合物1-3(140.0mg,415.7μmol)溶于1,4-二氧六环(10mL)，依次加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(37.6mg,41.5μmol)和碳酸铯(270.8mg,831.3μmol)，反应于100℃搅拌1.5小时。反应完毕后冷却过滤，滤液浓缩得化合物15-5粗品，直接用于下一步反应。MS ESI计算值MS ESI计算值C 26H 29F 2N 7O 3[M+H] +526，实测值526。
步骤5:将化合物15-5(100.0mg,159.8μmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(3mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Venusil ASB Phenyl250*50mm 10μm；流动相:[水(0.05％HCl)-乙腈]；乙腈％:20％-50％,10min.)分离得化合物15的盐酸盐。 1H NMR(400MHz,CD 3OD)δ8.61(s,1H),8.08(d,J＝3.8Hz,1H),7.52(dt,J＝6.7,8.5Hz,1H),7.03(d,J＝8.3Hz,1H),6.92(t,J＝8.9Hz,1H),6.82(d,J＝4.0Hz,1H),5.15-5.03(m,1H),3.93(s,3H),3.71-3.62(m,2H),3.32-3.25(m,2H),2.52-2.35(m,4H)。MS ESI计算值C 21H 21F 2N 7O[M+H] +426，实测值426。
实施例16
Figure PCTCN2020132306-appb-000052
步骤1:将化合物2-2(110.0mg,310.0μmol)和化合物7-4(95.3mg,310.0μmol)溶于1,4-二氧六环(10mL)，依次加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(28.1mg,31.0μmol)和碳酸铯(202.0mg,620.0μmol)，反应于100℃搅拌1.5小时。反应完毕后冷却过滤，滤液浓缩后得化合物16-1。MS ESI计算值C 31H 37F 2N 7O 5[M+H] +626，实测值626。
步骤4:将化合物16-1(50.0mg,79.9μmol)溶于二氯甲烷(2mL)中，加入三氟乙酸(2mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Venusil ASB Phenyl 250*50mm10μm；流动相:[水(0.05％HCl)-乙腈]；乙腈％:25％-55％,10min.)分离得化合物16的盐酸盐。 1H NMR(400MHz,CD 3OD)δ8.28(s,1H),7.66-7.58(m,2H),7.16(d,J＝8.8Hz,1H),7.07-7.00(m,1H),6.90(s,1H),5.51(br s,1H),4.12(s,3H),3.62(br s,4H),2.40-2.20(m,4H)。MS ESI计算值C 21H 21F 2N 7O[M+H] +426，实测值426。
实施例17
Figure PCTCN2020132306-appb-000053
步骤1:将化合物17-1(0.8g,3.8mmol)和化合物17-2(1.1g,3.5mmol)加入到甲苯(2mL)中，然后依次加入CuI(87.7mg,460.5μmol)，四(三苯基膦)钯(310.4mg,268.6μmol)，反应于60℃在氮气保护下搅拌12小时。反应完毕后过滤，滤液浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物17-3。MS ESI计算值C 7H 6ClN 3[M+H] +168，实测值168。
步骤2:将化合物17-3(0.4g,2.2mmol)溶于THF(2ml)，0℃下缓慢滴加叔丁醇钾的THF溶液(1M,3.2mL)，滴加完毕后升温至25℃下继续搅拌12小时，反应完毕后加水(2mL)，乙酸乙酯萃取(10mL)，有机相浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝2:1)得化合物17-4。 1H NMR(400MHz,CD 3OD)δ8.14(s,1H),6.26(s,1H),2.42(s,3H)。MS ESI计算值C 7H 6ClN 3[M+H] +168，实测值168。
步骤3：将化合物17-4(0.2g,1.2mmol)溶于DMF(2mL)，加入NaH(95.5mg,2.4mmol,纯度60％)后于25℃搅拌0.5小时，然后加入1-2(1.3g,4.8mmol)后升温至90℃搅拌16小时。反应完毕后加水(5mL)和乙酸乙酯(30mL)萃取，有机相浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物得化合物17-5。MS ESI计算值C 17H 23ClN 4O 2[M+H] +351，实测值351。
步骤4:将化合物17-5(100.0mg,370.5mg)和化合物7-4(113.8mg,370.5μmol)加入到无水1,4-二氧六环(2mL)中，然后依次加入甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(33.6mg,37.1μmol)和碳酸铯(241.4mg,741.0μmol)，反应于100℃搅拌1小时。反应完毕后加水(5mL)和乙酸乙酯(30mL)萃取，有机相浓缩后得化合物17-6粗品，直接用于下一步反应。MS ESI计算值C 32H 40FN 7O 5[M+H] +622，实测值622。
步骤5:将化合物17-6(0.1g,160.9μmol)溶于二氯甲烷(2ml)中，加入三氟乙酸(2mL)，25℃搅拌5分钟，反应完毕后浓缩，粗品经制备高效液相色谱(色谱柱:Venusil ASB Phenyl 250*50mm 10μm；流动相:[水(0.05％HCl)-乙腈]；乙腈％:20％-50％,10min.)分离得化合物17的盐酸盐。 1H NMR(400MHz,CD 3OD)δppm 1.11-1.23(m,2H)2.12(br d,J＝15.51Hz,2H)2.60(s,3H)2.91-3.01(m,2H)3.23-3.30(m,2H)3.55(br d,J＝12.26Hz,2H)3.93(s,3H)6.45(s,1H)6.84(dd,J＝10.94,8.44Hz,1H)6.95(d,J＝8.38Hz,1H)7.30-7.40(m,1H)8.59(s,1H)。MS ESI计算值C 22H 24FN 7O[M+H]+422，实测值422。
实施例18
Figure PCTCN2020132306-appb-000054
步骤1:将化合物1-3(150mg，0.445mmol)溶于DCM(20mL)中，加入HCl/MeOH(4M,20mL)，反应于25℃搅拌0.5小时后浓缩得化合物18-1粗品，直接用于下一步。MS ESI计算值C 11H 13ClN 4[M+H] +237，实测值237。
步骤2:将化合物18-1(100mg，0.422mmol)溶于DCM(10mL)和AcOH(1mL)中，加入4A分子筛(500mg)和甲醛水合物(0.314mL，4.22mmol)，反应于25℃搅拌0.5小时后加入醋酸硼氢化钠(179.08mg,0.845mmol)后继续搅拌2小时。反应完毕后过滤浓缩，粗品经制备薄层色谱纯化(石油醚：乙酸乙酯＝1:1)得化合物18-2。MS ESI计算值C 12H 15ClN 4[M+H] +251，实测值251。
步骤3:将化合物18-2(85.0mg，0.339mmol)和化合物1-8(108.2mg，0.339mmol)溶于二氧六环(10mL)，依次加入碳酸铯(165.69mg，0.508mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(30.73mg，0.034mmol)，95℃搅拌1小时，反应完毕后反应液过滤浓缩得化合物18-3粗品，直接用于下一步反应。MS ESI计算值C 27H 34N 8O 3[M+H] +519，实测值519。
步骤4:将化合物18-3(80.0mg，0.154mmol)溶于DCM(5mL)中，加入三氟乙酸(2.0mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Boston Green ODS150*30mm*5μm；流动相:[水(0.075％TFA)-乙腈]；乙腈％:18％-48％,8min.)分离得化合物18的三氟乙酸盐。 1H NMR(400MHz,CD 3OD)δ8.32(s,1H),8.03(d,J＝7.50Hz,1H),7.57(d,J＝3.63Hz,1H),6.99(d,J＝7.63Hz,1H),6.80(s,1H),6.64(d,J＝3.63Hz,1H),5.03-5.17(m,1H),4.11(s,3H),3.75(br d,J＝12.38Hz,2H),3.35-3.44(m,2H),3.00(s,3H),2.52(s,3H),2.34-2.47(m,4H).MS ESI计算值C 22H 26N 8O[M+H] +419，实测值419。
实施例19
Figure PCTCN2020132306-appb-000055
步骤1:将化合物2(4.8g,11.3mmol)溶于甲醇(100mL)和乙酸(1mL)中，加入甲醛水合物(1.0mL,13.5mmol，浓度37％)和氰基硼氢化钠(850.0mg,13.5mmol)，反应于25℃搅拌2小时。反应完毕后，用饱和碳酸氢钠水(20mL)溶液中和至pH＝7，过滤，滤饼用水(20mL)洗涤，乙腈(10mL)打浆后得化合物19。 1H NMR(400MHz,CD 3OD)δ8.27(s,1H),7.98(d,J＝7.63Hz,1H),7.32(d,J＝1.38Hz,1H),6.96 (d,J＝7.50Hz,1H),6.91(s,1H),4.86-4.80(brs,1H),4.10(s,3H),3.60-3.50(m,2H),3.01-3.21(m,2H),2.84(s,3H),2.51(s,3H),2.33(brs,4H)。MS ESI计算值C 22H 25FN 8O[M+H] +437，实测值437。
实施例20
Figure PCTCN2020132306-appb-000056
步骤1:将化合物1-4(10.0g,65.3mmol)，氘代碘甲烷(21.7g,150.2mmol)溶于氯仿(150mL)中，加入碳酸银(19.8g,71.8mmol)后反应于60℃搅拌16小时，反应完毕后冷却、过滤，滤液浓缩后粗品经柱层析纯化(石油醚：乙酸乙酯＝3:1)得化合物20-1。MS ESI计算值C 9H 5D 6NO 3[M+H] +188，实测值188。
步骤2:将乙腈(3.0mL,57.6mmol)加入到四氢呋喃(100mL)中，在-78℃下缓慢滴加正丁基锂(2.5M,24.6mL,61.5mmol)到反应液中，然后保持该温度继续反应30分钟后缓慢滴加化合物20-1(7.2g,38.46mmol)到反应液中，继续反应30分钟，然后升到室温，反应液倒入水(100mL)中，乙酸乙酯萃取(200mL)后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝2:1)得化合物20-2。MS ESI计算值C 10H 7D 3N 2O 2[M+H] +194，实测值194。
步骤3:将化合物20-2(7.4g,38.4mmol)溶于乙醇(100mL)，依次加入水合肼(4.5g,76.9mmol)和乙酸(6.6mL,115.3mmol)，反应于80℃下搅拌2小时，反应完毕后浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物20-3。MS ESI计算值C 10H 9D 3N 4O[M+H] +208，实测值208。
步骤4:将化合物20-3(7.0g,33.7mmol)溶于四氢呋喃(50mL)，在0℃下加入氢化钠(1.3g,33.7mmol,纯度60％)并搅拌0.5小时，然后将加入二碳酸二叔丁酯(7.3g,33.7mmol)，保持0℃下反应0.5小时后浓缩反应液，粗品经柱层析纯化(石油醚：乙酸乙酯＝1:1)得化合物20-4。MS ESI计算值C 15H 17D 3N 4O 3[M+H] +308，实测值308。
步骤5:将化合物20-4(4.3g，14.1mmol)和化合物2-2(5.0g，14.1mmol)溶于1,4-二氧六环(150mL)， 依次加入碳酸铯(9.218g，28.2mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(1.3g，1.4mmol)，反应于95℃搅拌3小时后过滤、滤液浓缩，粗品经柱层析纯化(石油醚：乙酸乙酯＝2:1)后得化合物20-5。MS ESI计算值C 31H 36D 3FN 8O 5[M+H] +626，实测值626。
步骤6:将化合物20-5(8.3g，13.2mmol)溶于二氯甲烷(100mL)中，加入三氟乙酸(10mL)，反应于25℃搅拌0.5小时，反应完毕后浓缩，用饱和碳酸氢钠水溶液中和至pH＝7，过滤，水洗涤，滤饼经乙腈打浆后得化合物20-6。MS ESI计算值C 21H 20D 3FN 8O[M+H] +426，实测值426。
步骤7:将化合物20-6(5.5g，12.9mmol)溶于MeOH(50mL)和AcOH(1mL)中，加入甲醛水合物(1.2mL，15.5mmol，浓度37％)和氰基硼氢化钠(975.0mg,15.5mmol)后25℃搅拌2小时。反应完毕后，用饱和碳酸氢钠水溶液中和至pH＝7，过滤，水(50mL)洗涤，滤饼经乙腈(10mL)打浆后得化合物20。 1H NMR(400MHz,CD 3OD)δ8.21(s,1H),7.96(d,J＝7.20Hz,1H),7.31(s,1H),7.12(brs,1H),6.93(d,J＝7.53Hz,1H),4.62(brs,1H),3.08(d,J＝11.80Hz,2H),2.50(s,3H),2.39(s,3H),2.33-2.27(m,2H),2.25-2.11(m,4H)。MS ESI计算值C 22H 22D 3FN 8O[M+H] +440，实测值440。
实施例21
Figure PCTCN2020132306-appb-000057
步骤1:将化合物18-1(1.0g，3.6mmol)溶于DMF(30mL)中，加入氘代碘甲烷(530.0mg，3.6mmol)和碳酸钾(1.5g，10.9mmol)，反应于25℃搅拌16小时。反应完毕后过滤后浓缩，粗品经柱层析纯化(二氯甲烷：甲醇＝10:1)得化合物21-1。MS ESI计算值C 12H 12D 3ClN 4[M+H]+254，实测值254。
步骤2:将化合物1-8(100.0mg，0.3mmol)和化合物21-1(100.0mg，0.4mmol)溶于二氧六环(10mL)，依次加入碳酸铯(214.1mg，0.7mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’- 联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(30.0mg，32.0μmol)，95℃搅拌1小时后反应液过滤浓缩得化合物21-2粗品，直接用于下一步反应。MS ESI计算值C 27H 31D 3N 8O 3[M+H] +522，实测值522。
步骤3:将化合物21-2(171.0mg，0.3mmol)溶于DCM(10mL)中，加入三氟乙酸(3.0mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Phenomenex Gemini-NX80*40mm*3μm；流动相:[水(0.05％氨水+10mM碳酸氢铵)-乙腈]；乙腈％:27％-57％,8min.)分离得化合物21。 1H NMR(400MHz,CD 3OD)δ8.23(brs,1H),7.98(d,J＝6.78Hz,1H),7.47(s,1H),7.12(brs,1H),6.94(d,J＝7.52Hz,1H),6.51(d,J＝3.02Hz,1H),4.70-4.58(m,1H),4.11(s,3H),3.10(d,J＝10.50Hz,2H),2.50(s,3H),2.37-2.10(m,6H).MS ESI计算值C 22H 23D 3N 8O[M+H] +422，实测值422。
实施例22
Figure PCTCN2020132306-appb-000058
步骤1:将化合物21-1(100.0mg，0.3mmol)和化合物20-4(99.0mg，0.4mmol)溶于二氧六环(10mL)，依次加入碳酸铯(214.1mg，0.6mmol)和甲磺酸(2-二环己基膦基-3,6-二甲氧基-2’,4’,6’-三异丙基-1,1’-联苯)(2’-氨基-1,1’-联苯基-2-基)钯(II)(30.0mg，32.0μmol)，95℃搅拌1小时后反应液过滤浓缩得化合物22-1粗品，直接用于下一步反应。MS ESI计算值C 27H 28D 6N 8O 3[M+H] +525，实测值525。
步骤2:将化合物22-1(170.0mg，0.3mmol)溶于DCM(10mL)中，加入三氟乙酸(3.0mL)，25℃搅拌0.5小时，反应完毕后反应液浓缩，粗品经制备高效液相色谱(色谱柱:Phenomenex Gemini-NX80*40mm*3μm；流动相:[水(0.05％氨水+10mM碳酸氢钠)-乙腈]；乙腈％:27％-57％,8min.)分离得化合物22。 1H NMR(400MHz,CD 3OD)δ8.27-8.17(m,1H),8.03-7.94(m,1H),7.50-7.45(m,1H),7.15-7.08(m,1H),6.94(d,J＝7.52Hz,1H),6.51(brd,J＝3.80Hz,1H),4.71-4.58(m,1H),3.11(brd,J＝11.04Hz,2H),2.50(s,3H),2.38-2.11(m,6H).MS ESI计算值C 22H 20D 6N 8O[M+H] +425，实测值425。
实验1：CHK1耦合反应系统中的化合物的活性测试
供实验用的本发明化合物为自制，其化学名称和结构式见各化合物的制备实施例。将含有本发明实施例化合物和Chk1和Chk2激酶的测定混合物在微量滴定板中混合在一起孵化，通过监测Chk1和Chk2激酶对具有特定氨基酸序列的合成肽底物(KKKVSRSGLYRSPSMPENLNRPR)的磷酸化来测 试化合物的CHK1抑制剂活性。实验测试在Eurofins公司KinaseProfiler TM蛋白激酶活性检测平台上进行，实验结果由该公司提供。过程如下：Chk1和Chk2激酶用20mM MOPS(吗啉丙烷磺酸)、1mM EDTA(乙二胺四乙酸)、0.04％Brij-35、5％丙三醇、0.1％2-巯基乙醇、1mg/mL BSA(牛血清蛋白)稀释后加入到反应体系中，反应体系包含实施例化合物、8mM MOPS pH 7.0、0.2mM EDTA、200μM多肽底物(KKKVSRSGLYRSPSMPENLNRPR)，10mM醋酸镁和一定浓度的[γ-33P]-ATP(强度大约500cpm/pmol)。加入Mg 2+和ATP(三磷酸腺苷)混合液后引发反应，室温孵育40min。加入0.5％磷酸缓冲液，终止反应。取10μL反应液在连续过滤机P30上过滤四次，用0.425磷酸缓冲液清洗三次，甲醇清洗一次，每次清洗4min。干燥后闪烁计数法读值。
化合物抑制活性结果见表3。
表3.本发明化合物Chk1/Chk2酶抑制活性结果
化合物 Chk1抑制IC 50(nM) Chk2抑制IC 50(nM)
1 <1 79
2的三氟乙酸盐 2 28
3的三氟乙酸盐 5 1966
4的三氟乙酸盐 3 333
5的三氟乙酸盐 3 375
6 <1 64
7 1 13
8的三氟乙酸盐 1 188
9的三氟乙酸盐 2 1011
10的盐酸盐 2 62
11的盐酸盐 3 99
12的三氟乙酸盐 1 32
13的三氟乙酸盐 2 84
14的盐酸盐 1 73
15的盐酸盐 2 24
16的盐酸盐 <1 2
17的盐酸盐 1 43
18的三氟乙酸盐 <1 257
19 2 166
20 2 282
21 1 362
22 1 386
实验结论：
本发明化合物展示了良好的Chk1抑制活性，具有用于治疗与Chk1相关的疾病(如细胞增殖相关疾病)的潜在应用价值。
实验例2：化合物药代动力学评价
实验目的：测试化合物在小鼠体内药代动力学
实验材料：
C57BL/6小鼠(雌性，7～9周龄，上海斯莱克)
实验操作：将试验化合物溶解后得到的澄清溶液分别经尾静脉注射和灌胃给予雌性C57BL/6小鼠体内(过夜禁食，7～9周龄)。给予受试化合物或对照化合物后，静脉注射组给药剂量为1mg/kg，在0.0833、0.25、0.5、1、2、4、6、8和24小时，灌胃组给药剂量为10mg/kg，在0.0833、0.25、0.5、1、2、4、6、8和24小时，分别从下颌静脉采血并离心后获得血浆。采用LC-MS/MS法测定血药浓度，使用WinNonlin TM Version 6.3药动学软件，以非房室模型线性对数梯形法计算相关药代动力学参数。测试结果如表4所示：
表4 化合物在小鼠中的PK测试结果
PK参数 化合物18 化合物19 化合物20
静脉注射T 1/2(h) 0.9 1.1 1.4
Vd(L/kg) 1.9 2.4 2.7
Cl(ml/min/kg) 35.3 33.1 30.9
静脉注射AUC 0-last(nM.hr) 1138 1148 1212
C max(nM) 1223 1215 1255
口服AUC 0-last(nM.hr) 3629 3912 5135
F(％) 33.2 39.1 41.9
注：T 1/2：半衰期；C max：达峰浓度；Vd(L/kg)：表观分布容积；Cl(ml/min/kg)：清除率；
AUC 0-last：从0时间到24h时的血浆浓度-时间曲线下面积；
F：生物利用度。
结论：本发明的化合物具良好的口服生物利用度，较高的暴露量，有利于产生良好的体内药效。
实验例3人卵巢癌OVCAR-3皮下异种移植肿瘤BALB/c nude裸小鼠模型的体内药效学研究
实验目的：对本发明化合物在人卵巢癌OVCAR-3皮下异种移植肿瘤BALB/c nude裸小鼠模型内的体内药效进行评估。
实验动物：雌性BALB/c Nude裸小鼠，6－8周龄，体重18-22克；供应商：北京维通利华实验动物技术有限公司实验方法与步骤：
3.1细胞培养
人卵巢癌OVCAR-3(ATCC-HTB-161)细胞体外单层培养，培养条件为RPMI1640培养基中加20％胎牛血清、0.01mg/mL牛胰岛素、加1％双抗，置37℃，5％CO 2孵箱培养。一周两次用胰酶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，接种。
3.2肿瘤细胞接种(肿瘤接种)
将0.1mL(10×10 6个)OVCAR-3细胞皮下接种于每只小鼠的右后背，肿瘤平均体积达到122mm 3时开始分组给药。
3.3受试物的配制
受试化合物18，化合物19配制成2mg/mL的澄清溶液，溶媒为10％二甲基亚砜-20％聚乙二醇400-5％吐温80-65％水。
3.4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×b 2，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(％)评价。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％。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＝V t/V 0，其中V 0是分组给药时(即D0)测量所得平均肿瘤体积，V t为某一次测量时的平均肿瘤体积。
3.5统计分析
统计分析基于试验结束时RTV的数据运用SPSS软件进行分析。两组间比较用T  test进行分析，三组或多组间比较用one-way ANOVA进行分析，如果方差齐(F值无显著性差异)，应用Tukey’s法进行分析，如果方差不齐(F值有显著性差异)，应用Games-Howell法进行检验。p<0.05认为有显著性差异。
3.6试验结果
给予人卵巢癌OVCAR-3皮下异种移植肿瘤BALB/c nude裸小鼠实施例18，实施例19治疗后各组肿瘤体积变化如表5所示。
表5 实施例化合物对OVCAR-3异种移植瘤模型的抑瘤效果
Figure PCTCN2020132306-appb-000059
注：a.平均值±SEM，n＝6。D0：分组给药当天，D21：给药第21天；
b.给药组与溶媒对照组相对肿瘤体积(RTV)比较的p值，运用one-way ANOVA进行分析肿瘤体积所得做，因方差不齐(F值有显著性差异)，用Games-Howell法进行post-hoc检验。p<0.05认为有显著性差异。
c.口服，75mg/kg，一天两次，第0～10天内，给3天停4天，第11～21天连续给药。
d.口服，25mg/kg，一天两次，第0～10天内，给3天停4天，第11～21天连续给药。
“--”不需计算。
3.7试验结论和讨论
在OVCAR-3人卵巢癌异种移植瘤模型中，开始给药后21天，溶媒对照组荷瘤鼠的瘤体积平均值达到885mm 3，化合物18与溶媒对照组相比TGI为92.3％(p＝0.024)，瘤体积平均值为180mm 3。化合物19与溶媒对照组相比TGI为98.8％(p＝0.018)，瘤体积平均值为131mm 3。上述结果表明：本发明实施例化合物在人OVCAR-3人卵巢癌异种移植瘤模型中具有显著的抑瘤药效。

Claims (15)

  1. 式(I)所示化合物或其药学上可接受的盐，
    Figure PCTCN2020132306-appb-100001
    其中，
    T 1选自CH和N；
    L选自单键和-CH 2-；
    m选自0、1、2、3和4；
    n和q分别独立地选自1和2；
    R 1选自H、F、Cl、Br和I；
    R 2选自H、D、F、Cl、Br和I；
    R 3选自H、F、Cl、Br、I、CN和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R a取代；
    各R 4分别独立地选自H、F、Cl、Br、I、C 1-3烷基和C 1-3烷氧基，所述C 1-3烷基和C 1-3烷氧基任选被1、2或3个R b取代；
    R 5选自H和C 1-3烷基，所述C 1-3烷基任选被1、2或3个R c取代；
    R a、R b和R c分别独立地选自H、D、F、Cl、Br和I。
  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R 1选自H和F。
  3. 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选自H和CH 3。
  4. 根据权利要求1所述化合物或其药学上可接受的盐，其中各R 4分别独立地选自H、F、Cl、CH 3和OCH 3，所述CH 3和OCH 3任选被1、2或3个R b取代。
  5. 根据权利要求1或4所述化合物或其药学上可接受的盐，其中各R 4分别独立地选自H、F、Cl、CH 3、CF 3、OCH 3、OCD 3和OCF 3。
  6. 根据权利要求1所述化合物或其药学上可接受的盐，其中结构片段
    Figure PCTCN2020132306-appb-100002
    选自
    Figure PCTCN2020132306-appb-100003
  7. 根据权利要求6所述化合物或其药学上可接受的盐，其中结构片段
    Figure PCTCN2020132306-appb-100004
    选自
    Figure PCTCN2020132306-appb-100005
  8. 根据权利要求7所述化合物或其药学上可接受的盐，其中结构片段
    Figure PCTCN2020132306-appb-100006
    选自
    Figure PCTCN2020132306-appb-100007
  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R 5选自H、CH 3和CD 3。
  10. 根据权利要求1或9所述化合物或其药学上可接受的盐，其中结构片段
    Figure PCTCN2020132306-appb-100008
    选自
    Figure PCTCN2020132306-appb-100009
  11. 根据权利要求10所述化合物或其药学上可接受的盐，其中结构片段
    Figure PCTCN2020132306-appb-100010
    选自
    Figure PCTCN2020132306-appb-100011
  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，其选自，
    Figure PCTCN2020132306-appb-100012
    Figure PCTCN2020132306-appb-100013
    其中，T 1、R 1、R 2、R 3、R 4、R 5、L和m如权利要求1～11任意一项所定义。
  13. 下式化合物或其药学上可接受的盐，
    Figure PCTCN2020132306-appb-100014
    Figure PCTCN2020132306-appb-100015
  14. 根据权利要求1～13任意一项所述的化合物或其药学上可接受的盐在制备与Chk1相关疾病的药物中的应用。
  15. 根据权利要求14所述的应用，其特征在于，所述药物是用于治疗胰腺癌等实体瘤的药物。
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