WO2021068953A1 - 作为prmt5抑制剂的取代三环类化合物及其应用 - Google Patents

作为prmt5抑制剂的取代三环类化合物及其应用 Download PDF

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WO2021068953A1
WO2021068953A1 PCT/CN2020/120284 CN2020120284W WO2021068953A1 WO 2021068953 A1 WO2021068953 A1 WO 2021068953A1 CN 2020120284 W CN2020120284 W CN 2020120284W WO 2021068953 A1 WO2021068953 A1 WO 2021068953A1
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alkyl
compound
amino
pharmaceutically acceptable
acyl
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PCT/CN2020/120284
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English (en)
French (fr)
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王勇
赵立文
全旭
郑国闯
孙韡
杨婷婷
詹康宁
施旗旗
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南京圣和药业股份有限公司
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Priority to KR1020227015656A priority Critical patent/KR20220080160A/ko
Priority to EP20873891.4A priority patent/EP4043459A4/en
Priority to AU2020363144A priority patent/AU2020363144B2/en
Priority to CN202080070137.8A priority patent/CN114466846A/zh
Priority to JP2022522055A priority patent/JP7301222B2/ja
Priority to US17/768,204 priority patent/US20240116918A1/en
Priority to CA3157682A priority patent/CA3157682A1/en
Publication of WO2021068953A1 publication Critical patent/WO2021068953A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems

Definitions

  • the present invention belongs to the field of medicinal chemistry, and specifically relates to substituted tricyclic compounds or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof as PRMT5 inhibitors, their preparation methods and drugs containing these compounds
  • DNA modification plays a central role in triggering gene expression programs at different stages of cell growth and development.
  • Arginine methylation plays an important role in cell processes, including signal transduction, transcription, RNA processing, DNA recombination and repair .
  • Protein arginine methyltransferases catalyze the methylation of specific arginine residues by transferring methyl groups from S-adenosylmethionine (SAM) to the guanidine nitrogen of arginine.
  • SAM S-adenosylmethionine
  • the different ways of arginine methylation can divide PRMTs into three categories: Type I (PRMT 1, 2, 3, 4, 6 and 8) catalyzes monomethylation and asymmetric dimethylation, and type II (PRMT5). And PRMT9) catalyze monomethylation and symmetric dimethylation, while type III (PRMT7) only performs monomethylation.
  • PRMT5 specifically binds to methyltransferase complex protein 50 (MEP50), which can symmetrically methylate histones H3 and H4, and regulate the transcription of specific target genomes.
  • MEP50 methyltransferase complex protein 50
  • the symmetric dimethylation of histone H3 arginine 8 (R8) and H4R3 catalyzed by PRMT5 has been shown to inhibit the expression of several tumor suppressor genes, such as tumor suppressor gene 7 (ST7) and retinoblastoma (RB) tumor suppressor genes Family and receptor type O protein tyrosine phosphatase (PTPROt).
  • T7 tumor suppressor gene 7
  • RB retinoblastoma
  • PTPROt Family and receptor type O protein tyrosine phosphatase
  • PRMT5 can also methylate several important transcription factors, making it play an important role in the process of cell regulation.
  • PRMT5 can methylate p53 and change its DNA binding activity, thereby triggering changes in the gene expression program controlled by p53.
  • PRMT5 has also been shown to methylate N-MYC and change its protein stability and enhance its oncogenic activity in neuroblastoma.
  • PRMT5 can also directly methylate transcription factors, including E2F-1 and NF- ⁇ B/p65, and induce the expression of its target genes.
  • PRMT5 can not only modify nuclear transcription factors, but also methylate cytoplasmic proteins such as golgin and ribosomal protein S10 (RPS10). Therefore, in addition to its ability to directly regulate its own target genes, PRMT5 can also indirectly affect global gene expression through symmetric methylation of key transcription factors, thereby affecting cell growth, proliferation and differentiation.
  • PRMT5 is overexpressed in different types and aggressive cancers, including B-cell and T-cell lymphoma, metastatic melanoma, neuroblastoma and glioblastoma, germ cell tumors, ovarian cancer, Nasopharyngeal cancer, breast cancer, colorectal cancer and gastric cancer.
  • Current research shows that PRMT5 plays an important role in controlling cell growth and proliferation, and its overexpression promotes cell transformation.
  • PRMT5 The enhanced expression of PRMT5 in cancer cells is related to the transcriptional silencing of its target tumor suppressor genes.
  • PRMT5 can promote the growth of cancer cells through the methylation of the promoter histones H3R8 and H4R3 and the modification of specific arginine residues of key transcription factors including E2F1 and NF-kB/p65 to cause global chromatin changes.
  • PRMT5 also interacts with programmed cell death 4 (PDCD4), causing it to become methylated at R110 and lose its tumor suppressor activity in MCF-7 cells.
  • PDCD4 programmed cell death 4
  • the overexpression of PRMT5 may make it interact with growth promoting proteins and tumor suppressor proteins to facilitate the growth, survival and metastasis of cancer cells.
  • PRMT5 inhibitors have a clear mechanism in the treatment of tumors and other related diseases, and have great potential to become a new treatment method in the field of tumor treatment. Therefore, it is necessary to develop safer and more effective PRMT5 inhibitors to meet clinical needs. demand.
  • An object of the present invention is to provide a class of compounds with PRMT5 inhibitory activity represented by general formula (A) or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof,
  • Cy is selected from a heterocyclic group
  • the heterocyclic group may be one or more selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, Carboxy, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkyl acyl, hydroxyl Group substitution of alkyl acyl, cycloalkyl acyl, heterocyclyl acyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and oxo groups.
  • Another object of the present invention is to provide a method for preparing the compound of general formula (A) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug.
  • Another object of the present invention is to provide a composition comprising the compound of the general formula (A) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug and a pharmaceutically acceptable carrier, and a composition comprising The compound of the general formula (A) of the present invention or its isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs and a combination of one or more drugs.
  • Another object of the present invention is to provide a method for the treatment of PRMT5-mediated diseases by the compound of general formula (A) or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof of the present invention, and the present invention Application of the compound of general formula (A) or its isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs in the preparation of drugs for the treatment of PRMT5-mediated diseases.
  • the present invention provides a compound represented by general formula (A) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof,
  • Cy is selected from a heterocyclic group
  • the heterocyclic group may be one or more selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, Carboxy, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, alkylsulfonyl, aminoacyl, alkylaminoacyl, dialkylamino, alkenyl, alkynyl, haloalkyl acyl, hydroxyl Group substitution of alkyl acyl, cycloalkyl acyl, heterocyclyl acyl, cycloalkyl, heterocyclyl, aryl, heteroaryl and oxo groups.
  • the compound of the present invention is a compound of general formula (A) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein:
  • Cy is selected from a 3-12 membered heterocyclic group, and the 3-12 membered heterocyclic group may be selected by one or more selected from halogen, hydroxy, C 1-6 alkyl, halogenated C 1-6 alkyl, hydroxy C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy, hydroxy C 1-6 alkoxy, nitro, carboxy, cyano, amino, mono C 1-6 alkyl Amino, C 1-6 alkyl acylamino, C 1-6 alkyl acyl, C 1-6 alkyl sulfonyl, amino acyl, C 1-6 alkyl amino acyl, bis C 1-6 alkyl amino, C 2-10 alkenyl, C 2-10 alkynyl, halogenated C 1-6 alkyl acyl, hydroxy C 1-6 alkyl acyl, C 3-12 cycloalkyl acyl, 3-12 membered heterocyclyl acyl, C 3-12
  • Cy is selected from 3-10 membered heterocyclic groups, and the 3-10 membered heterocyclic groups may be substituted by one or more selected from halogen, hydroxy, C 1-3 alkyl, and halogenated C 1-3 alkane.
  • Cy is selected from a 3-10 membered heterocyclic group, wherein the heterocyclic group further contains one or more N, O or S heteroatoms, and the 3-10 membered heterocyclic group may be substituted by one or A plurality of selected from halogen, hydroxy, C 1-3 alkyl, halogenated C 1-3 alkyl, hydroxy C 1-3 alkyl, C 1-3 alkoxy, halogenated C 1-3 alkoxy, Hydroxy C 1-3 alkoxy, nitro, carboxy, cyano, amino, mono C 1-3 alkylamino, C 1-3 alkyl acylamino, C 1-3 alkyl acyl, C 1-3 alkane Sulfonyl, aminoacyl, C 1-3 alkylamino acyl, bis C 1-3 alkylamino, C 2-6 alkenyl, C 2-6 alkynyl, halogenated C 1-3 alkyl acyl, hydroxyl C 1-3 alkyl acyl group, C
  • the present invention provides a compound represented by the general formula (A) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein the general formula (A) has the following general formula The structure of formula (I),
  • X is selected from O, S, C(R 1 )(R 2 ) and N(R 3 ), wherein R 1 , R 2 , and R 3 are each independently selected from hydrogen, halogen, hydroxy, alkyl, haloalkyl, and hydroxy Alkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxy, cyano, amino, monoalkylamino, alkylacylamino, alkylacyl, aminoacyl, alkylaminoacyl, dialkyl Group amino group and cycloalkyl group, and R 1 and R 2 together with the carbon atoms to which they are bound form a heterocyclic group, wherein the heterocyclic group is optionally selected from halogen, hydroxy, alkyl, haloalkane Group, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxy, cyano, amino, monoalkylamino, alkylacylamin
  • n 1, 2, 3 or 4;
  • n 0, 1, 2, 3, or 4.
  • the compound of the present invention is a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein:
  • R 1 , R 2 , and R 3 are each independently selected from hydrogen, halogen, hydroxy, C 1-6 alkyl, halogenated C 1-6 alkyl, hydroxy C 1-6 alkyl, C 1-6 alkoxy , Halogenated C 1-6 alkoxy, hydroxy C 1-6 alkoxy, nitro, carboxy, cyano, amino, mono C 1-6 alkylamino, C 1-6 alkyl acylamino, C 1 -6 alkyl acyl, amino acyl, C 1-6 alkylamino acyl, bis C 1-6 alkylamino and C 3-12 cycloalkyl;
  • R 1 , R 2 , and R 3 are each independently selected from hydrogen, halogen, hydroxy, C 1-3 alkyl, halogenated C 1-3 alkyl, hydroxy C 1-3 alkyl, C 1- alkoxy, a C 1-3 alkoxy group, halo, a C 1-3 alkoxy group, hydroxy, nitro, carboxy, cyano, amino, mono C 1-3 alkyl group, a C 1-3 alkyl group Amino, C 1-3 alkyl acyl, amino acyl, C 1-3 alkylamino acyl, bis C 1-3 alkylamino and C 3-8 cycloalkyl;
  • R 1 , R 2 , and R 3 are each independently selected from hydrogen, halogen, hydroxy, methyl, ethyl, propyl, isopropyl, halogenated C 1-3 alkyl, and hydroxy C 1- 3 alkyl, C 1-3 alkoxy, halogenated C 1-3 alkoxy, hydroxy C 1-3 alkoxy, nitro, carboxy, cyano, amino, mono C 1-3 alkylamino, C 1-3 alkyl acylamino, C 1-3 alkyl acyl, amino acyl, C 1-3 alkylamino acyl, bis C 1-3 alkylamino and C 3-8 cycloalkyl.
  • the compound of the present invention is a compound of general formula (I) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, wherein:
  • R 1 and R 2 together with the carbon atoms to which they are bonded form a 3-membered-8-membered heterocyclic group, wherein the heterocyclic group further contains one or more N, O or S heteroatoms, and the heterocyclic group is any One or more selected from halogen, hydroxy, alkyl, haloalkyl, hydroxyalkyl, alkoxy, haloalkoxy, hydroxyalkoxy, nitro, carboxy, cyano, amino, monoalkylamino, Alkyl acyl amino, alkyl acyl, amino acyl, alkyl amino acyl, dialkyl amino and cycloalkyl groups are substituted;
  • R 1 and R 2 together with the carbon atoms to which they are bound form a 3-membered-6-membered heterocyclic group, wherein the heterocyclic group further includes one or more N, O or S heteroatoms, and the The heterocyclic group is optionally selected by one or more selected from halogen, hydroxy, C 1-6 alkyl, halogenated C 1-6 alkyl, hydroxy C 1-6 alkyl, C 1-6 alkoxy, halogenated C 1-6 alkoxy, hydroxy C 1-6 alkoxy, nitro, carboxy, cyano, amino, mono C 1-6 alkylamino, C 1-6 alkyl acylamino, C 1-6 alkane Substitution of acyl group, aminoacyl group, C 1-6 alkylamino acyl group, bis C 1-6 alkylamino group and C 3-12 cycloalkyl group;
  • R 1 and R 2 together with the carbon atoms to which they are bonded form azetidinyl, azetidinyl, tetrahydropyrrolyl, piperidinyl, dihydropyrrolyl, tetrahydropyridyl, Pyrazolidinyl, dihydropyrazolyl, imidazolidinyl, dihydroimidazolyl, pyrazolyl, dihydropyrazolyl, oxazolidinyl, dihydrooxazolyl, thiazolyl, dihydrothiazolyl, Isoxazolidinyl, dihydroisoxazolyl, isothiazolidinyl, dihydroisothiazolyl, hexahydropyrimidinyl, tetrahydropyrimidinyl, dihydropyrimidinyl, hexahydropyridazinyl, tetrahydropyridazinyl
  • the present invention provides a compound of general formula (Ia) or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof,
  • X, m and n have the definitions described in the general formula (I) above.
  • the compound of general formula (I) or general formula (Ia) or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof according to the present invention wherein Selected from
  • the present invention provides the following specific compounds or isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs thereof:
  • the present invention provides a method for preparing the compound of general formula (A) of the present invention, including the step of reacting the compound of formula (1) with the compound of formula (2):
  • Cy has the definition described in the general formula (A), and the compound of formula (1) and the compound of formula (2) are commercially available compounds or can be synthesized by other technical means commonly used by those skilled in the art.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the present invention or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof.
  • the present invention provides the compound of the present invention or its isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs, and the compounds of the present invention or its isomers, pharmaceutically acceptable Pharmaceutical compositions of salts, solvates, crystals or prodrugs, said compounds or pharmaceutical compositions are used to treat PRMT5-mediated diseases.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the present invention or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, and a pharmaceutically acceptable carrier.
  • the compound of the present invention or its isomers, pharmaceutically acceptable salts, solvates, crystals or prodrugs can be mixed with pharmaceutically acceptable carriers, diluents or excipients to prepare pharmaceutical preparations suitable for oral administration or Parenteral administration.
  • Administration methods include, but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral routes.
  • the preparation can be administered by any route, for example, by infusion or bolus injection, by the route of absorption through the epithelium or skin mucosa (for example, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • preparations for oral administration include solid or liquid dosage forms, specifically, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, and the like.
  • the preparation can be prepared by a method known in the art, and contains a carrier, diluent or excipient conventionally used in the field of pharmaceutical preparations.
  • the present invention provides a compound represented by formula (A), formula (I) or (Ia) of the present invention or its isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug, or includes it
  • the pharmaceutical composition is used in the method of treating PRMT5-mediated diseases and the application in the preparation of medicines for treating PRMT5-mediated diseases.
  • the present invention provides a compound represented by formula (A), formula (I) or (Ia) of the present invention or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof , Or a pharmaceutical composition containing it for the treatment of PRMT5-mediated diseases and the use in the preparation of drugs for the treatment of PRMT5-mediated diseases, wherein the PRMT5-mediated diseases include but are not limited to: proliferative diseases , Metabolic disease or blood disease.
  • the PRMT5-mediated disease of the present invention is cancer.
  • PRMT5-mediated diseases of the present invention include but are not limited to: acoustic neuroma, adenocarcinoma, adrenal carcinoma, anal cancer, angiosarcoma (eg, lymphangiosarcoma, lymphatic endothelial sarcoma, angiosarcoma) , Accessory cancer, benign monoclonal gammopathy, bile cancer (for example, cholangiocarcinoma), bladder cancer, breast cancer (for example, breast adenocarcinoma, breast papillary cancer, breast cancer, breast medullary cancer, triple negative breast cancer ), brain cancer (e.g. meningioma; glioma, e.g.
  • angiosarcoma eg, lymphangiosarcoma, lymphatic endothelial sarcoma, angiosarcoma
  • Accessory cancer benign monoclonal gammopathy
  • bile cancer for example, cholangiocarcinoma
  • astrocytoma oligodendroglioma; medulloblastoma
  • bronchial carcinoma carcinoid tumor, cervical cancer (e.g. cervical adenocarcinoma) , Choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial cancer, ependymoma, endothelial sarcoma (e.g., Kaposi’s sarcoma ( Kaposi's sarcoma), multiple idiopathic hemorrhagic sarcoma), endometrial cancer (for example, uterine cancer, uterine sarcoma), esophageal cancer (for example, esophageal adenocarcinoma, Barrett's adenocarinoma), especially Due to sarcoma (Ewing sarcom
  • Gastrointestinal pancreatic neuroendocrine tumors GEP-NET
  • carcinoid tumors carcinoid tumors
  • osteosarcoma ovarian cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonic carcinoma, ovarian adenocarcinoma, ovarian clear cell carcinoma, ovarian serous cystadenocarcinoma ,
  • papillary adenocarcinoma pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary myxoma (IPMN), islet cell tumor), penile cancer (e.g., Paget's disease of the penis and scrotum), Pineal tumor, primary neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary duct cancer, skin cancer (e.g., squamous cell
  • the PRMT5-mediated diseases of the present invention include metabolic disorders such as diabetes or obesity.
  • the PRMT5-mediated diseases of the present invention include hemoglobinopathies such as sickle cell disease or ⁇ -thalassemia.
  • the PRMT5-mediated diseases of the present invention include inflammatory and autoimmune diseases.
  • the present invention provides a compound represented by general formula I of the present invention or an isomer, pharmaceutically acceptable salt, solvate, crystal or prodrug thereof, or a pharmaceutical composition containing it for use Method for treating PRMT5-mediated diseases and use in the preparation of drugs for treating PRMT5-mediated diseases, wherein the PRMT5-mediated diseases include, but are not limited to: breast cancer, esophageal cancer, bladder cancer, lung cancer, hematopoietic system Cancer, lymphoma, medulloblastoma, medulloblastoma, rectal adenocarcinoma, colon cancer, gastric cancer, pancreatic cancer, liver cancer, adenoid cystic carcinoma, prostate cancer, lung cancer, head and neck squamous cell carcinoma, brain Carcinoma, hepatocellular carcinoma, melanoma, oligodendroglioma, glioblastoma, testicular cancer, ovarian clear cell carcinoma, ovarian serous cystaden
  • the "hydrogen”, "carbon” and “oxygen” in the compounds of the present invention include all their isotopes. Isotopes should be understood to include those atoms that have the same atomic number but different mass numbers.
  • hydrogen isotopes include protium, tritium, and deuterium
  • carbon isotopes include 12 C, 13 C, and 14 C
  • oxygen isotopes include 16 O and 18 O.
  • “Isomers” in the present invention refer to molecules with the same atomic composition and connection mode but different three-dimensional arrangements, including but not limited to diastereomers, enantiomers, cis-trans isomers, and their Mixtures, such as racemic mixtures.
  • Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D, L or R, S are used to indicate the absolute configuration of the chiral center of the molecule.
  • the prefixes D, L or (+), (-) are used to name the symbols of the plane-polarized light rotation of the compound.
  • (-) or L means that the compound is levorotatory, and the prefix (+) or D means that the compound is dextrorotatory.
  • the chemical structures of these stereoisomers are the same, but their stereostructures are different.
  • a specific stereoisomer may be an enantiomer, and a mixture of isomers is usually called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is called a racemic mixture or a racemate, which may result in no stereoselectivity or stereospecificity in the chemical reaction process.
  • racemic mixture and “racemate” refer to an equimolar mixture of two enantiomers, lacking optical activity.
  • the compound of the present invention can be one of the possible isomers or a mixture of them, such as racemates and diastereomer mixtures (depending on the number of asymmetric carbon atoms) The form exists.
  • Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on differences in the physical and chemical properties of the components, for example, by chromatography Method and/or fractional crystallization method.
  • halogen in the present invention means fluorine, chlorine, bromine, and iodine.
  • halo in the present invention refers to substitution by fluorine, chlorine, bromine or iodine.
  • alkyl in the present invention refers to a linear or branched saturated aliphatic hydrocarbon group, preferably a linear or branched group containing 1 to 6 carbon atoms, and more preferably a linear or branched group containing 1 to 3 carbon atoms
  • Chain groups non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethyl Propyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, etc.
  • the alkyl group may be substituted or unsubstituted, and when substituted, the substituent may be at any available point of attachment.
  • the "sulfonyl group" in the present invention means -S(O) 2 -.
  • the "sulfonamide group" in the present invention refers to -S(O) 2 NH-.
  • haloalkyl in the present invention refers to an alkyl group substituted with at least one halogen.
  • hydroxyalkyl in the present invention refers to an alkyl group substituted with at least one hydroxy group.
  • alkoxy in the present invention refers to -O-alkyl.
  • alkoxy groups include: methoxy, ethoxy, propoxy, n-propoxy, isopropoxy, isobutoxy, sec-butoxy, and the like.
  • the alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituent may be at any available point of attachment.
  • cycloalkyl in the present invention refers to a cyclic saturated hydrocarbon group. Suitable cycloalkyl groups may be substituted or unsubstituted monocyclic, bicyclic or tricyclic saturated hydrocarbon groups with 3-12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • heterocyclic group in the present invention refers to a 3- to 12-membered non-aromatic group having 1 to 4 ring heteroatoms (wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon) Groups of ring systems ("3-12 membered heterocyclyl").
  • the point of attachment may be a carbon or nitrogen atom, as long as the valence permits.
  • Heterocyclyl groups can either be monocyclic ("monocyclic heterocyclyl") or fused, bridged or spiro ring systems (e.g., bicyclic ring systems (also known as "bicyclic heterocyclyl”)) And can be saturated or can be partially unsaturated.
  • Suitable heterocyclic groups include, but are not limited to, piperidinyl, azetidinyl, aziridinyl, tetrahydropyrrolyl, piperazinyl, dihydroquinazolinyl, oxepanyl, oxa Cyclobutyl, tetrahydrofuranyl, tetrahydropyranyl, Wait.
  • Each instance of a heterocyclic group may be optionally substituted or unsubstituted, and when substituted, the substituent may be at any available point of attachment.
  • Aryl in the present invention refers to an aromatic system that can contain a single ring or a fused polycyclic ring, preferably a single ring or a fused bicyclic aromatic system, which contains 6 to 12 carbon atoms, preferably about 6 to about 10 carbon atoms.
  • Suitable aryl groups include, but are not limited to, phenyl, naphthyl, anthryl, fluorenyl, indanyl.
  • the aryl group may be optionally substituted or unsubstituted, and when substituted, the substituent may be at any available point of attachment.
  • heteroaryl group in the present invention refers to an aryl group in which at least one carbon atom is replaced by a heteroatom, preferably composed of 5-12 atoms (5-12 membered heteroaryl), and more preferably composed of 5-10 atoms (5-10 membered heteroaryl), the heteroatoms are O, S, N.
  • heteroaryl groups include, but are not limited to, imidazolyl, pyrrolyl, furyl, thienyl, pyrazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, Tetrazolyl, indolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, isoindolyl, benzopyrazolyl, benzimidazolyl, benzofuranyl, benzopyridine Pyryl, benzothienyl, benzoxazolyl, benzothiazolyl, benzisoxazolyl, benzisothiazolyl, quinolinyl, isoquinolinyl, quinazolinyl, cinnolinyl, Quinoxalinyl, benzoxazinyl, benzothiazinyl
  • the “pharmaceutically acceptable salt” of the present invention refers to the salt of the compound of the present invention. Such salt is safe and effective when used in mammals, and has due biological activity.
  • the "solvate” in the present invention refers to a complex formed by a combination of a solute (such as an active compound, a salt of an active compound) and a solvent (such as water) in a conventional sense.
  • a solute such as an active compound, a salt of an active compound
  • a solvent such as water
  • the solvent refers to a solvent known or easily determined by those skilled in the art. If it is water, the solvate is usually referred to as a hydrate, such as hemihydrate, monohydrate, dihydrate, trihydrate, or alternative amounts thereof.
  • the in vivo effects of the compound of formula (A) can be partly exerted by one or more metabolites formed in the body of the human or animal after the compound of formula (A) is administered. As described above, the in vivo effects of the compound of formula (A) can also be exerted through the metabolism of precursor compounds ("prodrugs").
  • the "prodrug” of the present invention refers to a compound that is converted into a compound of the invention by reaction with enzymes, gastric acid, etc. under physiological conditions in an organism, that is, a compound that is converted into a compound of the invention by enzyme oxidation, reduction, hydrolysis, etc. /Or a compound or the like that is converted into a compound of the invention by a hydrolysis reaction of gastric acid or the like.
  • Crystal in the present invention refers to a solid whose internal structure is formed by regularly repeating constituent atoms (or groups thereof) in three dimensions, which is different from an amorphous solid that does not have such a regular internal structure.
  • the "pharmaceutical composition” of the present invention refers to comprising any one of the compounds of the present invention, including corresponding isomers, prodrugs, solvates, pharmaceutically acceptable salts or chemically protected forms thereof, and a Or a mixture of multiple pharmaceutically acceptable carriers and/or another one or more drugs.
  • the purpose of the pharmaceutical composition is to facilitate the administration of the compound to the organism.
  • the composition is generally used to prepare drugs for the treatment and/or prevention of diseases mediated by one or more kinases.
  • the "pharmaceutically acceptable carrier” in the present invention refers to a carrier that does not cause significant irritation to organisms and does not interfere with the biological activity and properties of the administered compound, and includes all solvents, diluents or other excipients, dispersants, and surface activities. Isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc. Unless any conventional carrier medium is incompatible with the compound of the present invention.
  • Some examples that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, And cellulose and cellulose acetate; malt, gelatin, etc.
  • excipient refers to an inert substance that is added to a pharmaceutical composition to further facilitate the administration of a compound.
  • Excipients may include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • the "PRMT5" of the present invention may be any mutant or variant of wild-type PRMT5 or PRMT5, and the mutant or variant of PRMT5 contains one or more mutations (for example, conservative substitutions).
  • Example 1 (R)-7'-((1-Acetylpiperidin-4-yl)amino)-2'-(3-(3,4-dihydroisoquinolin-2(1H)-yl )-2-hydroxypropyl)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-[2,6]naphthyridin]-1'-one
  • Methyl 5-bromo-2-chloroisonicotinate (10g, 51.8mmol) and tetrakis(triphenylphosphine) palladium (4.6g, 3.98mmol) were added to anhydrous N,N-dimethylformamide (DMF) , 25mL).
  • DMF N,N-dimethylformamide
  • trimethylaluminum (2M toluene solution, 51.9 mmol, 25.95 mL) was added.
  • the reaction system was heated to 80° C. for reaction and stirred overnight.
  • the reaction solution was poured into ice water (500 mL) for quenching, and ethyl acetate (500 mL) was added for extraction.
  • Step 5 Preparation of methyl 2-chloro-5-(1-cyanocyclopropyl)isonicotinate
  • Step 6 Preparation of methyl 2-((1-(tert-butoxycarbonyl)piperidin-4-yl)amino)-5-(1-cyanocyclopropyl)isonicotinate
  • Methyl 2-chloro-5-(1-cyanocyclopropyl)isonicotinate (0.1g, 0.38mmol) was dissolved in anhydrous tetrahydrofuran (THF, 3mL), and 4-aminopiperidine-1-carboxy was added Tert-butyl ester (114.1mg, 0.57mmol), cesium carbonate (247.6mg, 0.76mmol), chlorine (2-dicyclohexylphosphino-2',6'-diisopropyl-1,1'-biphenyl )[2-(2-Aminoethylphenyl]palladium(II) (31.82mg, 0.04mmol), N 2 protection, reacted overnight at 70°C.
  • Step 7 Preparation of methyl 2-((1-acetylpiperidin-4-yl)amino)-5-(1-cyanocyclopropyl)isonicotinate
  • Step 8 7'-((1-Acetylpiperidin-4-yl)amino)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-[2,6] Preparation of naphthyridin-1'-one
  • Step 9 (R)-7'-((1-Acetylpiperidin-4-yl)amino)-2'-(3-(3,4-dihydroisoquinolin-2(1H)-yl) -2-hydroxypropyl)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-[2,6]naphthyridin]-1'-one
  • the preparation method is the same as that in Example 1, except that 4-aminopiperidine-1-carboxylic acid tert-butyl ester in step 6 of Example 1 is replaced with 6-amino-2-azaspiro[3.3]heptane Tert-Butyl-2-carboxylate to give the title compound.
  • Example 1 of the present invention According to the synthesis method of Example 1 of the present invention, different commercially available raw materials were used to synthesize the compounds of Examples 3-9. The characterization parameters of these compounds are shown in Table 1:
  • Step 3 Preparation of 7'-nitro-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 4 (R)-2'-(3-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-7'-nitro-2',3' -Dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 5 (R)-7'-amino-2'-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-2',3'- Preparation of dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 6 (R)-6-((2'-(3-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1'-oxo-2 ',3'-Dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylic acid
  • tert-butyl ester (R)-6-((2'-(3-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-1'-oxo-2 ',3'-Dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-7'-yl)amino)-2-azaspiro[3.3]heptane-2-carboxylic acid
  • Step 7 (R)-7'-((2-Azaspiro[3.3]hepta-6-yl)amino)-2'-(3-(3,4-dihydroisoquinoline-2(1H) -Yl)-2-hydroxypropyl)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 8 (R)-7'-((2-Acetyl-2-azaspiro[3.3]hepta-6-yl)amino)-2'-(3-(3,4-dihydroisoquinoline) -2(1H)-yl)-2-hydroxypropyl)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • the compound A represented by the above formula was prepared with reference to the method disclosed by compound 208 in WO2014/100719 (PCT/US2013/077235), and was identified by hydrogen spectrum and mass spectrometry.
  • Step 3 Preparation of 7'-nitro-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 4 Preparation of 7'-amino-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one
  • Step 5 7'-((1-Acetylpiperidin-4-yl)amino)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]- Preparation of 1'-ketone
  • Step 6 (R)-7'-((1-Acetylpiperidin-4-yl)amino)-2'-(3-(3,4-dihydroisoquinolin-2(1H)-yl) -2-hydroxypropyl)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-isoquinoline]-1'-one (compound B)
  • Reagents and consumables PRMT5, purchased from Active Motif, item number 31921; peptide substrate H4(1-21)S1ac, purchased from Gil Biochemical (Shanghai) Co., Ltd., item number 342095; [ 3 H]-SAM, Purchased from PerkinElmer, the article number is NET155V001MC; SAM, purchased from Sigma, the article number is A7007-100MG; SAH, purchased from Sigma, the article number is A9384-25MG; DTT, purchased from Shenggong Bioengineering (Shanghai) Co., Ltd. Limited company, the article number is A620058-0005.
  • Corning-3657 purchased from Corning Company, the article number is 3657; Echo Qualified 384-Well, purchased from Labcyte Company, the article number is P-05525; FlashPlate, purchased from Perkin Elmer, the article number is SMP410J001PK.
  • reaction buffer and reaction termination solution 1x reaction buffer liquid components are 10mM Tris-HCl, pH 8.0; 0.01% Tween-20; 1mM DTT.
  • the composition of the reaction termination solution was 125 ⁇ M 3 H-SAM solution.
  • the compound was dissolved in 100% DMSO into a 10 mM stock solution, and then the compound was diluted to the required concentration on an Echo384-well plate.
  • the peptide substrate and [ 3 H]-SAM were added to the 1-fold reaction buffer to form a 2.5-fold substrate solution (final concentrations of 100 nM and 250 nM, respectively).
  • reaction stop solution 0.5 ⁇ L of reaction stop solution to each well of the 384-well reaction plate to stop the reaction. Take 25uL from each well of the test plate and transfer it to the Flashplate, and place it at room temperature for 1h. Then wash the Flashpate plate 3 times with 0.1% Tween-20 solution.
  • Inhibition rate (%) (maximum value-sample value)/(maximum value-minimum value) ⁇ 100%.
  • Test compound The compound of the present invention prepared in the above example, each compound was prepared with DMSO as a 10 mM stock solution, and the final dilution was 8 concentrations for detection.
  • the final concentration of the compound in the Z-138 cell experiment was 33333.00 nM, 6666.60 nM, 1333.32 nM, 266.66nM, 53.33nM, 10.67nM, 2.13nM, 0.43nM, MDA-MB-468 and NCI-H358 cell test compound final concentration is 50000nM, 10000nM, 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM.
  • Human mantle cell lymphoma cell Z-138, triple negative breast cancer cell MDA-MB-468, and human non-small cell lung cancer cell NCI-H358 were purchased from the American Type Culture Collection (ATCC).
  • Iscove's Modified Dulbecco's Medium (IMEM medium), article number is ATCC 30-2005; Leibovitz's L-15 Medium (L-15 medium), article number is Gibco 11415-064; 1640 medium, article number is Gibco 22400089; horse serum, The article number is Gibco 16050122; Fetal Bovine Serum, article number is Gibco 10099-141; Penicillin, article number is Gibco 15140-122; Sodium pyruvate, article number is Gibco 11360070; CellTiter-Glo Luminescent Cell Viability Assay, article number is Promega G7571. CCK-8 Proliferation Inhibition Detection Kit, the article number is KeyGEN KGA317.
  • MDA-MB-468 cell recovery Take out the MDA-MB-468 cell cryopreservation tube from the liquid nitrogen tank and place it in a 37°C water bath, shake it gently to thaw it as soon as possible. After thawing, take out the cryopreservation tube, disinfect with an alcohol cotton ball, unscrew the lid, aspirate the cell liquid into the centrifuge tube, and add 1 mL of L-15 medium containing 10% FBS, mix well, and place in a centrifuge at 1000 rpm and centrifuge 5min. Afterwards, the supernatant was discarded, and complete medium was added and pipetting repeatedly until the cells were completely blown out and resuspended. Inoculate in a petri dish at an appropriate concentration. Cultivate in a CO 2 free incubator at 37°C and 95% humid air.
  • NCI-H358 cells Take out the NCI-H358 cell cryopreservation tube from the liquid nitrogen tank and place it in a 37°C water bath, shake it gently to thaw it as soon as possible. After thawing, take out the cryopreservation tube, sterilize it with an alcohol cotton ball, unscrew the lid, aspirate the cell liquid into the centrifuge tube, and add 1 mL of 1640 medium containing 10% FBS, mix well, and place in a centrifuge at 1000 rpm for 5 min. Afterwards, the supernatant was discarded, and complete medium was added and pipetting repeatedly until the cells were completely blown out and resuspended. Inoculate in a petri dish at an appropriate concentration. Place it in a 37°C, 5% CO 2 , 95% CO 2 incubator with humid air.
  • MDA-MB-468 cell culture and passage cells grow to about 80-90%, transfer the medium (Leibovitz's L-15 Medium medium + 10% FBS + 1% penicillin) to a 15 mL centrifuge tube , 1000rpm, centrifugation for 5min. Remove the supernatant, resuspend the cells in complete medium, inoculate the cells in a petri dish at the required density, and place them in a CO 2 incubator at 37°C and 95% humid air. Change every 2-3 days depending on the cell growth. One culture medium or passage.
  • NCI-H358 cell culture and passage cells grow to about 80-90%, transfer the medium (1640 medium + 10% FBS + 1% penicillin + 1 mM sodium pyruvate) to a 15 mL centrifuge tube , 1000rpm, centrifugation for 5min. Remove the supernatant, resuspend the cells in complete medium, inoculate them in a petri dish at the required density, and place them in a CO 2 incubator at 37°C, 5% CO 2 , and 95% humid air, depending on the cell growth. -Change the culture medium once every 3 days or carry out subculture.
  • Z-138 cells were resuspended in complete medium at a density of 1000 cells/well and seeded in a 96-well culture plate: 36 wells in the outer circle of the 96-well plate were filled with 200 ⁇ L PBS to prevent the edge of the medium from evaporating Faster leads to large differences in the culture conditions of the inner plate holes; the leftmost column of the inner 60 holes is a blank hole, without cells, filled with an equal volume of PBS; the remaining 54 holes are plated with a row gun, and each hole is Contain 100 ⁇ L of medium containing the corresponding cells. After the plating is completed, tap the 96-well plate to suspend the cells uniformly, and place them in a 5% CO 2 incubator at 37°C for 24 hours.
  • MDA-MB-468 cells are resuspended in complete medium at the corresponding density, 2000 cells/well, inoculated in 96-well culture plate: fill the outer circle of cells with 200 ⁇ L PBS to prevent the edge of the medium from evaporating quickly As a result, the culture conditions of the inner plate wells are too different; the leftmost column of the inner 60 wells is a blank well, without cells, filled with an equal volume of PBS; the remaining 54 wells are plated with a row gun, and each well is 100 ⁇ L. Put it into a CO 2 incubator at 37°C for 24 hours.
  • NCI-H358 cells are resuspended in complete medium at the corresponding density, 1000 cells/well, and seeded in a 96-well culture plate:
  • the outer circle of the cells is filled with 200 ⁇ L PBS to prevent the edge of the medium from evaporating quickly and causing the inside
  • the culture conditions of the plate wells are too different;
  • the leftmost column of the inner 60 wells is a blank well, without cells, filled with an equal volume of PBS;
  • the remaining 54 wells are plated with a row gun, and each well is 100 ⁇ L, put in 5 Incubate at 37°C for 24h in a %CO 2 incubator.
  • Z-138 cells were added to the original medium (100 ⁇ L) with 50 ⁇ L (3 ⁇ ) of the drug, and each concentration group was set with two multiple holes, and the cells were placed in a CO 2 incubator and cultured for 7 days.
  • the compound is prepared as follows: Weigh 1-2 mg of compound in advance, and use DMSO to prepare a 10 mM stock solution. Use complete medium to dilute the drug.
  • the final concentration of the drug is 33333.00nM as the initial maximum concentration, and it is diluted in a 1:4 gradient to 7 concentration gradients: 6666.60nM, 1333.32nM, 266.66nM, 53.33nM, 10.67nM, 2.13nM, 0.43nM.
  • MDA-MB-468 cells were added to the original medium (100 ⁇ L) with 100 ⁇ L (2 ⁇ ) of the drug. Each concentration group was set with two multiple wells, and the cells were placed in a CO 2 free incubator and cultured for 7 days.
  • the compound is prepared as follows: Weigh 1-2 mg of compound in advance, and use DMSO to prepare a 10 mM stock solution. Use complete medium to dilute the drug.
  • the final concentration of the drug is 50,000nM as the initial maximum concentration, and it is diluted in a 1:4 gradient to 7 concentration gradients: 50,000nM, 10000nM, 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM.
  • NCI-H358 cells were added with 100 ⁇ L (2 ⁇ ) of the drug. Each concentration group was set with two multiple holes, and the cells were placed in a 5% CO 2 incubator for 7 days.
  • the compound is prepared as follows: Weigh 1-2 mg of compound in advance, and use DMSO to prepare a 10 mM stock solution. Use complete medium to dilute the drug.
  • the final concentration of the drug is 50,000nM as the initial maximum concentration, and it is diluted in a 1:4 gradient to 7 concentration gradients: 50,000nM, 10000nM, 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM.
  • the CellTiter-Glo Luminescent Cell Viabillity Assay was taken out 30 minutes in advance and equilibrated to room temperature. Aspirate PBS from the blank wells, add 150 ⁇ L of complete medium, then add 75uL Celltiter-Glo reagent to the blank wells, dosing wells and DMSO wells, and shake at room temperature for 2min. After continuing to incubate at room temperature for 10 minutes, draw 180 ⁇ L from each well and transfer to an opaque white board. After removing air bubbles, detect the chemiluminescence signal, shake, and the Read sample detection condition is 500 ms.
  • Inhibition(%) 100-(AU experimental well- AU blank well )/(AU solvent control well- AU blank well )* 100.
  • GraghPad 5.0 software was used to draw the IC 50 curve, analyze the data, and obtain the final IC 50 value. The experimental results are shown in Table 3.
  • the compound of the present invention has good inhibitory activity on human mantle cell lymphoma cell Z-138, triple negative breast cancer cell MDA-MB-468, and human non-small cell lung cancer cell NCI-H358. , It is very promising to become a therapeutic agent for lymphoma, triple negative breast cancer, and non-small cell lung cancer.
  • the hERG potassium channel is the standard for drug safety screening. Blocked hERG potassium channels can lead to cardiotoxicity and prolonged ventricular repolarization, and may lead to sudden death in severe cases. Drugs with inhibitory effects on hERG potassium channels may be a potential scourge of clinical medication. Therefore, the compound has a weak inhibitory effect on the hERG potassium ion channel and high safety.
  • the prolonged QT interval caused by drugs is related to the increased risk of fatal ventricular arrhythmia and sudden death.
  • Penicillin-streptomycin solution 100 ⁇
  • DMEM/F12 were purchased from Gibco
  • Fetal bovine serum was purchased from PAA
  • DMSO, EGTA, and MgATP were purchased from Sigma
  • KCl, CaCl 2 ⁇ 2H 2 O, MgCl 2. 6H 2 O and NaCl were purchased from Sinopharm Company
  • glucose was purchased from General-reagent Company
  • HEPES was purchased from Solarbio Company
  • Quinidine was purchased from Aladdin Company.
  • TI-S-FLU microscope was purchased from Nikon; SMZ-140/143 microscope was purchased from Motic; EPC-10 amplifier and Patchmaster V2X60 were purchased from HEKA; TMC-36 anti-vibration platform was purchased from TMC; MP-225, MPC-200 manipulator, ROE-200 micromanipulator, and P-97 electrode drawing instrument were purchased from Sutter Company; VC3-8PP perfusion drug delivery system was purchased from ALA Company.
  • Formulated test solvent extracellular fluid formulation (mM): 137NaCl, 4KCl, 1.8CaCl 2, 1MgCl 2, 10 glucose, and 10HEPES (pH 7.4); intracellular fluid formulation (mM): 130KCl, 1MgCl 2 , 5EGTA, 5MgATP and 10 HEPES (pH 7.2); negative control preparation: extracellular fluid + 0.3% DMSO; positive control: quinidine.
  • Compound treatment Weigh the compound and dissolve it in DMSO to prepare a 10 mM mother liquor, and dilute the mother liquor with DMSO to form a secondary mother liquor with concentrations of 3.3, 1.1, 0.37, and 0.12 mM. Take 90 ⁇ L of mother liquor and secondary mother liquor and dilute into 30mL extracellular fluid for electrophysiological detection. The final concentration of the compound is 30, 10, 3.3, 1.1, 0.37 ⁇ M, and the final concentration of DMSO is 3:1000.
  • Stable cell culture The cell line is derived from HEK-293 cells overexpressing the hERG potassium ion channel. It was established and verified by Creus Biosciences after providing technical support in the laboratory of Dr. Mohamed Boutjdir, New York University School of Medicine. The cells were cultured in a 37°C, 5% CO 2 incubator.
  • Cell culture medium composition DMEM, 15% fetal bovine serum and 1% 100 ⁇ penicillin-streptomycin.
  • Electrophysiological manual patch clamp system experiment inoculate the stable cells on a glass slide, the cell density is less than 50%, and culture overnight. Transfer the experimental cells to an approximately 1 mL bath embedded in the platform of an inverted microscope, and perfuse the extracellular fluid at a perfusion rate of 2.7 mL/min. The experiment can be started after 5 minutes of stability. Use HEKA EPC-10 patch clamp amplifier and PATCHMASTER acquisition system to record membrane current. All experiments were done at room temperature (22-24°C). In the experiment, the P-97 microelectrode drawing machine was used to straighten the electrode (BF150-110-10). The inner diameter of the electrode is 1-1.5mm, and the water resistance after being filled with the inner liquid is 2-4M ⁇ .
  • the electrophysiological stimulation scheme of hERG potassium channel is to first clamp the membrane voltage at -80mV, give the cells a continuous 2s, +20mV voltage stimulation to activate the hERG potassium channel, and then repolarize to -50mV for 5s to generate an outward tail current.
  • the stimulation frequency is once every 15s.
  • the current value is the peak value of the tail current.
  • the channel current was recorded in the whole cell recording mode. First, perfuse extracellular fluid (approximately 2 mL per minute) and continue recording, and wait for the current to stabilize (Run-Down is less than 5% within 5 minutes). At this time, the peak tail current is the control current value.
  • the extracellular fluid containing the drug to be tested is perfused and recording is continued until the inhibitory effect of the drug on the hERG current reaches a steady state, at which time the peak value of the tail current is the current value after the drug is added.
  • the standard of the steady state is judged by whether the last three consecutive current recording lines overlap. After reaching a stable state, if the hERG current is restored or close to the level before the drug is added after perfusion with extracellular fluid, then the perfusion can be continued to test other concentrations or drugs. 30 ⁇ M quinidine was used as a positive control in the experiment to ensure that the cells used had a normal response.
  • compound B showed a significant prolongation of the QT interval in an isolated heart experiment, while the compounds of Examples 1 and 2 of the present invention showed no effect on the QT interval in an isolated heart experiment.
  • the compound of the present invention has better cardiac safety.

Abstract

本发明属于医药化学领域，涉及一类作为PRMT5抑制剂的取代三环类化合物及其应用，具体地，本发明提供式(A)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，它们的制备方法以及含有这些化合物的药物组合物和这些化合物或组合物用于治疗PRMT5介导的疾病的用途。本发明化合物对PRMT5表现出显著的抑制活性。

Description

作为PRMT5抑制剂的取代三环类化合物及其应用 技术领域
本发明属于医药化学领域，具体涉及作为PRMT5抑制剂的取代三环类化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，它们的制备方法以及含有这些化合物的药物组合物和这些化合物或组合物用于治疗PRMT5介导的疾病的用途。
背景技术
DNA的修饰在触发细胞生长和发育的不同阶段的基因表达程序中起着核心作用，其中精氨酸甲基化在细胞进程中担任重要角色，包括信号传导，转录，RNA加工，DNA重组和修复。蛋白精氨酸甲基转移酶(PRMTs)通过将甲基从S-腺苷甲硫氨酸(SAM)转移到精氨酸的胍氮来催化特定精氨酸残基的甲基化，根据催化精氨酸甲基化方式的不同可将PRMTs分为三类：I型(PRMT 1,2,3,4,6和8)催化单甲基化和不对称二甲基化，II型(PRMT5和PRMT9)催化单甲基化和对称二甲基化，而III型(PRMT7)仅进行单甲基化。
其中，PRMT5与甲基转移酶复合体蛋白50(MEP50)特异性结合，可以对称甲基化组蛋白H3和H4，并调节特定靶基因组的转录。PRMT5催化的组蛋白H3精氨酸8(R8)和H4R3对称二甲基化已显示抑制几种肿瘤抑制基因的表达，例如抑癌基因7(ST7)，视网膜母细胞瘤(RB)肿瘤抑制基因家族和受体O型蛋白质酪氨酸磷酸酶(PTPROt)。
除了甲基化组蛋白的能力外，PRMT5还能够甲基化几种重要的转录因子，使其在细胞调节的过程发挥重要作用。PRMT5可以甲基化p53并改变其DNA结合活性，从而引发p53控制的基因表达程序的变化。PRMT5还显示甲基化N-MYC并改变其蛋白质稳定性以及增强其在神经母细胞瘤中的致癌活性。PRMT5还可直接甲基化转录因子，包括E2F-1和NF-κB/p65，诱导其靶基因表达。PRMT5不仅可修饰核转录因子，还可甲基化细胞质蛋白如golgin，核糖体蛋白S10(RPS10)。因此，除了其直接调节其自身靶基因的能力之外，PRMT5还能够通过关键转录因子的对称甲基化间接影响全局基因表达，从而影响细胞生长，增殖和分化。
大量研究已经证实PRMT5在不同类型和侵袭性的癌症中过度表达，包括B细胞和T细胞淋巴瘤，转移性黑素瘤，神经母细胞瘤和成胶质细胞瘤，生殖细胞肿瘤，卵巢癌，鼻咽癌，乳腺癌，结肠直肠癌和胃癌。目前研究表明PRMT5在控制细胞生长和增殖中起重要作用，并且其过表达促进细胞转化。
癌细胞中增强的PRMT5表达与其靶肿瘤抑制基因的转录沉默相关。PRMT5能够通过启动子组蛋白H3R8和H4R3的甲基化以及通过修饰包括E2F1和NF-kB/p65的关键转录因子的特定精氨酸残基引起 全局染色质变化来促进癌细胞生长。PRMT5还会与程序性细胞死亡4(PDCD4)相互作用，使其在R110处变为甲基化并且在MCF-7细胞中丧失其肿瘤抑制活性。总的来说，PRMT5过表达可能使其与生长促进蛋白和肿瘤抑制蛋白的相互作用，从而以有利于癌细胞生长，存活与转移。
综上所述，PRMT5抑制剂在治疗肿瘤等相关疾病方面有着明确的机制，有很大潜力可以成为肿瘤治疗领域新的治疗手段，因此，需要开发更安全、更有效的PRMT5抑制剂以满足临床需求。
发明内容
本发明的一个目的是提供通式(A)所示的一类具有PRMT5抑制活性的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，
Figure PCTCN2020120284-appb-000001
其中，
Cy选自杂环基，所述杂环基可被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、烷基磺酰基、氨基酰基、烷基氨基酰基、双烷基氨基、烯基、炔基、卤代烷基酰基、羟基烷基酰基、环烷基酰基、杂环基酰基、环烷基、杂环基、芳基、杂芳基和氧代基团的基团取代。
本发明的另一个目的是提供制备本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药的方法。
本发明的再一个目的是提供包含本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药和可药用载体的组合物，以及包含本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药和另一种或多种药物的组合物。
本发明的还一个目的是提供本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药治疗PRMT5介导的疾病的方法，以及本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药在制备用于治疗PRMT5介导的疾病的药物中的应用。
针对上述发明目的，本发明提供以下技术方案：
第一方面，本发明提供通式(A)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，
Figure PCTCN2020120284-appb-000002
其中，
Cy选自杂环基，所述杂环基可被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、烷基磺酰基、氨基酰基、烷基氨基酰基、双烷基氨基、烯基、炔基、卤代烷基酰基、羟基烷基酰基、环烷基酰基、杂环基酰基、环烷基、杂环基、芳基、杂芳基和氧代基团的基团取代。
在一些优选的实施方案中，本发明的化合物为通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中：
Cy选自3-12元杂环基，所述3-12元杂环基可被一个或多个选自卤素、羟基、C 1-6烷基、卤代C 1-6烷基、羟基C 1-6烷基、C 1-6烷氧基、卤代C 1-6烷氧基、羟基C 1-6烷氧基、硝基、羧基、氰基、氨基、单C 1-6烷基氨基、C 1-6烷基酰基氨基、C 1-6烷基酰基、C 1-6烷基磺酰基、氨基酰基、C 1-6烷基氨基酰基、双C 1-6烷基氨基、C 2-10烯基、C 2-10炔基、卤代C 1-6烷基酰基、羟基C 1-6烷基酰基、C 3-12环烷基酰基、3-12元杂环基酰基、C 3-12环烷基、3-12元杂环基、6-12元芳基、5-12元杂芳基和氧代基团的基团取代；
进一步优选地，Cy选自3-10元杂环基，所述3-10元杂环基可被一个或多个选自卤素、羟基、C 1-3烷基、卤代C 1-3烷基、羟基C 1-3烷基、C 1-3烷氧基、卤代C 1-3烷氧基、羟基C 1-3烷氧基、硝基、羧基、氰基、氨基、单C 1-3烷基氨基、C 1-3烷基酰基氨基、C 1-3烷基酰基、C 1-3烷基磺酰基、氨基酰基、C 1-3烷基氨基酰基、双C 1-3烷基氨基、C 2-6烯基、C 2-6炔基、卤代C 1-3烷基酰基、羟基C 1-3烷基酰基、C 3-8环烷基酰基、3-8元杂环基酰基、C 3-8环烷基、3-8元杂环基、6-8元芳基、5-8元杂芳基和氧代基团的基团取代；
更进一步优选地，Cy选自3-10元杂环基，其中所述杂环基进一步包含一个或多个N、O或S杂原子，且所述3-10元杂环基可被一个或多个选自卤素、羟基、C 1-3烷基、卤代C 1-3烷基、羟基C 1-3烷基、C 1-3烷氧基、卤代C 1-3烷氧基、羟基C 1-3烷氧基、硝基、羧基、氰基、氨基、单C 1-3烷基氨基、C 1-3烷基酰基氨基、C 1-3烷基酰基、C 1-3烷基磺酰基、氨基酰基、C 1-3烷基氨基酰基、双C 1-3烷基氨基、C 2-6烯基、C 2-6炔基、卤代C 1-3烷基酰基、羟基C 1-3烷基酰基、C 3-8环烷基酰基、3-8元杂环基酰基、C 3-8环烷基、3-8元杂环基、6-8元芳基、5-8元杂芳基和氧代基团的基团取代。
在一些优选的实施方案中，根据本发明的通式(A)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中Cy选自
Figure PCTCN2020120284-appb-000003
Figure PCTCN2020120284-appb-000004
在一些优选的实施方案中，本发明提供通式(A)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中通式(A)具有以下通式(I)的结构，
Figure PCTCN2020120284-appb-000005
其中，
X选自O、S、C(R 1)(R 2)和N(R 3)，其中R 1、R 2、R 3各自独立地选自氢、卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基，且R 1和R 2与它们所结合的碳原子一起形成杂环基，其中所述的杂环基任选被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基的基团取代；
m为1、2、3或4；和
n为0、1、2、3或4。
在一些优选的实施方案中，本发明的化合物为通式(I)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中：
R 1、R 2、R 3各自独立地选自氢、卤素、羟基、C 1-6烷基、卤代C 1-6烷基、羟基C 1-6烷基、C 1-6烷氧基、卤代C 1-6烷氧基、羟基C 1-6烷氧基、硝基、羧基、氰基、氨基、单C 1-6烷基氨基、C 1-6烷基酰基氨基、C 1-6烷基酰基、氨基酰基、C 1-6烷基氨基酰基、双C 1-6烷基氨基和C 3-12环烷基；
进一步优选地，R 1、R 2、R 3各自独立地选自氢、卤素、羟基、C 1-3烷基、卤代C 1-3烷基、羟基C 1-3烷基、C 1-3烷氧基、卤代C 1-3烷氧基、羟基C 1-3烷氧基、硝基、羧基、氰基、氨基、单C 1-3烷基氨基、C 1-3烷基酰基氨基、C 1-3烷基酰基、氨基酰基、C 1-3烷基氨基酰基、双C 1-3烷基氨基和C 3-8环烷基；
更进一步优选地，R 1、R 2、R 3各自独立地选自氢、卤素、羟基、甲基、乙基、丙基、异丙基、卤代C 1-3烷基、羟基C 1-3烷基、C 1-3烷氧基、卤代C 1-3烷氧基、羟基C 1-3烷氧基、硝基、羧基、氰基、氨基、单C 1-3烷基氨基、C 1-3烷基酰基氨基、C 1-3烷基酰基、氨基酰基、C 1-3烷基氨基酰基、双C 1-3烷基氨基和C 3-8环烷基。
在一些优选的实施方案中，本发明的化合物为通式(I)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中：
R 1和R 2与它们所结合的碳原子一起形成3元-8元杂环基，其中所述杂环基进一步包含一个或多个N、O或S杂原子，且所述杂环基任选被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基的基团取代；
进一步优选地，R 1和R 2与它们所结合的碳原子一起形成3元-6元杂环基，其中所述杂环基进一步包含一个或多个N、O或S杂原子，且所述杂环基任选被一个或多个选自卤素、羟基、C 1-6烷基、卤代C 1-6烷基、羟基C 1-6烷基、C 1-6烷氧基、卤代C 1-6烷氧基、羟基C 1-6烷氧基、硝基、羧基、氰基、氨基、单C 1-6烷基氨基、C 1-6烷基酰基氨基、C 1-6烷基酰基、氨基酰基、C 1-6烷基氨基酰基、双C 1-6烷基氨基和C 3-12环烷基的基团取代；
更进一步优选地，R 1和R 2与它们所结合的碳原子一起形成氮杂环丙基、氮杂环丁基、四氢吡咯基、哌啶基、二氢吡咯基、四氢吡啶基、吡唑烷基、二氢吡唑基、咪唑烷基、二氢咪唑基、吡唑基、二氢吡唑基、噁唑烷基、二氢噁唑基、噻唑烷基、二氢噻唑基、异噁唑烷基、二氢异噁唑基、异噻唑烷基、二氢异噻唑基、六氢嘧啶基、四氢嘧啶基、二氢嘧啶基、六氢哒嗪基、四氢哒嗪基、二氢哒嗪基、哌嗪基、四氢吡嗪基、二氢吡嗪基、吗啉基、硫代吗啉基或牛磺胺基，其中所述基团任选被一个或多个选自卤素、羟基、C 1-3烷基、卤代C 1-3烷基、羟基C 1-3烷基、C 1-3烷氧基、卤代C 1-3烷氧基、羟基C 1-3烷氧基、硝基、羧基、氰基、氨基、单C 1-3烷基氨基、C 1-3烷基酰基氨基、C 1-3烷基酰基、氨基酰基、C 1-3烷基氨基酰基、双C 1-3烷基氨基和C 3-12环烷基的基团取代。
在一些优选的实施方案中，本发明提供通式(Ia)的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，
Figure PCTCN2020120284-appb-000006
其中，X、m和n具有以上通式(I)所述的定义。
在一些优选的实施方案中，根据本发明的通式(I)或通式(Ia)的化合物或其异构体、药学上可接受 的盐、溶剂化物、结晶或前药，其中
Figure PCTCN2020120284-appb-000007
选自
Figure PCTCN2020120284-appb-000008
Figure PCTCN2020120284-appb-000009
本发明提供以下具体化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药：
Figure PCTCN2020120284-appb-000010
另一方面，本发明提供本发明的通式(A)的化合物的制备方法，包括使式(1)的化合物和式(2)的化合物反应的步骤：
Figure PCTCN2020120284-appb-000011
其中，Cy具有通式(A)所述的定义，式(1)的化合物和式(2)的化合物为市售化合物或可采用本领域技术人员惯用的其它技术手段进行合成。
第三方面，本发明提供药物组合物，其包含本发明的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药。
在一些实施方案中，本发明提供本发明的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药及包含本发明的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药的药物组合物，所述化合物或药物组合物用于治疗PRMT5介导的疾病。
在一些实施方案中，本发明提供药物组合物，其包含本发明的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药和可药用载体。
可以将本发明的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药与可药用载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于皮内、肌内、腹膜内、静脉内、皮下、鼻内和经口途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。经口施用制剂的实例包括固体或液体剂型，具体而言，包括片剂、丸剂、粒剂、粉剂、胶囊剂、糖浆、乳剂、混悬剂等。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
第四方面，本发明提供本发明式(A)、式(I)或(Ia)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，或包含其的药物组合物用于治疗PRMT5介导的疾病的方法以及在制备治疗PRMT5介导的疾病的药物中的用途。
在一些优选的实施方案中，本发明提供本发明式(A)、式(I)或(Ia)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，或包含其的药物组合物用于治疗PRMT5介导的疾病的方法以及在制备治疗PRMT5介导的疾病的药物中的用途，其中所述的PRMT5介导的疾病包括但不限于：增殖性疾病、代谢疾病或血液疾病。在一些实施方案中，本发明所述的PRMT5介导的疾病为癌症。
在一些实施方案中，本发明所述的PRMT5介导的疾病包括但不限于：听神经瘤、腺癌、肾上腺癌、肛门癌、血管肉瘤(例如，淋巴管肉瘤、淋巴管内皮肉瘤、血管肉瘤)、附件癌、良性单克隆性丙种球蛋白病、胆癌(例如，胆管癌)、膀胱癌、乳癌(例如，乳房腺癌、乳房乳头状癌、乳腺癌、乳房髓样癌、三阴性乳腺癌)、脑癌(例如，脑膜瘤；神经胶质瘤，例如星形细胞瘤、少突神经胶质瘤；成神经管细胞瘤)、支气管癌、类癌瘤、宫颈癌(例如宫颈腺癌)、绒毛膜癌、脊索瘤、颅咽管瘤、结肠直肠癌(例如，结肠癌、直肠癌、结肠直肠腺癌)、上皮癌、室管膜瘤、内皮肉瘤(例如，卡波西氏肉瘤(Kaposi's sarcoma)、多发性特发性出血性肉瘤)、子宫内膜癌(例如，子宫癌、子宫肉瘤)、食道癌(例如，食道腺癌、巴瑞特氏腺癌(Barrett’s adenocarinoma))、尤因肉瘤(Ewing sarcoma)、眼癌(例如，眼内黑素瘤、成 视网膜细胞瘤)、家族性嗜酸性粒细胞增多症、胆囊癌、胃癌(例如，胃腺癌)、胃肠道间质瘤(GIST)、头颈部癌(例如，头颈部鳞状细胞癌、口腔癌(例如，口腔鳞状细胞癌(OSCC)、咽喉癌(例如，喉癌、咽癌、鼻咽癌、口咽癌))、造血系统癌(例如，白血病如急性淋巴细胞性白血病(ALL)(例如，B-细胞ALL、T-细胞ALL)、急性髓细胞性白血病(AML)(例如，B-细胞AML、T-细胞AML)、慢性粒细胞性白血病(CML)(例如，B-细胞CML、T-细胞CML)以及慢性淋巴细胞性白血病(CLL)(例如，B-细胞CLL、T-细胞CLL)；淋巴瘤如霍奇金淋巴瘤(HL)(例如，B-细胞HL、T-细胞HL)以及非霍奇金淋巴瘤(NHL)(例如，B-细胞NHL如弥漫性大细胞淋巴瘤(DLCL)(例如，弥漫性大B-细胞淋巴瘤(DLBCL))、滤泡性淋巴瘤、慢性淋巴细胞性白血病/小淋巴细胞性淋巴瘤(CLL/SLL)、套细胞淋巴瘤(MCL)、边缘带B-细胞淋巴瘤(例如，粘膜相关淋巴样组织(MALT)淋巴瘤、结节边缘带B-细胞淋巴瘤、脾边缘带B-细胞淋巴瘤)、原发性纵隔B-细胞淋巴瘤、伯基特淋巴瘤(Burkittlymphoma)、淋巴浆细胞淋巴瘤(即，“沃尔丹斯特伦巨球蛋白血症(
Figure PCTCN2020120284-appb-000012
macroglobulinemia)”)、毛细胞白血病(HCL)、免疫母细胞性大细胞淋巴瘤、前体B-成淋巴细胞性淋巴瘤以及原发性中枢神经系统(CNS)淋巴瘤；以及T-细胞NHL如前体T-成淋巴细胞性淋巴瘤/白血病、外周T-细胞淋巴瘤(PTCL)(例如，皮肤T-细胞淋巴瘤(CTCL)(例如，蕈样真菌病(mycosis fungiodes)、西泽里综合征(Sezary syndrome))、血管免疫母细胞性T-细胞淋巴瘤、结节外天然杀伤T-细胞淋巴瘤、肠病类型T-细胞淋巴瘤、皮下脂膜炎样T-细胞淋巴瘤、间变性大细胞淋巴瘤)；如上所描述的一种或多种白血病/淋巴瘤的混合物；以及多发性骨髓瘤(MM))、重链病(例如，α链病、γ链病、μ链病)、成血管细胞瘤、炎性肌纤维母细胞瘤、免疫细胞淀粉样变性、肾癌(例如，肾母细胞瘤又称韦尔姆斯氏瘤(Wilms’tumor)、肾细胞癌)、肝癌(例如，肝细胞癌(HCC)、恶性肝细胞瘤)、肺癌(例如，支气管癌、小细胞肺癌(SCLC)、非小细胞肺癌(NSCLC)、肺腺癌)、平滑肌肉瘤(LMS)、肥大细胞增多症(例如，全身性肥大细胞增多症)、骨髓发育不良综合征(MDS)、间皮瘤、骨髓增殖性疾病(MPD)(例如，真性红细胞增多症(PV)、特发性血小板增多症(ET)、特发性骨髓外化生(AMM)又称为骨髓纤维变性(MF)、慢性特发性骨髓纤维变性、慢性骨髓性白血病(CML)、慢性嗜中性白血病(CNL)、嗜酸性白细胞增多综合征(HES))、成神经细胞瘤、神经纤维瘤(例如，1型或2型多发性神经纤维瘤(NF)、施旺细胞瘤病(schwannomatosis))、神经内分泌癌(例如，胃肠胰腺神经内分泌肿瘤(GEP-NET)、类癌瘤)、骨肉瘤、卵巢癌(例如，囊腺癌、卵巢胚胎性癌、卵巢腺癌、卵巢透明细胞癌、卵巢浆液性囊腺癌、)、乳头状腺癌、胰腺癌(例如，胰腺腺癌、管内乳头状粘液瘤(IPMN)、胰岛细胞肿瘤)、阴茎癌(例如，阴茎和阴囊佩吉特氏病(Paget’s disease))、松果体瘤、原发性神经外胚层瘤(PNT)、前列腺癌(例如，前列腺腺癌)、直肠癌、横纹肌肉瘤、唾液管癌、皮肤癌(例如，鳞状细胞癌(SCC)、角化棘皮瘤(KA)、黑素瘤、基底细胞癌(BCC))、小肠癌(例如，附件癌)、软组织肉瘤(例如，恶性纤维组织细胞瘤(MFH)、脂肪肉瘤、恶性外周神经鞘瘤(MPNST)、软骨肉瘤、纤维肉瘤、粘液肉瘤)、皮脂腺癌、汗腺癌、滑膜瘤、睾丸癌(例如，精原细胞瘤、睾丸胚胎性癌)、甲状腺癌(例如，甲状腺乳 头状癌、乳头状甲状腺癌(PTC)、髓样甲状腺癌)、尿道癌、阴道癌以及外阴癌(例如，外阴佩吉特氏病)、髓母细胞瘤、腺样囊性癌、黑色素瘤、胶质母细胞癌。
在一些实施方案中，本发明所述的PRMT5介导的疾病包括代谢性病症如糖尿病或肥胖症。
在一些实施方案中，本发明所述的PRMT5介导的疾病包括血红蛋白病如镰状细胞病或β-地中海贫血。
在一些实施方案中，本发明所述的PRMT5介导的疾病包括炎性和自身免疫性疾病。
在一些优选的实施方案中，本发明提供本发明通式I所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，或包含其的药物组合物用于治疗PRMT5介导的疾病的方法以及在制备治疗PRMT5介导的疾病的药物中的用途，其中所述的PRMT5介导的疾病包括但不限于：乳腺癌、食道癌、膀胱癌、肺癌、造血系统癌、淋巴瘤、髓母细胞瘤、成神经管细胞瘤、直肠腺癌、结肠癌、胃癌、胰腺癌、肝癌、腺样囊性癌、前列腺癌、肺癌、头颈部鳞状细胞癌、脑癌、肝细胞癌、黑色素瘤、少突神经胶质瘤、胶质母细胞癌、睾丸癌、卵巢透明细胞癌、卵巢浆液性囊腺癌、甲状腺癌、多发性骨髓瘤(AML)、肾细胞癌、套细胞淋巴瘤、三阴性乳腺癌、非小细胞肺癌、血红蛋白病、糖尿病和肥胖症。
术语定义
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
本发明化合物中的“氢”、“碳”、“氧”包括其所有同位素。同位素应理解为包括具有相同原子数但具有不同质量数的那些原子。举例来说，氢的同位素包括氕、氚和氘，碳的同位素包括 12C、 13C和 14C，氧的同位素包括 16O和 18O等。
本发明的“异构体”是指原子组成及连接方式相同，而其三维空间排列不同的分子，包括但不限于非对映体，对映异构体，顺反异构体，和它们的混合物，如外消旋混合物。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀D、L或(+)、(-)用来命名化合物平面偏振光旋转的符号，(-)或L是指化合物是左旋的，前缀(+)或D是指化合物是右旋的。这些立体异构体的化学结构是相同的，但其立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50:50的对映体混合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。
依据起始物料和方法的选择，本发明化合物可以以可能的异构体中的一个或它们的混合物，例如外消旋体和非对应异构体混合物(这取决于不对称碳原子的数量)的形式存在。光学活性的(R)-或(S)- 异构体可使用手性合成子或手性试剂制备，或使用常规技术拆分。
所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。
本发明的“卤素”是指氟、氯、溴、碘。本发明的“卤代”是指被氟、氯、溴或碘取代。
本发明的“烷基”指直链或支链的饱和脂肪烃基团，优选含1至6个碳原子的直链或支链基团，进一步优选含有1至3个碳原子的直链或支链基团，非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基等。烷基可以是取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上。
本发明的“羰基”、“酰基”均指-C(O)-。
本发明的“磺酰基”是指-S(O) 2-。
本发明的“磺酰胺基”是指-S(O) 2NH-。
本发明的“卤代烷基”是指至少被一个卤素取代的烷基。
本发明的“羟基烷基”是指至少被一个羟基取代的烷基。
本发明的“烷氧基”是指-O-烷基。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、正丙氧基、异丙氧基、异丁氧基、仲丁氧基等。烷氧基可以是任选取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上。
本发明的“环烷基”是指环状的饱和烃基。合适的环烷基可以为取代或未取代的具有3-12个碳原子的单环、二环或三环饱和烃基，例如环丙基、环丁基、环戊基、环己基。
本发明的“杂环基”是指具有1至4个环杂原子(其中每个杂原子独立地选自氮、氧、硫、硼、磷以及硅)的3-至12-元非芳香族环系统的基团(“3-12元杂环基”)。在包含一个或多个氮原子的杂环基基团中，连接点可以是碳或氮原子，只要化合价许可。杂环基基团或者可以是单环的(“单环杂环基”)或者是融合的、桥联的或螺的环系统(例如二环系统(又称“二环杂环基”))并且可以是饱和的或可以是部分不饱和的。合适的杂环基包括但不限于哌啶基、氮杂环丁烷基、氮杂环丙烷基、四氢吡咯基、哌嗪基、二氢喹唑啉基、氧杂环丙基、氧杂环丁基、四氢呋喃基、四氢吡喃基、
Figure PCTCN2020120284-appb-000013
Figure PCTCN2020120284-appb-000014
等。杂环基的每个实例可以是任选取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上。
本发明的“芳基”是指可以包含单环或稠合多环的芳香体系，优选包含单环或稠合双环的芳香体系，其含有6个至12个碳原子，优选含有约6至约10个碳原子。合适的芳基包括但不限于苯基、萘 基、蒽基、芴基、茚满基。芳基可以是任选取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上。
本发明的“杂芳基”是指至少有一个碳原子被杂原子替代的芳基，优选由5-12个原子构成(5-12元杂芳基)，进一步优选由5-10个原子组成(5-10元杂芳基)，所述的杂原子为O、S、N。所述杂芳基包括但不限于咪唑基、吡咯基、呋喃基、噻吩基、吡唑基、噁唑基、噻唑基、异噁唑基、异噻唑基、噁二唑基、三唑基、四唑基、吲哚基、吡啶基、嘧啶基、哒嗪基、吡嗪基、三嗪基、异吲哚基、苯并吡唑基、苯并咪唑基、苯并呋喃基、苯并吡喃基、苯并噻吩基、苯并噁唑基、苯并噻唑基、苯并异噁唑基、苯并异噻唑基、喹啉基、异喹啉基、喹唑啉基、噌啉基、喹喔啉基、苯并噁嗪基、苯并噻嗪基、咪唑并吡啶基、嘧啶并吡唑基、嘧啶并咪唑基等。杂芳基可以是任选取代的或未取代的，当被取代时，取代基可以在任何可使用的连接点上。
本发明的“药学上可接受的盐”是指本发明化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性。
本发明的“溶剂化物”在常规意义上是指溶质(如活性化合物、活性化合物的盐)和溶剂(如水)组合形成的复合物。溶剂是指本领域的技术人员所知的或容易确定的溶剂。如果是水，则溶剂化物通常被称作水合物，例如半水合物、一水合物、二水合物、三水合物或其替代量等。
具有化学式(A)的化合物的体内作用可以部分地由在给予具有化学式(A)的化合物之后在人体或动物体内形成的一种或多种代谢物来发挥。如上所述，具有化学式(A)的化合物的体内作用也可以经由前体化合物(“前药”)代谢来发挥。本发明的“前药”是指在生物体中的生理条件下，由于与酶、胃酸等反应而转化成本发明化合物的化合物，即通过酶的氧化、还原、水解等转化成本发明化合物的化合物和/或通过胃酸等的水解反应等转化成本发明化合物的化合物等。
本发明的“结晶”是指其内部结构是在三维上规律地重复构成原子(或其集团)而形成的固体，有别于不具有这种规律的内部结构的无定形固体。
本发明的“药物组合物”是指包含任何一种本发明所述的化合物，包括对应的异构体、前药、溶剂化物、药学上可接受的盐或其化学的保护形式，和一种或多种可药用载体和/或另一种或多种药物的混合物。药用组合物的目的是促进化合物对生物体的给药。所述组合物通常用于制备治疗和/或预防由一种或多种激酶介导的疾病的药物。
本发明的“可药用载体”是指对有机体不引起明显刺激性和不干扰所给予化合物的生物活性和性质的载体，包含所有的溶剂、稀释剂或其它赋形剂、分散剂、表面活性剂等渗剂、增稠剂或乳化剂、防腐剂、固体粘合剂、润滑剂等。除非任何常规载体介质与本发明化合物不相容。可以作为药学上可接受的载体的一些实例包括，但不限于糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，如羧甲基纤维素钠、以及纤维素和乙酸纤维素；麦芽、明胶等。
本发明的“赋形剂”指加入到药用组合物中以进一步促进给予化合物的惰性物质。赋形剂可以包括碳酸钙、磷酸钙、多种糖类和多种类型的淀粉、纤维素衍生物、明胶、植物油、聚乙二醇。
本发明的“PRMT5”可以是野生型PRMT5或PRMT5的任何突变体或变体，PRMT5的突变体或变体含有一个或多个突变(例如，保守取代)。
具体实施方式
下面结合实施例对本发明作进一步详细阐述，但本发明不限于这些实施例。以下实施例中使用的材料如无特殊说明均为商购获得。
实施例1：(R)-7’-((1-乙酰基哌啶-4-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟基丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮
Figure PCTCN2020120284-appb-000015
步骤1:5-溴-2-氯异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000016
将5-溴-2-氯异烟酸(10g，42.292mmol)溶于甲醇中，在0℃下加入9.2mL氯化亚砜(9.2mL,126.8mmol)。滴加完毕后，将反应液加热至80℃，反应10小时后，LCMS监测反应完全，加入乙酸乙酯(200mL)，饱和碳酸钠水溶液调至PH约8左右。分离有机相和水相后，水相用乙酸乙酯萃取3次，合并有机相用饱和氯化钠洗一次，用无水硫酸钠干燥有机相后浓缩得到标题化合物。LC-MS m/z:[M+H] +＝250.0,252.0。
步骤2:2-氯-5-甲基异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000017
将5-溴-2-氯异烟酸甲酯(10g，51.8mmol)和四(三苯基膦)钯(4.6g，3.98mmol)加入到无水N,N-二甲基甲酰胺(DMF，25mL)中。在氩气保护下加入三甲基铝(2M甲苯溶液，51.9mmol，25.95mL)，加入完毕后，将反应体系升温至80℃反应并搅拌过夜。反应完全后，将反应液倒入冰水中(500mL)淬灭，加入乙酸乙酯(500mL)萃取。有机相用饱和氯化钠水溶液水洗，无水硫酸钠干燥后减压蒸去溶剂。柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝186.0。
步骤3:5-(溴甲基)-2-氯异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000018
将2-氯-5-甲基异烟酸甲酯(5.14g，27.69mmol)溶于四氯化碳(50mL)中，加入N-溴代丁二酰亚胺(4.93g，27.7mmol)，过氧化苯甲酰(1g，4.13mmol)，80℃下搅拌反应过夜。LCMS监测反应完全，减压除去有机溶剂后，加入水和乙酸乙酯萃取，有机相用饱和氯化钠洗2次，无水硫酸钠干燥，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝264.0，266.1。
步骤4:2-氯-5-(氰基甲基)异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000019
将5-(溴甲基)-2-氯异烟酸甲酯(5.29g，20.1mmol)溶于无水乙腈(35mL)中，加入三甲基氰硅烷(2.20g，22.17mmol)，降温至-10℃。在该反应液中缓慢滴加四丁基氟化铵(7.88g，30.14mmol)，滴加完毕后升温至0℃反应3小时。LCMS监测反应完全，反应液用硅藻土抽滤后，加水，用乙酸乙酯萃取，有机相用饱和氯化钠洗2次，无水硫酸钠干燥，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝211.0。
步骤5:2-氯-5-(1-氰基环丙基)异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000020
将2-氯-5-(氰基甲基)异烟酸甲酯(1.5g，7.12mmol)溶于无水二甲基亚砜(DMSO，25mL)中，加入1,2-二溴乙烷(2.0g,10.65mmol)，碳酸铯(4.64g,14.24mmol)。在N 2保护，70℃反应1个小时。LCMS监测反应完全后，冷却至室温，加水，用乙酸乙酯萃取，有机相用层用饱和氯化钠洗2次，无水硫酸钠干燥，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝237.0。
步骤6:2-((1-(叔丁氧基羰基)哌啶-4-基)氨基)-5-(1-氰基环丙基)异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000021
将2-氯-5-(1-氰基环丙基)异烟酸甲酯(0.1g，0.38mmol)溶于无水四氢呋喃(THF，3mL)中，加入4-氨基哌啶-1-羧酸叔丁酯(114.1mg,0.57mmol)，碳酸铯(247.6mg,0.76mmol)，氯(2-二环己基膦基-2’,6’-二异丙基-1,1'-联苯)[2-(2-氨基乙基苯基]钯(II)(31.82mg,0.04mmol)，N 2保护，70℃反应过夜。LCMS监测反应完全，加水，用乙酸乙酯萃取，有机相用饱和氯化钠洗2次，无水硫酸钠干燥，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝401.2.
步骤7:2-((1-乙酰基哌啶-4-基)氨基)-5-(1-氰基环丙基)异烟酸甲酯的制备
Figure PCTCN2020120284-appb-000022
将2-((1-(叔丁氧基羰基)哌啶-4-基)氨基)-5-(1-氰基环丙基)异烟酸甲酯(162mg，0.405mmol)溶于无水二氯甲烷(4mL)中，冰浴至0℃缓慢滴加三氟乙酸(0.4mL，5.38mmol)，滴加完毕后撤去冰浴，室温搅拌过夜。减压蒸去有机溶剂后，加入无水二氯甲烷(5mL)。在该溶液中加入N,N-二异丙基乙胺至pH为7-8后再补加N,N-二异丙基乙胺(52.3mg,0.405mmol)，并加入乙酸酐(49.0mg,0.48mmol)，室温搅拌2小时。LCMS监测反应完全后，在反应液中加水淬灭后，并用二氯甲烷萃取，有机相用饱和氯化钠洗2次，无水硫酸钠干燥，浓缩后得到标题化合物。LC-MS m/z:[M+H] +＝343.2。
步骤8:7’-((1-乙酰基哌啶-4-基)氨基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的制备
Figure PCTCN2020120284-appb-000023
将2-((1-乙酰基哌啶-4-基)氨基)-5-(1-氰基环丙基)异烟酸甲酯(311.0mg，0.91mmol)溶于甲醇中，冰浴至0℃加入六水合二氯化钴(865.4mg，3.64mmol)，分批缓慢加入硼氢化钠(207.26mg，5.48mmol)，0℃下搅拌0.5小时后常温搅拌1小时。LCMS监测反应未完全，反应液加入饱和氯化铵水溶液淬灭后，乙酸乙酯萃取，有机相用饱和氯化钠洗2次，无水硫酸钠干燥，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝315.2
步骤9:(R)-7’-((1-乙酰基哌啶-4-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟基丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮的制备
Figure PCTCN2020120284-appb-000024
将7’-((1-乙酰基哌啶-4-基)氨基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮(45mg，0.175mmol)溶于无水DMF(5mL)中，在氮气保护下降温至0℃并缓慢加入NaH(11.45mg,0.48mmol)，0.5小时后加入(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉(32.53mg,0.172mmol)，室温搅拌过夜。LCMS监测反应未完全，补加10mg NaH，33mg(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉，继续反应2小时。LCMS监测反应完全，减压蒸去有机溶剂，柱层析分离得标题化合物。 1H NMR(400MHz,DMSO-d 6)δ8.12-8.23(m,1H),7.10-7.16(m,4H),6.65-6.80(m,1H),5.50-5.60(m,1H),4.72(s,1H),4.12-4.25(m,1H),3.94-4.10(m,1H),3.71-3.87(m,2H),3.62(s,2H),3.42-3.54(m,2H),3.36-3.41(m,1H),3.11-3.25(m,2H),2.75-2.94(m,3H),2.65-2.80(m,2H),2.36-2.48(m,2H),2.05(s,3H),1.80-2.00(m,2H),1.15-1.36(m,2H),0.80-0.97(m,4H).LC-MS m/z:[M+H] +＝504.3.
实施例2：(R)-7’-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2’-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2’,3’-二氢-1’H-螺[环丙烷-1,4’-[2,6]萘啶]-1’-酮
Figure PCTCN2020120284-appb-000025
制备方法同实施例1的制备方法，不同的是将实施例1步骤6中的4-氨基哌啶-1-羧酸叔丁酯替换成6-氨基-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯，得标题化合物。 1H NMR(400MHz,DMSO-d 6)δ7.71(s,1H),7.00-7.15(m,4H),6.87(m,1H),4.78(s,1H),4.16(s,1H),4.04-4.10(m,3H),3.88(m,1H),3.70-3.80(m,3H),3.61-3.63(m,2H),2.80-2.83(m,2H),2.70-2.72(m,2H),2.18-2.20(m,1H),1.98-2.02(m,4H),1.72(s,3H),1.24-1.30(m,4H),0.94-1.0(m,4H).LC-MS m/z:[M+H] +＝516.3.
按照本发明实施例1的合成方法，利用不同市售原料合成实施例3-9的化合物，这些化合物的表征参数如表1所示：
表1
Figure PCTCN2020120284-appb-000026
Figure PCTCN2020120284-appb-000027
实施例10：(R)-7'-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮
Figure PCTCN2020120284-appb-000028
步骤1:2-(1-氰基环丙基)苯甲酸甲酯的制备
Figure PCTCN2020120284-appb-000029
将氢化钠(4.46g，111mmol)置于三颈瓶中，0℃下加入20mL无水N,N-二甲基甲酰胺，搅拌5min，缓慢加入2-(氰基甲基)苯甲酸甲酯(7.80g，44.6mmol)的N,N-二甲基甲酰胺溶液(80mL)，0℃下搅拌30min，然后缓慢滴加1,2-二溴乙烷(10.0g，53.5mmol)，滴毕后移至室温反应2h。反应完全后，加入20mL饱和氯化铵溶液淬灭，乙酸乙酯(30mL×3)萃取，合并有机相，水洗(10mL×2)，饱和食盐水洗，经无水硫酸钠干燥，减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝202.
步骤2：2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000030
将2-(1-氰基环丙基)苯甲酸甲酯(13.3g，66.1mmol)溶解于150mL无水乙醇中，加入六水合氯化钴(31.5g，132mmol)，0℃下分批加入硼氢化钠(7.54g，198mmol)，移至室温反应1h后80℃反应2h。反应完全后，抽滤，滤液减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝174.
步骤3：7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000031
将2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(6.33g，36.6mmol)溶解于冰浴下冷却的浓硫酸(30 mL)中，-10℃下分批加入硝酸钾(3.69g，36.6mmol)，移至室温反应1h。反应完全后，倒入冰水中，有固体析出，抽滤，滤饼干燥得到标题化合物。LC-MS m/z:[M+H] +＝219.
步骤4:(R)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000032
将7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(300mg,1.38mmol)溶解于10mL DMSO中，加入碳酸铯(900mg,2.75mmol)后室温搅拌0.5h，加入(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉(520mg,2.75mmol)，100℃反应3h。反应完全后，加入水50mL，乙酸乙酯(30mL×3)萃取，无水硫酸钠干燥，减压蒸除溶剂，经柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝408.
步骤5:(R)-7'-氨基-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000033
将(R)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(500mg,1.23mmol)，还原铁粉(274mg,4.9mmol)，氯化铵(260mg,4.90mmol)，加入10mL乙醇和2mL水的混合溶液中，75℃反应2h。反应完全后，过滤，滤液减压蒸除溶剂，加入二氯甲烷20mL，过滤，滤液减压蒸除溶剂，得到标题化合物。LC-MS m/z:[M+H] +＝378.
步骤6:(R)-6-((2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-1'-氧代-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-7'-基)氨基)-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯的制备
Figure PCTCN2020120284-appb-000034
将(R)-7'-氨基-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(200mg,0.526mmol)，6-氧代-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯(65.06mg,0.582mmol)溶解于5mL甲醇中，加入一滴冰醋酸，室温搅拌0.5h，冰浴滴加吡啶硼烷(74.0mg,0.796mmol)，室温搅拌0.5h。反应完全后，减压蒸除溶剂，经柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝573.
步骤7:(R)-7'-((2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000035
将(R)-6-((2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-1'-氧代-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-7'-基)氨基)-2-氮杂螺[3.3]庚烷-2-羧酸叔丁酯(180mg，0.315mmol)溶解于10mL氯化氢甲醇(4.0mol/L)溶液中，室温搅拌0.5h。反应完全后，减压蒸除溶剂，得到标题化合物。LC-MS m/z:[M+H] +＝473.
步骤8:(R)-7'-((2-乙酰基-2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000036
将(R)-7'-((2-氮杂螺[3.3]庚-6-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(140mg,0.297mmol)加入10mL二氯甲烷中，加入三乙胺至溶液呈弱碱性，完全溶解后，加入醋酸酐(61.0mg,0.593mmol)，室温搅拌15min。反应完全后，减压蒸除溶剂，柱层析分离得标题化合物。 1H NMR(400MHz,MeOD)δ7.74(s,1H),7.31(m,1H),7.14-7.12(m,4H),7.06-7.00(m,1H),4.25-4.23(m,1H),4.05-3.83(m,5H),3.70-3.61(m,2H),3.45-3.37(m,2H),3.05-2.90(m,4H),2.87-2.72(m,2H),2.02-1.98(m,2H),1.93(s,3H),1.84-1.81(m,1H),1.79-1.76(m,3H),1.34-1.02(m,4H).LC-MS m/z:[M+HCO 2 -] -＝559.
比较例1：(S)-6-((1-乙酰基哌啶-4-基)氨基)-N-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟丙基)嘧啶-4-甲酰胺
Figure PCTCN2020120284-appb-000037
参照WO2014/100719(PCT/US2013/077235)中化合物208公开的方法制备上式代表的化合物A，并通过氢谱和质谱鉴定。
比较例2：(R)-7'-((1-乙酰基哌啶-4-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟基丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮
Figure PCTCN2020120284-appb-000038
步骤1:2-(1-氰基环丙基)苯甲酸甲酯的制备
Figure PCTCN2020120284-appb-000039
将氢化钠(4.46g，111mmol)置于三颈瓶中，0℃下加入20mL无水N,N-二甲基甲酰胺，搅拌5min，缓慢加入2-(氰基甲基)苯甲酸甲酯(7.80g，44.6mmol)的N,N-二甲基甲酰胺溶液(80mL)，0℃下搅拌30min，然后缓慢滴加1,2-二溴乙烷(10.0g，53.5mmol)，滴毕后移至室温反应2h。反应完全后，加入20mL饱和氯化铵溶液淬灭，乙酸乙酯(30mL×3)萃取，合并有机相，水洗(10mL×2)，饱和食盐水洗，经无水硫酸钠干燥，减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝202.
步骤2：2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000040
将2-(1-氰基环丙基)苯甲酸甲酯(13.3g，66.1mmol)溶解于150mL无水乙醇中，加入六水合氯化钴(31.5g，132mmol)，0℃下分批加入硼氢化钠(7.54g，198mmol)，移至室温反应1h后80℃反应2h。反应完全后，抽滤，滤液减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝174.
步骤3：7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000041
将2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(6.33g，36.6mmol)溶解于冰浴下冷却的浓硫酸(30mL)中，-10℃下分批加入硝酸钾(3.69g，36.6mmol)，移至室温反应1h。反应完全后，倒入冰水中，有固体析出，抽滤，滤饼干燥得到标题化合物。LC-MS m/z:[M+H] +＝219.
步骤4：7'-氨基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000042
将7'-硝基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(4.57g，210mmol)溶解于40mL乙醇和10mL水的混合溶剂中，加入铁粉(2.93g，52.4mmol)，氯化铵(3.33g，62.9mmol)，80℃反应2h。反应完全后，抽滤，滤液减压浓缩，二氯甲烷(30mL×3)萃取，合并有机相，经无水硫酸钠干燥，减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝189.
步骤5：7'-((1-乙酰基哌啶-4-基)氨基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮的制备
Figure PCTCN2020120284-appb-000043
将7'-氨基-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(1.55g，8.24mmol)，1-乙酰基-4-哌啶酮(1.16g，8.24mmol)溶解于20mL甲醇中，加入冰醋酸(0.472mL，8.24mmol)，室温反应2h。然后0℃下缓慢滴加硼烷吡啶络合物(1.24mL，12.4mmol)，移至室温2h。反应完全后，用饱和碳酸氢钠溶液调pH至碱性，二氯甲烷(20mL×3)萃取，合并有机相，经无水硫酸钠干燥，减压蒸除溶剂，柱层析分离得到标题化合物。LC-MS m/z:[M+H] +＝314.
步骤6：(R)-7'-((1-乙酰基哌啶-4-基)氨基)-2'-(3-(3,4-二氢异喹啉-2(1H)-基)-2-羟基丙基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(化合物B)的制备
Figure PCTCN2020120284-appb-000044
将7'-((1-乙酰基哌啶-4-基)氨基)-2',3'-二氢-1'H-螺[环丙烷-1,4'-异喹啉]-1'-酮(1.56g，4.98mmol)溶解于10mL无水N,N-二甲基甲酰胺溶液中，0℃下分批加入氢化钠(0.299g，7.48mmol)，搅拌30min，加入(R)-2-(环氧乙烷-2-基甲基)-1,2,3,4-四氢异喹啉(0.942g，4.98mmol)的N,N-二甲基甲酰胺溶液(5mL)，移至室温反应过夜。反应完全后，加入8mL饱和氯化铵溶液淬灭，减压蒸除溶剂，经柱层析分离，经prep-HPLC(制备液相)制备分离得到标题化合物。 1H NMR(400MHz,DMSO-d 6)δ7.16-7.23(m,1H),7.05-7.16(m,3H),6.99-7.05(m,1H),6.65-6.77(m,2H),5.57(d,1H),4.75(s,1H),4.19(d,1H),3.94-4.10(m,1H),3.71-3.87(m,2H),3.62(s,2H),3.42-3.54(m,2H),3.34-3.41(m,1H),3.11-3.25(m,2H),2.75-2.94(m,3H),2.62-2.74(m,2H),2.36-2.48(m,2H),2.00(s,3H),1.79-1.96(m,2H),1.12-1.36(m,2H),0.75-0.97(m,4H).LC-MS m/z:[M+H] +＝503.
实验例1 化合物体外激酶活性评价
1.实验材料
化合物：以上实施例制备的本发明的化合物，每个化合物用DMSO配制成10mM母液，最终稀释为10个浓度进行检测，终浓度为10000.00nM、3333.33nM、1111.11nM、370.37nM、123.46nM、41.15nM、13.72nM、4.57nM、1.52nM、0.51nM。
试剂与耗材：PRMT5，购自于Active Motif公司，货号为31921；多肽底物H4(1-21)S1ac，购自于吉尔生化(上海)有限公司，货号为342095；[ 3H]-SAM，购自于PerkinElmer公司，货号为NET155V001MC；SAM，购自于Sigma，货号为A7007-100MG；SAH，购自于Sigma公司，货号为A9384-25MG；DTT，购自于生工生物工程(上海)股份有限公司，货号为A620058-0005。Corning-3657，购自于Corning公司，货号为3657；Echo Qualified 384-Well，购自于Labcyte公司，货号为P-05525； FlashPlate，购自于Perkin Elmer，货号为SMP410J001PK。
仪器：闪烁计数仪，购自于PerkinElmer公司，型号为MicroBeta2；超声波纳升液体处理系统，购自于Labcyte公司，型号为Echo 550。
2.实验方法
2.1.反应缓冲液和反应终止液配制：1倍反应缓冲液体成分为10mM Tris-HCl,pH 8.0；0.01％Tween-20；1mM DTT。反应终止液成分为125μM的 3H-SAM溶液。
2.2 化合物配制
2.2.1 化合物稀释
化合物用100％DMSO溶解成10mM母液，再将化合物在Echo384孔板上稀释到所需要的浓度。
2.2.2 转移化合物到384反应板
用Echo550仪器从上述稀释好Echo384孔板中转移250nL化合物到384孔反应板中。
2.3 酶学反应
2.3.1 配制1.67倍酶溶液
将PRMT5加入1倍反应缓冲液，形成1.67倍酶溶液。
2.3.2 配制2.5倍的底物溶液
将多肽底物和[ 3H]-SAM加入1倍反应缓冲液，形成2.5倍底物溶液(终浓度分别为100nM和250nM)。
2.3.3 向384孔板中加入酶溶液
向384孔反应板孔中加入15μL的1.67倍酶溶液。对于无酶活对照孔，用15μL的1倍反应缓冲液替代酶溶液。1000rpm离心1min，室温下孵育15分钟。
2.3.4 向384孔板中加入底物溶液启动酶学反应
向384孔反应板每孔中加入10μL的2.5倍底物溶液。1000rpm离心1min。25℃反应60分钟。
2.3.5 酶学反应的终止
向384孔反应板每孔中加入5μL的反应终止液终止反应。从试验板中每孔取25uL转移到Flashplate中，在室温下放置1h。然后用0.1％的Tween-20溶液洗Flashpate板3次。
2.4 MicroBeta 2读取数据
2.4 抑制率计算
从Microbeta 2上复制数据。把数据转化成抑制率数据。其中最大值是指DMSO对照的转化率，最小值是指无酶活对照的转化率。抑制率(％)＝(最大值-样本值)/(最大值-最小值)×100％。
将数据导入GraphPad，并使用“log(inhibitor)vs.response--Variable slope”进行曲线拟合，得到IC 50。部分化合物的IC 50结果见表2。
表2
Figure PCTCN2020120284-appb-000045
实验例2 化合物体外细胞活性评价
1.实验材料
受试化合物：以上实施例制备的本发明的化合物，每个化合物用DMSO配制成10mM母液，最终稀释为8个浓度进行检测，Z-138细胞实验的化合物终浓度为33333.00nM、6666.60nM、1333.32nM、266.66nM、53.33nM、10.67nM、2.13nM、0.43nM，MDA-MB-468和NCI-H358细胞实验化合物终浓度为50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。
人套细胞淋巴瘤细胞Z-138、三阴性乳腺癌细胞MDA-MB-468、人非小细胞肺癌细胞NCI-H358购于美国典型培养物保藏中心(ATCC)。
试剂：Iscove's Modified Dulbecco's Medium(IMEM培养基)，货号为ATCC 30-2005；Leibovitz's L-15Medium(L-15培养基)，货号为Gibco 11415-064；1640培养基，货号为Gibco 22400089；马血清，货号为Gibco 16050122；Fetal Bovine Serum，货号为Gibco 10099-141；青链霉素，货号为Gibco15140-122；丙酮酸钠，货号为Gibco 11360070；CellTiter-Glo Luminescent Cell Viability Assay，货号为Promega G7571。CCK-8增殖抑制检测试剂盒，货号为KeyGEN KGA317。
2.实验方法
2.1 细胞复苏：
2.1.1 Z-138细胞复苏：从液氮罐中取出Z-138细胞冻存管置于37℃水浴锅中，轻轻摇动使其尽快解冻。解冻后取出冻存管，用酒精棉球消毒后旋开盖子，吸出细胞液注入离心管，并加入1mL含10％马血清的完全培养基，混匀后置于离心机中，1000rpm，离心5min。之后弃上清液，加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃，5％CO 2、95％潮湿空气的CO 2培养箱中培养。
2.1.2 MDA-MB-468细胞复苏：从液氮罐中取出MDA-MB-468细胞冻存管置于37℃水浴锅中，轻轻摇动使其尽快解冻。解冻后取出冻存管，用酒精棉球消毒后旋开盖子，吸出细胞液注入离心管，并加入1mL含10％FBS的L-15培养基，混匀后置于离心机中，1000rpm，离心5min。之后弃上清液，加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃，95％ 潮湿空气的无CO 2培养箱中培养。
2.1.3 NCI-H358细胞复苏：从液氮罐中取出NCI-H358细胞冻存管置于37℃水浴锅中，轻轻摇动使其尽快解冻。解冻后取出冻存管，用酒精棉球消毒后旋开盖子，吸出细胞液注入离心管，并加入1mL含10％FBS的1640培养基，混匀后置于离心机中，1000rpm，离心5min。之后弃上清液，加入完全培养基反复吹打至细胞完全吹散、重悬。以适宜浓度接种于培养皿中。置37℃，5％CO 2，95％潮湿空气的CO 2培养箱中培养。
2.2 细胞培养和传代：
2.2.1 Z-138细胞培养和传代：细胞生长至约80-90％，将培养基(IMDM培养基+10％马血清+1％青链霉素)转移至15mL离心管中，1000rpm，离心5min。去除上清，用完全培养基重悬细胞，按所需密度接种于培养皿中，置于37℃、5％CO 2、95％潮湿空气的培养箱中培养，视细胞生长情况每2-3天补一次培养液或进行传代。
2.2.2 MDA-MB-468细胞培养和传代：细胞生长至约80-90％，将培养基(Leibovitz's L-15Medium培养基+10％FBS+1％青链霉素)转移至15mL离心管中，1000rpm，离心5min。去除上清，用完全培养基重悬细胞，按所需密度接种于培养皿中，置于37℃、95％潮湿空气的无CO 2培养箱中培养，视细胞生长情况每2-3天换一次培养液或进行传代。
2.2.3 NCI-H358细胞培养和传代：细胞生长至约80-90％，将培养基(1640培养基+10％FBS+1％青链霉素+1mM丙酮酸钠)转移至15mL离心管中，1000rpm，离心5min。去除上清，用完全培养基重悬细胞，按所需密度接种于培养皿中，置于37℃、5％CO 2、95％潮湿空气的CO 2培养箱中培养，视细胞生长情况每2-3天换一次培养液或进行传代。
2.3 实验步骤：
实验第一天：
Z-138细胞传代后以1000个/孔的密度重悬于完全培养基中，接种于96孔培养板内：96孔板最外面一圈36个孔以200μL PBS填充，以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大；内部60个孔的最左列为空白孔，不加细胞，以等体积的PBS填充；其余的54个孔以排枪进行细胞铺板，每孔为含对应细胞的100μL培养基，铺板完成后，拍打96孔板使细胞均匀悬浮，放入5％CO 2培养箱内37℃培养24h。
MDA-MB-468细胞传代后以对应的密度重悬于完全培养基中，2000个/孔，接种于96孔培养板内：细胞外面一圈以200μL PBS填充，以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大；内部60个孔的最左列为空白孔，不加细胞，以等体积的PBS填充；其余的54个孔以排枪进行细胞铺板，每孔100μL，放入无CO 2培养箱内37℃培养24h。
NCI-H358细胞传代后以对应的密度重悬于完全培养基中，1000个/孔，接种于96孔培养板内： 细胞外面一圈以200μL PBS填充，以防边缘培养基蒸发较快导致内部板孔的培养条件差异过大；内部60个孔的最左列为空白孔，不加细胞，以等体积的PBS填充；其余的54个孔以排枪进行细胞铺板，每孔100μL，放入5％CO 2培养箱内37℃培养24h。
实验第二天：
Z-138细胞在原培养基(100μL)的基础上，加入50μL的(3×)药物，每个浓度组设置两个复孔，继续放入CO 2培养箱培养7天。化合物配制如下：提前称取化合物1-2mg，使用DMSO配置成10mM母液。使用完全培养基稀释药物，药物终浓度以33333.00nM为起始最高浓度，按1:4梯度依次稀释至7个浓度梯度：6666.60nM、1333.32nM、266.66nM、53.33nM、10.67nM、2.13nM、0.43nM。①取10mM的母液1:4稀释成对应的药液，共8个浓度(10μL母液+40μL DMSO)；②取5μL①的药物加入的495μL完全培养基，配制成对应的浓度(3×)(稀释100倍)。
MDA-MB-468细胞在原培养基(100μL)的基础上，加入100μL的(2×)药物，每个浓度组设置两个复孔，继续放入无CO 2培养箱培养7天。化合物配制如下：提前称取化合物1-2mg，使用DMSO配置成10mM母液。使用完全培养基稀释药物，药物终浓度以50000nM为起始最高浓度，按1:4梯度依次稀释至7个浓度梯度：50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。①取10mM的母液1:4稀释成对应的药液，共8个浓度(10μL药液+40μL DMSO)；②取5μL①的药物加入的495μL完全培养基，配制成对应的浓度(2×)(稀释100倍)。
NCI-H358细胞在原培养基(100μL)的基础上，加入100μL的(2×)药物，每个浓度组设置两个复孔，继续放入5％CO 2培养箱培养7天。化合物配制如下：提前称取化合物1-2mg，使用DMSO配置成10mM母液。使用完全培养基稀释药物，药物终浓度以50000nM为起始最高浓度，按1:4梯度依次稀释至7个浓度梯度：50000nM、10000nM、2000nM、400nM、80nM、16nM、3.2nM、0.64nM。①取10mM的母液1:4稀释成对应的药液，共8个浓度(10μL药液+40μL DMSO)；②取5μL①的药物加入的495μL完全培养基，配制成对应的浓度(2×)(稀释100倍)。
实验第八天：
Z-138细胞在药物处理7天后，提前30min将CellTiter-Glo Luminescent Cell Viabillity Assay取出，平衡至室温。空白孔将PBS吸弃，加入150μL的完全培养基，然后空白孔、给药孔和DMSO孔都加入75uL的Celltiter-Glo reagent，室温震荡2min。继续室温孵育10min后，每孔各吸取180μL转移至不透明白板，去除气泡后，检测化学发光信号，震荡，Read进样检测条件为500ms。根据酶标仪导出的A.U.值，计算每个孔相对于溶剂对照孔的抑制率：Inhibition(％)＝100-(A.U. 实验孔–A.U. 空白孔)/(A.U. 溶剂对照孔-A.U. 空白孔)*100。根据不同药物浓度及其所对应的抑制率，使用GraghPad 5.0软件进行IC 50曲线绘制，分析数据，得出最终IC 50值，实验结果见表3。
MDA-MB-468和NCI-H358细胞药物处理7天后吸弃孔内培养基，加入100μL已加入CCK-8的 完全培养基(CCK-8：完全培养基＝1:10)，第一竖排PBS作为空白对照孔，同步加入100μL的CCK-8，然后放入培养箱培养40min-2h左右，根据CCK-8的显色深浅决定最佳检测时间(DMSO组的OD值在1.0左右最佳)。待CCK-8显色至橙色，并且出现肉眼可分辨的一定梯度，将96孔板从培养箱中取出，置于室温中平衡5-10分钟；打开酶标仪软件，调整好检测参数，检测450nm处的吸光度(OD值)；将培养板的盖子取下，直接将培养板水平放置于板槽内，开始读数；读数完成后，保存程序，导出数据，关闭软件和电脑。根据酶标仪导出的OD值，计算每个孔相对于溶剂对照孔的抑制率：Inhibition(％)＝100-(OD 实验孔–OD 空白孔)/(OD 溶剂对照孔-OD 空白孔)*100。根据不同药物浓度及其所对应的抑制率，使用GraghPad 5.0软件进行IC 50曲线绘制，分析数据，得出最终IC 50值，实验结果见表3。
表3
Figure PCTCN2020120284-appb-000046
从以上实验可以看出，本发明的化合物对人套细胞淋巴瘤细胞Z-138、三阴性乳腺癌细胞MDA-MB-468、人非小细胞肺癌细胞NCI-H358均表现出了良好的抑制活性，非常有希望成为淋巴瘤、三阴性乳腺癌、非小细胞肺癌治疗剂。
实验例3 电生理手动膜片钳检测化合物对hERG钾通道的作用
hERG钾离子通道是药物安全筛查的标准。hERG钾离子通道被阻滞会导致心脏中毒和心室复极延长，严重时可能会导致猝死。具有hERG钾通道抑制作用的药物可能是临床用药的潜在祸患。因此，化合物对hERG钾离子通道抑制作用弱则安全性高。药物导致的QT间期延长与致命性室性心律失常和猝死的危险性增加有关。
1实验材料
主要试剂：青霉素-链霉素溶液(100×)、DMEM/F12购自Gibco公司；胎牛血清购自PAA公司；DMSO、EGTA、MgATP购自Sigma公司；KCl、CaCl 2·2H 2O、MgCl 2·6H 2O、NaCl购自Sinopharm公司；葡萄糖购自General-reagent公司；HEPES购自Solarbio公司；奎尼丁购自aladdin公司。
仪器：TI-S-FLU显微镜购自Nikon公司；SMZ-140/143显微镜购自Motic公司；EPC-10放大器、Patchmaster V2X60购自HEKA公司；TMC-36防震台购自TMC公司；MP-225、MPC-200操控器、ROE-200微操纵仪、P-97电极拉制仪购自Sutter公司；VC3-8PP灌流给药系统购自ALA公司。
2实验方法
测试溶剂配制：细胞外液配制(mM)：137NaCl、4KCl、1.8CaCl 2、1MgCl 2、10葡萄糖和10HEPES(pH 7.4)；细胞内液配制(mM)：130KCl、1MgCl 2、5EGTA、5MgATP和10HEPES(pH 7.2)；阴性对照配制：细胞外液+0.3％DMSO；阳性对照：奎尼丁。
化合物处理：称取化合物溶解于DMSO中，配制10mM母液，用DMSO稀释母液成次级母液，浓度为3.3，1.1，0.37，0.12mM。取母液和次级母液各90μL稀释至30mL细胞外液中，用于电生理检测。化合物的终浓度为30，10，3.3，1.1，0.37μM，DMSO的终浓度为3：1000。
稳转细胞培养：细胞株来源于过表达hERG钾离子通道HEK-293细胞，是在纽约大学医学院Mohamed Boutjdir博士实验室提供技术支持后科瑞斯生物与之合作建立并验证。细胞在37℃、5％CO 2培养箱中培养。当细胞密度达培养皿80％时，先用磷酸盐缓冲液(PBS)预清洗，然后用胰蛋白酶/EDTA消化细胞2-3min，加入细胞培养基停止消化，轻轻把细胞吹下来并转移到离心管中，1000rpm*3min，上清液弃置，加入细胞培养基，轻轻吹打将细胞混匀，随后转移到培养皿中进行传代培养，或将细胞滴于圆形玻片之上并置于培养皿中待细胞贴壁用于实验。
细胞培养基组成：DMEM、15％胎牛血清和1％100×青霉素-链霉素。
电生理手动膜片钳系统实验：将稳转的细胞接种于玻片上，细胞密度低于50％，培养过夜。将实验用细胞转移到一个嵌于倒置显微镜平台的约1mL的浴槽中，灌流细胞外液，灌流速度为2.7mL/min。稳定5分钟后即可开始实验。采用HEKA EPC-10膜片钳放大器和PATCHMASTER采集系统记录膜电流。所有实验均在室温(22-24℃)下完成。实验中使用P-97微电极拉制仪拉直电极(BF150-110-10)。电极内径为1-1.5mm，充满内液后的入水电阻为2-4MΩ。hERG钾通道的电生理刺激方案，是首先将膜电压钳制在-80mV，给予细胞持续2s、+20mV电压刺激，激活hERG钾通道，再复极化至-50mV、持续5s，产生外向尾电流，刺激频率每15s一次。电流值为尾电流的峰值。实验中采用全细胞记录模式记录通道电流。首先灌流细胞外液(大约每分钟2mL)并持续记录，并等待电流稳定(5分钟内电流衰减(Run-Down)小于5％)，此时尾电流峰值即为对照电流值。接着灌流含待测药物的细胞外液并持续记录直到药物对hERG电流的抑制作用到达稳定状态，此时尾电流峰值即为加药后电流值。稳定状态的标准以最近的连续3个电流记录线是否重合来判断。达到稳定态势以后如果以细胞外液灌流冲洗后hERG电流回复或接近加药物之前的大小，则可以继续灌流测试其它浓度或药物。30μM奎尼丁被用于实验中作为阳性对照以保证所使用的细胞反应正常。
3参数分析和数据分析统计
本研究通过测量对照组与药物处理组的电流最大值，计算处理组最大电流值所占对照组最大电流值的比率，评估待测化合物在测试浓度下对hERG钾离子通道的作用效果(Mean±SE)。
实验数据使用PATCHMASTER V2X60采集，并采用Origin 8.5软件以及Microsoft Excel进行分析和统计。实验结果见表4。
表4
Figure PCTCN2020120284-appb-000047
从以上实验可以看出，本发明实施例1和2的化合物对hERG钾通道抑制作用轻，对心脏的毒性低，优于化合物B。
此外，化合物B在离体心脏实验中表现出显著延长QT间期，而本发明实施例1和2的化合物在离体心脏实验中显示对QT间期无影响。本发明的化合物具有更好的心脏安全性。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (10)

  1. 一种通式(A)所示的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，
    Figure PCTCN2020120284-appb-100001
    其中，
    Cy选自杂环基，所述杂环基可被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、烷基磺酰基、氨基酰基、烷基氨基酰基、双烷基氨基、烯基、炔基、卤代烷基酰基、羟基烷基酰基、环烷基酰基、杂环基酰基、环烷基、杂环基、芳基、杂芳基和氧代基团的基团取代。
  2. 根据权利要求1所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中通式(A)具有以下通式(I)的结构，
    Figure PCTCN2020120284-appb-100002
    其中，
    X选自O、S、C(R 1)(R 2)和N(R 3)，其中R 1、R 2、R 3各自独立地选自氢、卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基，且R 1和R 2与它们所结合的碳原子一起形成杂环基，其中所述的杂环基任选被一个或多个选自卤素、羟基、烷基、卤代烷基、羟基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基的基团取代；
    m为1、2、3或4；和
    n为0、1、2、3或4。
  3. 根据权利要求2所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中R 1、R 2、R 3各自独立地选自氢、卤素、羟基、C 1-6烷基、卤代C 1-6烷基、羟基C 1-6烷基、C 1-6烷氧基、卤代C 1-6烷氧基、羟基C 1-6烷氧基、硝基、羧基、氰基、氨基、单C 1-6烷基氨基、C 1-6烷基酰基氨基、C 1-6烷基酰基、氨基酰基、C 1-6烷基氨基酰基、双C 1-6烷基氨基和C 3-12环烷基。
  4. 根据权利要求2-3之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中R 1和R 2与它们所结合的碳原子一起形成3元-8元杂环基，其中所述杂环基进一步包含一个或多个N、O或S杂原子，且所述杂环基任选被一个或多个选自卤素、羟基、烷基、卤代烷基、羟 基烷基、烷氧基、卤代烷氧基、羟基烷氧基、硝基、羧基、氰基、氨基、单烷基氨基、烷基酰基氨基、烷基酰基、氨基酰基、烷基氨基酰基、双烷基氨基和环烷基的基团取代。
  5. 根据权利要求2-4之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中R 1和R 2与它们所结合的碳原子一起形成3元-6元杂环基，其中所述杂环基进一步包含一个或多个N、O或S杂原子，且所述杂环基任选被一个或多个选自卤素、羟基、C 1-6烷基、卤代C 1-6烷基、羟基C 1-6烷基、C 1-6烷氧基、卤代C 1-6烷氧基、羟基C 1-6烷氧基、硝基、羧基、氰基、氨基、单C 1-6烷基氨基、C 1-6烷基酰基氨基、C 1-6烷基酰基、氨基酰基、C 1-6烷基氨基酰基、双C 1-6烷基氨基和C 3-12环烷基的基团取代。
  6. 根据权利要求2-5之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中通式(I)具有以下通式(Ia)的结构，
    Figure PCTCN2020120284-appb-100003
    其中，X、m和n具有权利要求2-5中所述的定义。
  7. 根据权利要求2-6之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中
    Figure PCTCN2020120284-appb-100004
    选自
    Figure PCTCN2020120284-appb-100005
    Figure PCTCN2020120284-appb-100006
  8. 根据权利要求1所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药，其中所述化合物为选自以下的化合物：
    Figure PCTCN2020120284-appb-100007
  9. 一种药物组合物，其包含权利要求1至8之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药和可药用载体。
  10. 权利要求1-8之任一项所述的化合物或其异构体、药学上可接受的盐、溶剂化物、结晶或前药或权利要求9所述的药物组合物在制备用于治疗PRMT5介导的疾病的药物中的应用。
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