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WO2023016511A1 - 氮杂芳基化合物、其制备方法及应用 - Google Patents

氮杂芳基化合物、其制备方法及应用 Download PDF

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Publication number
WO2023016511A1
WO2023016511A1 PCT/CN2022/111668 CN2022111668W WO2023016511A1 WO 2023016511 A1 WO2023016511 A1 WO 2023016511A1 CN 2022111668 W CN2022111668 W CN 2022111668W WO 2023016511 A1 WO2023016511 A1 WO 2023016511A1
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Prior art keywords
alkyl
compound
independently
cancer
methyl
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PCT/CN2022/111668
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English (en)
French (fr)
Inventor
马世超
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上海青煜医药科技有限公司
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Publication of WO2023016511A1 publication Critical patent/WO2023016511A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the invention relates to an azaaryl compound, its preparation method and application.
  • PcG proteins are an important class of chromatin modifying enzymes. It regulates the transcription of genes by modifying chromatin, and plays an important role in the growth, differentiation and long-term cell memory of stem cells.
  • PcG proteins are mainly divided into two types of transcriptional repression complexes, namely PRC1 (Polycomb Repressive Complex 1) and PRC2 (Polycomb Repressive Complex 2).
  • PRC2 inhibits the expression of related genes by methylating the 27th lysine (H3K27) of histone 3 in chromatin.
  • the PRC2 protein complex is mainly composed of core proteins such as EZH2 (Enhancer of Zeste Homolog 2) (or its very similar homologous protein EZH1), EED (Embryonic ectoderm Development) and SUZ12 (Suppressor of Zeste 12).
  • EZH2 has enzyme catalytic activity, and can transfer the methyl group of the substrate SAM (S-adenosyl-L-methionine) to H3K27 through the SET (Su(var), E(Z), and Trithorax) protein domain, thereby One- to three-methylation modification of H3K27 is achieved.
  • the enzymatic activity of EZH2 also depends on other components of PRC2, such as EED proteins belonging to the WD40 repeat structural protein family.
  • EED proteins belonging to the WD40 repeat structural protein family The combination of EED and trimethylated H3K27Me3 can greatly promote the enzymatic function of EZH2 on the one hand, and on the other hand, it can also position the PCR2 complex on the chromatin that needs to be modified.
  • Abnormal function of PRC2, such as EZH2 overexpression or gain-of-function mutation, is associated with many clinical tumor diseases, including lung cancer, breast cancer, rectal cancer, prostate cancer, bladder cancer, pancreatic cancer, sarcoma, and lymphoma.
  • PRC2 is also associated with a variety of cellular immune functions, such as EZH2 involved in the regulation of lymphocyte activation, and can also cooperate with glycolysis to promote the response of T cells to tumor cells. Therefore, the development of small molecule inhibitors of PRC2 has important and broad drug development value.
  • EZH2 inhibitors currently entering the clinic include EPZ-6438 (Epizyme, clinical phase II), GSK2816126 (GSK, clinical phase I), and CPI-1205 (Constellation, clinical phase I) and so on. Although several EZH2 inhibitors have entered the clinical research stage, these inhibitors all contain a common pharmacophore of 2-pyridone. Moreover, in clinical treatment with existing EZH2 inhibitors, secondary mutations have begun to appear. EED inhibitors have an allosteric inhibitory effect on the enzyme function of EZH2, and can achieve the same or similar biological functions as EZH2.
  • EED inhibitors can overcome the drug resistance problem of EZH2 well, and on the other hand, EED inhibitors can be used in combination with EZH2 inhibitors to achieve a better synergistic effect. Therefore, it is very important to develop new EED inhibitors. meaning.
  • the technical problem to be solved by the present invention is that the EED inhibitors in the prior art have a single structure. Therefore, the present invention provides an azaaryl compound, its preparation method and application.
  • the azaaryl compound of the invention exhibits good inhibitory activity on tumor cells and has broad prospects for drug development.
  • the present invention provides a compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer, solvate or their isotope-labeled compounds:
  • X is N or CR 2 ;
  • R 4a1 , R 4a2 and R 4b are independently H or C 1-4 alkyl
  • R 4c is independently H, C 1-4 alkyl or -OC 1-4 alkyl
  • R 5 is independently C 1-4 alkyl-O-, C 1-4 alkyl, C 1-4 haloalkyl, -N(R 5b R 5b ), amino protected by N protecting group, fluorine, hydroxyl;
  • R 5b , R 6 and R 7 are independently H, C 1-4 alkyl, C 1-4 haloalkyl;
  • X, R 1 , R 3 and R 4 are as defined above.
  • R 1 , R 2 , R 3 and R 4 are independently halogen
  • said halogen is fluorine, chlorine, bromine or iodine, preferably chlorine.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 1-4 alkyl
  • said C 1-4 alkyl is methyl, ethyl, n-propyl , isopropyl , n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 3-6 cycloalkyl groups
  • the C 3-6 cycloalkyl groups are cyclopropyl, cyclobutyl , cyclo Pentyl or cyclohexyl.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 1 -C 4 haloalkyl
  • the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 1 -C 4 haloalkyl
  • said C 1 -C 4 alkyl is methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 1 -C 4 haloalkyl groups, the number of halogenated groups is 1, 2, 3, 4 or 5; for example, 3 indivual.
  • R 1 , R 2 and R 3 are independently R 5 substituted or unsubstituted C 1 -C 4 haloalkyl
  • the C 1 -C 4 haloalkyl is trifluoromethyl.
  • R 4 , R 5 , R 5b , R 6 and R 7 are independently C 1 -C 4 haloalkyl
  • the halogen is fluorine, chlorine, bromine or iodine, preferably fluorine.
  • R 4 , R 5 , R 5b , R 6 and R 7 are independently C 1 -C 4 haloalkyl, wherein the C 1 -C 4 alkyl is methyl, ethyl, n-propyl, isopropyl , n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 5 is independently a C 1 -C 4 haloalkyl group, wherein the C 1 -C 4 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl , preferably methyl or ethyl.
  • R 4 , R 5b , R 6 and R 7 are independently C 1 -C 4 haloalkyl, wherein the C 1 -C 4 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl group, isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 4 , R 5 , R 5b , R 6 and R 7 are independently C 1 -C 4 haloalkyl, the number of said halo is 1, 2, 3, 4 or 5; for example, 3 .
  • R 4 , R 5 , R 5b , R 6 and R 7 are independently C 1 -C 4 haloalkyl, said C 1 -C 4 haloalkyl is trifluoromethyl.
  • R 4a1 , R 4a2 , R 4b , R 5b , R 6 and R 7 are independently C 1-4 alkyl
  • the C 1 -C 4 alkyl is methyl, ethyl, n-propyl, Isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 4c is independently C 1-4 alkyl or -OC 1-4 alkyl
  • said C 1 -C 4 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl or tert-butyl, preferably methyl or ethyl.
  • R 5 is independently C 1-4 alkyl, C 1-4 alkyl-O-, C 1-4 haloalkyl
  • said C 1-4 alkyl, C 1-4 alkyl-O- and C 1-4 alkyl in C 1-4 haloalkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.
  • R 5 is independently an amino group protected by an N protecting group
  • the protecting group can be R 5a is C 1-4 alkyl or C 1-4 alkyl-O-; the C 1-4 alkyl in C 1-4 alkyl and C 1-4 alkyl-O- can be independently methyl , ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl;
  • X is N.
  • R is selected from methyl, cyano, trifluoromethyl, difluoromethyl,
  • R2 is H.
  • R3 is selected from H, methyl, halo or cyano.
  • R 4a1 , R 4a2 , R 4b , R 4c , R 5b , R 6 and R 7 are independently methyl.
  • X is N
  • R is selected from methyl, cyano, trifluoromethyl, difluoromethyl,
  • R is selected from H, methyl, halogen or cyano
  • R 4a1 , R 4a2 , R 4b and R 4c are as defined above.
  • X is N
  • R 1 is cyano, R 5b substituted or unsubstituted amino, R 5 substituted or unsubstituted C 1-4 alkyl ; for example R 5 substituted or unsubstituted amino, R 5 substituted or unsubstituted C 1-4 alkyl;
  • R 3 is H, R 5 substituted or unsubstituted C 1-4 alkyl, halogen or cyano ; for example H;
  • R 5 is independently C 1-4 alkyl, -N(R 5b R 5b ), hydroxyl;
  • R 4b is independently C 1-4 alkyl;
  • R 5b is independently H or C 1-4 alkyl.
  • X is N
  • R 1 is R 5 substituted or unsubstituted C 1-4 alkyl ;
  • R3 is H
  • R 5 is independently C 1-4 alkyl, -N(R 5b R 5b ), hydroxyl;
  • R 4b is independently C 1-4 alkyl
  • R 5b is independently H or C 1-4 alkyl.
  • the compound represented by the formula (I) is selected from the compounds shown below:
  • R 1 is as defined above.
  • the compound described in formula (I) is selected from the compounds shown below:
  • the present invention also provides isotope-labeled compounds of the compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer or solvate thereof.
  • the isotope in the isotope-labeled compound is selected from 2 H, 3 H, 11 C, 13 C, 14 C , 15 N, 17 O, 18 O, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I.
  • the atoms that can be labeled with isotopes in the compound of formula (I) include but are not limited to hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, etc., which can be respectively labeled with isotopes 2 H, 3 H, 11 C, 13 C , 14 C, 15 N, 17 O, 18 O, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I etc. instead.
  • the present invention also provides the preparation method of the compound shown in formula (I), which comprises the following steps:
  • Halogenated intermediate B 0 and intermediate E 0 are subjected to a coupling reaction to obtain a compound shown in formula (I);
  • W represents halogen (such as Br);
  • R x is -B (OH) 2 or A, R 1 , R 3 , R 4 , and X are as defined above.
  • the present invention also provides a preparation method of intermediate compound B 0 , which can be any of the following schemes:
  • Option 1 includes the following steps:
  • Step (1) Compound SM-5 is ring-closed under the action of a catalyst (such as phosphorus oxychloride) to obtain compound SM-6,
  • Step (2) Compound SM-6 is reacted under the action of an oxidizing agent (such as m-chloroperoxybenzoic acid) to obtain compound SM-7,
  • an oxidizing agent such as m-chloroperoxybenzoic acid
  • Step (3) Reaction of compound SM-7 with H 2 N-CH 2 -A in the presence of a base (such as sodium carbonate) to obtain compound B 0 ;
  • a base such as sodium carbonate
  • Scheme two it is as shown in step (2) to step (3) in scheme one;
  • W, A, R 4 are as defined above.
  • the solvate is a hydrate.
  • the solvent involved in the present invention can be selected from, for example, methanol, ethanol, isopropanol, toluene, xylene, chlorobenzene, water, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, Dioxane, DMF, acetonitrile, DMSO, NMP, THF or combinations thereof; for example selected from dichloromethane, chloroform, 1,2-dichloroethane, dioxane, DMF, acetonitrile, DMSO, NMP, THF or a combination thereof.
  • the bases involved in the present invention may include organic bases and inorganic bases.
  • the organic base involved in the present invention can be selected from, for example, TEA, DIPEA or a combination thereof.
  • the inorganic base involved in the present invention can be selected from, for example, sodium hydride, sodium methoxide, potassium carbonate, sodium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, lithium tert-butoxide, LiHMDS, LDA, butyllithium , potassium hydroxide, potassium acetate, lithium aluminum hydride or combinations thereof; for example selected from sodium hydride, potassium carbonate, sodium carbonate, cesium carbonate, potassium tert-butoxide, sodium tert-butoxide, LiHMDS, LDA, butyllithium or combinations thereof .
  • the isotope-labeled compound of the compound shown in formula (I) according to the present invention can be prepared by a synthetic method similar to that of the unlabeled compound, the difference is that the unlabeled starting material and/or reagent are replaced by isotope-labeled Starting materials and/or reagents.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer, its solvate or their Isotope-labeled compounds and pharmaceutically acceptable auxiliary materials.
  • the pharmaceutically acceptable auxiliary material can be one or more of diluents, absorbents, wetting agents, binders, disintegrants and lubricants.
  • the present invention also provides the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer, its solvate or their isotope-labeled compound, or the above-mentioned pharmaceutical composition in Use in the preparation of medicines.
  • the medicine is preferably a medicine for treating cancer.
  • the present invention also provides the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer, its solvate or their isotope-labeled compound, or the above-mentioned pharmaceutical composition in Use of the invention in the preparation of cancer drugs mediated by EED protein and/or PRC2 protein complexes.
  • the cancer includes but is not limited to diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkinson lymphoma and other lymphoma, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumors, liver cancer, prostate cancer, breast cancer, brain tumors including neuroblastoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, Head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, kidney cancer, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcomas, etc.
  • diffuse large B-cell lymphoma follicular lymphoma, non-Hodgkinson lymphoma and other lymphoma
  • leukemia multiple myel
  • the compounds, their pharmaceutically acceptable salts, stereoisomers, solvates or their isotope-labeled compounds are used in combination with other drugs; more preferably, the other drugs are selected from anticancer drugs, tumor Immunological drugs, antiallergic drugs, antiemetics, analgesics, or cytoprotective drugs.
  • the present invention also provides a pharmaceutical preparation, which comprises the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer, its solvate or their isotope-labeled compound, or A pharmaceutical composition as described above.
  • the pharmaceutical preparation is preferably a tablet, capsule (e.g. sustained release or timed release capsule), pill, powder, granule (e.g. granule), elixir, tincture, suspension (e.g. nanosuspension, microsuspension) and spray-dried dispersions and other forms of suspensions, syrups, emulsions, solutions and the like.
  • the preferred mode of administration of the pharmaceutical preparation is oral administration, sublingual administration, injection including subcutaneous injection, intravenous injection, intramuscular injection, intrasternal injection, injection, etc., nasal administration (such as nasal membrane inhalation), topical ( Such as creams and ointments), rectal administration (such as suppositories) and so on.
  • the compounds disclosed in the present invention can be administered alone or together with appropriate pharmaceutical carriers.
  • the present invention also provides that the aforesaid pharmaceutical preparation can be formulated into an appropriate drug dose to facilitate and control the dosage of the drug.
  • Dosage regimens of the compounds disclosed in the present invention vary according to specific factors, such as pharmacodynamics and mode of administration, subject, gender, age, health status and body weight of the subject, disease characteristics, other simultaneous drug-taking conditions, and the duration of drug administration. Frequency, liver and kidney function, desired effect, etc.
  • the compounds disclosed in the present invention can be taken in a single dose per day, or the total dose can be divided into multiple doses (such as two to four times per day).
  • the present invention also provides a method for treating cancer, which comprises administering a therapeutically effective amount of the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer or their solvates or their isotope-labeled compounds, or pharmaceutical compositions as described above.
  • the cancers include, but are not limited to, diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkinson lymphoma and other lymphomas, leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, Liver cancer, prostate cancer, breast cancer, brain tumors including neuroblastoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer , lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, kidney cancer, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors and soft tissue sarcoma, etc.
  • the present invention also provides the compound represented by formula (I), its pharmaceutically acceptable salt, stereoisomer or solvate thereof, or the aforementioned isotope-labeled compound used in combination with other drugs, and the other drugs are selected from: anticancer Drugs, tumor immune drugs, anti-allergic drugs, antiemetic drugs, analgesics and cell protection drugs, etc., have a better effect when used together.
  • the cancer is preferably a cancer mediated by a complex with EED protein and/or PRC2 protein.
  • the present invention also provides a method for inhibiting the activity of EED protein and/or PRC2 protein complex, which comprises administering to the subject a therapeutically effective amount of the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salts, stereoisomers or solvates thereof, or their isotope-labeled compounds, or pharmaceutical compositions as described above.
  • a method for inhibiting the activity of EED protein and/or PRC2 protein complex which comprises administering to the subject a therapeutically effective amount of the above-mentioned compound represented by formula (I), its pharmaceutically acceptable salts, stereoisomers or solvates thereof, or their isotope-labeled compounds, or pharmaceutical compositions as described above.
  • the present invention also provides a method for blocking the combination of EED and H3K27 (such as H3K27me3), which includes administering to the subject a therapeutically effective amount of the above-mentioned compound represented by formula (I), its pharmaceutically acceptable Salts, stereoisomers or solvates thereof or their isotopically labeled compounds, or pharmaceutical compositions as described above.
  • the term "about” when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprises” or “includes (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of”, or “consisting of”.
  • reactions and purifications can be carried out using the manufacturer's instructions for the kit, or by methods known in the art or as described herein.
  • the techniques and methods described above can generally be performed according to conventional methods well known in the art as described in various general and more specific documents that are cited and discussed in this specification.
  • groups and substituents thereof can be selected by those skilled in the art to provide stable moieties and compounds.
  • substituent When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, -CH 2 O- is equivalent to -OCH 2 -.
  • C 1-6 alkyl refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms (eg, 1, 2, 3, 4, 5, 6 carbon atoms).
  • the total number of carbon atoms in the abbreviated notation does not include carbons that may be present in substituents of the stated group.
  • C 1 -C 6 alkyl or “C 1-6 alkyl” specifically refers to independently disclosed methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl and C 6 alkane "C 1-4 alkyl” or "C 1 -C 4 alkyl” specifically refers to independently disclosed methyl, ethyl, C 3 alkyl (ie propyl, including n-propyl and isopropyl), C4 alkyl (ie, butyl, including n-butyl, isobutyl, sec-butyl and tert-butyl).
  • linking substituents are described.
  • the Markush variables recited for that group are to be understood as linking groups.
  • the Markush group definition for that variable recites “alkyl,” it is understood that “alkyl” represents a linking alkylene group.
  • alkyl group when an alkyl group is clearly indicated as a linking group, then the alkyl group represents a linked alkylene group, for example, the group "halo-C 1 -C 6 alkane C 1 -C 6 alkyl in "group” should be understood as C 1 -C 6 alkylene.
  • halogen means fluorine, chlorine, bromine or iodine.
  • Haldroxy means an -OH group.
  • Amino refers to -NH2 .
  • R 5b substituted amino group R 5b can be one or two, when R 5b is two, they can be the same group or different groups.
  • Carboxy means -COOH.
  • alkyl refers to a fully saturated straight-chain or branched hydrocarbon chain group, Consisting solely of carbon and hydrogen atoms, having for example 1 to 12 (preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 4) carbon atoms, and attached to the remainder of the molecule by a single bond, Examples include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2- Dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl, decyl, etc.
  • haloalkyl as a group or part of another group, means that one or more hydrogen atoms in an alkyl group (as defined in this invention) are replaced by a halogen (as defined in this invention) ), the number of halogens can be one or more; when the number of halogens is multiple, the halogens are the same or different.
  • fluoroalkyl refers to an alkyl group substituted with one or more fluorines.
  • haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, and monofluoromethyl.
  • the number of carbon atoms in the "heterocycloalkyl" is 3, 4, 5 or 6, the heteroatoms are selected from N, O and S, and the number of heteroatoms is 1, 2, 3 or 4, Further preferably, the number of carbon atoms is 4 or 5, the heteroatoms are selected from N and O, and the number of heteroatoms is 1 or 2; for example
  • the number of carbon atoms in the "heterospirocycloalkyl" is 4, 5 or 6, the heteroatoms are selected from N, O and S, and the number of heteroatoms is 1, 2, 3 or 4, further Preferably, the heteroatoms are selected from N and O, and the number of heteroatoms is 1 or 2; for example
  • cyclohydrocarbyl as a group or part of another group, means a stable non-aromatic monocyclic or polycyclic hydrocarbon group composed only of carbon and hydrogen atoms, which may include fused rings system, bridged ring system or spiro ring system, has 3 to 15 carbon atoms, preferably has 3 to 10 carbon atoms, more preferably has 3 to 8 carbon atoms, and it is saturated or unsaturated and can be changed via any suitable
  • the carbon atoms are connected to the rest of the molecule by single bonds. Unless specifically stated otherwise in this specification, carbon atoms in a cycloalkyl group may be optionally oxidized.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, 1H-indene Base, 2,3-indanyl, 1,2,3,4-tetrahydro-naphthyl, 5,6,7,8-tetrahydro-naphthyl, 8,9-dihydro-7H-benzo Cyclohepten-6-yl, 6,7,8,9-tetrahydro-5H-benzocycloheptenyl, 5,6,7,8,9,10-hexahydro-benzocyclooctenyl, Fluorenyl, bicyclo[2.2.1]heptyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, bicyclo
  • cycloalkyl as a group or part of another group, means a saturated cyclic hydrocarbon group.
  • cycloalkenyl as a group or part of another group, means a cyclic hydrocarbon group having at least one double bond (eg, a carbon-carbon double bond).
  • a cycloalkenyl group can be attached to the rest of the molecule through an atom of a double bond within it.
  • heterocyclyl as a group or part of another group, means a group consisting of 2 to 14 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, phosphorus, oxygen and sulfur Stable 3- to 20-membered non-aromatic cyclic groups.
  • the heterocyclic group may be a monocyclic, bicyclic, tricyclic or multicyclic ring system, which may include a fused ring system, a bridged ring system or a spiro ring system;
  • the nitrogen, carbon, or sulfur atoms of can be optionally oxidized; the nitrogen atoms can be optionally quaternized; and the heterocyclyl can be partially or fully saturated.
  • a heterocyclyl group can be attached to the rest of the molecule via a carbon atom or a heteroatom and by a single bond.
  • one or more rings may be aryl or heteroaryl as defined below, provided that the point of attachment to the rest of the molecule is a non-aromatic ring atom.
  • heterocyclyl is preferably a stable 4- to 11-membered non-aromatic monocyclic, bicyclic, bridged or spirocyclic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur , more preferably a stable 4- to 8-membered non-aromatic monocyclic, bicyclic, bridged or spirocyclic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur.
  • heterocyclic groups include, but are not limited to: pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2,7-diaza-spiro[3.5]nonyl Alkane-7-yl, 2-oxa-6-aza-spiro[3.3]heptane-6-yl, 2,5-diaza-bicyclo[2.2.1]heptane-2-yl, aza Cyclobutanyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuryl, oxazinyl, dioxolyl, tetrahydroisoquinolyl, decahydroisoquinolyl, imidazolinyl, Imidazolidinyl, Quinazinyl, Thiazolidinyl, Isothiazolidinyl, Isoxazolidinyl, Indolinyl
  • aryl as a group or part of another group, means a conjugated hydrocarbon ring system group having 6 to 18 carbon atoms, preferably having 6 to 10 carbon atoms.
  • aryl can be a monocyclic, bicyclic, tricyclic or multicyclic ring system and can also be fused to a cycloalkyl or heterocyclyl as defined above, provided that the aryl is via The atoms on the aromatic ring are connected to the rest of the molecule by single bonds.
  • aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, fluorenyl, 2,3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-Benzoxazin-3(4H)-on-7-yl and the like.
  • arylalkyl refers to an alkyl group as defined above substituted with an aryl group as defined above.
  • heteroaryl as a group or part of another group, means having 1 to 15 carbon atoms (preferably 1 to 10 carbon atoms) and 1 to 6 carbon atoms selected from the group consisting of nitrogen A 5- to 16-membered conjugated ring system group of heteroatoms of oxygen and sulfur.
  • heteroaryl may be a monocyclic, bicyclic, tricyclic or multicyclic ring system, and may be fused to a cycloalkyl or heterocyclyl as defined above, provided that hetero An aryl group is connected to the rest of the molecule by a single bond through an atom on the aromatic ring.
  • heteroaryl is preferably a stable 5- to 12-membered aromatic group comprising 1 to 5 heteroatoms selected from nitrogen, oxygen and sulfur, more preferably 1 to 4 heteroatoms selected from nitrogen , a stable 5- to 10-membered aromatic group containing heteroatoms of nitrogen, oxygen and sulfur or a 5- to 6-membered aromatic group containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur (for example heteroaryl is a C 1 -C 5 heteroaryl group, wherein the heteroatoms are selected from N, O and S, and the number of heteroatoms is 1, 2, 3 or 4).
  • heteroaryl groups include, but are not limited to, thienyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, Benzimidazolyl, benzopyrazolyl, indolyl, furyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolyl, isoindolyl, indazolyl, isoindazolyl , Purinyl, quinolinyl, isoquinolinyl, diazinyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridine Base, phena
  • substituents described in the claims and description of the present invention are selected from the group consisting of alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl.
  • substituted or “substituent” means that one or more hydrogen atoms are replaced by the specified group.
  • substituents there may be one or more substituents; when the substitution position is not specified, the substitution may be at any position, but only when a stable or chemically feasible chemical compound is formed is considered Allowed.
  • any variable eg, R
  • its definition is independent at each occurrence.
  • said group may optionally be substituted with up to two R, with independent options for each occurrence of R.
  • R the benzene ring
  • each R has an independent option, that is, the 2 R can be the same or different.
  • substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
  • moiety As used herein, the terms “moiety”, “structural moiety”, “chemical moiety”, “group”, “chemical group” refer to a specific segment or functional group in a molecule. Chemical moieties are generally considered to be chemical entities embedded or attached to molecules.
  • the compounds of the present invention are intended to include both E- and Z-geometric isomers.
  • Tautomer refers to isomers formed by the transfer of a proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the invention are also intended to be within the scope of the invention.
  • the compounds of the present invention may contain one or more chiral carbon atoms, and thus may give rise to enantiomers, diastereoisomers and other stereoisomeric forms.
  • Each chiral carbon atom can be defined as (R)- or (S)- based on stereochemistry.
  • the present invention is intended to include all possible isomers, as well as their racemates and optically pure forms.
  • the preparation of the compounds of the present invention can select racemates, diastereomers or enantiomers as starting materials or intermediates.
  • Optically active isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.
  • Stereoisomers of the compounds of the present invention may exist as (R)- or (S)-isomers.
  • pharmaceutically acceptable salt includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to a salt formed with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects.
  • Inorganic acid salts include but not limited to hydrochloride, hydrobromide, sulfate, nitrate, phosphate, etc.
  • organic acid salts include but not limited to formate, acetate, 2,2-dichloroacetate , Trifluoroacetate, Propionate, Caproate, Caprylate, Caprate, Undecylenate, Glycolate, Gluconate, Lactate, Sebacate, Hexanoate glutarate, malonate, oxalate, maleate, succinate, fumarate, tartrate, citrate, palmitate, stearate, oleate , cinnamate, laurate, malate, glutamate, pyroglutamate, aspartate, benzoate, mesylate, benzenesulfonate, p
  • “Pharmaceutically acceptable base addition salt” refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects.
  • Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like.
  • Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, those of primary, secondary, and tertiary amines, substituted amines, including natural substituted amines, cyclic amines, and basic ion exchange resins , such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, bicyclic Hexylamine, Lysine, Arginine, Histidine, Caffeine, Procaine, Choline, Betaine, Ethylenediamine, Glucosamine, Methylglucamine, Theobromine, Purine, Piperazine, Piperazine Pyridine, N-ethylpiperidine, polyamine resin, etc.
  • Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine,
  • a "pharmaceutical composition” refers to a formulation of a compound of the present invention with a vehicle generally accepted in the art for the delivery of a biologically active compound to a mammal (eg, a human).
  • the medium includes a pharmaceutically acceptable carrier.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, facilitate the absorption of the active ingredient and thus exert its biological activity.
  • pharmaceutically acceptable refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the compounds of the present invention, and is relatively nontoxic, i.e., the substance can be administered to an individual without causing adverse biological effects. React or interact in an undesirable manner with any component contained in the composition.
  • pharmaceutically acceptable excipients include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners approved by the relevant government regulatory agencies as acceptable for human or livestock use , diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent or emulsifying agent.
  • prophylactic As used herein, the terms “prophylactic”, “prevention” and “prevention” include reducing the likelihood of a disease or condition occurring or worsening in a patient.
  • treatment and other similar synonyms include the following meanings:
  • a therapeutically effective amount refers to at least one agent or compound which, when administered, is sufficient to relieve to some extent one or more symptoms of the disease or condition being treated amount. The result may be a reduction and/or alleviation of a sign, symptom or cause, or any other desired change in a biological system.
  • a therapeutically “effective amount” is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant disease-modifying effect. Effective amounts suitable for any individual case can be determined using techniques such as dose escalation assays.
  • administering refers to methods capable of delivering a compound or composition to the desired site of biological action. These methods include, but are not limited to, oral routes, duodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration and rectal administration. Administration techniques useful for the compounds and methods described herein are well known to those skilled in the art, as described, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Those discussed in Easton, Pa. In preferred embodiments, the compounds and compositions discussed herein are administered orally.
  • the terms “pharmaceutical combination”, “drug combination”, “combination”, “administration of other treatments”, “administration of other therapeutic agents” and the like refer to drug treatments obtained by mixing or combining more than one active ingredient, It includes fixed and non-fixed combinations of active ingredients.
  • the term “fixed combination” refers to the simultaneous administration to a patient of at least one compound described herein and at least one co-agent in the form of a single entity or single dosage form.
  • the term “variable combination” refers to simultaneous, concomitant or sequential administration at variable intervals of at least one compound described herein and at least one synergistic agent as separate entities to a patient. These also apply to cocktail therapy, eg the administration of three or more active ingredients.
  • the functional groups of intermediate compounds may need to be protected by appropriate protecting groups.
  • Such functional groups include hydroxyl, amino, mercapto and carboxylic acid.
  • Suitable hydroxy protecting groups include trialkylsilyl or diarylalkylsilyl groups (eg tert-butyldimethylsilyl, tert-butyldiphenylsilyl or trimethylsilyl) , tetrahydropyranyl, benzyl, etc.
  • Suitable protecting groups for amino, amidino and guanidino include tert-butoxycarbonyl, benzyloxycarbonyl and the like.
  • Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like.
  • Suitable carboxy protecting groups include alkyl, aryl or aralkyl esters.
  • Protecting groups can be introduced and removed according to standard techniques known to those skilled in the art and as described herein. The use of protecting groups is described in detail in Greene, T.W. and P.G.M. Wuts, Protective Groups in Organi Synthesis, (1999), 4th Ed., Wiley.
  • the protecting group can also be a polymeric resin.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the positive progress effect of the present invention lies in: with US20160176882A1, compound N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridine- Compared with 3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine, the anti-cell proliferation activity of the compound of the present invention is increased by about 10 times.
  • the compound disclosed in the present invention is combined with the EED protein, the bicyclic structure outside the combined "pocket" can make the compound have better metabolic stability.
  • the starting materials used in the following examples can be purchased from chemical distributors such as Aldrich, TCI, Alfa Aesar, Bi De, Anaiji, etc., or can be synthesized by known methods.
  • the ice bath refers to -5 degrees Celsius to 0 degrees Celsius
  • the room temperature refers to 10 degrees Celsius to 30 degrees Celsius
  • the reflux temperature generally refers to the solvent reflux temperature under normal pressure. Reaction overnight means that the time is 8-15 hours.
  • the specific operating temperature is not limited, all carried out at room temperature.
  • the separation and purification of intermediates and final products are carried out by normal phase or reverse phase chromatographic column separation or other suitable methods.
  • the normal-phase flash chromatography column uses ethyl acetate and n-hexane or methanol and methylene chloride as mobile phases.
  • Reversed-phase preparative high-pressure liquid chromatography uses C18 column and detects with UV 214nm and 254nm, and its mobile phase is A (water and 0.1% formic acid), B (acetonitrile) or mobile phase A (water and 0.1% carbonic acid ammonium hydrogen), B (acetonitrile).
  • A-2 (20 g, 90.5 mmol) was added into a 2 L three-necked flask, and stirred and dissolved with anhydrous THF (1000 ml) and MeOH (200 ml). Sodium methoxide (5.87 g, 108.6 mmol) was added, and the reaction solution was stirred at 60° C. for 18 hours. Concentrate under reduced pressure to remove most of the solvent, and add 500 mL of water. The suspension was stirred for 30 minutes and filtered to collect the solid. The solid was slurried with a mixture of petroleum ether and ethyl acetate (5:1), filtered to obtain a solid, and dried under reduced pressure to obtain A-3 (18 g, 85% yield) as a yellow solid.
  • A-5 (2.7g, 11.6mmol), zinc cyanide (2.04g, 17.4mmol), DMF (50ml) and palladium tetrakistriphenylphosphine (1.34g, 1.16mmol) were successively added into a dry one-necked flask.
  • the reaction solution was heated to 120° C. under nitrogen protection and stirred for 18 hours.
  • the reaction solution was cooled to room temperature, extracted with ethyl acetate (200mL ⁇ 3) and water (200ml).
  • the organic phase was washed once with water and brine respectively, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • A-6 (1.55g, 8.65mmol), trifluoroacetic acid (1.09g, 8.65mmol), methanol (20mL) and 10% palladium carbon (2g, containing 50% water) were sequentially added into a 100mL single-necked flask.
  • the reaction mixture was sparged with hydrogen for 5 minutes, purged three times with a hydrogen balloon, and stirred at 60° C. for 48 hours under a hydrogen balloon.
  • N,N-dimethylformamide 60.0 mL
  • N-(dibenzylidene)aminoacetonitrile 1 10.0 g, 45.4mmol
  • N,N-dimethylformamide 50.0mL
  • 5-bromo-4-chloro-2-(methylthio)pyrimidine 10.8g, 45.4mmol
  • TLC monitors the end of the reaction, adjusts it to 8 with aqueous sodium hydroxide solution (1.00M) at zero degrees, extracts with ethyl acetate, dries, and selects dry to obtain the N-((5-bromo-2-( Methylthio)pyrimidin-4-yl)(cyano)methyl)formamide 4 (7.00 g, 22.6 mmol, 73.2% yield).
  • Step 4 8-bromo-5-(methylthio)imidazo[1,5-c]pyrimidine-1-carbonitrile (5)
  • Step 6 8-bromo-5-(((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)amino)imidazo[1,5-c]pyrimidine-1-carbonitrile ( 7)
  • Step 7 8-bromo-5-(bromomethyl)-[1,2,4]triazolo[1,5-a]pyridine (9)
  • Pure 8-bromo-5-(bromomethyl)-[1,2,4]triazolo[1,5-a]pyridine 9 was obtained after vacuum drying (1.7 g, 62% yield).
  • Step 8 1-(8-bromo-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-N,N-dimethylmethylamine (10)
  • Step 10 8-(5-((dimethylamino)methyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)-5-(((5-fluoro -2,3-dihydrobenzofuran-4-yl)methyl)amino)imidazo[1,5-c]pyrimidine-1-carbonitrile (BR-1)
  • Step 5 (5-(2-Hydroxypropan-2-yl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)boronic acid (12)
  • Step 6 5-(((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)amino)-8-(5-(2-hydroxypropan-2-yl)-[1 ,2,4]triazolo[1,5-a]pyridin-8-yl)imidazo[1,5-c]pyrimidine-1-carbonitrile (BR-2)
  • Step 1 8-iodo-N,N-dimethyl-[1,2,4]triazolo[1,5-a]pyridin-5-amine 14
  • Step 3 8-(5-(Dimethylamino)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)-5-(((5-fluoro-2,3 -Dihydrobenzofuran-4-yl)methyl)amino)imidazo[1,5-c]pyrimidine-1-carbonitrile
  • Step 1 8-bromo-N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)imidazo[1,5-c]pyrimidin-5-amine (17)
  • Step 2 Using the method of Step 10 of Example 2, 8-bromo-N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)imidazo[1,5-c ]pyrimidin-5-amine (17) and (5-((dimethylamino)methyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)boronic acid (11 ) as raw material can be obtained: 8-(5-((dimethylamine) methyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)-N-(( 5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)imidazo[1,5-c]pyrimidin-5-amine (18):
  • Step 3 8-(5-((dimethylamine)methyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)-N-((5-fluoro -2,3-Dihydrobenzofuran-4-yl)methyl)-1-iodoimidazo[1,5-c]pyrimidin-5-amine (19)
  • Step 4 8-(5-((dimethylamine)methyl)-[1,2,4]triazolo[1,5-a]pyridin-8-yl)-N-((5-fluoro -2,3-Dihydrobenzofuran-4-yl)methyl)-1-(methylsulfonyl)imidazo[1,5-c]pyrimidin-5-amine (BR-4)
  • EED plays an important role in the overall function of PRC2 although it does not have enzymatic activity.
  • the effect of EED on PRC2 is specifically manifested in two aspects: 1) EED directly binds to trimethylated H3K27Me3, which can locate the PCR2 complex on the chromatin that needs to be modified; 2) EED has a positive effect on the enzymatic function of EZH2.
  • Great allosteric boost Therefore, the development of compounds that are targets of EED as an allosteric protein provides a new strategy for providing inhibition of EZH2 enzymatic activity. Moreover, such inhibitors have better or complementary advantages than EZH2 enzyme catalytic site inhibitors.
  • EED inhibitors when patients develop resistance to EZH2 enzyme inhibitors, EED inhibitors can also inhibit EZH2 enzyme activity.
  • the invention discloses that the compound can be used as an EED target inhibitor, and has a therapeutic effect on diseases related to the mechanism of action of EED and/or PRC2.
  • the biological functions of the compounds disclosed in the present invention have been proved in biochemical and cell level tests.
  • the compound disclosed in the present invention can have a strong competitive binding effect with the H3K27Me3 polypeptide binding to the EED protein (IC 50 can reach ⁇ 1nM).
  • the compounds disclosed in the present invention can not only inhibit the methylation level of histone H3K27 but also inhibit the proliferation of cancer cells through this effect (IC 50 can reach ⁇ 1nM).
  • Representative compounds of the disclosure were serially diluted in 3-fold in DMSO, and 10 concentration gradients were tested per compound, up to a maximum assay concentration of 10 ⁇ .
  • Compounds were diluted 200-fold into G401 cells cultured in 96-well plates (DMSO final concentration 0.5%). After the cells were cultured for 72 hours, the trimethylation level of histone H3K27 was detected by ELISA method.
  • Histone extraction Compound-treated cells in 96-well plates were washed with 1x PBS (10x PBS buffer (80g NaCl (Sigma, product number S3014), 2g KCl (Sigma, product number 60128), 14.4g Na2HP04 (Sigma, Product No. S5136), 2.4g KH 2 P04 (Sigma, Product No. P9791) into 1L water, pH to 7.4), wash three times, add 100 ⁇ L 0.4N HCl to each well, place at 4°C, gently shake for 2 hours to lyse the cells.
  • 1x PBS 10x PBS buffer (80g NaCl (Sigma, product number S3014), 2g KCl (Sigma, product number 60128), 14.4g Na2HP04 (Sigma, Product No. S5136), 2.4g KH 2 P04 (Sigma, Product No. P9791) into 1L water, pH to 7.4), wash three times, add 100 ⁇ L 0.4N HCl to each well, place at
  • the lysate was then neutralized with 80 ⁇ L of neutralization buffer (0.5 M disodium hydrogen phosphate, pH 12.5, 2.5 mM DTT; 1% cocktail (Sigma, product number P8340) (mix the cell lysate and neutralization buffer thoroughly).
  • neutralization buffer 0.5 M disodium hydrogen phosphate, pH 12.5, 2.5 mM DTT; 1% cocktail (Sigma, product number P8340) (mix the cell lysate and neutralization buffer thoroughly).
  • ELISA detection method transfer the cell lysate in parallel to two 384-well detection plates (PerkinElmer, OptiPlate-384HB, product number 6007290), one plate is used to detect the trimethylation level of H3K27, and the other plate is used to measure H3 adjusted to a final volume of 50 ⁇ L/well with PBS, and coated overnight at 4°C. The next day, discard the solution in the well, and use TBST buffer (lxTBS (10x TBS: 24.2g Tris (Sigma, product number T6066), 80g NaCl (Sigma, product number S3014) in 1L water, adjust the pH to 7.6 with HCl) ,0.1% Tween-20) washed 5 times, and dried on absorbent paper.
  • TBST buffer lxTBS (10x TBS: 24.2g Tris (Sigma, product number T6066), 80g NaCl (Sigma, product number S3014) in 1L water, adjust the pH to 7.6 with HCl) ,0.1% T
  • the secondary antibody (anti-rabbit antibody (Jackson ImmunoResearch, product number 111-035-003)) was diluted 2000 times with blocking buffer before use. After 1 hour, wash with TBST and dry. Add 30 ⁇ L of ECL substrate (Pierce, product number 34080) to each well, and centrifuge at 2000 rpm for 30 seconds. The signal of each sample was detected by using Molecular Devices, SpectraMax. Data processing: H3K27 methylation reads were first normalized by H3 signal, 0.5% DMSO-treated samples were used as a control, and the percentage inhibition of the compound was calculated. The data were fitted to a dose-response curve using the GraphPad prisim5 program to obtain the IC50 values of the test compounds.
  • Table 1 shows the IC50 values of some compounds.
  • Human B-cell non-Hodgkin's lymphoma cells KARPAS-422 S were cultured in culture flasks using standard cell culture conditions.
  • the culture medium is containing 15% fetal bovine serum (FBS, Invitrogen, product number 10099-141), 1% penicillin/streptomycin solution (P/S) RPMI-1640 (Invitrogen, product number 11875), and the culture bottle is placed at temperature Cultured in a sterile incubator at 37°C, 95% relative humidity, 5% CO2 .
  • the cells counted each time were inoculated into a new 96-well plate at the same density (1 ⁇ 10 4 cells/well), supplemented with fresh medium to 100 ⁇ L, and added different concentrations of compounds at the same time.
  • After culturing until the 13th day add 100 ⁇ L of CellTiter-Glo (CellTiter-GloCellTiter-GloCellTiter-GloCTG) (Promega, Product No. G7573) to each well, place it in the dark at room temperature for 10-20 minutes, and use Molecular Devices, SpectraMax i3X to read the luminescent signal .
  • the data were fitted to dose-response curves using GraphPad prisim5 to obtain IC50 values for test compounds.
  • Table 5 shows the IC50 values of some compounds.
  • the compounds disclosed in the present invention above can be used to treat cancers related to the mechanism of action of EED protein and/or PRC2 protein complex, including but not limited to diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkinson lymphoma, etc.
  • Isolymphoma leukemia, multiple myeloma, mesothelioma, gastric cancer, malignant rhabdoid tumor, liver cancer, prostate cancer, breast cancer, brain tumors including neuroblastoma, glioma, glioblastoma, and astrocytoma Cytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, kidney cancer, rectal cancer, thyroid cancer, parathyroid tumors, uterus Tumors and soft tissue sarcomas, among others.

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Abstract

提供了一种如式(I)所示的氮杂芳基化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物。此外也提供了该类化合物的制备方法、含有该类化合物的组合物以及该类化合物用于制备治疗与EED蛋白和/或PRC2蛋白复合物作用机理相关疾病或障碍的药物用途。

Description

氮杂芳基化合物、其制备方法及应用
本申请要求申请日为2021/8/11的中国专利申请2021109212000的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及一种氮杂芳基化合物、其制备方法及应用。
背景技术
PcG(Polycomb Group)蛋白是一类重要的染色质修饰酶。它通过对染色质的修饰达到调控基因的转录，从而对干细胞的生长、分化及长期的细胞记忆有重要作用。在哺乳动物细胞中，PcG蛋白主要分为两类转录抑制复合物，分别是PRC1(Polycomb Repressive Complex 1)和PRC2(Polycomb Repressive Complex 2)。其中，PRC2是通过对染色质中组蛋白3的27位赖氨酸(H3K27)的甲基化修饰来抑制相关基因的表达。PRC2蛋白复合物主要由EZH2(Enhancer of Zeste Homolog 2)(或其非常类似的同源蛋白EZH1)，EED(Embryonic ectoderm Development)以及SUZ12(Suppressor of Zeste 12)等核心蛋白组成。其中，EZH2具有酶催化活性，通过SET(Su(var),E(Z)，and Trithorax)蛋白结构域能够把底物SAM(S-adenosyl-L-methionine)的甲基转移到H3K27上，从而达到H3K27的一到三甲基化修饰。EZH2的酶催化活性也依赖于PRC2其他组成部分，比如属于WD40重复结构蛋白家族的EED蛋白。EED与三甲基化的H3K27Me3的结合作用一方面对EZH2的酶催化功能有很大的变构促进作用，另一方面也能把PCR2复合物定位在需要修饰的染色质上。PRC2的功能异常，比如EZH2的过表达或功能获得性突变，与临床上许多肿瘤疾病相关，包括肺癌，乳腺癌，直肠癌，前列腺癌，膀胱癌，胰腺癌，肉瘤以及淋巴癌等等。PRC2也与多种细胞免疫功能相关，比如EZH2参与调节淋巴细胞活化，也能与糖酵解共同促进T细胞对肿瘤细胞的应答。因此，研发PRC2小分子抑制剂有重要且广阔的药物开发价值。
围绕PRC2抑制剂的研发主要是开发EZH2抑制剂以及EED抑制剂两个策略。目前进入临床的EZH2抑制剂有EPZ-6438(Epizyme，临床二期)，GSK2816126(GSK，临床一期)，以及CPI-1205(Constellation,临床一期)等等。尽管EZH2抑制剂研发有多个进入临床研究阶段，但这些抑制剂都含有一个共同的2-吡啶酮的药效团。并且，在用已有EZH2抑制剂于临床治疗中，已经开始出现二次突变。EED抑制剂对EZH2酶功能有变构抑制的作用，可以达到与EZH2相同或类似的生物功能。而且一方面EED抑制剂很好的克服了EZH2的耐药性问题，另一方面EED抑制剂可以与EZH2抑制剂联用达到更好的协同作用效果，因此，开发新的EED抑制剂具有非常重要的意义。
发明内容
本发明所要解决的技术问题在于现有技术中的EED抑制剂结构单一。为此，本发明提供了一种 氮杂芳基化合物、其制备方法及应用。本发明的氮杂芳基化合物表现出对肿瘤细胞很好的抑制活性，具有广阔的药物开发前景。
本发明提供了一种如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物：
Figure PCTCN2022111668-appb-000001
其中，A独立地为
Figure PCTCN2022111668-appb-000002
X为N或CR 2；
R 1、R 2和R 3独立地为H、卤素、氰基、R 5b取代或未取代的氨基、R 5b-O-、R 5取代或未取代的C 1- 4烷基、R 5取代或未取代的C 3- 6的环烷基、R 5取代或未取代的C 1-C 4卤代烷基、-C(＝O)R 6、-CO 2R 6、-C(＝O)NR 6R 7、-SO 2R 6或-SO 2NR 6R 7；当取代基为多个时，相同或不同；
R 4独立地为H、卤素、氰基、C 1-4卤代烷基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c；
R 4a1、R 4a2和R 4b独立地为H或C 1-4烷基；
R 4c独立地为H、C 1-4烷基或-O-C 1-4烷基；
R 5独立地为C 1-4烷基-O-、C 1-4烷基、C 1-4卤代烷基、-N(R 5bR 5b)、N保护基团保护的氨基、氟、羟基；
R 5b、R 6和R 7独立地为H、C 1-4烷基、C 1-4卤代烷基；
“*”表示带“*”的碳原子为手性碳原子时，其为R构型、S构型或其混合。
在本发明某些优选实施方案中，所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物中的某些基团如下定义，未提及的基团同本申请任一方案所述(简称“在本发明某些优选实施方案中”)：
式(I)所示的化合物选自如下所示的化合物：
Figure PCTCN2022111668-appb-000003
上述化合物中，X、R 1、R 3和R 4的定义如上所述。
在本发明某些优选实施方案中：
Figure PCTCN2022111668-appb-000004
为
Figure PCTCN2022111668-appb-000005
或其混合物。
在本发明某些优选实施方案中：
当R 1、R 2、R 3和R 4独立地为卤素时，所述的卤素为氟、氯、溴或碘，优选氯。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 1- 4烷基时，所述的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 3- 6的环烷基时，所述的C 3- 6的环烷基为环丙基、环丁基、环戊基或环己基。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-C 4卤代烷基时，所述的卤为氟、氯、溴或碘，优选氟。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-C 4卤代烷基时，所述的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-C 4卤代烷基时，所述的卤代的个数为1、2、3、4或5个；例如3个。
在本发明某些优选实施方案中：
当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-C 4卤代烷基时，所述C 1-C 4卤代烷基为三氟甲基。
在本发明某些优选实施方案中：
当R 4、R 5、R 5b、R 6和R 7独立地为C 1-C 4卤代烷基时，所述的卤为氟、氯、溴或碘，优选氟。
在本发明某些优选实施方案中：
在本发明某些优选实施方案中：
当R 4、R 5、R 5b、R 6和R 7独立地为C 1-C 4卤代烷基时，其中的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 5独立地为C 1-C 4卤代烷基时，其中的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
当R 4、R 5b、R 6和R 7独立地为C 1-C 4卤代烷基时，其中的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 4、R 5、R 5b、R 6和R 7独立地为C 1-C 4卤代烷基时，所述的卤代的个数为1、2、3、4或5个；例如3个。
在本发明某些优选实施方案中：
当R 4、R 5、R 5b、R 6和R 7独立地为C 1-C 4卤代烷基时，所述的C 1-C 4卤代烷基为三氟甲基。
在本发明某些优选实施方案中：
当R 4a1、R 4a2、R 4b、R 5b、R 6和R 7独立地为C 1-4烷基时，所述的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 4c独立地为C 1-4烷基或-O-C 1-4烷基时，所述的C 1-C 4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基或乙基。
在本发明某些优选实施方案中：
当R 5独立地为C 1-4烷基、C 1-4烷基-O-、C 1-4卤代烷基时，所述的C 1-4烷基、C 1-4烷基-O-和C 1-4卤代烷基里的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基，优选甲基。
在本发明某些优选实施方案中：
当R 5独立地为N保护基团保护的氨基时，所述的保护基团可以为
Figure PCTCN2022111668-appb-000006
R 5a为C 1-4烷基或C 1-4烷基-O-；C 1-4烷基和C 1-4烷基-O-里的C 1-4烷基可独立地为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
例如
Figure PCTCN2022111668-appb-000007
可以为叔丁氧羰基。
在本发明某些优选实施方案中：
X为N。
在本发明某些优选实施方案中：
R 1选自甲基、氰基、三氟甲基、二氟甲基、
Figure PCTCN2022111668-appb-000008
在本发明某些优选实施方案中：
R 2为H。
在本发明某些优选实施方案中：
R 3选自H、甲基、卤素或氰基。
在本发明某些优选实施方案中：
R 4选自氰基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c。
在本发明某些优选实施方案中：
R 4a1、R 4a2、R 4b、R 4c、R 5b、R 6和R 7独立地为甲基。
在本发明某些优选实施方案中：
X为N；
R 1选自甲基、氰基、三氟甲基、二氟甲基、
Figure PCTCN2022111668-appb-000009
R 3选自H、甲基、卤素或氰基；
R 4选自氰基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c；
R 4a1、R 4a2、R 4b和R 4c的定义如上所述。
在本发明某些优选实施方案中：
X为N；
R 1为氰基、R 5b取代或未取代的氨基、R 5取代或未取代的C 1- 4烷基；例如R 5取代或未取代的氨基、R 5取代或未取代的C 1- 4烷基；
R 3为H、R 5取代或未取代的C 1- 4烷基、卤素或氰基；例如H；
R 5独立地为C 1-4烷基、-N(R 5bR 5b)、羟基；
R 4选自氰基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c；例如R 4选自氰基或-S(＝O) 2R 4b；例如R 4b独立地为C 1-4烷基；
R 5b独立地为H或C 1-4烷基。
在本发明某些优选实施方案中：
X为N；
R 1为R 5取代或未取代的C 1- 4烷基；
R 3为H；
R 5独立地为C 1-4烷基、-N(R 5bR 5b)、羟基；
R 4选自氰基或-S(＝O) 2R 4b；
R 4b独立地为C 1-4烷基；
R 5b独立地为H或C 1-4烷基。
在本发明某些优选实施方案中，所述式(I)所示的化合物选自如下所示的化合物：
Figure PCTCN2022111668-appb-000010
上述化合物中，R 1的定义如上所述。
在本发明某些优选实施方案中，式(I)所述的化合物选自如下所示的化合物：
Figure PCTCN2022111668-appb-000011
Figure PCTCN2022111668-appb-000012
本发明还提供所述如式(I)所示的化合物、其药学上可接受的盐、立体异构体或其溶剂化物的同位素标记化合物。所述同位素标记化合物中所述同位素选自 2H、 3H、 11C、 13C、 14C、 15N、 17O、 18O、 18F、 31P、 32P、 35S、 36Cl和 125I。式(I)化合物中能够被同位素标记的原子包括但不局限于氢、碳、氮、氧、磷、氟、氯和碘等，它们可分别被同位素 2H、 3H、 11C、 13C、 14C、 15N、 17O、 18O、 18F、 31P、 32P、 35S、 36Cl和 125I等代替。
本发明还提供所述如式(I)所示的化合物的制备方法，其包含如下步骤：
将卤代中间体B 0与中间体E 0进行偶联反应得到如式(I)所示的化合物；
Figure PCTCN2022111668-appb-000013
其中，W代表卤素(例如Br)；R x为-B(OH) 2或
Figure PCTCN2022111668-appb-000014
A、R 1、R 3、R 4、X的定义如上所述。
本发明还提供一种中间体化合物B 0的制备方法，其可为如下任一方案：
方案一、包括以下步骤：
步骤(1)化合物SM-5在催化剂(例如三氯氧磷)作用下关环得到化合物SM-6，
步骤(2)化合物SM-6在氧化剂(例如间氯过氧苯甲酸)作用下反应得到化合物SM-7，
步骤(3)化合物SM-7与H 2N-CH 2-A在碱(例如碳酸钠)存在下反应得到化合物B 0；
方案二、其如方案一中步骤(2)至步骤(3)所示；
方案三、其如方案一中步骤(3)所示；
反应方程式如下：
Figure PCTCN2022111668-appb-000015
其中，W、A、R 4的定义如上所述。
在本发明某些优选实施方案中：所述的溶剂化物为水合物。
本发明所涉及到的溶剂例如可选自：甲醇、乙醇、异丙醇、甲苯、二甲苯、氯苯、水、二氯甲烷、氯仿、四氯化碳、1,2-二氯乙烷、二氧六环、DMF、乙腈、DMSO、NMP、THF或其组合；例如选自二氯甲烷、氯仿、1,2-二氯乙烷、二氧六环、DMF、乙腈、DMSO、NMP、THF或其组合。
本发明所涉及到的碱可包括有机碱和无机碱。
本发明所涉及到的有机碱例如可选自：TEA、DIPEA或其组合。
本发明所涉及到的无机碱例如可选自：氢化钠、甲醇钠、碳酸钾、碳酸钠、碳酸铯、叔丁醇钾、叔丁醇钠、叔丁醇锂、LiHMDS、LDA、丁基锂、氢氧化钾、醋酸钾、氢化铝锂或其组合；例如选自氢化钠、碳酸钾、碳酸钠、碳酸铯、叔丁醇钾、叔丁醇钠、LiHMDS、LDA、丁基锂或其组合。
本发明所述的如式(I)所示化合物的同位素标记化合物可通过与未标记化合物类似的合成方法来制备，所不同的是把未标记的起始原料和/或试剂换成同位素标记的起始原料和/或试剂。
本发明还提供了一种药物组合物，其包含治疗有效量的如上所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物以及药学上可接受的辅料。所述药学上可接受的辅料可以为稀释剂、吸收剂、润湿剂、粘合剂、崩解剂和润滑剂中的一种或多种。
本发明还提供了如上所述的式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物在制备药物中的用途。所述的药物优选治疗癌症的药物。
本发明还提供了如上所述的式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物在制备治疗与EED蛋白和/或PRC2蛋白复合物介导的癌症药物方面的用途。
优选地，所述癌症包括但不局限于扩散大B细胞淋巴癌、滤泡性淋巴瘤、非霍奇金森淋巴瘤等淋巴癌、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌样瘤、肝癌、前列腺癌、乳腺癌、脑瘤包括神经母细胞瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤等等。
优选地，所述化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物与其他药物联合使用；更优选地，所述其他药物选自抗癌药、肿瘤免疫药物、抗过敏药、止吐药、镇痛药或细胞保护药物。
本发明还提供了一种药物制剂，其包含如上所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物。所述药物制剂优选为片剂、胶囊(如持续释放或定时释放的胶囊)、药丸、粉末、颗粒(如小颗粒)、酏剂、酊剂、悬浮液(如纳米混悬液、微悬浮液)和喷雾干燥的分散体等形式的悬浮物、糖浆、乳液、溶液等形式。所述药物制剂优选的给药方式为口服、舌下含服、包括皮下注射、静脉注射、肌肉注射、胸骨内注射、注入等形式的注射、鼻部服用(比如鼻膜吸入)、局部表面(如乳霜和药膏)给药、直肠给药(如栓剂)等等方式。本发明公开的化合物可以单独服用也可以与适当的药物载体一起服用。
本发明还提供了前述的药物制剂可配方成适当的药物剂量以方便并控制药物的服用量。本发明公开的化合物的剂量方案根据具体的因素有所不同，比如药效学及服用的方式、服用对象、性别、年龄、健康状况以及服药对象的体重、病情特征、其它同时服药状况、服药的频率、肝肾功能以及想达到的效果等等。本发明公开的化合物可以每天单剂量的服用，也可以总剂量分多次服用(比如每天两至四次)。
本发明还提供了一种治疗癌症的方法，其包括向需要此治疗的患者给予治疗有效量的如上所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体或其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物。所述的癌症包括但不局限于扩散大B细胞淋巴癌、滤泡性淋巴瘤、非霍奇金森淋巴瘤等淋巴癌、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌样瘤、肝癌、前列腺癌、乳腺癌、脑瘤包括神经母细胞瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色 素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤等等。
本发明还提供了式(I)所示的化合物、其药学上可接受的盐、立体异构体或其溶剂化物或前述同位素标记化合物与其他药物联合使用，所述其他药物选自：抗癌药、肿瘤免疫药物、抗过敏药、止吐药、镇痛药和细胞保护药物等等，一起联用具有更好的效果。
本发明中，所述的癌症优选与EED蛋白和/或PRC2蛋白复合物介导的癌症。
本发明还提供了一种抑制EED蛋白和/或PRC2蛋白复合物活性的方法，其包括给予受试者治疗有效量的如上所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体或其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物。
本发明还提供了一种阻断EED与H3K27(例如H3K27me3)结合的方法，其包括给予受试者治疗有效量的如上所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体或其溶剂化物或它们的同位素标记化合物，或如上所述的药物组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
基团定义
可在参考文献(包括Carey and Sundberg"ADVANCED ORGANIC CHEMISTRY 4TH ED."Vols.A(2000)and B(2001),Plenum Press,New York)中找到对标准化学术语的定义。除非另有说明，否则采用本领域技术范围内的常规方法，如质谱、NMR、IR和UV/VIS光谱法和药理学方法。除非提出具体定义，否则本文在分析化学、有机合成化学以及药物和药物化学的有关描述中采用的术语是本领域已知的。可在化学合成、化学分析、药物制备、制剂和递送，以及对患者的治疗中使用标准技术。例如，可利用厂商对试剂盒的使用说明，或者按照本领域公知的方式或本发明的说明来实施反应和进行纯化。通常可根据本说明书中引用和讨论的多个概要性和较具体的文献中的描述，按照本领域熟知的常规方法实施上述技术和方法。在本说明书中，可由本领域技术人员选择基团及其取代基以提供稳定的结构部分和化合物。
当通过从左向右书写的常规化学式描述取代基时，该取代基也同样包括从右向左书写结构式时所得到的在化学上等同的取代基。举例而言，-CH 2O-等同于-OCH 2-。
本文所用的章节标题仅用于组织文章的目的，而不应被解释为对所述主题的限制。本申请中引用 的所有文献或文献部分包括但不限于专利、专利申请、文章、书籍、操作手册和论文，均通过引用方式整体并入本文。
在本文中定义的某些化学基团前面通过简化符号来表示该基团中存在的碳原子总数。例如，C 1-6烷基是指具有总共1至6个碳原子(如1,2,3,4,5,6个碳原子)的如下文所定义的烷基。简化符号中的碳原子总数不包括可能存在于所述基团的取代基中的碳。
当所列举的取代基中没有指明其通过哪一个原子连接到化学结构通式(包括但未具体提及的化合物)中时，这种取代基可以通过其任何原子相键合。取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除前述以外，当用于本申请的说明书及权利要求书中时，除非另外特别指明，否则以下术语具有如下所示的含义。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。术语“C x-C y烷基"或“C x-y烷基"是指含有x至y个碳原子的直链或支链饱和烃。例如，术语“C 1-C 6烷基”或“C 1-6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基；“C 1-4烷基”或“C 1-C  4烷基”特指独立公开的甲基、乙基、C 3烷基(即丙基，包括正丙基和异丙基)、C 4烷基(即丁基，包括正丁基、异丁基、仲丁基和叔丁基)。
当所列举的基团中没有明确指明其具有取代基时，这种基团仅指未被取代。例如当“C 1-C 4烷基”前没有“取代或未取代的”的限定时，仅指“C 1-C 4烷基”本身或“未取代的C 1-C 4烷基”。
在本发明的各部分，描述了连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”，则应该理解，该“烷基”代表连接的亚烷基基团。
在一些具体的结构中，当烷基基团清楚地表示为连接基团时，则该烷基基团代表连接的亚烷基基团，例如，基团“卤代-C 1-C 6烷基”中的C 1-C 6烷基应当理解为C 1-C 6亚烷基。
另外，需要说明的是，除非以其他方式明确指出，在本发明中所采用的描述方式“…独立地为”应做广义理解，是指所描述的各个个体之间是相互独立的，可以独立地为相同或不同的具体基团。更详细地，描述方式“…独立地为”既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响；也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
应该理解，在本发明中使用的单数形式，如“一种”，包括复数指代，除非另有规定。
术语“一种(个)或多种(个)”或“一种(个)或两种(个)以上”是指即1、2、3、4、5、6、7、8、9或更多。
在本申请中，术语“卤素”是指氟、氯、溴或碘。
Figure PCTCN2022111668-appb-000016
表示单键时，是指
Figure PCTCN2022111668-appb-000017
的混合。
本领域技术人员可以理解，根据本领域中使用的惯例，本申请描述基团的结构式中所使用的
Figure PCTCN2022111668-appb-000018
及
Figure PCTCN2022111668-appb-000019
是指，相应的基团R通过该位点与化合物中的其它片段、基团进行连接。
“羟基”是指-OH基团。
“羰基”是指-C(＝O)-基团。当
Figure PCTCN2022111668-appb-000020
中的R为羰基时，
Figure PCTCN2022111668-appb-000021
为
Figure PCTCN2022111668-appb-000022
“氰基”是指-CN。
“氨基”是指-NH 2。
“R 5b取代的氨基”，R 5b可以为一个或两个，当R 5b为两个时，可以是相同基团，也可以是不同基团。
“羧基”是指-COOH。
在本申请中，作为基团或是其它基团的一部分(例如用在卤素取代的烷基等基团中)，术语“烷基”是指完全饱和的直链或支链的烃链基，仅由碳原子和氢原子组成、具有例如1至12个(优选1至8个，更优选1至6个，更优选1至4个)碳原子，且通过单键与分子的其余部分连接，例如包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、2-甲基丁基、2,2-二甲基丙基、正己基、庚基、2-甲基己基、3-甲基己基、辛基、壬基和癸基等。
在本申请中，作为基团或是其它基团的一部分，术语“卤代烷基”是指烷基(如本发明中所定义)中的一个或多个氢原子被卤素(如本发明中所定义)所取代，卤素的个数可以为一个或多个；当卤素的个数为多个时，卤素相同或不同。例如，氟代烷基是指烷基被一个或多个氟取代。卤代烷基的例子包括但不限于三氟甲基、二氟甲基和一氟甲基。
本申请中，优选地，“杂环烷基”中碳原子数为3、4、5或6个，杂原子选自N、O和S，杂原子数为1、2、3或4个，进一步优选地，碳原子数为4或5个，杂原子选自N和O，杂原子数为1或2个；例如
Figure PCTCN2022111668-appb-000023
本申请中，优选地，“杂螺环烷基”中碳原子数为4、5或6个，杂原子选自N、O和S，杂原子数为1、2、3或4个，进一步优选地，杂原子选自N和O，杂原子数为1或2个；例如
Figure PCTCN2022111668-appb-000024
在本申请中，作为基团或是其它基团的一部分，术语“环烃基”意指仅由碳原子和氢原子组成的稳定的非芳香族单环或多环烃基，其可包括稠合环体系、桥环体系或螺环体系，具有3至15个碳原子，优选具有3至10个碳原子，更优选具有3至8个碳原子，且其为饱和或不饱和并可经由任何适宜的碳原子通过单键与分子的其余部分连接。除非本说明书中另外特别指明，环烃基中的碳原子可以任选地被氧化。环烃基的实例包括但不限于环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环辛基、1H-茚基、2,3-二氢化茚基、1,2,3,4-四氢-萘基、5,6,7,8-四氢-萘基、8,9-二氢-7H-苯并环庚烯-6-基、6,7,8,9-四氢-5H-苯并环庚烯基、5,6,7,8,9,10-六氢-苯并环辛烯基、芴基、二环[2.2.1]庚基、7,7-二甲基-二环[2.2.1]庚基、二环[2.2.1]庚烯基、二环[2.2.2]辛基、二环[3.1.1]庚基、二环 [3.2.1]辛基、二环[2.2.2]辛烯基、二环[3.2.1]辛烯基、金刚烷基、八氢-4,7-亚甲基-1H-茚基和八氢-2,5-亚甲基-并环戊二烯基等。
在本申请中，作为基团或是其它基团的一部分，术语“环烷基”意指饱和的环烃基。
在本申请中，作为基团或是其它基团的一部分，术语“环烯基”意指具有至少一个双键(如碳碳双键)的环烃基。环烯基可以通过其中的双键的原子与分子的其余部分连接。
在本申请中，作为基团或是其它基团的一部分，术语“杂环基”意指由2至14个碳原子以及1至6个选自氮、磷、氧和硫的杂原子组成的稳定的3元至20元非芳香族环状基团。除非本说明书中另外特别指明，否则杂环基可以为单环、双环、三环或更多环的环体系，其可包括稠合环体系、桥环体系或螺环体系；其杂环基中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化；且杂环基可为部分或完全饱和。杂环基可以经由碳原子或者杂原子并通过单键与分子其余部分连接。在包含稠环的杂环基中，一个或多个环可以是下文所定义的芳基或杂芳基，条件是与分子其余部分的连接点为非芳香族环原子。就本发明的目的而言，杂环基优选包含1至3个选自氮、氧和硫的杂原子的稳定的4元至11元非芳香性单环、双环、桥环或螺环基团，更优选包含1至3个选自氮、氧和硫的杂原子的稳定的4元至8元非芳香性单环、双环、桥环或螺环基团。杂环基的实例包括但不限于：吡咯烷基、吗啉基、哌嗪基、高哌嗪基、哌啶基、硫代吗啉基、2,7-二氮杂-螺[3.5]壬烷-7-基、2-氧杂-6-氮杂-螺[3.3]庚烷-6-基、2,5-二氮杂-双环[2.2.1]庚烷-2-基、氮杂环丁烷基、吡喃基、四氢吡喃基、噻喃基、四氢呋喃基、噁嗪基、二氧环戊基、四氢异喹啉基、十氢异喹啉基、咪唑啉基、咪唑烷基、喹嗪基、噻唑烷基、异噻唑烷基、异噁唑烷基、二氢吲哚基、八氢吲哚基、八氢异吲哚基、吡咯烷基、吡唑烷基、邻苯二甲酰亚氨基等。
在本申请中，作为基团或是其它基团的一部分，术语“芳基”意指具有6至18个碳原子(优选具有6至10个碳原子)的共轭烃环体系基团。就本发明的目的而言，芳基可以为单环、双环、三环或更多环的环体系，还可以与上文所定义的环烷基或杂环基稠合，条件是芳基经由芳香环上的原子通过单键与分子的其余部分连接。芳基的实例包括但不限于苯基、萘基、蒽基、菲基、芴基、2,3-二氢-1H-异吲哚基、2-苯并噁唑啉酮、2H-1,4-苯并噁嗪-3(4H)-酮-7-基等。
在本申请中，术语“芳基烷基”是指被上文所定义的芳基所取代的上文所定义的烷基。
在本申请中，作为基团或是其它基团的一部分，术语“杂芳基”意指环内具有1至15个碳原子(优选具有1至10个碳原子)和1至6个选自氮、氧和硫的杂原子的5元至16元共轭环系基团。除非本说明书中另外特别指明，否则杂芳基可为单环、双环、三环或更多环的环体系，还可以与上文所定义的环烷基或杂环基稠合，条件是杂芳基经由芳香环上的原子通过单键与分子的其余部分连接。杂芳基中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化。就本发明的目的而言，杂芳基优选包含1至5个选自氮、氧和硫的杂原子的稳定的5元至12元芳香性基团，更优选包含1至4个选自氮、氧和硫的杂原子的稳定的5元至10元芳香性基团或者包含1至3个选自氮、氧和硫的杂原子的5元至6元芳香性基团(例如杂芳基为C 1-C 5的杂芳基，其中杂原子选自N、O和S，杂原子数为1、2、3或4)。杂芳基的实例包括但不限于噻吩基、咪唑基、吡唑基、噻唑基、噁唑基、噁二唑基、异噁 唑基、吡啶基、嘧啶基、吡嗪基、哒嗪基、苯并咪唑基、苯并吡唑基、吲哚基、呋喃基、吡咯基、三唑基、四唑基、三嗪基、吲嗪基、异吲哚基、吲唑基、异吲唑基、嘌呤基、喹啉基、异喹啉基、二氮萘基、萘啶基、喹噁啉基、蝶啶基、咔唑基、咔啉基、菲啶基、菲咯啉基、吖啶基、吩嗪基、异噻唑基、苯并噻唑基、苯并噻吩基、噁三唑基、噌啉基、喹唑啉基、苯硫基、中氮茚基、邻二氮杂菲基、异噁唑基、吩噁嗪基、吩噻嗪基、4,5,6,7-四氢苯并[b]噻吩基、萘并吡啶基、[1,2,4]三唑并[4,3-b]哒嗪、[1,2,4]三唑并[4,3-a]吡嗪、[1,2,4]三唑并[4,3-c]嘧啶、[1,2,4]三唑并[4,3-a]吡啶、咪唑并[1,2-a]吡啶、咪唑并[1,2-b]哒嗪、咪唑并[1,2-a]吡嗪等。
在本申请中，“任选地”表示随后描述的事件或状况可能发生也可能不发生，且该描述同时包括该事件或状况发生和不发生的情况。例如，“任选地被取代的芳基”表示芳基被取代或未被取代，且该描述同时包括被取代的芳基与未被取代的芳基。本发明权利要求书和说明书部分所述的“任选地”的取代基选自烷基、烯基、炔基、卤素、卤代烷基、卤代烯基、卤代炔基、氰基、硝基、任选取代的芳基、任选取代的杂芳基、任选取代的环烃基、任选取代的杂环烃基。
在本发明中，术语“取代”或“取代基”是指一个或多个氢原子被指定的基团所代替。当没有指明取代基的个数时，取代基可以为一个或多个；当没有指明取代位置时，取代可以在任何位置，但是只有形成一个稳定的或者是化学意义上可行的化学物才是被允许的。
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。例如，在
Figure PCTCN2022111668-appb-000025
中，当n为2时，表示苯环被2个R取代，并且每个R都有独立的选项，即2个R可以相同，也可以不同。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。
本文所用术语“部分”、“结构部分”、“化学部分”、“基团”、“化学基团”是指分子中的特定片段或官能团。化学部分通常被认为是嵌入或附加到分子上的化学实体。
当本发明的化合物中含有烯双键时，除非另有说明，否则本发明的化合物旨在包含E-和Z-几何异构体。
“互变异构体”是指质子从分子的一个原子转移至相同分子的另一个原子而形成的异构体。本发明的化合物的所有互变异构形式也将包含在本发明的范围内。
本发明的化合物或其药学上可接受的盐可能含有一个或多个手性碳原子，且因此可产生对映异构体、非对映异构体及其它立体异构形式。每个手性碳原子可以基于立体化学而被定义为(R)-或(S)-。本发明旨在包括所有可能的异构体，以及其外消旋体和光学纯形式。本发明的化合物的制备可以选择外消旋体、非对映异构体或对映异构体作为原料或中间体。光学活性的异构体可以使用手性合成子或手性试剂来制备，或者使用常规技术进行拆分，例如采用结晶以及手性色谱等方法。本发明的化合物的立体异构体可有为(R)-或(S)-异构体。
制备/分离个别异构体的常规技术包括由合适的光学纯前体的手性合成，或者使用例如手性高效液相色谱法拆分外消旋体(或盐或衍生物的外消旋体)，例如可参见Gerald Gübitz and Martin G.Schmid(Eds.),Chiral Separations,Methods and Protocols,Methods in Molecular Biology,Vol.243,2004；A.M.Stalcup,Chiral Separations,Annu.Rev.Anal.Chem.3:341-63,2010；Fumiss et al.(eds.),VOGEL’S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED.,Longman Scientific and Technical Ltd.,Essex,1991,809-816；Heller,Acc.Chem.Res.1990,23,128。
在本申请中，术语“药学上可接受的盐”包括药学上可接受的酸加成盐和药学上可接受的碱加成盐。
“药学上可接受的酸加成盐”是指能够保留游离碱的生物有效性而无其它副作用的，与无机酸或有机酸所形成的盐。无机酸盐包括但不限于盐酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐等；有机酸盐包括但不限于甲酸盐、乙酸盐、2,2-二氯乙酸盐、三氟乙酸盐、丙酸盐、己酸盐、辛酸盐、癸酸盐、十一碳烯酸盐、乙醇酸盐、葡糖酸盐、乳酸盐、癸二酸盐、己二酸盐、戊二酸盐、丙二酸盐、草酸盐、马来酸盐、琥珀酸盐、富马酸盐、酒石酸盐、柠檬酸盐、棕榈酸盐、硬脂酸盐、油酸盐、肉桂酸盐、月桂酸盐、苹果酸盐、谷氨酸盐、焦谷氨酸盐、天冬氨酸盐、苯甲酸盐、甲磺酸盐、苯磺酸盐、对甲苯磺酸盐、海藻酸盐、抗坏血酸盐、水杨酸盐、4-氨基水杨酸盐、萘二磺酸盐等。这些盐可通过本专业已知的方法制备。
“药学上可接受的碱加成盐”是指能够保持游离酸的生物有效性而无其它副作用的、与无机碱或有机碱所形成的盐。衍生自无机碱的盐包括但不限于钠盐、钾盐、锂盐、铵盐、钙盐、镁盐、铁盐、锌盐、铜盐、锰盐、铝盐等。优选的无机盐为铵盐、钠盐、钾盐、钙盐及镁盐。衍生自有机碱的盐包括但不限于以下的盐：伯胺类、仲胺类及叔胺类，被取代的胺类，包括天然的被取代胺类、环状胺类及碱性离子交换树脂，例如氨、异丙胺、三甲胺、二乙胺、三乙胺、三丙胺、乙醇胺、二乙醇胺、三乙醇胺、二甲基乙醇胺、2-二甲氨基乙醇、2-二乙氨基乙醇、二环己胺、赖氨酸、精氨酸、组氨酸、咖啡因、普鲁卡因、胆碱、甜菜碱、乙二胺、葡萄糖胺、甲基葡萄糖胺、可可碱、嘌呤、哌嗪、哌啶、N-乙基哌啶、聚胺树脂等。优选的有机碱包括异丙胺、二乙胺、乙醇胺、三甲胺、二环己基胺、胆碱及咖啡因。这些盐可通过本专业已知的方法制备。
在本申请中，“药物组合物”是指本发明化合物与本领域通常接受的用于将生物活性化合物输送至哺乳动物(例如人)的介质的制剂。该介质包括药学上可接受的载体。药物组合物的目的是促进生物体的给药，利于活性成分的吸收进而发挥生物活性。
本文所用术语“药学上可接受的”是指不影响本发明化合物的生物活性或性质的物质(如载体或稀释剂)，并且相对无毒，即该物质可施用于个体而不造成不良的生物反应或以不良方式与组合物中包含的任意组分相互作用。
在本申请中，“药学上可接受的辅料”包括但不限于任何被相关的政府管理部门许可为可接受供人类或家畜使用的佐剂、载体、赋形剂、助流剂、增甜剂、稀释剂、防腐剂、染料/着色剂、矫味剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗剂、溶剂或乳化剂。
本文所用术语“预防的”、“预防”和“防止”包括使病患减少疾病或病症的发生或恶化的可能性。
本文所用的术语“治疗”和其它类似的同义词包括以下含义：
(i)预防疾病或病症在哺乳动物中出现，特别是当这类哺乳动物易患有该疾病或病症，但尚未被诊断为已患有该疾病或病症时；
(ii)抑制疾病或病症，即遏制其发展；
(iii)缓解疾病或病症，即，使该疾病或病症的状态消退；或者
(iv)减轻该疾病或病症所造成的症状。
本文所使用术语“有效量”、“治疗有效量”或“药学有效量”是指服用后足以在某种程度上缓解所治疗的疾病或病症的一个或多个症状的至少一种药剂或化合物的量。其结果可以为迹象、症状或病因的消减和/或缓解，或生物系统的任何其它所需变化。例如，用于治疗的“有效量”是在临床上提供显著的病症缓解效果所需的包含本文公开化合物的组合物的量。可使用诸如剂量递增试验的技术测定适合于任意个体病例中的有效量。
本文所用术语“服用”、“施用”、“给药”等是指能够将化合物或组合物递送到进行生物作用的所需位点的方法。这些方法包括但不限于口服途径、经十二指肠途径、胃肠外注射(包括静脉内、皮下、腹膜内、肌内、动脉内注射或输注)、局部给药和经直肠给药。本领域技术人员熟知可用于本文所述化合物和方法的施用技术，例如在Goodman and Gilman,The Pharmacological Basis of Therapeutics,current ed.；Pergamon；and Remington’s,Pharmaceutical Sciences(current edition),Mack Publishing Co.,Easton,Pa中讨论的那些。在优选的实施方案中，本文讨论的化合物和组合物通过口服施用。
本文所使用术语“药物组合”、“药物联用”、“联合用药”、“施用其它治疗”、“施用其它治疗剂”等是指通过混合或组合不止一种活性成分而获得的药物治疗，其包括活性成分的固定和不固定组合。术语“固定组合”是指以单个实体或单个剂型的形式向患者同时施用至少一种本文所述的化合物和至少一种协同药剂。术语“不固定组合”是指以单独实体的形式向患者同时施用、合用或以可变的间隔时间顺次施用至少一种本文所述的化合物和至少一种协同制剂。这些也应用到鸡尾酒疗法中，例如施用三种或更多种活性成分。
本领域技术人员还应当理解，在下文所述的方法中，中间体化合物官能团可能需要由适当的保护基保护。这样的官能团包括羟基、氨基、巯基及羧酸。合适的羟基保护基包括三烷基甲硅烷基或二芳基烷基甲硅烷基(例如叔丁基二甲基甲硅烷基、叔丁基二苯基甲硅烷基或三甲基甲硅烷基)、四氢吡喃基、苄基等。合适的氨基、脒基及胍基的保护基包括叔丁氧羰基、苄氧羰基等。合适的巯基保护基包括-C(O)-R”(其中R”为烷基、芳基或芳烷基)、对甲氧基苄基、三苯甲基等。合适的羧基保护基包括烷基、芳基或芳烷基酯类。
保护基可根据本领域技术人员已知的和如本文所述的标准技术来引入和除去。保护基的使用详述于Greene,T.W.与P.G.M.Wuts,Protective Groups in Organi Synthesis,(1999),4th Ed.,Wiley中。保护基还可为聚合物树脂。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：与US20160176882A1，WO2017219948A1中的化合物N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-8-(2-甲基吡啶-3-基)-[1,2,4]三唑并[4,3-c]嘧啶-5-胺相对比，本发明的化合物抗细胞增殖活性增加了约10倍。本发明公开的化合物在与EED蛋白的结合时，在结合的“口袋”外侧的双环结构能使化合物具有更好的代谢稳定性。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
下述实施例中所用的起始物可由化学品销售商如Aldrich、TCI、Alfa Aesar、毕得、安耐吉等处购得，或者可通过已知的方法来合成。
下述实施例中，冰浴是指-5摄氏度至0摄氏度，室温是指10摄氏度至30摄氏度，回流温度一般是指常压下溶剂回流温度。反应过夜是指时间为8-15小时。下述实施例中，未限定具体操作温度的，均在室温下进行。
下述实施例中，中间体和最终产物的分离提纯是通过正相或反相色谱柱分离或者其它合适的方法。正相快速色谱柱是用乙酸乙酯和正己烷或甲醇和二氯甲烷等作为流动相。反相制备性高压液相色谱(HPLC)是用C18柱并用UV 214nm和254nm来检测，其流动相为A(水和0.1％甲酸)、B(乙腈)或者流动相A(水和0.1％碳酸氢铵)、B(乙腈)。
各实施例中：
LCMS仪器：Pump Agilent 1260UV检测器：Agilent 1260 DAD
Mass Spectrometer API 3000
层析柱：Waters sunfire C18,4.6×50mm,5μm
流动相：A-H 2O(0.1％HCOOH)；B-乙腈
NMR仪器：Bruker Ascend 400M( 1H NMR:400MHz； 13C NMR:100MHz)。
实施例1
中间体4-(氨基甲基)-5-氟-2,3-二氢苯并呋喃-3-醇(A)：
步骤一：中间体2-溴-3,6-二氟苯甲醛(A-2)：
Figure PCTCN2022111668-appb-000026
在500mL干燥的三口烧瓶中加入A-1(22g,114mmol)和干燥的THF(200mL)，冷却至–70℃。二异丙基氨基锂(2M,68.4mL)缓慢滴加入反应液中。反应液在相同温度下搅拌45分钟后，加入DMF(17.8mL,228mmol)。反应液在相同温度下搅拌两小时后升至0℃。饱和氯化铵(200mL) 加入到反应液中。反应液用EtOAc(200mL X 2)萃取。合并的有机相用盐水洗涤一次，经无水硫酸钠干燥，过滤，在减压下浓缩。残余物在硅胶上纯化(石油醚：乙酸乙酯＝100：1)，得到A-2(20g，79.4％产率)，为淡黄色固体。
1H NMR(400MHz,CDCl 3)δ10.34(s,1H),7.34(ddd,J＝9.2,7.4,4.5Hz,1H),7.16(td,J＝9.3,4.0Hz,1H)ppm.
步骤二：中间体2-溴-3-氟-6-甲氧基苯甲醛(A-3)：
Figure PCTCN2022111668-appb-000027
在2L的三口烧瓶中加入A-2(20g，90.5mmol)，用无水THF(1000ml)和MeOH(200ml)搅拌溶解。加入甲醇钠(5.87g，108.6mmol)，反应液在60℃.搅拌18小时。减压浓缩除去大部分溶剂，加入500mL水。悬浊液搅拌30分钟后过滤，收集固体。固体用石油醚和乙酸乙酯(5:1)混合液打浆，过滤得到固体，减压干燥得到A-3(18g,85％产率)，为黄色固体。
LC-MS:m/z 233.1[M+H] +.
步骤三：中间体2-溴-3-氟-6-羟基苯甲醛(A-4)：
Figure PCTCN2022111668-appb-000028
在1L的单口烧瓶中加入A-3(16.8g，72.1mmol)和二氯甲烷(300mL)。在-78℃下缓慢滴加三溴化硼(21.7g,86.5mmol),反应液升至室温，并搅拌18小时。反应液用二氯甲烷(300mL)稀释，缓慢加入饱和碳酸氢钠(300mL)。有机相用盐水洗涤两次，经无水硫酸钠干燥，过滤，在减压下浓缩。残余物在硅胶上纯化(石油醚：乙酸乙酯＝50：1)，得到A-4(10g，63.3％产率)，为淡黄色固体。
1H NMR(400MHz,CDCl 3)δ11.77(s,1H),10.34(s,1H),7.29(dt,J＝12.7,6.3Hz,1H),6.94(dd,J＝9.3,4.1Hz,1H)ppm.
步骤四：中间体4-溴-5-氟-2,3-二氢苯并呋喃-3-醇(A-5)：
Figure PCTCN2022111668-appb-000029
在干燥的三口烧瓶中加入三甲基碘化亚砜(9.73g，44.2mmol)和DMSO(50ml)。在冰水浴下加入叔丁醇钠(4.25g，44.2mmol)。反应液在室温下搅拌2小时，加入A-4(8.8g，40.2mmol)。反应液在室温下搅拌18小时后，加入乙酸乙酯(250ml)和水(250ml)，用乙酸乙酯萃取(250ml X 2)。有机相用水和盐水分别洗涤一次，经无水硫酸钠干燥，过滤，在减压下浓缩。残余物在硅胶上纯化(石油醚：乙酸乙酯＝10：1)，得到A-5(6.2g，66.2％产率)，为白色固体。
1H NMR(400MHz,CDCl 3)δ7.03(t,J＝8.7Hz,1H),6.76(dd,J＝8.8,3.5Hz,1H),5.51-5.40(m,1H),4.58(ddd,J＝13.4,10.8,4.6Hz,2H),2.33(d,J＝4.9Hz,1H)ppm.
步骤五：中间体5-氟-3-羟基-2,3-二氢苯并呋喃-4-甲腈(A-6)：
Figure PCTCN2022111668-appb-000030
在干燥的单口烧瓶中依次加入A-5(2.7g，11.6mmol)，氰化锌(2.04g，17.4mmol)，DMF(50ml)和四三苯基膦钯(1.34g，1.16mmol)。反应液在氮气保护下加热至120℃，搅拌18小时。反应液冷却至室温，用乙酸乙酯(200mL X 3)和水(200ml)萃取。有机相用水和盐水分别洗涤一次，经无水硫酸钠干燥，过滤，在减压下浓缩。残余物在硅胶上纯化(石油醚：乙酸乙酯＝5：1)，得到A-6(1.6g，77％产率)，为白色固体。
1H NMR(400MHz,CDCl 3)δ7.20-6.99(m,2H),5.63(dd,J＝6.9,2.9Hz,1H),4.69(dd,J＝10.8,7.1Hz,1H),4.55(dd,J＝10.8,3.1Hz,1H),2.74(s,1H)ppm.
步骤六：中间体(5-氟-2,3-二氢苯并呋喃-4-基)甲胺(A2)：
Figure PCTCN2022111668-appb-000031
在100mL单口瓶中依次加入A-6(1.55g，8.65mmol)，三氟乙酸(1.09g，8.65mmol)，甲醇(20mL)和10％钯碳(2g，含50％水)。反应混合物用氢气鼓气5分钟，用氢气球换气三次，在氢气球下60℃搅拌48小时。混合物经硅藻土过滤，用甲醇(50mL X 2)洗涤，滤液减压浓缩加入二氧六环(10mL)和10M氢氧化钠水溶液(1mL)，用二氧六环(10mL X 2)萃取，有机相经无水硫酸钠干燥，过滤，在减压下浓缩。得到化合物A2(1.4g，90％纯度，87％产率)，直接用于下一步。
1H NMR(400MHz,CD 3OD)δ3.27(t,2H),3.77(s,2H),4.56(t,2H),6.59(dd 1H),6.81(dd,1H)ppm；LC-MS:m/z 168.1[M+H] +.
实施例2
8-(5-((二甲氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈(BR-1)
步骤一：2-(5-溴-2-(甲硫基)嘧啶-4-基)-2-((二苯亚甲基)氨基)乙腈(2)
Figure PCTCN2022111668-appb-000032
在零度下往氢化钠(3.63g，90.8mmol，60％纯度)的N,N-二甲基甲酰胺(60.0mL)溶液中加入N-(二苯亚甲基)氨基乙腈1(10.0g，45.4mmol)的N,N-二甲基甲酰胺(50.0mL)溶液，2分钟之后零度下加入5-溴-4-氯-2-(甲硫基)嘧啶(10.8g，45.4mmol)N,N-二甲基甲酰胺(30.0mL)，于40℃反应2小时。LCMS监测反应结束，氯化铵水溶液淬灭，乙酸乙酯萃取旋干得到粗品2-(5-溴-2-(甲硫基)嘧啶-4-基)-2-((二苯亚甲基)氨基)乙腈2(15.0g，粗品)，直接用于下一步。
1H NMR(400MHz,DMSO-d 6)δ7.58-7.52(m,7H),7.44-7.40(m,3H),7.26-7.23(m,2H),4.37(s,3H)ppm；LCMS m/z 424.7[M+H] +.
步骤二：2-氨基-2-(5-溴-2-(甲硫基)嘧啶-4-基)乙腈(3)
Figure PCTCN2022111668-appb-000033
在零度的条件下将2-(5-溴-2-(甲硫基)嘧啶-4-基)-2-((二苯亚甲基)氨基)乙腈2(15.0g，粗品)，的四氢呋喃(200mL)和水溶液(150mL)加入浓盐酸(12M，23.6mL)，于20℃反应2小时。TLC监测反应结束，反应液旋掉四氢呋喃后加入氢氧化钠水溶液(1.00M)调节pH到8.0左右，用乙酸乙酯萃取，干燥，选干得到粗品2-氨基-2-(5-溴-2-(甲硫基)嘧啶-4-基)乙腈3(9.00g，粗品)直接用于下一步。
1H NMR(400MHz,DMSO-d 6)δ8.86(s,1H),5.28(s,1H),2.89(s,2H),2.57(s,3H)ppm；LCMS m/z 260.9[M+H] +.
步骤三：N-((5-溴-2-(甲硫基)嘧啶-4-基)(氰基)甲基)甲酰胺(4)
Figure PCTCN2022111668-appb-000034
将甲酸(39.0g，848mmol)和醋酐(34.8g,341mmol)混合后在50℃反应1小时，冷到0度后加入到2-氨基-2-(5-溴-2-(甲硫基)嘧啶-4-基)乙腈3(9.00g，粗品)二氯甲烷(100mL)溶液中。在20度下反应2h。TLC监测反应结束，在零度下用氢氧化钠水溶液(1.00M)调减到8，乙酸乙酯萃取，干燥，选干得到粗品后柱层析得到的N-((5-溴-2-(甲硫基)嘧啶-4-基)(氰基)甲基)甲酰胺4(7.00g，22.6mmol，73.2％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.86(s,1H),5.28(s,1H),2.89(s,2H),2.57(s,3H)ppm；LCMS m/z 260.9[M+H] +.
步骤四：8-溴-5-(甲硫基)咪唑并[1,5-c]嘧啶-1-腈(5)
Figure PCTCN2022111668-appb-000035
往N-((5-溴-2-(甲硫基)嘧啶-4-基)(氰基)甲基)甲酰胺4(7.00g，24.3mmol)加入三氯氧磷(28.8g，188mmol)，在110度反应1小时。TLC监测反应结束，反应液选掉一部分三氯氧磷，然后在<30度下用水淬灭，最后用NaOH水溶液(6.00M)调减到8，乙酸乙酯萃取，干燥，选干得到粗品用石油醚/乙酸乙酯(5:1，100mL)打浆，滤出固体得到纯品，滤液柱层析得到少量产品，最后合并得到8-溴-5-(甲硫基)咪唑并[1,5-c]嘧啶-1-腈5(3.20g,11.7mmol,48.1％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.81(s,1H),8.06(s,1H),2.76(s,3H)ppm；LCMS m/z 270.9[M+H] +.
步骤五：8-溴-5-(甲磺酰基)咪唑并[1,5-c]嘧啶-1-腈(6)
Figure PCTCN2022111668-appb-000036
将8-溴-5-(甲硫基)咪唑并[1,5-c]嘧啶-1-腈5(0.60g，2.23mmol)溶于二氯甲烷(5mL)，在0℃下加入85％的间氯过氧苯甲酸(1.40g，6.91mmol)在20℃搅拌12小时。LCMS监测反应结束，反应液直接用于下一步。
LCMS m/z 302.9[M+H] +.
步骤六：8-溴-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈(7)
Figure PCTCN2022111668-appb-000037
将8-溴-5-(甲磺酰基)咪唑并[1,5-c]嘧啶-1-腈6(671mg，2.23mmol)以及(5-氟-2,3-二氢苯并呋喃-4-基)甲胺A(453mg，2.23mmol)溶在二氯甲烷(5mL)，加入碳酸钠(1.18g，11.1mmol)，在20℃搅拌4小时。LCMS监测反应结束，反应液用二氯甲烷稀释，盐水洗涤，有机相干燥旋干后经过柱层析得到棕色固体产物8-溴-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈7(400mg，46.2％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.90-8.77(m,2H),7.75(s,1H),6.93(t,J＝9.4Hz,1H),6.69(dd,J＝4.0,8.8Hz,1H),4.67(s,2H),4.53(t,J＝8.8Hz,2H),3.29-3.24(m,3H)ppm；LCMS m/z 388.0[M+H] +.
步骤七：8-溴-5-(溴甲基)-[1,2,4]三唑并[1,5-a]吡啶(9)
Figure PCTCN2022111668-appb-000038
在100mL圆底烧瓶中加入8-溴-5-甲基-[1,2,4]三唑并[1,5-a]吡啶8(2g，9.43mmol)和四氯化碳(20ml)，在冰水浴下加入NBS(2g，11.3mmol)和AIBN(310mg，0.2mmol)。加料完成后，将反应常温升至80加，保持反应16h过夜。反应液用亚硫酸钠水溶液洗涤后旋干后得到红褐色固体，用快速分离柱(洗脱剂为石油醚：乙酸乙酯＝3:1)纯化得到红褐色固体。真空干燥后得到纯的8-溴-5-(溴甲基)-[1,2,4]三唑并[1,5-a]吡啶9(1.7g，62％收率)。
1H NMR(400MHz,CDCl 3)δ8.48(s,1H),7.77(d,J＝7.7Hz,1H),7.07(d,J＝7.7Hz,1H),4.90(s,2H)ppm；LCMS:m/z 291.9[M+H] +.
步骤八：1-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)-N,N-二甲基甲胺(10)
Figure PCTCN2022111668-appb-000039
在100mL圆底烧瓶中加入8-溴-5-(溴甲基)-[1,2,4]三唑并[1,5-a]吡啶9(1g，3.44mmol)和四氢呋喃(15ml)，在冰水浴下加入1M的二甲胺四氢呋喃溶液(17ml，17.2mmol)和碳酸钾(0.59g，1.43mmol)。 加料完成后，将反应常温升至80℃，保持反应3h。反应完成后，液旋干后得到黄褐色固体，用快速分离柱(石油醚：乙酸乙酯＝5:1)洗脱纯化后得到黄色固体1-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)-N,N-二甲基甲胺10,(0.6g，68.3％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.74-8.38(m,1H),8.14-7.85(m,1H),7.31-6.90(m,1H),4.03-3.78(m,2H),2.38-2.20(m,6H)ppm；LCMS:m/z 255.0,257.0[M+H] +.
步骤九：(5-((二甲基氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(11)
Figure PCTCN2022111668-appb-000040
将1-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)-N,N-二甲基甲胺10(100mg，391μmol)，联硼酸频那醇酯(159mg，627μmol)，醋酸钾(115mg，1.18mmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(64.0mg，78.4μmol)在手套箱中溶于二氧六环(6mL)，在125℃反应5小时。LCMS监测反应结束，反应液直接用于下一步反应。
LCMS m/z 221.1[M+H] +.
步骤十：8-(5-((二甲氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈(BR-1)
Figure PCTCN2022111668-appb-000041
向上一步的硼酸反应液中加入8-溴-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈7(151mg，390μmol)，碳酸钾(108mg，781μmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(31.9mg，39.0μmol)，水(1mL)，二氧六环(3mL)，在100℃反应2小时。LCMS监测反应结束，反应液浓缩后通过柱层析纯化和perp-HPLC得到的溶液用饱和碳酸氢钠水溶液调节至pH为8，浓缩除去有机溶剂，过滤得到白色固体8-(5-((二甲氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈BR-1(70.0mg，36.8％收率)。
1H NMR(400MHz,DMSO-d 6)δ＝8.93(s,1H),8.83(s,1H),8.50(s,1H),7.85-7.76(m,2H),7.32(d,J＝7.6Hz,1H),6.96(t,J＝9.6Hz,1H),6.72(dd,J＝4.0,8.8Hz,1H),4.77(s,2H),4.56(t,J＝8.8Hz,2H),4.03(s,2H),3.35-3.35(m,2H),2.34(s,6H)ppm；LCMS m/z 484.4[M+H] +.
实施例3
5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)-8-(5-(2-羟基丙烷-2-基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)咪唑并[1,5-c]嘧啶-1-腈(BR-2)
步骤一：(E)-5-溴-6-(((二甲胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-2
Figure PCTCN2022111668-appb-000042
将(E)-5-溴-6-(((二甲胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯(5.0g,21.6mmol)，1,1-二甲氧基-N,N-二甲基甲胺(5.16g，43.3mmol)溶于甲苯(100mL)，反应液升温至110℃下反应4小时，TLC检测无原料剩余，反应液浓缩得到粗品(E)-5-溴-6-(((二甲胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-2(6.00g)。
LCMS:m/z 585.7[M+H] +.
步骤二：(E)-5-溴-6-(((羟胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-3
Figure PCTCN2022111668-appb-000043
在反应瓶中依次加入(E)-5-溴-6-(((二甲胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-2(6.0g,20.97mmol),盐酸羟胺(2.91g,41.94mmol),乙酸钠(3.44g，41.9mmol)，乙醇(60mL)。反应液于氮气氛围下升温至50℃反4小时，LCMS监测反应结束，降至室温，过滤得到(E)-5-溴-6-(((羟胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-3(5.9g，85.0％收率)。
1H NMR(400MHz,CDCl 3)δ8.27(br d,J＝8.5Hz,1H),8.19-8.13(m,1H),7.85(d,J＝7.9Hz,1H),7.50(d,J＝7.9Hz,1H),3.90(s,3H)ppm；LCMS:m/z 273.7[M+H] +.
步骤三：8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-甲酸甲酯E-4
Figure PCTCN2022111668-appb-000044
在室温下往(E)-5-溴-6-(((羟胺基)亚甲基)氨基)-吡啶-2-甲酸甲酯E-3(10g,36.49mmol)的四氢呋 喃(100mL)溶液中逐滴加入三氟乙酸酐(23.0g，109mmol)。然后缓慢升至75℃，并搅拌反应3小时。反应结束后，在0℃向混合物中加入饱和的碳酸氢钠水溶液(100mL)进行淬灭，用乙酸乙酯(100mL*3)进行萃取，合并的有机相干燥，浓缩。浓缩物用硅胶柱(石油醚：乙酸乙酯＝5:1到4:1)纯化得到白色固体产物8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-甲酸甲酯E-4(5.00g,收率53.5％)。
步骤四：2-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)丙烷-2-醇E-5
Figure PCTCN2022111668-appb-000045
在-30℃下，往8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-甲酸甲酯E-4(500mg,1.95mmol)的四氢呋喃溶液中(10mL)滴加甲基溴化镁(3M,2.60mL)并在此温度下搅拌三小时。反应结束后，向反应液中加入水(10mL)进行淬灭，用乙酸乙酯(10mL*3)进行萃取，合并的有机相用干燥，浓缩后用快速硅胶柱纯化得到2-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)丙烷-2-醇E-5(400mg，80.0％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.61(s,1H),8.05(d,J＝8.0Hz,1H),7.28(d,J＝8.0Hz,1H),5.91(s,1H),1.72(s,6H)ppm；LCMS:m/z 255.9[M+H] +.
步骤五：(5-(2-羟基丙烷-2-基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(12)
Figure PCTCN2022111668-appb-000046
将2-(8-溴-[1,2,4]三唑并[1,5-a]吡啶-5-基)叔醇E-5(100mg，390μmol)，联硼酸频那醇酯(158mg，624μmol)，醋酸钾(114mg，1.17mmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(63.7mg，78.0μmol)在手套箱中溶于二氧六环(6mL)，在125℃反应6小时。LCMS监测反应结束，反应液直接用于下一步反应。
LCMS m/z 222.2[M+H] +.
步骤六：5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)-8-(5-(2-羟基丙烷-2-基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)咪唑并[1,5-c]嘧啶-1-腈(BR-2)
Figure PCTCN2022111668-appb-000047
向上一步的硼酸12的反应液中加入8-溴-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈7(151mg，389μmol)，碳酸钾(107mg，778μmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(31.7mg，38.9μmol)，水(1mL)，二氧六环(3mL)，在100℃反应2小时。LCMS监测反应结束，反应液浓缩后通过柱层析纯化和perp-HPLC得到的溶液用饱和碳酸氢钠水溶液调节至pH为8，浓缩除去有机溶剂，过滤得到白色固体5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)-8-(5-(2-羟基丙烷-2-基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)咪唑并[1,5-c]嘧啶-1-腈(50.0mg，26.0％收率)。
1H NMR(400MHz,DMSO-d 6)δ＝8.90(s,1H),8.84(s,1H),8.51(s,1H),7.83-7.78(m,2H),7.44(d,J＝7.6Hz,1H),6.95(t,J＝9.4Hz,1H),6.75-6.67(m,1H),5.86(s,1H),4.77(s,2H),4.56(t,J＝8.6Hz,2H),3.37-3.32(m,2H),1.77(s,6H)ppm；LCMS m/z 485.4[M+H] +.
实施例4
8-(5-(二甲氨基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-甲腈(BR-3)
步骤一：8-碘-N,N-二甲基-[1,2,4]三唑并[1,5-a]吡啶-5-胺14
Figure PCTCN2022111668-appb-000048
向二甲胺的四氢呋喃溶液(4mL，2M/L)中加入5-氯-8-碘-[1,2,4]三唑并[1,5-a]吡啶13(200mg，0.72mmol)。混合物在90℃搅拌16h。LCMS显示反应结束，浓缩并纯化(石油醚：乙酸乙酯＝1:1)，得到8-碘-N,N-二甲基-[1,2,4]三唑并[1,5-a]吡啶-5-胺14(190mg，90％)。
LCMS:m/z 288.9[M+H] +.
步骤二：(5-(二甲氨基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(15)
Figure PCTCN2022111668-appb-000049
将8-碘-N,N-二甲基-[1,2,4]三唑并[1,5-a]吡啶-5-胺14(100mg，414μmol)，联硼酸频那醇酯(168mg，663μmol)，醋酸钾(122mg，1.24mmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(67.7mg，82.9μmol)在手套箱中溶于二氧六环(5mL)，在125℃反应6小时。LCMS监测反应结束，反应液直接用于下一步反应。
LCMS m/z 207.2[M+H] +.
步骤三：8-(5-(二甲氨基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈
Figure PCTCN2022111668-appb-000050
向上一步的硼酸15反应液中加入8-溴-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈(160mg，412μmol)，碳酸钾(114mg，825μmol)，[1,1-双(二苯基膦)二茂铁]二氯化钯二氯甲烷(33.6mg，41.2μmol)，水(1mL)，二氧六环(3mL)在100℃反应2小时。LCMS监测反应结束，反应液浓缩后通过柱层析纯化和perp-HPLC得到的溶液用饱和碳酸氢钠水溶液调节至pH为8，浓缩除去有机溶剂，过滤得到白色固体8-(5-(二甲氨基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)咪唑并[1,5-c]嘧啶-1-腈BR-3(13.0mg，6.61％收率)。
1H NMR(400MHz,DMSO-d 6)δ＝8.90-8.68(m,2H),8.40(s,1H),7.70-7.61(m,2H),6.94(t,J＝9.2Hz,1H),6.74-6.66(m,1H),6.56(d,J＝8.0Hz,1H),4.74(s,2H),4.55(t,J＝8.6Hz,2H),3.37-3.33(m,2H),3.17(s,6H)ppm；LCMS m/z 470.4[M+H] +.
实施例5
8-(5-((二甲氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-(甲磺酰)咪唑并[1,5-c]嘧啶-5-胺(BR-4)
步骤一:8-溴-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)咪唑并[1,5-c]嘧啶-5-胺(17)
Figure PCTCN2022111668-appb-000051
将8-溴-5-(甲硫基)咪唑并[1,5-c]嘧啶16(4.0g，16.4mmol)溶于二氯甲烷(60ml)中，在0℃加入间氯过氧苯甲酸(4.99g，24.6mmol，85％纯度)并在0℃下反应55分钟，然后在0℃滴加三乙胺(6.63g，65.5mmol)并搅拌5分钟，最后将5-氟-2,3-二氢苯并呋喃-4-基)甲胺A(3.34g，16.4mmol，HCl)加入反应液中，升温至20℃搅拌3小时，TLC显示无原料剩余，向反应液中加入水淬灭，用二氯甲烷萃取，将有机相洗涤，干燥，浓缩，粗品用快速硅胶柱纯化得到8-溴-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)咪唑并[1,5-c]嘧啶-5-胺17(4.0g，40.3％收率)。
1H NMR(400MHz,CDCl 3)δ8.23(s,1H),7.41-7.30(m,2H),6.87-6.77(m,1H),6.71-6.58(m,1H),6.10(br s,1H),4.79-4.71(m,2H),4.60(t,J＝8.8Hz,2H),3.37(t,J＝8.8Hz,2H)ppm；LCMS:m/z 363.0[M+H] +
步骤二：用实施例2步骤十的方法，以8-溴-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)咪唑并[1,5-c]嘧啶-5-胺(17)和(5-((二甲基氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(11)为原料可以得到：8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)咪唑并[1,5-c]嘧啶-5-胺(18)：
Figure PCTCN2022111668-appb-000052
1H NMR(400MHz,CDCl 3)δ8.22(s,1H),8.03(s,1H),7.78(s,1H),7.71(d,J＝7.4Hz,1H),7.30-7.27(m,1H),7.07(s,1H),6.87-6.72(m,1H),6.65(dd,J＝3.9,8.6Hz,1H),4.82(s,2H),4.60(t,J＝8.8Hz,2H),4.16(s,2H),3.67(t,J＝6.0Hz,1H),3.43(br t,J＝8.6Hz,2H),2.52(s,6H))ppm；LCMS:m/z 459.2[M+H] +
步骤三：8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-碘代咪唑并[1,5-c]嘧啶-5-胺(19)
Figure PCTCN2022111668-appb-000053
将8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)咪唑并[1,5-c]嘧啶-5-胺(400mg，0.782mmol)溶于醋酸(2ml)，在0℃加入N-碘代丁二酰亚胺(137mg，0.62mmol)，升温至20℃反应0.25小时，TLC和LCMS监测反应结束，将反应液的pH调至7，用乙酸乙酯萃取，有机相洗涤，干燥浓缩，粗品用快速硅胶柱纯化得到8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-碘代咪唑并[1,5-c]嘧啶-5-胺(190mg，33.9％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.73(s,1H),8.53-8.47(m,1H),8.47-8.43(m,1H),7.64(d,J＝7.3Hz,1H),7.33-7.26(m,2H),6.95(t,J＝9.4Hz,1H),6.74-6.66(m,1H),4.74(d,J＝4.9Hz,2H),4.55(t,J＝8.8Hz,2H),4.03(s,2H),3.30(br s,2H),2.35(s,6H)ppm；LCMS:m/z 585.5[M+H] +
步骤四：8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-(甲磺酰基)咪唑并[1,5-c]嘧啶-5-胺(BR-4)
Figure PCTCN2022111668-appb-000054
将8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-碘代咪唑并[1,5-c]嘧啶-5-胺(100mg，0.172mmol)，碘化亚铜(98mg，0.513mmol)，甲基亚磺酸钠(52.4mg，0.513mmol)溶于二甲基亚砜(1ml)，鼓氮气5分钟，在120℃微波20分钟，然后降至100℃反应3小时LCMS监测反应结束，将反应液浓缩，粗品用Pre-HPLC纯化得到8-(5-((二甲基胺)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)-N-((5-氟-2,3-二氢苯并呋喃-4-基)甲基)-1-(甲磺酰基)咪唑并[1,5-c]嘧啶-5-胺(30mg，32.3％收率)。
1H NMR(400MHz,DMSO-d 6)δ8.94(s,1H),8.89-8.80(m,1H),8.36(br s,1H),7.66-7.56(m,2H),7.26-7.15(m,1H),6.96(t,J＝9.4Hz,1H),6.75-6.67(m,1H),4.82-4.72(m,2H),4.56(t,J＝8.7Hz,2H),3.99(br s,2H),3.31-3.31(m,2H),2.87(s,3H),2.33(br s,6H)ppm；LCMS:m/z 537.2[M+H] +.
实施例6
按照实施例5的方法，用(5-(2-羟基丙烷-2-基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(12)替换(5- ((二甲基氨基)甲基)-[1,2,4]三唑并[1,5-a]吡啶-8-基)硼酸(11)可以得到如下化合物：
2-(8-(5-(((5-氟-2,3-二氢苯并呋喃-4-基)甲基)氨基)-1-(甲磺酰)咪唑并[1,5-c]嘧啶-8-基)-[1,2,4]三唑并[1,5-a]吡啶-5-基)丙烷-2-醇(BR-5) 1H NMR(400MHz,DMSO-d 6)δ8.94(s,1H),8.88-8.76(m,1H),8.38(s,1H),7.65-7.57(m,2H),7.34(d,J＝7.4Hz,1H),6.96(t,J＝9.4Hz,1H),6.80-6.62(m,1H),5.82(s,1H),4.85-4.68(m,2H),4.56(t,J＝8.7Hz,2H),3.32-3.31(m,2H),2.88(s,3H),1.76(s,6H)ppm；LCMS:m/z 538.2[M+H] +
Figure PCTCN2022111668-appb-000055
药理学及应用
EED作为PRC2蛋白复合物的主要组成部分之一，虽然并不具有酶催化的活性，但是它对PRC2的整体功能具有重要的作用。EED对PRC2的作用具体表现为两个方面：1)EED直接与三甲基化的H3K27Me3结合，这样能够把PCR2复合物定位在需要修饰的染色质上；2)EED对EZH2的酶催化功能有很大的变构促进作用。因此，开发作为变构蛋白EED的靶点化合物为提供抑制EZH2酶活性提供了新策略。而且这样的抑制剂具有比EZH2酶催化位点抑制剂具有更好或互补的优势，比如当病人对于EZH2酶抑制剂产生耐药性时，EED抑制剂同样可以起到抑制EZH2酶活性的作用。本发明公开了化合物可以作为EED靶点抑制剂，并对与EED和/或PRC2作用机理相关的疾病有治疗作用。
本发明公开化合物的生物学功能在生化以及细胞水平的测试中得到了证明。比如在生化测试中，本发明公开的化合物能够与和EED蛋白结合的H3K27Me3多肽有很强的竞争结合作用(IC 50可达<1nM)。在细胞水平上，本发明公开化合物不仅可以抑制组蛋白H3K27的甲基化水平而且也可以通过这一作用抑制癌细胞的增殖(IC 50可达<1nM)。
实施例7
ELISA(H3K27三甲基化)分析
将代表性的本公开内容的化合物用DMSO中进行3-倍梯度稀释，每化合物检测10个浓度梯度，最高测定浓度为10μM。化合物200-倍稀释到在96-孔板中培养的G401细胞中(DMSO终浓度为0.5％)。给药细胞培养72小时后，ELISA方法检测组蛋白H3K27三甲基化水平。
组蛋白提取:96-孔板中化合物处理的细胞用1x PBS(10x PBS缓冲液(80g NaCl(Sigma,产品号S3014),2g KCl(Sigma,产品号60128),14.4g Na 2HP04(Sigma,产品号S5136),2.4g KH 2P04(Sigma,产品号P9791)至1L水中，pH至7.4)洗涤三次，每孔加入100μL 0.4N HCl，置于4℃，温和振摇2小时裂解细胞。细胞裂解液再用80μL中和缓冲液(0.5M磷酸氢二钠，pH 12.5,2.5mM DTT；1％cocktail(Sigma，产品号P8340)中和(充分混匀细胞裂解液与中和缓冲液)。
ELISA检测方法:将细胞裂解液平行转移至2个384-孔检测板(PerkinElmer，OptiPlate-384HB,产品号6007290)中，一块板用于检测H3K27三甲基化水平，另一块板用于测定H3的水平，PBS调至终体积为50μL/孔，4℃包被过夜。次日，弃去孔内溶液，用TBST缓冲液(l xTBS(10x TBS:24.2g Tris(Sigma,产品号T6066),80g NaCl(Sigma，产品号S3014)至1L水中，HCl调pH至7.6),0.1％Tween-20)洗涤5次，于吸水纸上扣干水分。将70μL封闭缓冲液(TBST,5％BSA)加入已包被之反应孔中，于室温孵育1小时。弃去封闭缓冲液，加入一级抗体(30μL/孔)。所需一级抗体均用封闭缓冲液稀释，稀释倍数如下：抗H3K27me3抗体(Cell Signaling Technology,产品号9733)，1:2000稀释；抗H3抗体(Cell Signaling Technology,产品号4499),1：10000稀释。加入一抗后，置室温孵育1小时。TBST洗涤5次后，扣干水分，各反应孔加入二级抗体(30μL/孔)，室温孵育1小时。二级抗体(抗兔抗体(Jackson ImmunoResearch,产品号111-035-003))用封闭缓冲液稀释2000倍后使用。1小时后，TBST洗涤，扣干水分。于各孔中加入30μL的ECL底物(Pierce，产品号34080)，2000rpm离心30秒。利用Molecular Devices，SpectraMax检测各样品信号。数据处理：H3K27甲基化读数先用H3信号进行标准化，0.5％DMSO处理的样品作为对照，计算出化合物的抑制百分率。采用GraphPad prisim5程序将数据拟合为剂量响应曲线，得到测试化合物的IC 50值。
下表1显示了部分化合物的IC 50值。
表1
Figure PCTCN2022111668-appb-000056
实施例8
细胞增殖分析
采用标准细胞培养条件，人类B细胞非霍奇金淋巴瘤细胞KARPAS-422 S培养在培养瓶中。培养基为含15％胎牛血清(FBS，Invitrogen，产品号10099-141)，1％青霉素/链霉素溶液(P/S)RPMI-1640 (Invitrogen，产品号11875)，培养瓶置于温度37℃，相对湿度95％，5％CO 2的无菌培养箱中培养。为了检测PRC2抑制剂对细胞增殖的影响，取指数生长期的细胞，以1x10 4细胞/孔的密度接种到96孔板(Corning，产品号3904)中，每孔加入100μL培养基。随后，将本文公开内容的、不同浓度的化合物加入到已接种细胞的孔中(每个化合物设置9个浓度梯度，最高检测浓度为10μM,3-倍梯度稀释)，每个处理浓度设置2个平行重复，DMSO终浓度为0.5％。之后每隔3～4天用Vi-CELL(Beckman Coulter)测定存活细胞数。每次计过数的细胞以同等密度(1X 10 4个细胞/孔)接种到新的96-孔板中，补充新鲜培养基至100μL，同时加入不同浓度的化合物。培养至第13天，每孔加入100μL的CellTiter-Glo(CellTiter-GloCellTiter-GloCellTiter-GloCTG)(Promega,产品号G7573)，室温避光放置10～20分钟，利用Molecular Devices,SpectraMax i3X读取发光信号。采用GraphPad prisim5将数据拟合为剂量响应曲线，从而得到测试化合物的IC 50值。
下表5显示了部分化合物的IC 50值。
表2
Figure PCTCN2022111668-appb-000057
上述本发明公开的化合物可用于治疗与EED蛋白和/或PRC2蛋白复合物作用机理相关的癌症，包括但不局限于扩散大B细胞淋巴癌、滤泡性淋巴瘤、非霍奇金森淋巴瘤等等淋巴癌、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌样瘤、肝癌、前列腺癌、乳腺癌、脑瘤包括神经母细胞瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤等等。

Claims (12)

  1. 一种如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：
    Figure PCTCN2022111668-appb-100001
    其中，A独立地为
    Figure PCTCN2022111668-appb-100002
    X为N或CR 2；
    R 1、R 2和R 3独立地为H、卤素、氰基、R 5b取代或未取代的氨基、R 5b-O-、R 5取代或未取代的C 1- 4烷基、R 5取代或未取代的C 3- 6的环烷基、R 5取代或未取代的C 1- 4卤代烷基、-C(＝O)R 6、-CO 2R 6、-C(＝O)NR 6R 7、-SO 2R 6或-SO 2NR 6R 7；当取代基为多个时，相同或不同；
    R 4独立地为H、卤素、氰基、C 1-4卤代烷基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c；
    R 4a1、R 4a2和R 4b独立地为H或C 1-4烷基；
    R 4c独立地为H、C 1-4烷基或-O-C 1-4烷基；
    R 5独立地为C 1-4烷基-O-、C 1-4烷基、C 1-4卤代烷基、-N(R 5bR 5b)、N保护基团保护的氨基、氟、羟基；
    R 5b、R 6和R 7独立地为H、C 1-4烷基或C 1-4卤代烷基；
    “*”表示带“*”的碳原子为手性碳原子时，其为R构型、S构型或其混合。
  2. 如权利要求1所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：
    所述式(I)所示的化合物选自如下所示的化合物：
    Figure PCTCN2022111668-appb-100003
    和/或，当R 1、R 2、R 3和R 4独立地为卤素时，所述的卤素为氟、氯、溴或碘；
    和/或，当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-4烷基时，所述的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 1、R 2和R 3独立地为R 5取代或未取代的C 3-6的环烷基时，所述的C 3-6的环烷基为环丙基、环丁基、环戊基或环己基；
    和/或，当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-4卤代烷基时，所述的卤为氟、氯、溴或碘；
    和/或，当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-4卤代烷基时，所述的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 1、R 2和R 3独立地为R 5取代或未取代的C 1-4卤代烷基时，所述的卤代的个数为1、2、3、4或5个；
    和/或，当R 4、R 5、R 5b、R 6和R 7独立地为C 1-4卤代烷基时，所述的卤为氟、氯、溴或碘；
    和/或，当R 4、R 5、R 5b、R 6和R 7独立地为C 1-4卤代烷基时，其中的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 4、R 5、R 5b、R 6和R 7独立地为C 1-4卤代烷基时，所述的卤代的个数为1、2、3、4或5个；
    和/或，当R 4a1、R 4a2、R 4b、R 5b、R 6和R 7独立地为C 1-4烷基时，所述的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 4c独立地为C 1-4烷基或-O-C 1-4烷基时，所述的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 5独立地为C 1-4烷基、C 1-4烷基-O-、C 1-4卤代烷基时，所述的C 1-4烷基、C 1-4烷基-O-和C 1-4卤代烷基里的C 1-4烷基为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，当R 5独立地为N保护基团保护的氨基时，所述的保护基团为
    Figure PCTCN2022111668-appb-100004
    R 5a为C 1-4烷基或C 1-4烷基-O-。
  3. 如权利要求1或2所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：
    当R 5独立地为N保护基团保护的氨基、所述的保护基团为
    Figure PCTCN2022111668-appb-100005
    R 5a为C 1-4烷基或C 1-4烷基-O-时，C 1-4烷基和C 1-4烷基-O-里的C 1-4烷基独立地为甲基、乙基、正丙基、异丙基、正丁基、异丁基或叔丁基；
    和/或，X为N；
    和/或，R 1选自甲基、氰基、三氟甲基、二氟甲基、
    Figure PCTCN2022111668-appb-100006
    和/或，R 2为H；
    和/或，R 3选自H、甲基、卤素或氰基；
    和/或，R 4选自氰基、-C(＝O)NR 4a1R 4a2、-S(＝O) 2R 4b或-C(＝O)R 4c；
    和/或，R 4a1、R 4a2、R 4b、R 4c、R 5b、R 6和R 7独立地为甲基。
  4. 如权利要求1所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：其中，所述如式(I)所述的化合物选自如下所示的化合物：
    Figure PCTCN2022111668-appb-100007
    上述化合物中，R 1的定义如权利要求1所示；
    和/或，
    Figure PCTCN2022111668-appb-100008
    为
    Figure PCTCN2022111668-appb-100009
    或其混合物。
  5. 如权利要求1至4任一项所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：所述式(I)所示的化合物选自如下所示的化合物：
    Figure PCTCN2022111668-appb-100010
    Figure PCTCN2022111668-appb-100011
  6. 一种如权利要求1-5任一项所述的式(I)所示的化合物、其药学上可接受的盐、立体异构体、溶剂化物或它们的同位素标记化合物，其特征在于：所述同位素选自 2H、 3H、 11C、 13C、 14C、 15N、 18F、 31P、 32P、 35S、 36Cl和 125I。
  7. 一种如权利要求1-5任一项所述如式(I)所示的化合物的制备方法，其特征在于：其包含如下步骤：
    将卤代中间体B 0与中间体E 0进行偶联反应得到如式(I)所示的化合物；
    Figure PCTCN2022111668-appb-100012
    其中，W代表卤素，例如Br；R x为-B(OH) 2或
    Figure PCTCN2022111668-appb-100013
    A、R 1、R 3、R 4、X的定义如权利要 求1-5任一项所述。
  8. 一种中间体化合物B 0的制备方法，其特征在于：其为如下任一方案：
    方案一、包括以下步骤：
    步骤(1)化合物SM-5在催化剂作用下关环得到化合物SM-6，
    步骤(2)化合物SM-6在氧化剂作用下反应得到化合物SM-7，
    步骤(3)化合物SM-7与H 2N-CH 2-A在碱存在下反应得到化合物B 0；
    方案二、其如方案一中步骤(2)至步骤(3)所示；
    方案三、其如方案一中步骤(3)所示；
    反应方程式如下：
    Figure PCTCN2022111668-appb-100014
    其中，W、A、R 4的定义如权利要求1-5任一项所述。
  9. 一种药物组合物，其特征在于：其包含治疗有效量的如权利要求1-6中任一项所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物以及药学上可接受的辅料。
  10. 一种物质A在制备药物中的应用，其特征在于：所述的物质A为如权利要求1-6中任一项所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物或如权利要求9所述的药物组合物；所述的药物优选为治疗癌症的药物；所述的药物更优选为治疗与EED蛋白和/或PRC2蛋白复合物介导的癌症的药物。
  11. 如权利要求10所述的应用，其特征在于：
    所述癌症选自扩散大B细胞淋巴癌、滤泡性淋巴瘤等淋巴癌、白血病、多发性骨髓瘤、间皮瘤、胃癌、恶性横纹肌样瘤、肝癌、前列腺癌、乳腺癌、脑瘤包括神经母细胞瘤、胶质瘤、胶质母细胞瘤和星形细胞瘤、宫颈癌、结肠癌、黑色素瘤、子宫内膜癌、食管癌、头颈癌、肺癌、鼻咽癌、卵巢癌、胰腺癌、肾癌、直肠癌、甲状腺癌、甲状旁腺肿瘤、子宫肿瘤和软组织肉瘤；优选扩散大B细胞淋巴癌、滤泡性淋巴瘤等淋巴癌；
    和/或，所述的物质A与其他药物联合使用；优选地，所述其他药物选自抗癌药、肿瘤免疫药物、抗过敏药、止吐药、镇痛药和细胞保护药物。
  12. 一种药物制剂，其特征在于：其包含如权利要求1-6中任一项所述的如式(I)所示的化合物、其药学上可接受的盐、立体异构体、其溶剂化物或它们的同位素标记化合物或如权利要求9所述的药物组合物，所述药物制剂优选片剂、胶囊、药丸、粉末、颗粒、酏剂、酊剂、悬浮物、糖浆、乳液和溶液；更优选地，所述药物制剂的给予方式选自口服、舌下含服、皮下注射、静脉注射、肌肉注射、胸骨内注射、鼻部服用、局部表面给药和直肠给药；
    和/或，所述药物制剂每天单次服用或多次服用。
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