EZH2-specific inhibitor, preparation method therefor and use thereof

Abstract

The purpose of the present invention is to provide compounds represented by formula 1 capable of inhibiting EZH2, stereoisomers, enantiomers, diastereomers, or mixtures of different stereoisomers thereof, and pharmaceutically acceptable salts thereof, as well as a preparation method therefor and the use thereof. The compounds have very good inhibitory activity on PRC2-dependent tumor cells, and have the advantages of good pharmacokinetic properties, good solubility, good stability, basically no induction effect on hepatic drug metabolizing enzymes, and low toxicity. The compounds have good application prospects in drug treatment of diseases (such as tumors) caused by PRC2.

Description

An EZH2-specific inhibitor, its preparation method and uses

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 202410986475.6, filed on July 23, 2024, entitled "An EZH2-Specific Inhibitor and Its Preparation Method and Use", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of chemical drugs, specifically to EZH2-specific inhibitors, their preparation methods, and uses. Background Technology

[0004] The human homolog of the Drosophila zeste gene enhancer 2 (EZH2) is a core component of the epigenetic control factor polycomb repressive complex 2 (PRC2). It catalyzes the methylation of the amino group in histone H3 lysine K27 by S-adenosyl-L-methionine (SAM), achieving monomethylation, dimethylation, and trimethylation (H3K27me3) of H3K27, thereby inhibiting the expression of tumor suppressor genes and participating in the regulation of physiological or pathological processes such as cell cycle, cellular senescence, differentiation, and cancer. Multiple studies have shown that EZH2 is highly expressed in many solid tumors (including breast cancer, prostate cancer, bladder cancer, skin cancer, liver cancer, pancreatic cancer, lung cancer, gastric cancer, and ovarian cancer), and this high expression is closely related to the progression, metastasis, and poor prognosis of these cancers. Furthermore, multiple gain-of-function mutations exist at multiple active sites in the SET catalytic domain of EZH2. For example, over 20% of diffuse large B-cell lymphomas and 7% of follicular lymphomas contain tyrosine 641 mutations in EZH2 (Y641C, Y641F, Y641N, Y641S, Y641N, and Y641H). These mutations increase the trimethylation level of H3K27, leading to the silencing of tumor suppressor genes. Besides this EZH2-dependent histone methylation mechanism for tumor suppressor gene silencing, EZH2 can also function by methylating non-histone substrates in a PRC2-independent manner, or by forming transcriptional complexes with other factors to activate the transcription of downstream target genes, increasing their expression. For instance, in castration-resistant prostate cancer cells (CRPC), phosphorylated EZH2 can assist the androgen receptor-associated complex in activating androgen gene expression, leading to the further development of castration-resistant prostate cancer cells. This evidence suggests that EZH2 is a very promising therapeutic target. Currently, several EZH2 inhibitors, such as Tazemetostat, have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of epithelioid sarcoma and follicular lymphoma; Valemetostat has been approved by the Japanese Ministry of Health, Labour and Welfare (MHLW) for the treatment of adult T-cell lymphoma. However, these SAM competitive inhibitors all have their own limitations, such as insufficient inhibitory activity against EZH1 / 2, low selectivity, the existence of drug resistance mutations, and high toxicity.

[0005] Therefore, there is a need to develop a new type of EZH2 inhibitor with a novel target-binding mode. This inhibitor should possess higher catalytic activity against EZH2, fewer toxic side effects, and better drug-like properties, playing an important role in cancer treatment. Summary of the Invention

[0006] The present invention aims to provide a class of compounds capable of inhibiting EZH2, their stereoisomers, enantiomers, diastereomers, or mixtures of different stereoisomers, and pharmaceutically acceptable salts thereof, as well as methods for their preparation. It also provides pharmaceutical compositions comprising said compounds, their stereoisomers, enantiomers, diastereomers, or mixtures of different stereoisomers, and pharmaceutically acceptable salts thereof, and the use of said compounds or pharmaceutical compositions in the preparation of medicaments for treating or preventing diseases or symptoms related to EZH2 and / or PRC2.

[0007] According to a first aspect of the invention, an object of the invention is to provide a compound represented by Formula 1 or a stereoisomer, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt thereof:

[0008] Among them, R 1 The positionally connected chiral atoms have an S configuration, OR 2 The positionally connected chiral atoms are in either the R or S configuration;

[0009] R 1 The group is selected from H, nitro, amino, hydroxyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the hydrogen atom on the corresponding group being replaced by one or more substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, SF5, C1-C3 alkoxy, C1-C3 aldehyde, amino, C1-C3 amide, nitro, cyano, and C1-C3 alkoxycarbonyl.

[0010] R 2 The group is selected from H, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted carbonyl, and substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the substitution of the hydrogen atom on the corresponding group by one or more substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, SF5, C1-C3 alkoxy, C1-C3 aldehyde, amino, C1-C3 amide, nitro, cyano, and C1-C3 alkoxycarbonyl.

[0011] Preferably, OR 2 The positionally connected chiral atoms have an S configuration.

[0012] Preferably, R 1The group is selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the hydrogen atom on the corresponding group being replaced by 1 to 3 substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, SF5, C1-C3 alkoxy, C1-C3 aldehyde, amino, C1-C3 amide, nitro, cyano, and C1-C3 alkoxycarbonyl.

[0013] Preferably, R 1 The group is selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the hydrogen atom on the corresponding group being replaced by one or two substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 aldehyde, amino, and C1-C3 amide.

[0014] Preferably, R 1 Selected from methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0015] Preferably, R 2 The group is selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted carbonyl, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the hydrogen atom on the corresponding group being replaced by 1 to 3 substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, mercapto, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 aldehyde, amino, and C1-C3 amide.

[0016] Preferably, R 2 The group is selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted carbonyl, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution refers to the hydrogen atom on the corresponding group being replaced by one or two substituents selected from the group consisting of: deuterium, tritium, halogen, hydroxyl, carboxyl, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 aldehyde.

[0017] Preferably, R 2 Selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, deuterated methyl, deuterated ethyl, deuterated propyl, methyl carbonyl, ethyl carbonyl, n-propyl carbonyl, hydroxymethyl, hydroxyethyl, hydroxy n-propyl, halomethyl, haloethyl, halon-propyl.

[0018] Preferably, the compound represented by Formula 1 according to the present invention, or its stereoisomers, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts, have a structure selected from the following:

[0019] According to a second aspect of the invention, another object of the invention is to provide a method for preparing the compound represented by Formula 1 or its stereoisomers, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts, said method being represented by the following reaction formula 1:

[0020] 1) Tert-butyl sulfinamide intermediate 1 (INT-1) was obtained by dehydration condensation of trans-disubstituted cyclohexane and optically pure tert-butyl sulfinamide.

[0021] 2) INT-1 is then reduced to obtain tert-butylsulfinamide intermediate 2 (INT-2);

[0022] 3) INT-2 is then selectively de-tert-butylsulfinyl group to obtain intermediate 3 (INT-3);

[0023] 4) INT3 reacts directly with heterocyclic aryl halides via electrophilic aryl substitution or metal-catalyzed coupling to give the corresponding intermediate 4 (INT-4);

[0024] 5) After removing the Boc, INT-4 is directly coupled with a nitrogen-containing heterobicyclic aryl halide through a metal-catalyzed coupling reaction or an electrophilic aryl substitution reaction to obtain intermediate 5 (INT-5).

[0025] 6) After hydrolysis of INT-5 ester to form free carboxylic acid, it directly undergoes acid-amine condensation reaction with oxetane-3-amine to obtain intermediate 6 (INT-6);

[0026] 7) INT-6 in relation to R 2 X undergoes an alkyl substitution reaction to yield the compound represented by Formula 1.

[0027] Preferably, the compound represented by Formula 1 can be separated by supercritical fluid chromatography (SFC) to obtain compounds of Formula 1-1 and Formula 1-2.

[0028] According to a third aspect of the invention, another object of the invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by Formula 1 or a stereoisomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

[0029] Preferably, the pharmaceutical composition according to the invention may further comprise additional active pharmaceutical agents.

[0030] Preferably, the pharmaceutically acceptable carrier is selected from pharmaceutically acceptable mediators and pharmaceutically acceptable excipients. In some embodiments, the pharmaceutically acceptable carrier is selected from diluents, solvents, dispersants, antioxidants, preservatives, buffers, emulsifiers, pharmaceutically acceptable fillers, disintegrants, surfactants, binders, flavoring agents, dyes, and lubricants, and combinations thereof.

[0031] Preferably, the additional active pharmaceutical agent may include one or more of the following: anticancer agents, biological agents, radiopharmaceuticals, and hormonal drugs.

[0032] According to a third aspect of the invention, another object of the invention is to provide the use of a compound represented by Formula 1 or a stereoisomer, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt or said pharmaceutical composition in the preparation of a medicament for treating and / or mediated by at least one of PRC2, EZH1 and EZH2.

[0033] Preferably, the disease mediated by at least one of PRC2, EZH1, and EZH2 is cancer, selected from breast cancer, glioblastoma, prostate cancer, uterine cancer, ovarian cancer, pancreatic cancer, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, lymphoma, leukemia, malignant rhabdoid tumor, mesothelioma, myeloma, liver cancer, lung cancer, or oropharyngeal cancer, etc.

[0034] According to a fourth aspect of the invention, another object of the invention is to provide a method for treating a disease or symptom mediated by at least one of PRC2, EZH1 and EZH2, the method comprising administering to a subject requiring the treatment a therapeutically effective amount of a compound represented by Formula 1 or a stereoisomer, enantiomer, diastereomer, mixture thereof, or pharmaceutically acceptable salt or said pharmaceutical composition thereof.

[0035] Preferably, the disease mediated by at least one of PRC2, EZH1, and EZH2 is cancer, selected from breast cancer, glioblastoma, prostate cancer, uterine cancer, ovarian cancer, pancreatic cancer, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, lymphoma, leukemia, malignant rhabdoid tumor, mesothelioma, myeloma, liver cancer, lung cancer, or oropharyngeal cancer, etc. Beneficial effects

[0036] This invention unexpectedly discovered a class of SAM non-competitive EZH2 inhibitors, which have very good inhibitory activity against PRC2-dependent tumor cells. Furthermore, they possess advantages such as good pharmacokinetic properties, good solubility, good stability, minimal induction of hepatic drug-metabolizing enzymes, and low toxicity. They show great promise for the drug treatment of PRC2-induced diseases (such as tumors). Attached Figure Description

[0037] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1 shows the ee value detection spectrum of compound 1S;

[0039] Figure 2 shows the ee value detection spectrum of compound 1R;

[0040] Figure 3 shows the ee value detection spectrum of compound 2S;

[0041] Figure 4 shows the ee value detection spectrum of compound 2R;

[0042] Figure 5 shows the ee value detection spectrum of compound 3S;

[0043] Figure 6 shows the ee value detection spectrum of compound 3R;

[0044] Figure 7 shows the calculation of binding free energy for monomethyl, monomethoxy-substituted and disubstituted series of compounds;

[0045] Figure 8 shows the target binding mode and calculated binding free energy of the 3R compound;

[0046] Figure 9 shows the target binding mode and the calculated binding free energy of the 3S compound. Detailed Implementation

[0047] The present invention will now be described in detail. Before proceeding with the description, it should be understood that the terminology used in this specification and the appended claims should not be construed as limited to its general or dictionary meaning, but rather should be interpreted according to the meaning and concept corresponding to the technical aspects of the invention, based on the principle that the inventors are allowed to appropriately define the terms for the best interpretation. Therefore, the description presented herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention. It should be understood that other equivalents or modifications can be obtained from it without departing from the spirit and scope of the invention.

[0048] In this document, the terms “comprising,” “including,” “having,” “containing,” or any other similar terms are open-ended conjunctions intended to cover non-exclusive inclusions. For example, a composition or article containing a plurality of elements is not limited to those listed herein, but may also include other elements not explicitly listed but typically inherent to the composition or article. Furthermore, unless explicitly stated to the contrary, the term “or” is inclusive, not exclusive. For example, the condition “A or B” is satisfied in any of the following cases: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); A and B are both true (or exist). Moreover, in this document, the terms “comprising,” “including,” “having,” and “containing” should be interpreted as specifically disclosed and simultaneously cover closed or semi-closed conjunctions such as “composed of” and “substantially composed of.”

[0049] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual values ​​within those ranges, particularly integer values. For example, a range description of "1 to 8" should be considered as specifically disclosing all secondary ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., particularly secondary ranges defined by all integer values, and should be considered as specifically disclosing individual values ​​within those ranges such as 1, 2, 3, 4, 5, 6, 7, 8, etc. Unless otherwise specified, the foregoing interpretation applies to all content throughout this invention, regardless of its scope.

[0050] If a quantity or other numerical value or parameter is expressed as a range, a preferred range, or a series of upper and lower limits, it should be understood that this document has specifically disclosed all ranges consisting of any upper or preferred value of that range and the lower or preferred value of that range, regardless of whether such ranges are separately disclosed. Furthermore, when a range of numerical values ​​is mentioned herein, unless otherwise stated, the range shall include its endpoints and all integers and fractions within the range.

[0051] In this document, numerical values ​​are to be understood as having a precision with significant digits, provided that the purpose of the invention can be achieved. For example, the number 40.0 should be understood to cover a range from 39.50 to 40.49.

[0052] In this document, when Markush groups or alternative terms are used to describe features or examples of the invention, those skilled in the art should understand that subgroups of all elements within a Markush group or option list, or any individual element, can also be used to describe the invention. For example, if X is described as "selected from the group consisting of X1, X2, and X3," it also indicates that the claim that X is X1 and the claim that X is X1 and / or X2 have been fully described. Furthermore, when Markush groups or alternative terms are used to describe features or examples of the invention, those skilled in the art should understand that any combination of subgroups of all elements within a Markush group or option list, or any combination of individual elements, can also be used to describe the invention. Accordingly, for example, if X is described as "selected from the group consisting of X1, X2, and X3," and Y is described as "selected from the group consisting of Y1, Y2, and Y3," it indicates that the claim that X is X1 or X2 or X3 and Y is Y1 or Y2 or Y3 has been fully described.

[0053] Chiral drugs are drugs whose molecular structure contains a chiral center (also called an asymmetric center). They include single stereoisomers, mixtures of two or more stereoisomers in unequal amounts, and racemates. Different stereoisomer configurations may also have different biological activities.

[0054] The compounds of the present invention can be prepared using the methods described herein or by other similar methods as understood by those skilled in the art of organic synthesis. Compounds of Formula 1 having a chiral center, or preferred specific compounds thereof, can be prepared in a substantially optically pure form using substantially optically pure starting materials or by separation chromatography, recrystallization, or other separation techniques well known in the art. Terminology Definitions

[0055] The term "an" or "a" in this invention includes "at least one" or "at least one", and the nouns have both singular and plural forms. For example, "another pharmaceutical agent" means one, two or more pharmaceutical agents.

[0056] In this invention, the term "PRC2" refers to the epigenetic control factor polycomb repressive complex 2.

[0057] In this invention, the term "EZH2" refers to human homolog 2 of the Drosophila zeste gene enhancer, a core component of PRC2, which regulates the transcription of target genes by introducing 3-methylation (H3K27me3) into histone H3 lysine 27.

[0058] The term "compound" as used in this invention refers to a collection of molecules having the same chemical structure, unless otherwise specified as a collection of stereoisomers (e.g., a collection of racemic compounds, a collection of cis / trans stereoisomers, or a collection of (E) and (Z) stereoisomers), but does not exclude isotopes contained in the atoms constituting the molecules. Therefore, those skilled in the art will understand that when a compound's specific chemical structure contains deuterium atoms, the compound also contains small amounts of isotopes where one or more deuterium sites are actually hydrogen atoms. In the compounds of this invention, the relative amount of such isotopes will depend on many factors, including, for example, the isotopic purity of the reagents used to prepare the compound and the efficiency of isotopic incorporation in the various synthetic steps used to prepare the compound. However, as described above, the total relative amount of such isotopes will be less than 49.9% of the compound. In other embodiments, the total relative amount of such isotopes will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% of the compound.

[0059] The term "stereoisomer" in this invention includes cis-trans isomers, enantiomers, and diastereomers.

[0060] The term "pharmaceutical-grade salt" or "pharmaceutical-acceptable salt" in this invention refers to the formation between an acid and a basic group of a compound, such as an amino functional group, or between a base and an acidic group of a compound, such as a carboxyl functional group.

[0061] The term "medicinal" refers to a component that, within reasonable medical judgment, is suitable for contact with the tissues of humans and other mammals without excessive toxicity, irritation, allergic reactions, etc., and is commensurate with a reasonable benefit / risk ratio.

[0062] The term "medicinally acceptable salt" or "pharmaceutically acceptable salt" refers to any non-toxic salt that, when administered to a recipient, can directly or indirectly provide the contents of this invention. Suitable medicinally acceptable salts are, for example, those disclosed by SMBerge et al. in J. Pharmaceutical Sciences, 1977, 66, pp. 1-19.

[0063] Acids commonly used to form pharmaceutically acceptable salts include inorganic acids such as hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, as well as organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, as well as related inorganic and organic acids. Therefore, such medicinal salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, decanoates, heptanoates, propynylates, oxalates, malonates, succinates, caprylates, sebacic acid, fumarates, maleates, and butyn-1,4-dicarboxylic acid. Salts, including hexyne-1, 6-diacidates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolate salts, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, and other salts. In some embodiments, pharmaceutically acceptable acid addition salts include salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and salts formed with organic acids such as maleic acid.

[0064] Pharmaceutically usable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Alkyl)4 salts. The present invention also contemplates the quaternization of any basic nitrogen-containing group in the compounds disclosed herein. Suitable non-limiting examples of alkali metal and alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Further non-limiting examples of pharmaceutically usable salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate ions. Other suitable non-limiting examples of pharmaceutically usable salts include benzenesulfonates and glucosamine salts.

[0065] The active pharmaceutical ingredient described in this invention may include one or more of the following: anticancer agents, biological agents, hormonal drugs, and radiopharmaceuticals:

[0066] Anticancer agents, such as azacitadine, doxil, all-trans retinoic acid, blenoxane, Xeloda, 5-FU, Ifex, Gleevec, Ellence, Targretin, CeeNU, Platinol, Valstar, Clolar, 6-mercaptopurine, Ara-C, Eloxatin, Leukeran, Panretin, Gemzar, Novantrone, Abraxane, 2CdA, Emcyt, nilotinib, L-PAM, DaunoXome, Paraplatin, and hexamethyltrimer. Cyanamide, Mitomycin C, Oncaspar, Hycamtin, Vincristine Sulfate, Fludara, 6-TG, Daunorubicin Hydrochloride, Nipent, Camptosar, Neutrexin, DIC, Accutane, Tespa, Vidaza, Temodar, Matulane, Cytoxan, Adriamycin, Mithracin, Alanta, Lysodren, Ixempra, Etoposide Phosphate, Velban, Trisenox, Ixabepilone, Actinomycin D, Sandostatin, VM 26. Zanosar, Dacogen, nitrogen mustard, FUDR, Liposomal Ara-C, Idamycin, Taxotere, L-asparaginase, Hydrea, BCNU, prolifeprospan20 with carbochlor mustard implant, Pazopanib, Sorafenib, Erlotinib, Osimertinib.

[0067] Biologics, such as interferon, Herceptin, lenalidomide, Tarceva, Velcade, BCG, Iressa, Revlimid, Zevalin, Bevacizumab, Pembrolizumab, Nimotuzumab, Bexxar, interleukin-2, Ontak, Camppath, Rituxan, Trastuzumab Deruxtecan, Erlotinib, Mylotarg, Abraxane, Ergamisol, Tykerb, Pegasys, and Thalomid.

[0068] Hormonal drugs, such as Arimidex, Cytadren, Evista, Delta-Cortef, Eligard, Faslodex, Femara, Halotetin, Megace, Nilandron, Nolvadex, Plenaxis, Zoladex, dexamethasone sodium phosphate, DeltaSone, medroxyprogesterone acetate, leuprolide acetate, fulvestrant, and exemestane.

[0069] Radiopharmaceuticals, such as Phosphocol, Iodotope, Samarium SM-153, and Metastron.

[0070] The diluents, solvents, dispersants, antioxidants, preservatives, buffers, emulsifiers, pharmaceutically acceptable fillers, disintegrants, surfactants, adhesives, flavorings, dyes, and lubricants used in this invention have their meanings as known in the art.

[0071] The term "halogen" in this invention includes F, Cl, Br, and I, which are respectively fluorine, chlorine, bromine, and iodine. In some embodiments, the halogen is preferably F, Cl, or Br.

[0072] The term "alkyl" as used in this invention refers to a fully saturated, straight-chain, branched, or cyclic hydrocarbon chain, substituted or unsubstituted. For example, "C1-C3 alkyl" is an alkyl group comprising 1 to 3 carbon atoms, namely C1, C2, or C3, which are methyl, ethyl, propyl, isopropyl, or cyclopropyl, respectively. In some embodiments, the alkyl group is substituted.

[0073] In this invention, the term "straight-chain alkyl" refers to an alkyl group without side chains or branches.

[0074] In this invention, the term "branched alkyl" refers to an alkyl group in which one or more carbon atoms on the alkyl main chain are connected to a side chain.

[0075] In this invention, the term "cycloalkyl" refers to an alkyl group in which carbon atoms form a closed cyclic structure through single bonds.

[0076] In this invention, the term "substituted" means that one or more H atoms in a group are replaced by other atoms or groups, such as "C1-C3 alkyl group substituted with 1-3 halogens", which means that 1-3 H atoms in a C1-C3 alkyl group are replaced by halogens.

[0077] The cancers or tumors in this invention include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, leukemia, multiple myeloma, gastric cancer, malignant rhabdoid tumor, hepatocellular carcinoma, prostate cancer, breast cancer, bile duct and gallbladder cancer, bladder cancer, neuroblastoma, glioma, glioblastoma and astrocytoma, cervical cancer, colon cancer, melanoma, endometrial cancer, esophageal cancer, head and neck cancer, lung cancer, nasopharyngeal carcinoma, ovarian cancer, pancreatic cancer, renal cell carcinoma, rectal cancer, thyroid cancer, parathyroid tumors, uterine tumors, rhabdomyosarcoma, Kaposi's sarcoma, synovial sarcoma, osteosarcoma, and Ewing's sarcoma.

[0078] Breast tumors include, for example, breast cancer with a positive hormone receptor status, breast cancer with a negative hormone receptor status, Her-2 positive breast cancer, hormone receptor and Her-2 negative breast cancer, BRCA-associated breast cancer, and inflammatory breast cancer. Respiratory tract tumors include, for example, non-small cell bronchial carcinoma and small cell bronchial carcinoma, non-small cell lung cancer, and small cell lung cancer.

[0079] Brain tumors include, for example, gliomas, glioblastomas, astrocytomas, meningiomas, and medulloblastomas. Male reproductive organ tumors include, for example, prostate cancer, malignant epididymal tumors, malignant testicular tumors, and penile cancer.

[0080] Tumors of the female reproductive organs include, for example, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer.

[0081] Gastrointestinal tumors include, for example, colorectal cancer, anal cancer, stomach cancer, pancreatic cancer, esophageal cancer, gallbladder cancer, small bowel cancer, salivary gland cancer, neuroendocrine tumors, and gastrointestinal stromal tumors.

[0082] Genitourinary tract tumors include, for example, bladder cancer, renal cell carcinoma, and cancers of the renal pelvis and urinary tract. Eye tumors include, for example, retinoblastoma and intraocular melanoma.

[0083] Liver tumors include, for example, hepatocellular carcinoma and cholangiocarcinoma.

[0084] Skin tumors include, for example, malignant melanoma, basal tumor, spinal cord tumor, Kaposi's sarcoma, and Merkel cell carcinoma.

[0085] Tumors of the head and neck include, for example, laryngeal cancer, as well as pharyngeal and oral cancer.

[0086] Sarcomas include, for example, soft tissue sarcomas, synovial sarcomas, rhabdomyosarcomas, and osteosarcomas. Lymphomas include, for example, non-Hodgkin lymphoma, Hodgkin lymphoma, cutaneous lymphoma, central nervous system lymphoma, and AIDS-related lymphoma.

[0087] Bladder cancer, brain cancer, breast cancer, colorectal cancer, chronic myelomonocytic leukemia, MLL rearrangement leukemia, lung adenocarcinoma, lymphoma, medulloblastoma, melanoma, multiple cancers, myeloma, prostate cancer, malignant rhabdoid tumor, synovial sarcoma, teratoma / rhabdoid tumor, or T-cell acute lymphoblastic leukemia.

[0088] The following embodiments are merely examples illustrating implementations of the present invention and do not constitute any limitation on the present invention. Those skilled in the art will understand that modifications made without departing from the spirit and concept of the present invention fall within the protection scope of the present invention. Unless otherwise specified, the reagents and instruments used in the following embodiments are commercially available products.

[0089] The raw materials can be obtained commercially or prepared using methods known or disclosed in the art.

[0090] Purification of intermediates and compounds was performed using conventional chemical experimental procedures such as normal-phase or reverse-phase chromatography or recrystallization. Normal-phase chromatography used pre-packed silica gel columns or preparative thin-layer chromatography. Silica gel columns were primarily glass columns or used in rapid preparative chromatographs. The mobile phase for normal-phase chromatography was selected and proportioned from petroleum ether / ethyl acetate, dichloromethane / methanol, or other suitable solvents for elution. Reversed-phase preparative liquid chromatography used a C18 column and was performed using a preparative liquid chromatograph or a rapid preparative chromatograph, with detection at 220 nM and 254 nM or using preparative liquid chromatography-mass spectrometry. Gradient elution was performed using water / acetonitrile containing 0.1% hydrochloric acid, water / acetonitrile containing 0.1% formic acid, water / acetonitrile containing 0.1% ammonia, water / acetonitrile containing 0.1% trifluoroacetic acid, or other suitable solvent systems as the mobile phase.

[0091] The structures of intermediates and compounds were characterized using nuclear magnetic resonance (NMR) and LC-MS. A Bruker Ascend 500 NMR spectrometer was used. The solvents used were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, or other labeled deuterated solvents. Spectral data are reported in the following mode: chemical shift δ (number of peak splits, coupling constant J (Hz), number of hydrogen atoms). Tetramethylsilane was used as the internal standard for the chemical shift, and its chemical shift was set to zero (δ, 0 ppm). Some abbreviations are used: s (singleton), d (doublet), t (triplet), q (quartet), m (multiplex), br (broad peak).

[0092] Representative methods of liquid chromatography-mass spectrometry (LCMS) for the structural characterization of intermediates and compounds are as follows:

[0093] Instruments and equipment: Agilent LC1260 system coupled with 6125 single quadrupole mass spectrometer

[0094] Column: Agilent ZORBAX SB-C18, 1.8μm, 2.1*50mm.

[0095] Mobile phase: A: 0.1% formic acid in acetonitrile solution, B: 0.1% formic acid in aqueous solution.

[0096] Gradient elution: 0-0.25 min, 80% B; 0.25-3 min, 0% B; 3-4.5 min, 0% B; 4.5-6.0 min, 80% B.

[0097] Flow rate: 0.4 mL / min.

[0098] Column temperature: room temperature (25°C).

[0099] General synthesis method for the examples:

[0100] Synthesis of key starting material (S)-(4-(1-aminoethyl)cyclohexyl)carbamate tert-butyl ester (A):

[0101] Step 1: Synthesis of intermediate A1

[0102] To a reaction flask equipped with a stir bar, tert-butyl (trans-4-acetylcyclohexyl)carbamate (4.82 g, 20 mmol, 1.0 equivalent) and (S)-tert-butylsulfinamide (2.91 g, 24 mmol, 1.2 equivalent) were added sequentially. The mixture was purged with nitrogen three times. Tetrahydrofuran (100 mL) and tetraethyl titanate (6.84 g, 30 mmol, 1.5 equivalent) were then added. The mixture was heated to reflux for 20 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature. Under vigorous stirring, the reaction solution was poured into a container saturated with sodium bicarbonate (100 mL), filtered, and the filter cake was washed with ethyl acetate (100 mL). The organic phases were combined, and the solvent was removed by rotary evaporation under reduced pressure to obtain intermediate A1, which was then directly proceeded to the next step.

[0103] Step 2: Synthesis of intermediate A2

[0104] The above-mentioned oily substance A1 was dissolved in tetrahydrofuran (50 mL), and sodium borohydride (1.51 g, 40 mmol, 2.0 equivalent) was added with stirring. The mixture was stirred at room temperature for 20 h, and the reaction was monitored by TLC. After the reaction was completed, water (40 mL) was added to quench the reaction. The above mixed solution was extracted three times with ethyl acetate (10.0 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediate A2 (4.29 g, 12.4 mmol, yield 62%), a white solid. 1H NMR(500MHz,CD3OD)δ4.60(brs,1H),3.25-3.12(m,1H),1.96-1.78(m,4H),1.43(s,9H),1.23(s,9H),1.22-1.13(m,7H).LCMS[M+H] + m / z:347.2.

[0105] Step 3: Synthesis of Key Starting Material A

[0106] The above-mentioned solid intermediate A2 (4.29 g, 12.4 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (30 mL) and water (6 mL). Iodine (2.20 g, 8.68 mmol, 0.7 equivalent) was added with stirring, and the mixture was heated to 70 °C for 4 h. The reaction was monitored by TLC. After the reaction was completed, the mixture was extracted three times with saturated sodium thiosulfate solution (30.0 mL) and ethyl acetate (10.0 mL). The organic phases were combined, washed twice with saturated sodium carbonate solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give compound A (2.57 g, 10.6 mmol, yield 86%), a white solid. 1 H NMR(500MHz, CDCl3)δ4.41(brs,1H),3.35(brs,1H),2.73-2.68(m,1H),2.04-2 .02(m,2H),1.85-1.76(m,4H),1.45-1.42(m,9H),1.09-1.02(m,7H).LCMS[M+H] + m / z:243.2.

[0107] Example 1: Synthesis of 2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-hydroxy-N-(oxetane-3-yl)acetamide (1)

[0108] Step 1: Synthesis of intermediates 1-2

[0109] Under a nitrogen atmosphere, 5-bromo-2-methylmercaptopyrimidine (1-1) (10.00 g, 48.78 mmol, 1.0 equivalent), Pd2(dba)3 (4.47 g, 4.88 mmol, 0.1 equivalent), X-Phos (4.65 g, 9.76 mmol, 0.2 equivalent), and potassium tert-butoxide (16.39 g, 146.34 mmol, 3.0 equivalent) were dissolved in mesitylene (300 mL). Dimethyl malonate (19.32 g, 146.34 mmol, 3.0 equivalents) was added with stirring, and the reaction was carried out overnight at 120 °C. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to room temperature, slurried with petroleum ether, and filtered to obtain a solid. The solid was washed with ethyl acetate, and the filtrate was used to remove the solvent by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to obtain intermediates 1-2 (5.00 g, 19.51 mmol, 40% yield). LC-MS [M+H] + m / z:257.0.

[0110] Step 2: Synthesis of intermediates 1-3

[0111] Compounds 1-2 (5.00 g, 19.51 mmol, 1.0 equivalent) were dissolved in dichloromethane (50 mL). m-CPBA (6.73 g, 39.02 mmol, 2.0 equivalent) was added in portions at 0 °C. After addition, the mixture was allowed to warm to room temperature overnight. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to 0 °C, and m-CPBA precipitated. The precipitate was quenched with 10% sodium sulfite solution (50 mL), and extracted with dichloromethane (50 mL x 3). The organic phase was washed with saturated sodium bicarbonate solution (50 mL) and sodium chloride solution (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediate 1-3 (4.05 g, 14.04 mmol, 72% yield). LC-MS [M+H] + m / z:289.0.

[0112] Step 3: Synthesis of intermediates 1-4

[0113] Intermediate 1-3 (3.20 g, 11.11 mmol, 1.0 equivalent) was dissolved in dimethyl sulfoxide (30 mL), and compound A (2.96 g, 12.22 mmol, 1.1 equivalent), DIPEA (7.17 g, 55.55 mmol, 5.0 equivalent), and cesium fluoride (2.53 g, 16.67 mmol, 1.5 equivalent) were added. The resulting reaction solution was reacted at 100 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction was quenched with saturated ammonium chloride solution (50 mL). The mixture was extracted with ethyl acetate (50 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride solution (50 mL x 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediate 1-4 (2.10 g, 4.67 mmol, yield 42%). LCMS[M+H]+m / z:451.2.

[0114] Step 4: Synthesis of intermediates 1-5

[0115] Intermediate 1-4 (1.04 g, 2.31 mmol, 1.0 equivalent) was dissolved in dimethyl sulfoxide (5 mL) and water (0.5 mL), and lithium chloride powder (196 mg, 4.62 mmol, 2.0 equivalent) was added. The reaction was carried out overnight at 100 °C. The reaction was monitored by TLC. After completion, the reaction was quenched with water (10 mL), extracted with ethyl acetate (10 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride solution (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediate 1-5 (525 mg, 1.34 mmol, yield 58%). LCMS [M+H]+ m / z: 393.3.

[0116] Step 5: Synthesis of intermediates 1-6

[0117] Intermediate 1-5 (500 mg, 1.28 mmol, 1.0 equivalent) was dissolved in methanol (2.0 mL), and 1,4-dioxane solution (0.5 mL) of 4 M hydrochloric acid was added. The reaction was carried out at room temperature for 3 h, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed under reduced pressure, and the resulting oily compound was directly used for the next step.

[0118] The above-mentioned oily compound and intermediates 1-6 (380 mg, 1.92 mmol, 1.5 equivalents) were dissolved in N,N-dimethylformamide (5.0 mL), and potassium carbonate (530 mg, 3.84 mmol, 3.0 equivalents) was added. The mixture was stirred overnight at 100 °C. The reaction was monitored by TLC. After completion, the mixture was cooled to room temperature, and the reaction was quenched by adding a saturated ammonium chloride solution (5.0 mL). Extraction was performed with ethyl acetate (10 mL x 3). The organic phases were combined, washed with a saturated sodium chloride solution (10 mL x 3), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediates 1-7 (372 mg, 0.82 mmol, 64% yield). LC-MS [M+H] + m / z:455.1.

[0119] Step Six: Synthesis of Intermediates 1-8

[0120] Under a nitrogen atmosphere, 1 M NaHMDS tetrahydrofuran solution (1.23 mL, 1.23 mmol, 1.5 equivalents) and tetrahydrofuran (2.0 mL) were added. After cooling to -78 °C, a tetrahydrofuran solution of intermediate 1-7 (372 mg, 0.82 mmol, 1.0 equivalents) (2.0 mL) was slowly added, and the mixture was stirred for 30 minutes. A tetrahydrofuran solution of Davis reagent (321 mg, 1.23 mmol, 1.5 equivalents) (2.0 mL) was added, and the reaction was carried out at -78 °C for 30 minutes. The reaction was monitored by TLC. After completion, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL) at -78 °C. The mixture was extracted with ethyl acetate (10 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride aqueous solution (5.0 mL x 3), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure. The resulting mixture was purified by silica gel column chromatography to give intermediates 1-8 (139 mg, 0.30 mmol, yield 36%). LC-MS [M+H] + m / z:471.2.

[0121] Step 7: Synthesis of Compound 1

[0122] Compound 3-oxacyclobutamine (0.25 mL) was added to a reaction flask containing intermediate 1-8 (139 mg, 0.30 mmol) and reacted overnight at 65 °C. The reaction was monitored by TLC. After completion, the reaction was quenched with water (2.0 mL), extracted with ethyl acetate (5.0 mL x 3), and the organic phases were combined. The mixture was washed three times with saturated sodium chloride aqueous solution (5.0 mL x 3), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give the final product compound 1 (110 mg, 0.22 mmol, yield 72%). 1H NMR (500MHz, CDCl3) δ8.29(s,2H),8.00(d,J=5.5Hz,1H),7.75(d,J=9.0Hz,1H),7.48(d,J=8.0Hz,1H),7 .34(d,J=5.5Hz,1H),6.80(d,J=9.0Hz,1H),5.37–5.32(m,1H),5.13(brs,1H),5.09–5.02(m,1H),5.00(s ,1H),4.95–4.90(m,2H),4.58–4.52(m,2H),4.06–3.98(m,1H),3.85(brs,1H),2.29–2.20(m,2H),1.94– 1.90(m,1H),1.86–1.82(m,1H),1.52–1.44(m,1H),1.30–1.22(m,4H),1.18(d,J=6.5Hz,3H).LC-MS[M+H] + m / z:512.3.

[0123] Examples 2 and 3: Preparation of (S)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-hydroxy-N-(oxetane-3-yl)acetamide (1S) and (R)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-hydroxy-N-(oxetane-3-yl)acetamide (1R)

[0124] Chiral isomers of compound 1 were separated using SFC (spatial fractionation), and the corresponding fractions were collected. The solvent was removed by rotary evaporation to obtain pure optical isomers. The ee value and chemical purity of the final product were determined using analytical SFC and LCMS. Preparation method:

[0125] SFC device: Waters SFC Prep 80Q

[0126] Column: REGIS(s,s)WHELK-O1 (250mm*30mm, 10um)

[0127] Mobile phase: A: CO2, B: EtOH (0.1% NH3H2O)

[0128] Elution gradient: B% = 65.00%, isogradient elution

[0129] Flow rate: 80 ml / min

[0130] Detection wavelengths: 220 & 254nm

[0131] Column temperature: 40℃

[0132] System pressure: 100 bar

[0133] Analysis method:

[0134] SFC device: SHIMADZU LC-30ADsf

[0135] Chromatographic column: (S,S)Whelk-O1 50*4.6mm ID, 3.5um

[0136] Mobile phase: A: CO2, B: EtOH (0.05% DEA)

[0137] Elution gradient: gradient elution

[0138] Flow rate: 3.0 mL / min

[0139] Detection wavelength: 220nm

[0140] Column temperature: 35℃

[0141] System pressure: 100 bar

[0142] Preparation results

[0143] After SFC resolution, reverse-phase purification was performed to obtain a total sample of 329.8 mg, with a target analyte yield of 68%. The ee values ​​of compounds 1S and 1R are shown in Figures 1 and 2.

[0144] Example 4: Synthesis of 2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-methoxy-N-(oxetane-3-yl)acetamide (2)

[0145] Under a nitrogen atmosphere, compound 1 (110 mg, 0.22 mmol, 1.0 equivalent) was dissolved in DMF (2.0 mL), cooled to -30 °C, and NaH (13.2 mg, 0.33 mmol, 1.5 equivalent) was added. The mixture was stirred for 30 minutes, followed by the addition of iodomethane (34 mg, 0.24 mmol, 1.1 equivalent), and the reaction was carried out at -30 °C for 2 hours. The reaction was monitored by TLC. After completion, the reaction was quenched with saturated ammonium chloride aqueous solution (5.0 mL), extracted with ethyl acetate (5.0 mL x 3), and the organic phases were combined. The mixture was washed with saturated sodium chloride aqueous solution (5.0 mL x 3), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to obtain the final product compound 2 (84 mg, 0.16 mmol, yield 73%). 1 H NMR (500MHz, DMSO-d6) δ8.83(d,J=7.0Hz,1H),8.15(s,2H),7.89(d,J=5.0Hz,1H),7.84(d,J=9.0Hz,1H ),7.49(d,J=5.0Hz,1H),7.44(d,J=7.0Hz,1H),7.14(d,J=9.0Hz,1H),6.93(d,J=8.5Hz,1H),4.81–4.73 (m,1H),4.61(t,J=6.9Hz,2H),4.48–4.45(m,2H),4.44(s,1H),3.88–3.72(m,2H),3.22(s,3H),2.14–2 .02(m,2H),1.83–1.73(m,2H),1.44–1.36(m,1H),1.15–1.06(m,4H),1.04(d,J=6.5Hz,3H).LC-MS[M+H] + m / z:526.2.

[0146] Examples 5 and 6: Preparation of (S)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthid-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-methoxy-N-(oxetane-3-yl)acetamide (2S) and (R)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthid-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-methoxy-N-(oxetane-3-yl)acetamide (2R)

[0147] Chiral isomers of compound 2 were separated using SFC (Sequencing Filtration-Fluid Conversion), and the corresponding fractions were collected. The solvent was removed by rotary evaporation to obtain pure optical isomers. The ee value and chemical purity of the final product were determined using analytical SFC and LCMS.

[0148] Preparation method:

[0149] SFC device: Waters SFC Prep 150

[0150] Column: REGIS(s,s)WHELK-O1 (250mm*30mm, 10um)

[0151] Mobile phase: A: CO2, B: EtOH:ACN = 4:1 (0.1% NH3H2O)

[0152] Elution gradient: B% = 35.00%, isogradient elution

[0153] Flow rate: 150 ml / min

[0154] Detection wavelengths: 220 & 254nm

[0155] Column temperature: 40℃

[0156] System pressure: 100 bar

[0157] Analysis method:

[0158] SFC device: SHIMADZU LC-30ADsf

[0159] Chromatographic column: (S,S)Whelk-O1 50*4.6mm ID, 3.5μm

[0160] Mobile phase: A: CO2, B: EtOH:ACN = 4:1 (0.05% DEA)

[0161] Elution gradient:

[0162] Flow rate: 3.0 mL / min

[0163] Detection wavelength: 220nm

[0164] Column temperature: 35℃

[0165] System pressure: 100 bar

[0166] Preparation results

[0167] After SFC resolution, a total of 2.5 g of sample was obtained, with a target analyte yield of 74%. The ee values ​​of compounds 2S and 2R are shown in Figures 3 and 4.

[0168] Example 7: 2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-(methoxy-d3)-N-(oxetane-3-yl)acetamide (3)

[0169] Under a nitrogen atmosphere, compound 1 (100 mg, 0.20 mmol, 1.0 equivalent) was dissolved in DMF (2.0 mL), cooled to -30 °C, and NaH (12 mg, 0.29 mmol, 1.5 equivalent) was added. The mixture was stirred for 30 minutes, followed by the addition of deuterated iodomethane (31 mg, 0.22 mmol, 1.1 equivalent), and the reaction was carried out at -30 °C for 2 hours. The reaction was monitored by TLC. After completion, the reaction was quenched with saturated ammonium chloride aqueous solution (2.0 mL), and the mixture was extracted three times with ethyl acetate (2.0 mL). The organic phases were combined, washed three times with saturated sodium chloride aqueous solution (2.0 mL), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give final product 3 (79 mg, 0.15 mmol, 75% yield). 1 H NMR (500MHz, CDCl3) δ8.27(s,2H),8.05(d,J=5.0Hz,1H),7.77(d,J=9.0Hz,1H),7.51(d,J=7.5Hz,1H),7.36 (d,J=5.0Hz,1H),6.84(d,J=8.5Hz,1H),5.50–5.40(m,1H),5.27(s,1H),5.15–5.06(m,1H),5.01–4.92(m,2 H),4.60(dt,J=12.0,6.0Hz,2H),4.51(s,1H),4.11–4.03(m,1H),3.97–3.83(m,1H),2.31–2.26(m,2H),2.0 0–1.94(m,1H),1.92–1.85(m,1H),1.55–1.46(m,1H),1.39–1.25(m,4H),1.22(d,J=6.5Hz,3H).LC-MS[M+H] + m / z:529.3.

[0170] Examples 8 and 9: Preparation of (S)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-(methoxy-d3)-N-(oxetane-3-yl)acetamide (3S) and (R)-2-(2-(S)-1-((1,4-trans)-4-((8-chloro-1,7-naphthidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-(methoxy-d3)-N-(oxetane-3-yl)acetamide (3R)

[0171] Chiral isomers of compound 3 were separated using SFC (Sequencing Fractional Filtration), and the corresponding fractions were collected. The solvent was removed by rotary evaporation to obtain pure optical isomers. The ee value and chemical purity of the final product were determined by analytical SFC and LCMS.

[0172] Preparation method:

[0173] SFC device: Waters SFC Prep 150

[0174] Column: DAICEL CHIRALPAK IK (250mm*25mm, 10um)

[0175] Mobile phase: A: CO2, B: EtOH (0.1% NH3H2O)

[0176] Elution gradient: B% = 50.00%, isogradient elution

[0177] Flow rate: 120 ml / min

[0178] Detection wavelengths: 220 & 254nm

[0179] Column temperature: 40℃

[0180] System pressure: 100 bar

[0181] Analysis method:

[0182] SFC device: SHIMADZU LC-30AD

[0183] Chromatographic column: Chiralpak IK-3 50×4.6mm ID, particle size 3μm

[0184] Mobile phase: A: CO2, B: EtOH (0.05% DEA)

[0185] Elution gradient:

[0186] Flow rate: 3.0 mL / min

[0187] Detection wavelength: 220nm

[0188] Column temperature: 35℃

[0189] System pressure: 10MPa

[0190] Preparation results

[0191] After SFC resolution, a total of 58.5 mg of sample was obtained, with a target analyte yield of 74%. The ee values ​​of compounds 3S and 3R are shown in Figures 5 and 6.

[0192] Biological assay

[0193] The ability of the compounds of the present invention to inhibit EZH2 can be evaluated using the assays described below and other assays known in the art.

[0194] Main instruments and equipment for the experiment:

[0195] Centrifuge (Beckman Avanti J-15R); Ultra-low temperature freezer (Thermo FDE60086FV); Carbon dioxide incubator (ESCO CLM-170B-CN); Microplate reader (Spark Tecan); Multichannel pipette (20-200μL); Cell counter (Bio-Rad TC20).

[0196] Example 10: Determination of H3K27me3 protein levels in cell lines (G401, WSU-DLCL2, Karpas-422) Step 1

[0197] G401 cells (2500 / well) were seeded in standard 96-well cell culture plates (corning, 3599), while WSU-DLCL2 cells (2000 / well) and Karpas 422 cells (2000 / well) were seeded in V-type 96-well plates (corning, 3894) and cultured for 24 hours. Cells were then seeded into the middle 60 wells of the 96-well plates at 100 μL / well, with 100 μL of PBS added to each well for four weeks.

[0198] Step 2

[0199] Based on the sensitivity of the cell line and the activity of the test compound, the treatment concentration of the test compound was determined through preliminary experiments, with the highest concentration being 37 nM or 1000 nM containing 0.2% DMSO. Ten concentrations of the test compound were prepared by isotropic (3-fold) dilution to treat the cells. Before the ELISA procedure, the cells were cultured at 37°C in a 5% CO2 incubator for 48 h.

[0200] Step 3

[0201] Cells in 96-well plates were washed with 1×PBS buffer and lysed with 100 μL / well of lysis buffer (0.4N HCl). The plates were then incubated on a shaker at 150 rpm and 4°C for 30 min. Neutralization buffer (0.5M disodium hydrogen phosphate, pH 12.5, 1mM DTT, proteasome inhibitor cocktail; 80 μL / well) was added, and the plates were pipetted approximately 20 times to thoroughly mix the solution. After neutralization, the 96-well plates were centrifuged at 2000 rpm for 2 min.

[0202] Step 4

[0203] Transfer 10-50 μL of cell lysis buffer from the centrifuged 96-well plate to each well of a 384-well plate, and adjust the volume to 50 μL with 0-40 μL of 1×PBS. Seal the plate, centrifuge at 2000 rpm for 2 min, and incubate at 4°C for approximately 16 h.

[0204] Step 5

[0205] After incubation, wash five times with TBST: the washing procedure is to add 60 μL of TBST buffer to each well and wash for 5 min at 200 rpm and 25°C on a shaker.

[0206] Step 6

[0207] Add 50 μL of blocking buffer (TBST, 2% BSA) to each well, centrifuge the plate at 2000 rpm for 2 min, and incubate on a shaker at 200 rpm and 25°C for 1 h.

[0208] Step 7

[0209] Remove the blocking buffer and add 30 μL of primary antibody (anti-H3K27me3 antibody [1:1500] or anti-H3 antibody [1:2000]) to each well. Centrifuge the plate at 2000 rpm for 2 min and incubate it on a shaker at 150 rpm and 25 °C for 1 h.

[0210] Step 8

[0211] Wash five times with TBST to remove primary antibody, add 30 μL of secondary antibody (1:2500) prepared with blocking buffer to each well, centrifuge the 384-well plate at 2000 rpm for 2 min, and incubate on a shaker at 150 rpm and 25°C for 1 h.

[0212] Step 9

[0213] Secondary antibody was removed by washing five times with TBST. 30 μL of ECL substrate was added to each well, and the plate was centrifuged at 2000 rpm for 2 min.

[0214] Step 10

[0215] After incubation at room temperature for 5 minutes, the signal was read on a microplate reader. The H3 K27me3 methylation reading was normalized using the H3 reading signal, and then the inhibition percentage was calculated for the DMSO-treated sample. The data were fitted to a dose-response curve using the GraphPad Prism program to obtain the IC50 of the test compound. 50 value.

[0216] The compounds of this invention were tested according to the above experimental methods, and the results are shown in Table 1. The compounds exhibit methylation inhibitory activity at the cellular level, primarily at the nM level, demonstrating significant target inhibitory activity.

[0217] This invention uses Tazemetostat (EPZ-6438), the first marketed drug targeting EZH2, as Comparative Example 1, and compound C36, the compound with the best activity in patent CN114746414A, as Comparative Example 2. The compounds in this invention exhibit significant advantages. Furthermore, the IC50 values ​​of the compounds separated from chiral fractions are [not specified in the original text]. 50 Looking up, -OR 2 When the connected chiral carbon atom is in the S configuration, its IC 50 It is significantly superior to the R configuration.

[0218] Table 1. Methylation inhibition activity at the cellular level

[0219] Compared to Comparative Example 2, the compound of the present invention exhibits better performance in R... 1 The introduction of alkyl substitution resulted in an unexpected increase in activity.

[0220] Example 11: Tumor cell proliferation inhibition activity test

[0221] KARPAS-422 is a diffuse large B-cell lymphoma cell line carrying an activating mutation at the Y641N site of the EZH2 gene, while WSU-DLCL2 cells are a non-lytic diffuse large cell lymphoma cell line carrying an activating mutation at the Y641F site of the EZH2 gene. Both cell lines have been shown to be sensitive to selective EZH2 inhibitors and are commonly used as models for evaluating EZH2-targeted antitumor activity at the cellular level.

[0222] The following experimental methods were used for testing.

[0223] 1) Culture medium:

[0224] Suspension cells WSU-DLCL2 / KARPAS-422: 10% FBS (gibico) + 89% 1640 (Cat: PM150110; Pricella) + 1% Pen Strep bispecific antibody (Cat: 15140; Gibco).

[0225] 2) Before seeding WSU-DLCL2 / KARPAS-422 suspension cells into 96-well plates, treat with 50 μL / well PDL for at least one hour, wash with 100 μL / well PBS, and air dry. Seed WSU-DLCL2 / KARPAS-422 cells with viability above 90% into 96-well plates (2000 cells / well, clear 96-well plates), and administer medication the following day.

[0226] 3) On the second day, prepare the test compound solutions. Ten concentrations of the test compound solutions were prepared by serially diluting them with DMSO (3-fold). After dilution, 2 μL of the test compound solution was added to 198 μL of culture medium and thoroughly mixed (pipe-pipette at least 20 times). Then, 25 μL of culture medium was added to each well of 96 cells and pipetted 10 times. Based on the activity of the test compounds, the maximum treatment concentration of the test compounds was determined through preliminary experiments, ultimately resulting in ten test compound treatment concentrations: a maximum treatment concentration of 37 nM or 1000 nM containing 0.2% DMSO, serially diluted 3-fold.

[0227] 4) Cells treated with different concentrations of the test compound solution or control solution were then cultured in a 37°C incubator for 3-4 days, followed by a change of medium. During the change, 80 μL of fresh medium was added to each 96-well plate (pre-treated with PDL). The test compound solution was then prepared as before, with 25 μL added to each well and mixed by pipetting 4 times using a pipette. For suspension cells WSU-DLCL2 / KARPAS-422, the cells were first mixed by pipetting 10 times using a pipette, and 20 μL / well was transferred to a 96-well plate, mixed for 4 eight-beat cycles without changing the pipette tip. This same change of medium was performed every 3-4 days, for a total of 4 treatments.

[0228] 5) Cell viability was assessed 14 days after treatment. Using a live cell count, the culture medium in the plate was first agitated 10-20 times with a pipette to thoroughly disperse the cells. 20 μL of the culture medium containing cells was taken per well, and 20 μL of trypan blue was added.

[0229] Mix thoroughly and then count the cells.

[0230] Calculation formula: Cell viability (%) = 100% * drug-treated group / DMSO group

[0231] Importing cell viability data into GraphPad Prism allows for the generation of viability curves under small molecule inhibition, thereby yielding the IC50 value. 50 Numerical value.

[0232] The compounds of this invention were tested according to the above experimental methods and showed significant antitumor activity. Some results are shown in Table 2.

[0233] Table 2. Inhibitory activity of compounds on the proliferation of tumor KARPAS422 / WSU-DLCL2 cells.

[0234] Example 12: Pharmacokinetic Properties Testing in a Mouse Model

[0235] Using male ICR mice (3 mice / group) as test animals, grade: SPF, the pharmacokinetic behavior of the compound of this invention in male ICR mice after oral / intravenous administration was studied.

[0236] Source: Laboratory animal reserve (999M-018).

[0237] Oral administration: The compound was administered in a solution of 5% NMP + 10% solubil + 85% (6% HP-b-CD) saline at a dose of 10 mg / kg. Blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration.

[0238] Intravenous administration: The compound was administered in a solution of 5% NMP + 10% solubil + 85% (6% HP-b-CD) saline at a dose of 5 mg / kg. Blood samples were collected at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration, via the submandibular vein or other suitable method.

[0239] Approximately 30 μL of each sample was collected at each time point, anticoagulated with K2-EDTA, and placed on ice after collection. Plasma was centrifuged within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8℃). Plasma samples were stored at -80℃ for LC-MS / MS analysis prior to analysis.

[0240] Based on the obtained blood drug concentration-time data, pharmacokinetic parameters were calculated using WinNonlin software. The compound showed good bioavailability and excellent drug development potential.

[0241] Table 3 Pharmacokinetic parameters of ICR mice after intravenous administration

[0242] Table 4 Pharmacokinetic parameters of ICR mice after oral administration

[0243] Example 13: Pharmacokinetic Properties Tested in a Rat Model

[0244] Using male SD rats (3 rats / group) as test animals, the pharmacokinetic behavior of the compound of the present invention in male SD rats after oral / intravenous administration was studied.

[0245] Rating: SPF

[0246] Source: Laboratory animal reserve (999M-017).

[0247] Oral administration: The compound was administered in a solution of 5% NMP + 10% solubil + 85% (6% HP-β-CD) saline at a dose of 20 mg / kg. Blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration.

[0248] Intravenous administration: The compound was administered at a dose of 5 mg / kg using 5% NMP + 10% solubil + 85% (6% HP-β-CD) saline at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after administration. Blood was collected via the jugular vein or other suitable method, with approximately 200 μL collected per time point. K2-EDTA was used for anticoagulation, and the collected samples were placed on ice.

[0249] Blood samples were placed on ice after collection and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8℃). Plasma samples were stored at -80℃ before analysis, pending LC-MS / MS analysis. The analytical methods for biological samples and the analysis of all samples were performed by the analytical laboratory. Intra-day accuracy evaluation of quality control samples was conducted simultaneously with sample analysis, requiring that the accuracy of more than 66.7% of the quality control samples be between 80-120%.

[0250] Based on the obtained blood drug concentration-time data, pharmacokinetic parameters were calculated using WinNonlin software. The compound showed good bioavailability and excellent drug development potential.

[0251] Table 5 Pharmacokinetic parameters of intravenous administration in SD rats

[0252] Table 6 Pharmacokinetic parameters of SD rats after oral administration

[0253] The PK data of the compound show that R 2The key substitution significantly improves its oral in vivo exposure and bioavailability. This is because, compared to 1S, compound 2S: 1. Methyl substitution enhances lipophilicity, potentially increasing the drug's passive diffusion through gastrointestinal epithelial cells, thereby increasing absorption and bioavailability; 2. Hydroxyl groups are easily metabolized, and methyl groups can block metabolic sites. Hydroxyl groups are common sites of drug metabolism and are easily oxidized to aldehydes, ketones, or further bound (e.g., glucuronidation) by cytochrome P450 enzymes (CYP) in the liver (such as CYP3A4, CYP2D6), leading to a significant first-pass effect and reduced bioavailability. Methyl substitution of the hydroxyl group, if directly covering the metabolic site, can reduce hepatic metabolism, increase the proportion of drug reaching systemic circulation, and thus increase bioavailability; 3. Exposure reflects the area under the concentration-time curve (AUC) and peak concentration (C) of the drug in vivo. max Absorption rate, volume of distribution, and elimination rate are all affected by the methyl group. Methyl substitution of the hydroxyl group simultaneously increases both absorption and absorption rate; however, this may lead to oral C... max As the AUC increases, the AUC also increases.

[0254] Example 14

[0255] The relative binding free energies between methyl substituents, methoxy substituents, and disubstituents and Comparative Example 2 were calculated using the free energy perturbation method to quantitatively verify the strength of the synergistic effect.

[0256] Compared to the compound of Comparative Example 2, the compound in this invention, except for the presence of an S-type methyl group (R) on the carbon atom between the cyclohexyl group and the amino group, is different. 1 The introduction of ) also introduces new chiral substituents (OR) on the carbon atom between the pyrimidine ring and the amide group. 2 ).

[0257] Compared with Comparative Example 2, compound 3-methyl substitution (R 1 ) and methoxy substitution (OR) 2 The introduction of the two substituents simultaneously resulted in an unexpected increase in activity, suggesting a synergistic effect between the two substituents, making the inhibition effect of the two substituents significantly better than that of the single methyl-substituted or methoxy-substituted compounds.

[0258] To perform molecular dynamics simulations, the initial crystal structure was manually preprocessed to reconstruct the missing flexible regions of the protein, determine the small molecule tautomer states, and add hydrogen atoms. The complex was then inserted at an edge distance of [missing information]. The simulation was performed in a cube-shaped TIP3P water box. Additional sodium and chloride ions were added to maintain the electroneutrality of the simulation system and the ion concentration at 0.15 M. Calculations were performed using the CHARMM36m molecular force field to describe proteins and the CGenFF force field to describe small molecules. Molecular dynamics sampling was performed at 10 ns intervals using 16 replicas on the CUDA platform, and the free energy was calculated using the MBAR method. The calculation results are shown in Figure 7.

[0259] All three sets of calculations showed good convergence, and the binding free energy changes caused by single methyl substitution (-0.4kT) and single methoxy substitution (0.3kT) were worse than those of the disubstituted result (-1.5kT). At the same time, the sum of the free energy changes of the two monosubstituted results was still worse than that of the disubstituted result, which confirmed the synergistic advantage of the disubstituted result.

[0260] Furthermore, in these compounds, the synergistic effect of the S configuration on activity unexpectedly contributes far more than that of the R configuration when a new chiral substituent is introduced on the carbon atom between the pyrimidine ring and the amide group. We also conducted systematic target kinetic studies on this, using compounds 3S and 3R as examples:

[0261] CHARMM36m and CGenFF force fields were used for protein and ligand molecules. The simulation system underwent a series of pre-equilibrium simulations, including energy minimization and force constants from 2000 kJ / mol / nm. 2 Up to 20 kJ / mol / nm 2 Constrained equilibrium simulations and final unconstrained simulations were performed. Simulations of a total duration of 100 ns were conducted using Langevin dynamics and Monte-Carlo Barostat at 300 K and 1 atm. The trajectories were then aligned with the first frame, and the RMSD of the ligands was calculated using Ambertools.

[0262] The R and S configurations exhibit different behaviors in simulations. For R configuration substitution, RMSD shows... and This indicates that the fluctuation of 3R is relatively large during the 100 ns simulation. For S-configuration substitution, the RMSD is... The RMSD comparison further confirms that 3S is a more favorable configuration, allowing for more stable interactions with the protein. Further quantitative evaluation of 3R and 3S was performed on the FEP platform, calculating the relative binding free energies between compound 8 and 3R, and between compound 8 and 3S in CN202410986475.6. The initial CHARMM file for FEP was extracted from the last frame of the previous molecular dynamics simulation. Basic settings such as the integrator and Barostat were identical to those in the previous molecular dynamics simulation. Twelve replicas were initialized with lambda values ​​of 0.0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0. The 10 ns FEP results demonstrate that the R configuration is an unfavorable substitution, increasing the binding free energy of 3R by 3.8 kT relative to compound 8 in CN202410986475.6, while the S configuration of 3S decreases the binding free energy by 1.0 kT, thereby improving the inhibitory activity. The calculation results are shown in Figures 8 and 9.

[0263] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A compound represented by Formula 1 or a stereoisomer, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof: wherein wherein R 1 the chiral atom in the 2-position is in the S configuration, OR 2 the chiral atom in the 2-position is in the R configuration or the S configuration; R 1 selected from the group consisting of H, nitro, amino, hydroxyl, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C1-C6alkoxy, substituted or unsubstituted C3-C6cycloalkyl, wherein the substitution denotes that a hydrogen atom on the corresponding group is replaced with one or more substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, SF5, C1-C3alkoxy, C1-C3alkanoyl, amino, C1-C3amido, nitro, cyano, C1-C3alkoxycarbonyl; R 2 is selected from the group consisting of H, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted carbonyl, substituted or unsubstituted C3-C6cycloalkyl, wherein said substitutions indicate that the hydrogen atoms of the corresponding group are replaced by one or more substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, SF5, C1-C3alkoxy, C1-C3alkanoyl, amino, C1-C3amido, nitro, cyano, C1-C3alkoxycarbonyl.

2. The compound of claim 1 represented by Formula 1 or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, OR 2 The chiral atom attached to the position is in the S configuration. 3.The compound represented by Formula 1 according to claim 1, or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, R 1 is selected from H, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy, substituted or unsubstituted C3-C6 cycloalkyl, wherein the substitution denotes that a hydrogen atom on the corresponding group is replaced by one to three substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, SF5, C1-C3 alkoxy, C1-C3 aldehyde, amino, C1-C3 amido, nitro, cyano, C1-C3 alkoxycarbonyl; Preferably, R 1 is selected from H, substituted or unsubstituted C1-C3alkyl, substituted or unsubstituted C1-C3alkoxy, substituted or unsubstituted C3-C6cycloalkyl, wherein the substitution means that a hydrogen atom in the corresponding group is replaced by 1 or 2 substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, C1-C3alkyl, C1-C3alkoxy, C1-C3aldehyde, amino, C1-C3amide; Preferably, R 1 is selected from methyl, ethyl, n-propyl, i-propyl, methoxy, ethoxy, n-propoxy, i-propoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. 4.The compound represented by Formula 1 according to claim 1, or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, characterized in that, R 2 selected from the group consisting of H, substituted or unsubstituted C1-C3alkyl, substituted or unsubstituted carbonyl, substituted or unsubstituted C3-C6cycloalkyl, wherein the substitution means that a hydrogen atom on the corresponding group is replaced with one to three substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, thiol, C1-C3alkyl, C1-C3alkoxy, C1-C3aldehyde, amino, C1-C3amide; Preferably, R 2 is selected from H, substituted or unsubstituted C1-C3alkyl, substituted or unsubstituted carbonyl, substituted or unsubstituted C3-C6cycloalkyl, wherein the substitution means that a hydrogen atom on the corresponding group is replaced by 1 or 2 substituents selected from the group consisting of deuterium, tritium, halogen, hydroxyl, carboxyl, C1-C3alkyl, C1-C3alkoxy, C1-C3aldehyde; Preferably, R 2 is selected from H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, deuterated methyl, deuterated ethyl, deuterated propyl, methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, hydroxymethyl, hydroxyethyl, hydroxy-n-propyl, halogenated methyl, halogenated ethyl, halogenated n-propyl.

5. The compound of claim 1 represented by Formula 1 or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, having a structure selected from the group consisting of: ​ 6.A pharmaceutical composition comprising a therapeutically effective amount of the compound represented by Formula 1 according to any one of claims 1 to 4, or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein, The pharmaceutically acceptable carrier is selected from pharmaceutically acceptable vehicles and pharmaceutically acceptable adjuvants; Preferably, the pharmaceutically acceptable carrier is selected from diluents, solvents, dispersants, antioxidants, preservatives, buffers, emulsifiers, pharmaceutically acceptable fillers, disintegrants, surfactants, binders, flavoring agents, dyes, and lubricants, and combinations thereof.

8. The pharmaceutical composition of claim 5, wherein, The pharmaceutical composition can further comprise an additional active pharmaceutical agent; Preferably, the additional active pharmaceutical agent comprises one or more of an anticancer agent, a biologic, a radiopharmaceutical, a hormonal agent. 9.Use of the compound represented by Formula 1 according to any one of claims 1 to 5, or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 6 in the manufacture of a medicament for treating and / or a disease or condition mediated by at least one of PRC2, EZH1 and EZH2. Preferably, the disease mediated by at least one of PRC2, EZH1 and EZH2 is cancer selected from breast cancer, glioblastoma, prostate cancer, uterine cancer, ovarian cancer, pancreatic cancer, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, lymphoma, leukemia, malignant rhabdoid tumor, mesothelioma, myeloma, liver cancer, lung cancer, or oropharyngeal cancer. 10.A method of treating a disease or condition mediated by at least one of PRC2, EZH1 and EZH2, the method comprising administering to a subject in need of the treatment a therapeutically effective amount of the compound represented by Formula 1 according to any one of claims 1 to 5, or a stereoisomer, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 6.

11. The method of treating a disease or condition mediated by at least one of PRC2, EZH1 and EZH2 according to claim 10, wherein, The disease mediated by at least one of PRC2, EZH1 and EZH2 is cancer selected from breast cancer, glioblastoma, prostate cancer, uterine cancer, ovarian cancer, pancreatic cancer, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, lymphoma, leukemia, malignant rhabdoid tumor, mesothelioma, myeloma, liver cancer, lung cancer, or oropharyngeal cancer.

Images