EZH2 specific inhibitor and application thereof

Abstract

The invention provides a compound with EZH2 specific inhibitor activity and application of the compound. The compound provided by the invention has extremely strong inhibitory activity and good drug properties, and can be used for treating diseases and / or symptoms of the diseases mediated by EZH1, EZH2 and / or PRC2.

Description

Technical Field

[0001] This invention relates to the field of chemical drugs, specifically to EZH2-specific inhibitors and their uses. Background Technology

[0002] 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, there are acquired mutations 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 FDA for the treatment of epithelioid sarcoma and follicular lymphoma; Valemetostat has been approved by the 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, resistance mutations, and high toxicity.

[0003] Novartis has discovered a class of non-competitive inhibitors of SAM, namely azirquinoline compounds (CN114746414A and CN114555589A). The main characteristic of these compounds is the linker between the azirquinoline and pyrimidine rings, with p-aminomethylenecyclohexylamine acting as a linker. A typical example is compound C13 reported in CN114746414A. Due to the need for further improvement in their overall properties, including antitumor activity and druggability, Novartis has not yet brought these compounds to clinical trials.

[0004]

[0005] Shanghai Sailan Biotechnology has discovered a class of compounds (CN117384153A) characterized by a bridged ring or aryl (hetero) ring as the linker site. These compounds use p-aminomethylaniline or p-aminomethylaniline with further methyl substitution as the linker to connect the aziridine ring and the pyrimidine ring. Representative compounds include PA001 and PA062 from the aforementioned patent. However, from a drug development perspective, there is still room for improvement in their activity.

[0006]

[0007] Therefore, there is a need to develop a novel class of EZH2 inhibitors. These inhibitors should possess higher catalytic activity against EZH2, fewer toxic side effects, and excellent pharmacometological stability to play an important role in cancer treatment. This invention, through exploration, discovered that introducing substituents (e.g., methyl groups) at the amino group linked to the linker and pyrimidine ring resulted in an unexpectedly high activity enhancement of tens of times, achieving a very high level of druggable activity. This is likely because the sterically hindered alkyl substituents on the nitrogen atom significantly alter the molecule's three-dimensional structure, allowing for better interaction with the amino acid residues surrounding the target protein pocket. Furthermore, these compounds also exhibited excellent metabolic stability in liver microsomes from various species, suggesting the potential for further drug development. Summary of the Invention

[0008] This invention provides a class of compounds that inhibit EZH2, pharmaceutical salts thereof, and methods and pharmaceutical compositions for treating or preventing diseases or symptoms related to EZH2 and PRC2.

[0009] In one aspect, the present invention provides a compound having Formula 1 or a stereoisomer, enantiomer, or pharmaceutically acceptable salt thereof:

[0010]

[0011] Among them, A 1 Selected from N or CR 11 A 3 Selected from N or CR13 A 5 Selected from N or CR 15 And A 1 A 3 A 5 The number of N in the text is 1 or 2;

[0012] R 12 Selected from H, halogens, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl substituted with 1-3 halogens, and C1-C3 alkoxy substituted with 1-3 halogens;

[0013] R 11 R 13 R 14 R 15 R 16 R 17 Each is independently selected from H, halogen, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 alkyl substituted with 1-3 halogens;

[0014] R 2 Selected from H or methyl;

[0015] In the group, It is a single bond or a double bond, and all of them It can be a single bond or a double bond at the same time;

[0016] when In When it is a single bond, B 1 For NH or CHR 21 B 2 For NH or CHR 22 B 3 For NH or CHR 23 B 4 For NH or CHR 24 R 3 For H;

[0017] when In When it is a double bond, B 1 For N or CR 21 B 2 For N or CR 22 B 3 For N or CR 23 B 4 For N or CR 24 R 3 It is H or C1-C3 alkyl;

[0018] B 1 B 2 B3 and B 4 In this context, the number of N or NH is 0 or 1.

[0019] R 21 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens;

[0020] R 22 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens;

[0021] R 23 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens;

[0022] R 24 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens;

[0023] R 4 Selected from C1-C3 alkyl groups or 1-3 deuterated C1-C3 alkyl groups;

[0024] C 1 Selected from N or CH;

[0025] L is selected from -C(R) 6 R 7 -, -C(=CH2)-;

[0026] R 6 R 7 Each is independently selected from H, halogen, hydroxyl, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, C1-C3 alkyl substituted with 1-3 halogens, and C1-C3 alkyl substituted with hydroxyl and 1-3 halogens;

[0027] R 5 Selected from R a -C(=O)R a -CH2R a ;

[0028] R a Selected from -NHR c ,

[0029]

[0030] In the group, For single or double bonds, R a1 R a2 R a3Each is independently selected from -CH2-, -CH=, =CH-, -CH(CH3)-, =C(CH3)-, -C(CH3)=, or -C(=O)-;

[0031] R b Selected from H, halogens, C1-C3 alkyl groups, and cyano-substituted C1-C3 alkyl groups;

[0032] R c Selected from C1-C4 alkyl groups substituted with hydroxyl groups and C2-C3 alkyl groups substituted with C1-C3 alkoxy groups;

[0033] D 1 -C(R) d R e )-;

[0034] D 2 Selected from -O-, -SO 2 -、-CH(OH)-、-S-、-C(R f R g )-;

[0035] R d R e Each is independently selected from H, halogens, and C1-C3 alkyl groups;

[0036] R f R g Each is independently selected from H and halogens;

[0037] E 1 Selected from -C(=O)- or -C(R) h R i )-;

[0038] E 2 Selected from -NH- or -O-;

[0039] E 3 Selected from -NH-, -O-, or -CH(N(CH3)2)-;

[0040] R h R i Each is independently selected from H or halogen;

[0041] F 1 Selected from -N(R) j - or -O-;

[0042] Among them, R j Selected from H or C1-C3 alkyl groups.

[0043] In some implementations, the formula in Equation 1 The group structure is selected from:

[0044]

[0045] In some implementations, R 21 Selected from H, halogens, methyl groups, or CF3;

[0046] In some implementations, R 22 Selected from H, halogens, methyl groups, or CF3;

[0047] In some implementations, R 23 Selected from H, halogens, methyl groups, or CF3;

[0048] In some implementations, R 24 Selected from H, halogens, methyl groups, or CF3;

[0049] In some embodiments, the compound has the structure of Formula 2:

[0050]

[0051] In some embodiments, R in the compound of formula 2 3 For H.

[0052] In some embodiments, R in the compound of formula 2 3 It is a C1-C3 alkyl group. In some embodiments, R in the compound of formula 2 3 It is a methyl group.

[0053] In some implementations... The group structure is selected from:

[0054]

[0055] In some embodiments, the compound has the structure of Formula 3:

[0056]

[0057] In some embodiments, the compound has the structure of Formula 4:

[0058]

[0059] In some implementations, R 4 Selected from methyl, ethyl or -CD3.

[0060] In some implementations, R 4 It is a methyl group.

[0061] In some implementations, R a Selected from

[0062]

[0063]

[0064] In some embodiments of the present invention, the compound is selected from the following structures:

[0065]

[0066]

[0067]

[0068] A second aspect of the invention provides a pharmaceutical composition comprising a compound of any one of formulas 1-4 or any one of compounds 1-67, a stereoisomer thereof, an enantiomer thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise additional active pharmaceutical agents.

[0069] In some embodiments, 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, flavorings, dyes, and lubricants, and combinations thereof.

[0070] In some embodiments, additional active pharmaceutical agents may include one or more of the following: anticancer agents, biological agents, radiopharmaceuticals, and hormonal drugs.

[0071] A third aspect of the present invention provides a method for treating a disease or symptom mediated by at least one of PRC2, EZH1, and EZH2, characterized in that a therapeutically effective amount of a compound of one of formulas 1-4 or one of compounds 1-67, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of one of formulas 1-4 or one of compounds 1-67, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt thereof, is administered to a subject requiring the treatment.

[0072] In some implementations, the disease mediated by at least one of PRC2, EZH1, and EZH2 is cancer, including at least breast cancer (e.g., triple-negative breast cancer), glioblastoma, prostate cancer, uterine cancer, ovarian cancer, pancreatic cancer, melanoma, renal cell carcinoma, bladder cancer, colorectal cancer, lymphoma, leukemia, malignant rhabdomyosarcoma, or oropharyngeal cancer.

[0073] In a fourth aspect, the present invention provides a pharmaceutically acceptable salt of a compound of any one of Formulas 1-4 or Compounds 1-67, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a mixture of the above-mentioned compounds, stereoisomers, or mixtures of different stereoisomers of the compound, or a pharmaceutical composition containing the thereof, for use in the preparation of a medicament for treating diseases or symptoms mediated by at least one of PRC2, EZH1, and EZH2.

[0074] In a fifth aspect, the present invention provides a method for preparing a compound of any one of Formulas 1-4 or any one of Compounds 1-67 and its stereoisomers.

[0075] The compounds of the present invention can be prepared using the methods described below or other similar methods as understood by those skilled in the art of organic synthesis. Compounds of one of compounds 1-67 having a chiral center 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.

[0076]

[0077] A bromine- or chlorine-substituted nitrogen-containing heterocyclic quinoline ring undergoes a CN-bond coupling reaction with an aryl or alkylamine under palladium or copper catalysis to generate intermediate INT-1. After deprotection of the Boc group, INT-1 undergoes an electrophilic aryl substitution reaction with a halogen-substituted nitrogen-containing aryl compound A to generate a CN-bonded compound INT2. INT2 is then hydrolyzed to convert its L-terminal ester to the corresponding carboxylic acid, or reduced to convert its L-terminal ester to the corresponding aldehyde, followed by an acid-amine condensation or reductive amination reaction with the corresponding amino compound to prepare compounds 1-67.

[0078] The compounds of the present invention, as effective EZH2 inhibitors, have stronger inhibitory activity and suitable pharmaceutical properties, and can be used to treat diseases and / or symptoms mediated by EZH1, EZH2 and / or PRC2. Attached Figure Description

[0079] Figure 1 The activity (IC50) of Comparative Examples 2, 3 and 1 50 )contrast Detailed Implementation

[0080] Terminology Definition

[0081] 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.

[0082] In this invention, the term "PRC2" refers to the epigenetic control factor polycombrepressive complex 2.

[0083] 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.

[0084] In this invention, the term "subject" refers to an animal including a human.

[0085] The term "therapeutic effective amount" in this invention refers to the amount of a compound that produces the intended effect of its application (e.g., improvement of disease and / or disease symptoms mediated by EZH1, EZH2, and / or PRC2, reduction of the severity of the corresponding disease and / or disease symptoms, and / or slowing of the progression of the corresponding disease and / or symptoms). The precise amount of the therapeutic effective amount will depend on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

[0086] As used herein, the term "treatment" and its cognates refer to slowing or stopping disease progression. As used herein, "treatment" and its cognates include, but are not limited to, complete or partial remission, cure of disease and / or symptoms mediated by EZH1, EZH2, and / or PRC2, and reduction of the risk of the corresponding disease and / or symptoms. Improvement or reduction in the severity of any of these symptoms can be evaluated using methods and techniques known in the art.

[0087] 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.

[0088] The term "stereoisomer" in this invention includes cis-trans isomers, enantiomers, and diastereomers.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] The active pharmaceutical ingredient described in this invention may include one or more of the following: anticancer agents, biological agents, hormonal drugs, and radiopharmaceuticals:

[0095] 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.

[0096] 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.

[0097] 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.

[0098] Radiopharmaceuticals, such as Phosphocol, Iodotope, Samarium SM-153, and Metastron.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] In this invention, the term "straight-chain alkyl" refers to an alkyl group without side chains or branches.

[0103] 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.

[0104] In this invention, the term "cycloalkyl" refers to an alkyl group in which carbon atoms form a closed cyclic structure through single bonds.

[0105] 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.

[0106] In this invention, the term "deuterated" refers to the substitution of one or more H atoms in a group by D atoms.

[0107] In this invention, the term "alkoxy" refers to a group having an -OR structure, where R is an alkyl group as defined above. For example, "C1-C3 alkoxy" is a group having an -OC1, -OC2, or -OC3 structure.

[0108] The description in the structural formula of this invention This indicates that the chemical bond can optionally be a single bond "-" or a double bond.

[0109] In this invention, the terms "cyano" and "nitrile" mean "-CN" groups.

[0110] In this invention, the term "hydroxyl group" means "-OH" group.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Tumors of the female reproductive organs include, for example, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer.

[0115] 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.

[0116] 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.

[0117] Liver tumors include, for example, hepatocellular carcinoma and cholangiocarcinoma.

[0118] Skin tumors include, for example, malignant melanoma, basal tumor, spinal cord tumor, Kaposi's sarcoma, and Merkel cell carcinoma.

[0119] Tumors of the head and neck include, for example, laryngeal cancer, as well as pharyngeal and oral cancer.

[0120] 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.

[0121] 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.

[0122] The abbreviations of this invention have their common meanings in the art or the definitions in Table 1 below:

[0123] Table 1 List of Abbreviations

[0124]

[0125]

[0126] Method for preparing the compounds of the present invention

[0127] The compounds of the present invention can be prepared using the methods of Examples 1-67, or other similar methods as understood by those skilled in the art.

[0128] Example 1: Synthesis of 2-(2-((4-((8-methoxy-1,7-naphthidin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 1)

[0129]

[0130] Preparation route of compound 1:

[0131]

[0132] Step 1: Synthesis of compounds 1-3

[0133] Compound 1-1 (211 mg, 1.00 mmol, 1.0 equivalent), compound 1-2 (236 mg, 1.00 mmol, 1.0 equivalent), BINAP (125 mg, 0.20 mmol, 0.2 equivalent), Pd2(dba)3 (92 mg, 0.10 mmol, 0.1 equivalent), and sodium tert-butoxide (192 mg, 2.00 mmol, 2.0 equivalent) were added to a 25 mL double-necked flask. Under a nitrogen atmosphere, the atmosphere was purged three times. Tetrahydrofuran (5.0 mL) was added, and the reaction mixture was heated and stirred overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched with a saturated ammonium chloride aqueous solution (5.0 mL). The mixture was extracted three times with ethyl acetate (5.0 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give intermediate compounds 1-3 (303 mg, 0.77 mmol, 77% yield). 1 H NMR(500MHz, CDCl3)δ7.90(d,J=5.5Hz,1H),7.85(d,J=9.0Hz,1H),7.42–7.34(m,2H),7.27–7.24(m,2H ),7.14(s,1H),7.09(d,J=5.5Hz,1H),4.42(s,2H),4.19(s,3H),2.85(s,3H),1.50(s,9H).LC-MS[M+H] + m / z:395.4.

[0134] Step 2: Synthesis of compounds 1-4

[0135] Compounds 1-3 (303 mg, 0.77 mmol, 1.0 equivalent) were dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was reacted at room temperature for 3 hours and monitored by TLC. After the starting material was completely reacted, the solvent was evaporated and the resulting oily compound was directly used for the next step.

[0136] The above-mentioned oily compound and compound A1 (163 mg, 0.77 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (3.0 mL), and N,N-diisopropylethylamine (498 mg, 3.85 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL), yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phases were combined. The mixture was washed three times with saturated sodium chloride solution (10 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 intermediate compounds 1-4 (250 mg, 0.51 mmol, yield 67%). LC-MS [M+H] + m / z:487.3.

[0137] Step 3: Synthesis of Compound 1

[0138] Intermediate compound 1-4 (49 mg, 0.10 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.4 mL) was added. The reaction was carried out at room temperature for 2 h, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed by rotary evaporation under reduced pressure. The resulting oily compound was directly used for the next step.

[0139] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (2.0 mL), and N,N-diisopropylethylamine (52 mg, 0.40 mmol, 4.0 equivalent), HATU (76 mg, 0.20 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (15 mg, 0.20 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 h. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL) to obtain a mixed solution. The above mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 obtain compound 1 (13 mg, 0.03 mmol, yield 27%), a white solid compound. 1H NMR (500MHz, DMSO-d6) δ9.54 (s, 1H), 8.75 (d, J = 6.5Hz, 1H), 8.18 (s, 2H), 7.95 ( d,J=9.0Hz,1H),7.89(d,J=8.5Hz,2H),7.77(d,J=5.5Hz,1H),7.17(t,J=7.5Hz ,2H),7.13(d,J=8.5Hz,2H),4.75(s,2H),4.74-4.67(m,1H),4.63(t,J=6.5Hz, 2H),4.35(t,J=6.5Hz,2H),3.96(s,3H),3.24(s,3H),3.00(s,2H).LC-MS[M+H] + m / z:486.4.

[0140] Example 2: Synthesis of 2-(2-((4-((8-chloro-1,7-naphthidin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 2)

[0141]

[0142] Preparation route of compound 2:

[0143]

[0144] Step 1: Synthesis of Compound 2-1

[0145] Intermediate 1-4 (49 mg, 0.10 mmol, 1.0 equivalent) was dissolved in ethyl acetate (1.0 mL), followed by the addition of 4 M ethyl hydrochloride solution (0.1 mL, 0.40 mmol, 4.0 equivalent). The reaction was carried out at 40 °C for 20 h, and then ethanol (1.0 mL) was added to the reaction solution, and the mixture was refluxed at 90 °C for 20 h. TLC monitoring showed that the reaction proceeded completely. The solvent was removed under reduced pressure, and the resulting oily compound was directly used in the next step.

[0146] The above-mentioned oily compound was dissolved in phosphorus oxychloride (0.5 mL) and reacted at 110 °C for 1.5 h. The reaction was monitored by TLC. After the reaction was complete, phosphorus oxychloride was removed under reduced pressure. The reaction solution was diluted with ethyl acetate and then adjusted to neutral with a saturated aqueous solution of NaHCO3 at 0 °C to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (4.0 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain intermediate compound 2-1. This was then directly proceeded to the next reaction step.

[0147] Step 2: Synthesis of Compound 2

[0148] Intermediate 2-1 (0.10 mmol) was dissolved in a mixed solvent of tetrahydrofuran (1.0 mL) and methanol (1.0 mL), followed by the addition of 2M sodium hydroxide aqueous solution (0.1 mL). The reaction was carried out at room temperature for 2 h, monitored by TLC. After the reactants had reacted completely, the mixture was neutralized with 1M HCl aqueous solution to obtain a mixed solution. The mixed solution was extracted three times with dichloromethane (5.0 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting oily compound was directly used in the next reaction.

[0149] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (2.0 mL), and N,N-diisopropylethylamine (52 mg, 0.40 mmol, 4.0 equivalent), HATU (76 mg, 0.20 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (15 mg, 0.20 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 h. After the reaction was completed, the reaction was quenched with saturated ammonium chloride aqueous solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution, 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 obtain compound 2 (19 mg, 0.04 mmol, yield 39%), a white solid compound. 1 H NMR(500MHz,DMSO-d6)δ9.88(s,1H),8.74(d,J=6.5Hz,1H),8.18(s,2H),8.0 7(dd,J=12.5,7.0Hz,2H),8.01(d,J=8.5Hz,2H),7.64(d,J=5.0Hz,1H),7.27( d,J=9.0Hz,1H),7.18(d,J=8.5Hz,2H),4.76(s,2H),4.74-4.67(m,1H),4.63( t,J=7.0Hz,2H),4.35(t,J=6.5Hz,2H),3.23(s,2H),3.00(s,3H).LC-MS[M+H] + m / z:490.2.

[0150] Example 3: Synthesis of 2-(2-((4-((8-chloro-1,5-naphthidin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 3)

[0151]

[0152] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 2,8-dichloro-1,5-naphthidine, and other operating steps were similar, resulting in compound 3 as a white solid with a yield of 73%. 1 H NMR(500MHz,DMSO-d6)δ9.92(s,1H),8.82(d,J=6.5Hz,1H),8.54(d,J=5.0Hz,1H) ,8.25(s,2H),8.13(d,J=9.0Hz,1H),8.09(d,J=8.5Hz,2H),7.81(d,J=5.0Hz,1H) ,7.37(d,J=9.0Hz,1H),7.24(d,J=8.5Hz,2H),4.83(s,2H),4.81-4.75(m,1H),4. 71(t,J=7.0Hz,2H),4.43(t,J=6.5Hz,2H),3.31(s,2H),3.08(s,3H).LC-MS[M+H] + m / z:490.3.

[0153] Example 4: Synthesis of 2-(2-(((5-((8-chloro-1,7-naphthidin-2-yl)amino)pyridin-2-yl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 4)

[0154]

[0155] Preparation route of compound 4:

[0156]

[0157] Step 1: Synthesis of compound 4-2

[0158] Compound 1-1 (194 mg, 1.00 mmol, 1.0 equivalent), compound 4-1 (237 mg, 1.00 mmol, 1.0 equivalent), BINAP (125 mg, 0.20 mmol, 0.2 equivalent), Pd2(dba)3 (92 mg, 0.10 mmol, 0.1 equivalent), and sodium tert-butoxide (192 mg, 2.00 mmol, 2.0 equivalent) were added to a 25 mL double-necked flask. Under a nitrogen atmosphere, the atmosphere was purged three times. Tetrahydrofuran (10 mL) was added, and the reaction mixture was heated and stirred overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give intermediate compound 4-2 (383 m, 0.97 mmol, 97% yield). LC-MS [M+H] + m / z:396.3.

[0159] Step 2: Synthesis of Compound 4-3

[0160] Compound 4-2 (290 mg, 0.73 mmol, 1.0 equivalent) was dissolved in dichloromethane (3.0 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction of the starting materials was completed, the solvent was evaporated and the resulting oily compound was directly used for the next step.

[0161] The above-mentioned oily compound and compound A1 (155 mg, 0.73 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (5.0 mL), and N,N-diisopropylethylamine (474 ​​mg, 3.67 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution (10 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 intermediate compound 4-3 (225 mg, 0.46 mmol, yield 63%). LC-MS [M+H] + m / z:488.3.

[0162] Step 3: Synthesis of Compound 4-4

[0163] Intermediate compound 4-3 (97 mg, 0.20 mmol, 1.0 equivalent) was dissolved in ethyl acetate (4.0 mL), followed by the addition of 4 M ethyl hydrochloride solution (0.20 mL, 0.80 mmol, 4.0 equivalent). The reaction was carried out at 40 °C for 20 hours. Subsequently, ethanol (4.0 mL) was added to the reaction solution, and the mixture was refluxed at 90 °C for 20 hours. TLC monitoring was performed. After the starting material had completely reacted, the solvent was removed under reduced pressure, and the resulting oily compound was directly used in the next step.

[0164] The above-mentioned oily compound was dissolved in phosphorus oxychloride (2.0 mL) and reacted at 110 °C for 1.5 h. The reaction was monitored by TLC. After the reaction was complete, phosphorus oxychloride was removed under reduced pressure. The reaction solution was diluted with ethyl acetate and then adjusted to neutral with a saturated aqueous solution of NaHCO3 at 0 °C to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (4.0 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 compound 4-4 (60 mg, 0.13 mmol, yield 65%). LC-MS [M+H] + m / z:464.2.

[0165] Step 4: Synthesis of Compound 4

[0166] Intermediate 4-4 (60 mg, 0.13 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (2.0 mL) and methanol (2.0 mL), followed by the addition of 2 M sodium hydroxide aqueous solution (0.5 mL). The reaction was carried out at room temperature for 2 hours, monitored by TLC. After the reactants had reacted completely, the mixture was neutralized with 1 M hydrochloric acid aqueous solution to obtain a mixed solution. The mixed solution was extracted three times with dichloromethane (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting oily compound was directly used in the next reaction.

[0167] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (5.0 mL), and N,N-diisopropylethylamine (67 mg, 0.52 mmol, 4.0 equivalent), EDCI (50 mg, 0.26 mmol, 2.0 equivalent), HOBt (35 mg, 0.26 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (19 mg, 0.26 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL) to obtain a mixed solution. The above mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 obtain 4 (42 mg, 0.09 mmol, yield 66%), a white solid compound. 1 H NMR(500MHz,DMSO-d6)δ10.21(s,1H),9.18(d,J=2.0Hz,1H),8.85(d,J=6.5Hz,1H),8.58 (dd,J=8.5,2.5Hz,1H),8.24(s,2H),8.21(d,J=9.0Hz,1H),8.16(d,J=5.0Hz,1H),7.74( d,J=5.0Hz,1H),7.39(d,J=9.0Hz,1H),7.18(d,J=8.5Hz,1H),4.90(s,2H),4.80–4.73(m ,1H),4.70(t,J=6.5Hz,2H),4.42(t,J=6.5Hz,2H),3.30(s,2H),3.17(s,3H).LC-MS[M+H] + m / z:491.2.

[0168] Example 5: Synthesis of 2-(2-(((5-(((1-chloroisoquinoline-7-yl)amino)pyridin-2-yl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 5)

[0169]

[0170] Referring to the preparation process of compound 4 in Example 4, ((6-aminopyridin-3-yl)methyl(methyl)carbamate tert-butyl ester (4-1) was replaced with ((5-aminopyridin-2-yl)methyl(methyl)carbamate tert-butyl ester, and the other operation steps were similar, to obtain compound 5 as a white solid with a yield of 20%. 1H NMR(500MHz,DMSO-d6)δ9.92(s,1H),8.82(d,J=6.5Hz,1H),8.54(d,J=5.0Hz,1H) ,8.25(s,2H),8.13(d,J=9.0Hz,1H),8.09(d,J=8.5Hz,2H),7.81(d,J=5.0Hz,1H) ,7.37(d,J=9.0Hz,1H),7.24(d,J=8.5Hz,2H),4.83(s,2H),4.81-4.75(m,1H),4. 71(t,J=7.0Hz,2H),4.43(t,J=6.5Hz,2H),3.31(s,2H),3.08(s,3H).LC-MS[M+H] + m / z:490.3.

[0171] Example 6: Synthesis of 2-(2-((4-((8-fluoroquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 6)

[0172]

[0173] Following the preparation process of compound 1 in Example 1, 2-chloro-8-fluoroquinoline was used to replace 2-chloro-8-methoxy-1,7-naphthidine (1-1), and other operating steps were similar, to obtain compound 6 as a white solid with a yield of 28%. 1 H NMR (500MHz, DMSO-d6) δ9.54 (s, 1H), 8.74 (d, J = 7.0Hz, 1H), 8.18 (s, 2H), 8.02 (dd, J = 9. 0,1.5Hz,1H),7.89(d,J=8.5Hz,2H),7.48(dd,J=8.0,1.5Hz,1H),7.33(ddd,J=11.0,8. 0,1.5Hz,1H),7.22–7.11(m,3H),7.04(d,J=9.0Hz,1H),4.74(s,2H),4.71(q,J=7.0Hz, 1H),4.63(t,J=6.0Hz,2H),4.36(t,J=6.0Hz,2H),3.23(s,2H),3.00(s,3H).LC-MS[M+H] + m / z:473.3.

[0174] Example 7: Synthesis of 2-(2-((4-((8-chloroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 7)

[0175]

[0176] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 2,8-dichloroquinoline, and other operating steps were similar, resulting in compound 7 as a white solid with a yield of 42%. 1 H NMR (500MHz, DMSO-d6) δ9.75 (s, 1H), 8.87 (d, J = 6.5Hz, 1H), 8.31 (s, 2H), 8. 23–8.12(m,3H),7.81(dd,J=8.0,1.5Hz,1H),7.77(dd,J=8.0,1.5Hz,1H),7. 34–7.23(m,3H),7.18(d,J=9.0Hz,1H),4.87(s,2H),4.86-4.80(m,1H),4.7 8–4.72(m,2H),4.48(t,J=6.5Hz,2H),3.36(s,2H),3.12(s,3H).LC-MS[M+H] + m / z:489.2.

[0177] Example 8: Synthesis of 2-(2-((4-((8-bromoquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 8)

[0178]

[0179] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 8-bromo-2-chloroquinoline, and other operating steps were similar, to obtain compound 8 as a white solid with a yield of 45%. 1 H NMR (500MHz, DMSO) δ9.73 (s, 1H), 8.84 (d, J = 6.5Hz, 1H), 8.28 (s, 2H), 8.19 (d, J = 8. 5Hz,2H),8.11(d,J=9.0Hz,1H),7.96(dd,J=7.5,1.0Hz,1H),7.78(dd,J=8.0,1.1H z,1H),7.27–7.21(m,3H),7.14(d,J=9.0Hz,1H),4.85(s,2H),4.84–4.77(m,1H),4 .73(t,J=7.0Hz,2H),4.45(t,J=6.5Hz,2H),3.37(s,2H),3.10(s,3H).LC-MS[M+H] + m / z:533.2.

[0180] Example 9: Synthesis of 2-(2-(methyl(4-((8-methylquinolin-2-yl)amino)benzyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 9)

[0181]

[0182] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 2-chloro-8-methylquinoline, and other operating steps were similar, to obtain compound 9 as a white solid with a yield of 33%. 1 H NMR (500MHz, DMSO) δ9.37 (s, 1H), 8.74 (d, J = 6.5Hz, 1H), 8.18 (s, 2H), 7.96-7.92 (m,3H),7.49(d,J=8.0Hz,1H),7.39(d,J=7.0Hz,1H),7.14(d,J=8.5Hz,2H),7.1 2-7.09(m,1H),6.97(d,J=9.0Hz,1H),4.74(s,2H),4.73–4.67(m,1H),4.63(t,J =7.0Hz,2H),4.35(t,J=6.5Hz,2H),3.24-3.22(m,5H),3.00(s,3H).LC-MS[M+H] + m / z:469.3.

[0183] Example 10: Synthesis of 2-(2-(methyl(4-((8-(trifluoromethyl)quinolin-2-yl)amino)benzyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 10)

[0184]

[0185] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 2-chloro-8-trifluoromethylquinoline, and other operating steps were similar, to obtain compound 10 as a white solid with a yield of 54%. 1H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.75(d,J=6.5Hz,1H),8.19(s,2H),8.08(d ,J=9.0Hz,1H),7.96-7.91(m,3H),7.88(d,J=7.5Hz,1H),7.31(t,J=7.5Hz,1H), 7.12(d,J=8.5Hz,2H),7.08(d,J=9.0Hz,1H),4.75(s,2H),4.74-4.67(m,1H),4. 64(t,J=6.5Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,2H),3.00(s,3H).LC-MS[M+H] + m / z:523.3.

[0186] Example 11: Synthesis of 2-(2-((4-((8-chloroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)acetamide (compound 11)

[0187]

[0188] Following the preparation process of compound 7 in Example 7, oxetane-3-methylamine was replaced with 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one, and other operating steps were similar, resulting in compound 11 as a white solid with a yield of 47%. 1 HNMR(500MHz,DMSO-d6)δ11.49(s,1H),9.70(s,1H),8.23(s,2H),8.14-8.10(m, 3H),8.03(s,1H),7.76(dd,J=7.5,1.5Hz,1H),7.72(dd,J=8.0,1.5Hz,1H),7.26 (t,J=8.0Hz,1H),7.23–7.19(m,2H),7.13(d,J=9.0Hz,1H),5.84(s,1H),4.82(s ,2H),4.08(d,J=5.0Hz,2H),3.32(s,2H),3.07(s,3H),2.10(s,6H).LC-MS[M+H] + m / z:568.3.

[0189] Example 12: Synthesis of 2-(2-((methyl-d3)(4-(((7-methylquinolin-2-yl)amino)benzyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 12)

[0190]

[0191] Preparation route of compound 12:

[0192]

[0193] Step 1: Synthesis of Compound 12-3

[0194] Compound 12-1 (300 mg, 1.42 mmol, 1.0 equivalent), compound 12-2 (315 mg, 1.42 mmol, 1.0 equivalent), BINAP (174 mg, 0.28 mmol, 0.2 equivalent), Pd2(dba)3 (128 mg, 0.14 mmol, 0.1 equivalent), and sodium tert-butoxide (273 mg, 2.84 mmol, 2.0 equivalent) were added to a 25 mL double-necked flask. Under a nitrogen atmosphere, the atmosphere was purged three times. Tetrahydrofuran (10 mL) was added, and the reaction mixture was heated and stirred overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give intermediate compound 12-3 (340 mg, 0.86 mmol, 60% yield). LC-MS [M+H] + m / z:398.2.

[0195] Step 2: Synthesis of compound 12-4:

[0196] Compound 12-3 (270 mg, 0.68 mmol, 1.0 eq) was added to a 25 mL double-necked flask. The flask was purged three times under a nitrogen atmosphere. Tetrahydrofuran (10 mL) was added, followed by 1 M lithium aluminum hydride tetrahydrofuran solution (3.4 mL, 3.40 mmol, 5.0 eq). The reaction was carried out at 60 °C for 5 hours, monitored by TLC. After the reactants had reacted completely, water (5.0 mL) was added dropwise, followed by 15% sodium hydroxide solution (5.0 mL). The lithium aluminum hydride was then quenched with water (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting oily compound was directly used in the next step.

[0197] The above-mentioned oily compound and compound A1 (144 mg, 0.68 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (5.0 mL), and N,N-diisopropylethylamine (439 mg, 3.40 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution (10 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 intermediate compound 12-4 (132 mg, 0.28 mmol, yield 41%). LC-MS [M+H] + m / z:473.4.

[0198] Step 3: Synthesis of Compound 12

[0199] Intermediate compound 12-4 (51 mg, 0.11 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed by rotary evaporation under reduced pressure. The resulting oily compound was directly used for the next step.

[0200] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (2.0 mL), and N,N-diisopropylethylamine (56 mg, 0.43 mmol, 4.0 equivalent), HATU (84 mg, 0.22 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (16 mg, 0.22 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL), and the above mixed solution was extracted three times with ethyl acetate (5.0 mL). The organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 12 (23 mg, 0.05 mmol, yield 44%), a white solid. 1HNMR (500MHz, CDCl3) δ9.32 (s, 1H), 8.84 (d, J = 6.5 Hz, 1H), 8.24 (s, 2H), 7.96 (d, J = 9. 0Hz,1H),7.90(d,J=8.5Hz,2H),7.59(d,J=8.0Hz,1H),7.46(s,1H),7.18(d,J=8.5Hz, 2H),7.10(dd,J=8.0,1.0Hz,1H),6.94(d,J=9.0Hz,1H),4.79(s,2H),4.78–4.74(m,1H ),4.70(t,J=7.0Hz,2H),4.42(t,J=6.5Hz,2H),3.30(s,2H),2.43(s,3H).LC-MS[M+H] + m / z:472.3.

[0201] Example 13: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 13)

[0202]

[0203] Following the preparation process of compound 1 in Example 1, 2-chloro-8-methoxy-1,7-naphthidine (1-1) was replaced with 2,8-dichloro-7-methylquinoline, and other operating steps were similar, resulting in compound 13 as a white solid with a yield of 71%. 1 H NMR(500MHz,DMSO-d6)δ9.56(s,1H),8.74(d,J=5.5Hz,1H),8.18(s,2H),8.06(d,J= 8.0Hz,2H),7.98(d,J=8.5Hz,1H),7.54(d,J=8.0Hz,1H),7.19(d,J=8.0Hz,1H),7.15 (d,J=8.0Hz,2H),6.99(d,J=8.5Hz,1H),4.75(s,2H),4.73–4.68(m,1H),4.63(t,J=6 .5Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,3H),3.00(s,3H),2.46(s,2H).LC-MS[M+H] + m / z:503.3.

[0204] Example 14: Synthesis of 2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl-d3)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 14)

[0205]

[0206] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl-D3)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 14 as a white solid with a yield of 79%. 1 H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.84(d,J=6.5Hz,1H),8.25(s,2H),8.13(d, J=8.5Hz,2H),8.05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H) ,7.21(d,J=8.5Hz,2H),7.05(d,J=9.0Hz,1H),4.81(s,2H),4.80–4.74(m,1H),4. 71(t,J=7.0Hz,2H),4.42(t,J=6.5Hz,2H),3.30(s,2H),2.53(s,3H).LC-MS[M+H] + m / z:506.3.

[0207] Example 15: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 15)

[0208]

[0209] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(ethyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 15 as a white solid with a yield of 44%. 1H NMR(500MHz,DMSO-d6)δ9.68(s,1H),8.88(d,J=6.5Hz,1H),8.29(s,2H),8.18(d,J=8 .5Hz,2H),8.10(d,J=9.0Hz,1H),7.66(d,J=8.0Hz,1H),7.33-7.27m,3H),7.11(d,J=9 .0Hz,1H),4.85(s,2H),4.84-4.80(m,1H),4.76(t,J=6.5Hz,2H),4.48(t,J=6.5Hz,2H ),3.63(q,J=7.0Hz,2H),3.35(s,2H),2.58(s,3H),1.13(t,J=7.0Hz,3H).LC-MS[M+H] + m / z:517.3.

[0210] Example 16: Synthesis of 2-(2-((4-((8-bromo-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 16)

[0211]

[0212] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 8-bromo-2-chloro-7-methylquinoline, and other operating steps were similar, resulting in compound 16 as a white solid with a yield of 31%. 1 H NMR(500MHz,DMSO-d6)δ9.71(s,1H),8.88(d,J=6.5Hz,1H),8.31(s,2H),8.24(d,J= 8.5Hz,2H),8.10(d,J=9.0Hz,1H),7.71(d,J=8.0Hz,1H),7.33(d,J=8.0Hz,1H),7.28 (d,J=8.5Hz,2H),7.11(d,J=9.0Hz,1H),4.88(s,2H),4.87-4.80(m,1H),4.76(t,J=7 .0Hz,2H),4.48(t,J=7.0Hz,2H),3.36(s,2H),3.13(s,3H),2.63(s,3H).LC-MS[M+H] + m / z:547.2.

[0213] Example 17: Synthesis of 2-(2-((4-((8-chloro-7-fluoroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 17)

[0214]

[0215] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 2,8-dichloro-7-fluoroquinoline, and other operating steps were similar, resulting in compound 17 as a white solid with a yield of 49%. 1 H NMR (500MHz, DMSO-d6) δ9.84 (s, 1H), 8.87 (d, J = 6.5Hz, 1H), 8.31 (s, 2H), 8. 20-8.14(m,3H),7.84(dd,J=9.0,6.0Hz,1H),7.40(t,J=9.0Hz,1H),7.29(d, J=8.5Hz,2H),7.15(d,J=9.0Hz,1H),4.88(s,2H),4.87-4.80(m,1H),4.76(t ,J=7.0Hz,2H),4.48(t,J=6.5Hz,2H),3.36(s,2H),3.13(s,3H).LC-MS[M+H] + m / z:507.2.

[0216] Example 18: Synthesis of 2-(2-((4-((7,8-dichloroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 18)

[0217]

[0218] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 2,7,8-trichloroquinoline, and other operating steps were similar, resulting in compound 18 as a white solid with a yield of 36%. 1 H NMR(500MHz,DMSO-d6)δ9.74(s,1H),8.75(d,J=6.5Hz,1H),8.18(s,2H),8 .08-7.99(m,3H),7.67(d,J=8.5Hz,1H),7.39(d,J=8.5Hz,1H),7.17(d,J=8 .5Hz,2H),7.06(d,J=9.0Hz,1H),4.76(s,2H),4.74-4.68(m,1H),4.64(t, J=6.5Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,2H),3.00(s,3H).LC-MS[M+H] + m / z:523.2.

[0219] Example 19: Synthesis of 2-(2-((4-((6,8-dichloroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 19)

[0220]

[0221] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 2,6,8-trichloroquinoline, and other operating steps were similar, resulting in compound 19 as a white solid with a yield of 44%. 1 H NMR (500MHz, DMSO) δ9.80 (s, 1H), 8.83 (d, J = 6.5Hz, 1H), 8.25 (s, 2H), 8.26-8.06 (m, 3H), 7.85 (d, J = 4.0Hz, 2H), 7.22 (d, J = 8.5Hz, 2H), 7. 16(d,J=9.0Hz,1H),4.82(s,2H),4.81-4.74(m,1H),4.70(t,J=6.5Hz,2H),4.42(t,J=6.5Hz,2H),3.30(s,2H),3.07(s,3H).LC-MS:[M+H] + :523.3.

[0222] Example 20: Synthesis of 2-(2-((4-((5,8-dichloroquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 20)

[0223]

[0224] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 2,5,8-trichloroquinoline, and other operating steps were similar, resulting in compound 20 as a white solid with a yield of 46%. 1H NMR(500MHz,DMSO-d6)δ9.92(s,1H),8.83(d,J=7.0Hz,1H),8.30(d,J=9.0Hz,1H) ,8.25(s,2H),8.11(d,J=8.0Hz,2H),7.77(d,J=8.0Hz,1H),7.41(d,J=8.0Hz,1H) ,7.27(d,J=9.0Hz,1H),7.23(d,J=8.0Hz,2H),4.83(s,2H),4.81-4.784(m,1H),4 .71(t,J=7.0Hz,2H),4.42(t,J=7.0Hz,2H),3.31(s,2H),3.07(s,3H).LC-MS[M+H] + 523.2.

[0225] Example 21: Synthesis of 2-(2-((4-((4,8-dichloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 21)

[0226]

[0227] Following the preparation process of compound 13 in Example 13, 2,8-dichloro-7-methylquinoline was replaced with 2,4,8-trichloro-7-methylquinoline, and other operating steps were similar, resulting in compound 21 as a white solid with a yield of 44%. 1 H NMR (500MHz, DMSO) δ9.80 (s, 1H), 8.83 (d, J = 6.5Hz, 1H), 8.25 (s, 2H), 8.26-8.06 (m, 3H), 7.85 (d, J = 4.0Hz, 2H), 7.22 (d, J = 8.5Hz, 2H), 7. 16(d,J=9.0Hz,1H),4.82(s,2H),4.81-4.74(m,1H),4.70(t,J=6.5Hz,2H),4.42(t,J=6.5Hz,2H),3.30(s,2H),3.07(s,3H).LC-MS:[M+H] + :523.3.

[0228] Example 22: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)(methyl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 22)

[0229]

[0230] Following the preparation process of compound 13 in Example 13, tert-butyl methyl (4-aminobenzyl)(methyl)carbamate was replaced with tert-butyl methyl (4-(methylamino)benzyl)carbamate, and other operating steps were similar, resulting in compound 22 as a white solid with a yield of 39%. 1 H NMR (500MHz, DMSO-d6) δ8.75 (d, J = 6.5 Hz, 1H), 8.25-8.15 (s, 2H), 7.82 (d, J = 9. 0Hz,1H),7.50(d,J=8.0Hz,1H),7.29-7.25(m,3H),7.14(d,J=8.0Hz,1H),6.62( d,J=9.0Hz,1H),4.83(s,2H),4.74-4.67(m,1H),4.63(t,J=7.0Hz,2H),4.35(t ,J=6.5Hz,2H),3.49(s,3H),3.24(s,2H),3.06(s,3H),2.45(s,3H).LC-MS[M+H] + m / z:517.3.

[0231] Example 23: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)-3-methylbenzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 23)

[0232]

[0233] Following the preparation procedure of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was substituted with (4-aminobenzyl)(methyl)carbamate tert-butyl ester. Other operational steps were similar, yielding compound 23 as a white solid with a yield of 20%. LC-MS [M+H] + m / z:517.2

[0234] Example 24: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)-2-methylbenzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 24)

[0235]

[0236] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 23 as a white solid with a yield of 23%. 1H NMR(500MHz,DMSO-d6)δ9.51(s,1H),8.75(d,J=6.5Hz,1H),8.21(d,J=1.5Hz,1H),8.18(s,2H) ,7.97(d,J=9.0Hz,1H),7.71(dd,J=8.5,2.0Hz,1H),7.54(d,J=8.0Hz,1H),7.18(d,J=8.0Hz,1H ),6.98(d,J=9.0Hz,1H),6.88(d,J=8.5Hz,1H),4.74(s,2H),4.73-4.67(m,1H),4.63(t,J=7.0 Hz,2H),4.35(t,J=6.5Hz,2H),3.23(s,2H),2.99(s,3H),2.46(s,3H),2.22(s,3H).LC-MS[M+H] + m / z:517.3.

[0237] Example 25: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)-3-fluorobenzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 25)

[0238]

[0239] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 25 as a white solid with a yield of 40%. 1 H NMR(500MHz,DMSO-d6)δ9.20(s,1H),9.09(t,J=8.0Hz,1H),8.75(d,J=6.0Hz,1H),8 .20(s,2H),8.02(d,J=9.0Hz,1H),7.56(d,J=8.0Hz,1H),7.29(d,J=9.0Hz,1H),7.2 2(d,J=8.0Hz,1H),7.05-7.03(m,2H),4.77(s,2H),4.74-4.68(m,1H),4.63(t,J=6. 5Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,2H),3.03(s,3H),2.46(s,3H).LC-MS[M+H] + m / z:521.3.

[0240] Example 26: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)-2-fluorobenzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 26)

[0241]

[0242] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 26 as a white solid with a yield of 21%. 1 H NMR(500MHz,DMSO-d6)δ9.86(s,1H),8.78(d,J=6.5Hz,1H),8.63(dd,J=14.0,2.0Hz,1H),8.1 9(s,2H),8.03(d,J=9.0Hz,1H),7.57(d,J=8.0Hz,1H),7.37(dd,J=8.5,2.0Hz,1H),7.23(d,J =8.0Hz,1H),7.09(t,J=8.5Hz,1H),7.02(d,J=9.0Hz,1H),4.79(s,2H),4.75-4.67(m,1H),4. 63(t,J=7.0Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,2H),3.03(s,3H),2.48(s,3H).LC-MS[M+H] + m / z:521.3.

[0243] Example 27: Synthesis of 2-(2-((3-chloro-4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 27)

[0244]

[0245] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 27 as a white solid with a yield of 19%. 1H NMR(500MHz,DMSO-d6)δ8.85(d,J=8.5Hz,1H),8.78-8.74(m,2H),8.20(s,2H),8.04(d ,J=9.0Hz,1H),7.57(d,J=8.0Hz,1H),7.30(d,J=9.0Hz,1H),7.27(d,J=1.5Hz,1H),7. 22(d,J=8.0Hz,1H),7.18(d,J=8.5Hz,1H),4.77(s,2H),4.74-4.67(m,1H),4.63(t,J= 7.0Hz,2H),4.35(t,J=6.5Hz,2H),3.24(s,2H),3.04(s,3H),2.45(s,3H).LC-MS[M+H] + m / z:537.2.

[0246] Example 28: Synthesis of 2-(2-((2-chloro-4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 28)

[0247]

[0248] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 28 as a white solid with a yield of 17%. 1 H NMR (500MHz, DMSO-d6) δ9.78(s,1H),8.93(d,J=2.0Hz,1H),8.75(d,J=6.5Hz,1H),8.18(s, 2H),8.04(d,J=9.0Hz,1H),7.58(d,J=8.0Hz,1H),7.52(dd,J=8.5,2.0Hz,1H),7.23(d,J=8. 0Hz,1H),7.00(d,J=9.0Hz,1H),6.98(d,J=8.5Hz,1H),4.82(s,2H),4.73-4.66(m,1H),4.63 (t,J=7.0Hz,2H),4.35(t,J=6.5Hz,2H),3.23(s,2H),3.07(s,3H),2.48(s,3H).LC-MS[M+H] + m / z:537.3.

[0249] Example 29: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)-3-(trifluoromethyl)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 29)

[0250]

[0251] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate tert-butyl ester was replaced with (4-aminobenzyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 29 as a white solid with a yield of 50%. 1 H NMR(500MHz,DMSO-d6)δ8.75(d,J=6.5Hz,1H),8.59(s,1H),8.20(s,2H),8.01(d,J= 9.0Hz,1H),7.55(d,J=8.0Hz,1H),7.52(d,J=2.0Hz,1H),7.46(d,J=8.5Hz,1H),7.19 (d,J=8.0Hz,1H),7.09(d,J=9.0Hz,1H),4.85(s,2H),4.75–4.67(m,1H),4.63(t,J=7 .0Hz,2H),4.35(t,J=6.5Hz,2H),3.25(s,3H),3.06(s,3H),2.42(s,2H).LC-MS[M+H] + m / z:571.3.

[0252] Example 30: Synthesis of 2-(2-(((6-((8-chloro-7-methylquinoline-2-yl)amino)pyridin-3-yl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 30)

[0253]

[0254] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate was replaced with ((6-aminopyridin-3-yl)methyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 30 as a white solid with a yield of 30%. 1H NMR (500MHz, CDCl3) δ8.27 (s, 2H), 8.25–8.19 (m, 2H), 7.95 (d, J = 9.0Hz, 1H), 7. 67(dd,J=8.5,2.0Hz,1H),7.60(s,1H),7.57(d,J=8.0Hz,1H),7.19(d,J=8.0Hz, 1H),6.26(d,J=5.5Hz,1H),5.07–5.00(m,1H),4.91(t,J=6.5Hz,2H),4.85(s,2H ),4.48(t,J=6.5Hz,2H),3.38(s,2H),3.16(s,3H),2.52(s,3H).LC-MS[M+H-Cl] + m / z:470.0.

[0255] Example 31: Synthesis of 2-(2-(((5-((8-chloro-7-methylquinoline-2-yl)amino)pyridin-2-yl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 31)

[0256]

[0257] Following the preparation process of compound 13 in Example 13, (4-aminobenzyl)(methyl)carbamate was replaced with ((5-aminopyridin-2-yl)methyl)(methyl)carbamate tert-butyl ester, and other operating steps were similar, to obtain compound 31 as a white solid with a yield of 8%. 1 H NMR (500MHz, DMSO) δ9.80(s,1H),9.17(d,J=2.0Hz,1H),8.77(d,J=6.5Hz,1H),8.57(dd,J= 8.5,2.5Hz,1H),8.18(s,2H),8.04(d,J=9.0Hz,1H),7.57(d,J=8.0Hz,1H),7.22(d,J=8.0H z,1H),7.10(d,J=8.5Hz,1H),7.03(d,J=9.0Hz,1H),4.83(s,2H),4.76–4.67(m,1H),4.63( t,J=6.5Hz,2H),4.36(t,J=6.5Hz,2H),3.24(s,2H),3.10(s,3H),2.47(s,3H).LC-MS[M+H] + m / z:504.3.

[0258] Example 32: Synthesis of 2-(6-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyridin-3-yl)-N-(oxetane-3-yl)acetamide (compound 32)

[0259]

[0260] Preparation route of compound 32:

[0261]

[0262] Step 1: Synthesis of Compound 32-3

[0263] Compound 32-1 (1.00 g, 4.65 mmol, 1.0 equivalent), compound 32-2 (865 mg, 4.65 mmol, 1.0 equivalent), and potassium carbonate (3.21 g, 23.25 mmol, 5.0 equivalent) were added to a 100 mL double-necked flask equipped with a stirrer. Under a nitrogen atmosphere, the mixture was purged three times, followed by the addition of acetonitrile (20 mL). The reaction mixture was stirred overnight at 90 °C. TLC monitoring was performed. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated ammonium chloride solution (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (20 mL), and 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 obtain intermediate compound 32-3 (460 mg, 1.43 mmol, yield 31%). LC-MS [M+H] + m / z:322.1.

[0264] Step 2: Synthesis of compound 32-4

[0265] Compound 32-3 (460 mg, 1.43 mmol, 1.0 equivalent), Xphos (138 mg, 0.29 mmol, 0.2 equivalent), and Pd2(dba)3 (128 mg, 0.14 mmol, 0.1 equivalent) were added to a 50 mL double-necked flask equipped with a stirrer. The mixture was purged three times under a nitrogen atmosphere. Then, a solution of compound A2 tetrahydrofuran (20 mL, 3.58 mmol, 0.175 M, 2.5 equivalent) was added. The reaction mixture was stirred overnight at 60 °C. The reaction was monitored by TLC. After completion, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated ammonium chloride solution (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (20 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 obtain intermediate 32-4 (500 mg, 1.40 mmol, 98% yield). LC-MS[M+H] +m / z: 358.2. Step 3: Synthesis of compound 32-5

[0266] Intermediate 32-4 (500 mg, 1.40 mmol, 1.0 equivalent) was dissolved in a mixed solution of methanol (5.0 mL) and dichloromethane (5.0 mL), and palladium on carbon (30 mg, 0.28 mmol, 0.2 equivalent, 20%) was added. The mixture was reacted overnight at room temperature and monitored by TLC. After the reaction of the starting material was complete, palladium on carbon was filtered off with diatomaceous earth, and the mixture was washed with dichloromethane (10 mL). The filtrate was collected, and the solvent was removed under reduced pressure. The resulting oily compound was directly used for the next step.

[0267] The above-mentioned oily compound, compound 12-1 (266 mg, 1.26 mmol, 0.9 equivalents), BINAP (174 mg, 0.28 mmol, 0.2 equivalents), Pd2(dba)3 (128 mg, 0.14 mmol, 0.1 equivalents), and sodium tert-butoxide (269 mg, 2.80 mmol, 2.0 equivalents) were added to a 50 mL double-necked flask. Under a nitrogen atmosphere, the atmosphere was purged three times. Tetrahydrofuran (15 mL) was added, and the reaction mixture was stirred overnight at room temperature. After the reaction was complete, a saturated ammonium chloride aqueous solution (15 mL) was added to quench the reaction, yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give intermediate compound 32-5 (422 mg, 0.84 mmol, 60% yield). LC-MS [M+H] + m / z:503.3.

[0268] Step 4: Synthesis of Compound 32

[0269] Intermediate compound 32-5 (60 mg, 0.12 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (2.0 mL) and methanol (2.0 mL), followed by the addition of 2 M sodium hydroxide aqueous solution (0.30 mL, 0.60 mmol, 5.0 equivalent). The reaction was carried out at room temperature for 2 hours, monitored by TLC. After the starting material had reacted completely, the mixture was neutralized with 1 M HCl aqueous solution to obtain a mixed solution. The mixed solution was extracted three times with dichloromethane (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting oily compound was directly used in the next reaction.

[0270] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (4.0 mL), and N,N-diisopropylethylamine (62 mg, 0.48 mmol, 4.0 equivalent), HATU (91 mg, 0.24 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (18 mg, 0.24 mmol, 2.0 equivalent) were added sequentially. The resulting reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, a saturated ammonium chloride solution (5.0 mL) was added to quench the reaction, yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, washed with a saturated sodium chloride aqueous solution (10 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 32 (20 mg, 0.04 mmol, yield 33%), a white solid. 1 H NMR (500MHz, CDCl3) δ8.05 (s, 1H), 7.86 (d, J = 9.0Hz, 1H), 7.78 (d, J = 8.5Hz, 2H), 7.44 (d, J = 8. 0Hz,1H),7.37(dd,J=9.0,2.0Hz,1H),7.25(d,J=8.5Hz,2H),7.17(d,J=8.0Hz,1H),6.88(d,J =9.0Hz,1H),6.55(d,J=9.0Hz,1H),6.09(d,J=7.0Hz,1H),5.06–4.96(m,1H),4.87(t,J=6.5H z,2H),4.78(s,2H),4.43(t,J=6.5Hz,2H),3.43(s,2H),3.10(s,3H),2.59(s,3H).LC-MS[M+H] + m / z:502.3.

[0271] Example 33: Synthesis of R)-2(-(2-((1-(1-((8-chloro-1,6-naphthid-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 33)

[0272]

[0273] Following the preparation process of compound 13 in Example 13, N-methyloxetane-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 33 as a white solid with a yield of 37%. 1H NMR (500MHz, DMSO-d6) δ9.63 (s, 1H), 8.19-8.16 (m, 2H), 8.12 (d, J = 8.5Hz, 2H), 8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.21(d,J= 8.5Hz,2H),7.05(d,J=8.5Hz,1H),5.29-5.25(m,1H),4.82(s,2H),4.74-4.57( m,4H),3.58-3.53(m,2H),3.31(s,3H),3.06(s,3H),2.52(s,3H).LC-MS:[M+H] + =517.3.

[0274] Example 34: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2-cyanoethyl)-N-(oxetane-3-yl)acetamide (compound 34)

[0275]

[0276] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 3-(oxetane-3-ylamino)propionitrile, and other operating steps were similar, resulting in compound 34 as a white solid with a yield of 49%. 1 H NMR(500MHz,DMSO-d6)δ9.57(s,1H),8.13(m,2H),8.06(d,J=8.5Hz,2H),7.98(d,J=9.0 Hz,1H),7.54(d,J=8.0Hz,1H),7.19(d,J=8.0Hz,1H),7.15(d,J=7.5Hz,2H),6.99(d,J=9 .0Hz,1H),5.35-5.13(m,1H),4.75(s,2H),4.70-4.62(m,2H),4.54(d,J=7.0Hz,2H),3. 76-3.68(m,2H),3.24(s,2H),3.00(s,3H),2.79-2.71(m,2H),2.46(s,3H).LC-MS:[M+H] + =556.3.

[0277] Example 35: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(1,1-dioxothiophen-3-yl)acetamide (compound 35)

[0278]

[0279] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 3-aminothiohexacyclobutane-1,1-dioxide, and other operating steps were similar, resulting in compound 35 as a white solid with a yield of 45%. 1 H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.85(d,J=5.0Hz,1H),8.26(s,2H),8.13(d,J =8.5Hz,2H),8.05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7. 22(d,J=8.5Hz,2H),7.06(d,J=9.0Hz,1H),4.82(s,2H),4.57-4.46(m,2H),4.40-4 .31(m,1H),4.11-4.03(m,2H),3.31(s,2H),3.07(s,3H),2.53(s,3H).LC-MS:[M+H] + =551.3.

[0280] Example 36: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(3,3-difluorocyclobutyl)acetamide (compound 36)

[0281]

[0282] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 3,3-difluorocyclobutane-1-amine, and other operating steps were similar, resulting in compound 36 as a white solid with a yield of 43%. 1 H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.52(d,J=6.5Hz,1H),8.25(s,2H),8.14(d,J =8.5Hz,2H),8.05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7. 22(d,J=8.5Hz,2H),7.06(d,J=9.0Hz,1H),4.82(s,2H),4.10-3.99(m,1H),3.28(s ,2H),3.07(s,3H),2.91-2.88(m,2H),2.61-2.54(m,2H),2.53(s,3H).LC-MS:[M+H] + =537.3.

[0283] Example 37: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(thiophen-3-yl)acetamide (compound 37)

[0284]

[0285] Following the preparation process of compound 13 in Example 13, thiocyclobutane-3-amine was used instead of oxocyclobutane-3-amine, and other operating steps were similar, resulting in compound 37 as a white solid with a yield of 56%. 1 H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.72(d,J=7.5Hz,1H),8.24(s,2H),8.13(d,J =8.5Hz,2H),8.05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7. 22(d,J=8.5Hz,2H),7.06(d,J=9.0Hz,1H),5.03-4.91(m,1H),4.82(s,2H),3.38-3 .35(m,2H),3.25(s,2H),3.23-3.17(m,2H),3.07(s,3H),2.53(s,3H).LC-MS:[M+H] + =519.3.

[0286] Example 38: Synthesis of 2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-1-(3-hydroxyazacyclobutane-1-yl)acet-1-one (compound 38)

[0287]

[0288] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with aziridine-3-ol, and other operating steps were similar, resulting in compound 38 as a white solid with a yield of 53%. 1H NMR(500MHz,DMSO-d6)δ9.63(s,1H),8.23(s,2H),8.13(d,J=8.3Hz,2H),8.05(d,J=8.9Hz,1H),7.6 1(d,J=8.0Hz,1H),7.25(d,J=8.1Hz,1H),7.22(d,J=8.3Hz,2H),7.06(d,J=8.9Hz,1H),4.82(s,2H), 4.46(ddd,J=11.1,6.8,4.3Hz,1H),4.39(t,J=7.8Hz,1H),4.04(dd,J=10.1,6.8Hz,1H),3.94(dd,J =8.9,4.3Hz,1H),3.59(dd,J=10.1,4.4Hz,1H),3.28(s,2H),3.07(s,3H),2.53(s,3H).LC-MS:[M+H] + :503.2.

[0289] Example 39: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(tetrahydrofuran-3-yl)acetamide (compound 39)

[0290]

[0291] Following the preparation process of compound 13 in Example 13, tetrahydrofuran-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 39 as a white solid with a yield of 48%. 1 H NMR(500MHz,DMSO-d6)δ9.57(s,1H),8.26(d,J=6.5Hz,1H),8.19(s,2H),8.07(d,J=8.5 Hz,2H),7.97(d,J=9.0Hz,1H),7.53(d,J=8.0Hz,1H),7.17(d,J=8.0Hz,1H),7.15(d,J=8 .5Hz,2H),7.00(d,J=8.5Hz,1H),4.75(s,2H),4.16(m,1H),3.76–3.56(m,3H),3.39(m, 1H),3.21(s,2H),3.00(s,3H),2.46(s,3H),2.01(m,1H),1.72–1.62(m,1H).LC-MS[M+H] + 517.3.

[0292] Example 40: Synthesis of 2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-1-morpholinyl ethyl-1-one (compound 40)

[0293]

[0294] Following the preparation process of compound 13 in Example 13, morpholine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 40 as a white solid with a yield of 53%. 1 H NMR(500MHz,DMSO-d6)δ9.63(s,1H),8.19(d,J=12.0Hz,2H),8.13(d,J=8.5Hz,2H) ,8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.21(d,J=7.5 Hz,2H),7.05(d,J=9.0Hz,1H),4.82(s,2H),4.77-4.68(m,2H),4.63-4.60(m,2H),3 .78(m,2H),3.30(s,2H),3.06(s,3H),2.83-2.81(m,2H),2.52(s,3H).LC-MS:[M+H] + :517.3.

[0295] Example 41: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2,6-dioxopiperidin-3-yl)acetamide (compound 41)

[0296]

[0297] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 3-aminopiperidine-2,6-dione, and other steps were similar, resulting in compound 41 as a white solid with a yield of 55%. 1H NMR(500MHz,DMSO-d6)δ10.82(s,1H),9.63(s,1H),8.43(d,J=8.0Hz,1H),8.28 (s,2H),8.13(d,J=8.5Hz,2H),8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.2 6–7.20(m,3H),7.06(d,J=9.0Hz,1H),4.82(s,2H),3.36(s,2H),3.07(s,3H),2. 76–2.70(m,1H),2.52(s,3H),2.49–2.45(m,2H),1.98–1.90(m,2H).LC-MS[M+H] + :558.3.

[0298] Example 42: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2-oxopyrrolidone-3-yl)acetamide (compound 42)

[0299]

[0300] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 3-aminopyrrolidone-2-one, and other steps were similar, resulting in compound 42 as a white solid with a yield of 55%. 1 H NMR(500MHz,DMSO-d6)δ9.57(s,1H),8.28(d,J=8.0Hz,1H),8.20(s,2H),8.07(d,J=8.5Hz, 2H),7.98(d,J=9.0Hz,1H),7.77(s,1H),7.54(d,J=8.0Hz,1H),7.18(d,J=8.0Hz,1H),7.15 (d,J=8.5Hz,2H),6.99(d,J=9.0Hz,1H),4.75(s,2H),4.31-4.17(m,1H),3.25(s,2H),3.12 -3.09(m,2H),3.00(s,3H),2.46(s,3H),2.28-2.20(m,1H),1.80-1.62(m,1H).LC-MS:[M+H] + :530.3.

[0301] Example 43: Synthesis of (R)-2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-1-(3-(dimethylamino)pyrrolidine-1-yl)acet-1-one (compound 43)

[0302]

[0303] Following the preparation process of compound 13 in Example 13, (R)-N,N-dimethylpyrrolidine-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 43 as a white solid with a yield of 47%. 1 H NMR(500MHz,DMSO-d6)δ9.67(s,1H),8.22(d,J=3.5Hz,2H),8.14(d,J=8.5Hz,2H),8 .05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7.22(d,J=8.5Hz, 2H),7.07(d,J=9.0Hz,1H),4.75(s,2H),3.83-3.69(m,1H),3.54-3.46(m,4H),3.31( s,2H),3.07(s,3H),2.54(s,3H),2.21-2.17(m,6H),2.04-1.97(m,2H).LC-MS:[M+H] + =544.4.

[0304] Example 44: Synthesis of (S)-2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-1-(3-(dimethylamino)pyrrolidone-1-yl)acet-1-one (compound 44)

[0305]

[0306] Following the preparation process of compound 13 in Example 13, (S)-N,N-dimethylpyrrolidine-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 44 as a white solid with a yield of 50%. 1H NMR(500MHz,DMSO-d6)δ9.67(s,1H),8.22(d,J=3.5Hz,2H),8.14(d,J=8.5Hz,2H),8 .05(d,J=9.0Hz,1H),7.61(d,J=8.0Hz,1H),7.26(d,J=8.0Hz,1H),7.22(d,J=8.5Hz, 2H),7.07(d,J=9.0Hz,1H),4.75(s,2H),3.83-3.69(m,1H),3.54-3.46(m,4H),3.31( s,2H),3.07(s,3H),2.54(s,3H),2.21-2.17(m,6H),2.04-1.97(m,2H).LC-MS:[M+H] + =544.4.

[0307] Example 45: Synthesis of 2-(2-((4-((8-chloro-7-methylquinolin-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-1-(4-methylpiperazin-1-yl)acet-1-one (compound 45)

[0308]

[0309] Following the preparation process of compound 13 in Example 13, 1-methylpiperazine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 45 as a white solid with a yield of 45%. 1 H NMR(500MHz,DMSO-d6)δ9.70(s,1H),8.27(s,2H),8.19(d,J=8.5Hz,2H),8.11(d,J=9 .0Hz,1H),7.67(d,J=8.0Hz,1H),7.31(d,J=8.0Hz,1H),7.28(d,J=8.5Hz,2H),7.12(d ,J=9.0Hz,1H),4.88(s,2H),3.62(s,2H),3.61-3.58(m,2H),3.55-3.49(m,2H),3.13 (s,3H),2.59(s,3H),2.39-2.35(m,2H),2.34-2.29(m,2H),2.24(s,3H).LC-MS:[M+H] + =530.3.

[0310] Example 46: Synthesis of N-(azacyclobutane-3-yl)-2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)acetamide (compound 46)

[0311]

[0312] Referring to the preparation process of compound 13 in Example 13, tert-butyl aziridine-3-carbamate was used instead of oxaziridine-3-amine. Other operation steps were similar. Then, the Boc protecting group was removed to obtain compound 46 as a white solid with a yield of 88%. 1 HNMR(500MHz,DMSO-d6)δ9.66(d,J=5.5Hz,1H),8.89(d,J=7.0Hz,1H),8.20(s,2H),8 .08(d,J=8.5Hz,2H),7.98(d,J=9.0Hz,1H),7.54(d,J=8.0Hz,1H),7.19(d,J=8.0Hz, 1H),7.15(d,J=8.5Hz,2H),7.06–7.00(m,1H),4.75(s,2H),4.55–4.47(m,1H),4.00– 3.96(m,2H),3.86–3.82(m,2H),3.27(s,2H),3.01(s,3H),2.46(s,3H).LC-MS:[M+H] + :502.3.

[0313] Example 47: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2,2-dimethyloxetane-3-yl)acetamide (compound 47)

[0314]

[0315] Following the preparation process of compound 13 in Example 13, 2,2-dimethyloxetane-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 47 as a white solid with a yield of 44%. 1 H NMR(500MHz,DMSO-d6)δ9.65(s,1H),8.64(d,J=7.0Hz,1H),8.26(s,2H),8.16–8.12( m,2H),8.04(d,J=9.0Hz,1H),7.59(d,J=8.0Hz,1H),7.24(d,J=8.0Hz,1H),7.21(d,J =8.5Hz,2H),7.06(d,J=9.0Hz,1H),4.81(s,2H),4.50–4.42(m,2H),4.24(t,J=6.0Hz ,1H),3.37(s,2H),3.06(s,3H),2.52(s,3H),1.33(s,3H),1.16(s,3H).LC-MS:[M+H] +=531.3.

[0316] Example 48: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2-oxotetrahydrofuran-3-yl)acetamide (compound 48)

[0317]

[0318] Following the preparation process of compound 13 in Example 13, (R)-3-aminodihydrofuran-2(3H)-one was used to replace oxetane-3-amine, and other operating steps were similar, to obtain compound 48 as a white solid with a yield of 51%. 1 H NMR(500MHz,DMSO-d6)δ9.64(s,1H),8.58(d,J=8.0Hz,1H),8.26(s,2H),8.13(d,J=8.5Hz, 2H),8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.22(d,J=8.5Hz, 2H),7.06(d,J=9.0Hz,1H),4.82(s,2H),4.61-4.55(m,1H),4.37–4.30(m,1H),4.21(m,1H) ,3.32(s,2H),3.07(s,3H),2.52(s,3H),2.44–2.35(m,1H),2.21–2.11(m,1H).LC-MS:[M+H] + =531.3.

[0319] Example 49: Synthesis of (S)-2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(5-oxotetrahydrofuran-3-yl)acetamide (compound 49)

[0320]

[0321] Following the preparation process of compound 13 in Example 13, (S)-4-aminodihydrofuran-2(3H)-one was used to replace oxetane-3-amine, and other operating steps were similar, to obtain compound 49 as a white solid with a yield of 47%. 1H NMR (500MHz, DMSO-d6) δ9.68(s,1H),8.68(d,J=5.5Hz,1H),8.24(d,J=9.0Hz,2H),8.14(d ,J=8.5Hz,2H),8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.23–7 .19(m,2H),7.07(d,J=9.0Hz,1H),4.81(s,2H),4.46–4.41(m,2H),4.08(d,J=6.5Hz,1H), 3.29(s,2H),3.06(s,3H),2.90-2.85(m,1H),2.52(s,3H),2.40-2.34(m,1H).LC-MS:[M+H] + =531.3.

[0322] Example 50: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(2-methoxyethyl)acetamide (Compound 50)

[0323]

[0324] Following the preparation process of compound 13 in Example 13, 2-methoxyethane-1-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 50 as a white solid with a yield of 36%. 1 H NMR (500MHz, DMSO-d6) δ9.57(s,1H),8.18(s,2H),8.06(m,3H),7.98(d,J=9.0Hz,1H),7.54(d,J=8.0Hz,1H),7.18(d,J=8.0Hz,1H),7.15(d,J=8.5H z,2H),6.99(d,J=9.0Hz,1H),4.75(s,2H),3.30-3.27(m,2H),3.21(s,2H) ),3.18(s,3H),3.17-3.13(m,2H),3.00(s,3H),2.46(s,3H).LC-MS:[M+H] + =505.3.

[0325] Example 51: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(1-hydroxypropyl-2-yl)acetamide (compound 51)

[0326]

[0327] Following the preparation process of compound 13 in Example 13, 2-amino-1-propanol was used to replace oxetane-3-amine, and other steps were similar, resulting in compound 51 as a white solid with a yield of 33%. 1 H NMR (500MHz, CDCl3) δ8.19(s,2H),7.78(d,J=9.0Hz,1H),7.64(d,J=8.5Hz,2H),7.37(d,J =8.0Hz,1H),7.20(d,J=8.5Hz,2H),7.09(d,J=8.0Hz,1H),6.82(d,J=8.5Hz,1H),5.70(d, J=7.0Hz,1H),4.81(s,2H),4.08-3.96(m,1H),3.59(dd,J=11.0,4.0Hz,1H),3.45(dd,J=1 1.0,4.0Hz,1H),3.29(s,2H),3.07(s,3H),2.52(s,3H),1.09(d,J=6.5Hz,3H).LCMS[M+H] + m / z:505.3.

[0328] Example 52: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(6-oxo-1,6-dihydropyridin-3-yl)acetamide (compound 52)

[0329]

[0330] Following the preparation process of compound 13 in Example 13, oxetane-3-amine was replaced with 2-amino-1-propanol, and other steps were similar, resulting in compound 52 as a white solid with a yield of 62%. 1 H NMR(500MHz,DMSO-d6)δ9.75(s,1H),8.19(s,2H),8.12–8.07(m,2H),7.98(d,J=9.0Hz,1H),7.54(d,J=8.0Hz,1H),7.49(s,1H),7.39(s,1H), 7.20-7.17(m,3H),7.15(d,J=8.0Hz,2H),7.06(d,J=8.5Hz,1H),6.87(s,1H),4.76(s,2H),3.19(s,2H),3.00(s,3H),2.46(s,3H).LC-MS[M+H] + :540.3.

[0331] Example 53: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-3-hydroxy-N-(oxetane-3-yl)propionamide (compound 53)

[0332]

[0333] Preparation route of compound 53:

[0334]

[0335] Compound 13 (20 mg, 0.04 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (2.0 mL), and potassium carbonate (17 mg, 0.12 mmol, 3.0 equivalent) and paraformaldehyde (2 mg, 0.08 mmol, 2.0 equivalent) were added. The reaction was carried out at 30 °C for 4 hours, and the reaction was monitored by TLC. After the reaction was completed, saturated ammonium chloride aqueous solution (5.0 mL) was added to quench the reaction, and a mixed solution was obtained. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 obtain compound 53 (6 mg, 0.01 mmol, yield 28%), a white solid. 1 HNMR(500MHz, CDCl3)δ8.26(s,2H),7.80(d,J=9.0Hz,1H),7.68(d,J=8.0Hz,2H) ,7.40(d,J=8.0Hz,1H),7.24-7.21(m,3H),7.13(d,J=8.0Hz,1H),6.86(d,J=9.0H z,1H),5.00–4.93(m,1H),4.89–4.81(m,4H),4.46(t,J=6.0Hz,1H),4.41(t,J=6 .5Hz,1H),4.02–3.95(m,1H),3.45(s,2H),3.11(s,3H),2.56(s,3H).LC-MS[M+H] + m / z:533.3.

[0336] Example 54: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-3-hydroxy-2-(hydroxymethyl)-N-(oxetane-3-yl)propionamide (compound 54)

[0337]

[0338] Preparation route of compound 54:

[0339]

[0340] Compound 13 (20 mg, 0.04 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (2.0 mL), and potassium carbonate (17 mg, 0.12 mmol, 3.0 equivalent) and paraformaldehyde (2 mg, 0.08 mmol, 2.0 equivalent) were added. The mixture was reacted at 30 °C for 4 hours, and the reaction was monitored by TLC. After the reaction was completed, saturated ammonium chloride aqueous solution (5.0 mL) was added to quench the reaction, resulting in a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phases were combined. The organic phase was washed with saturated sodium chloride aqueous solution (10 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 54 (2 mg, 0.004 mmol, yield 9%) as a white solid. LC-MS [M+H] + m / z:563.3.

[0341] Example 55: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-2,2-difluoro-N-(oxetane-3-yl)acetamide (compound 55)

[0342]

[0343] Preparation route of compound 55:

[0344]

[0345] Step 1: Synthesis of Compound 55-2

[0346] Compound 31-1 (99 mg, 0.24 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was evaporated and the resulting oily compound was directly used for the next step.

[0347] The above-mentioned oily compound and compound A3 (57 mg, 0.24 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (2.0 mL), and N,N-diisopropylethylamine (155 mg, 1.20 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution (10 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 intermediate compound 55-2 (50 mg, 0.10 mmol, yield 41%). LC-MS [M+H] + m / z:513.1.

[0348] Step 2: Synthesis of Compound 55

[0349] Intermediate compound 55-2 (50 mg, 0.10 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed by rotary evaporation under reduced pressure. The resulting oily compound was directly used for the next step.

[0350] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (2.0 mL), and N,N-diisopropylethylamine (50 mg, 0.40 mmol, 4.0 equivalent), HATU (76 mg, 0.20 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (15 mg, 0.20 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL) to obtain a mixed solution. The above mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 obtain compound 55 (25 mg, 0.05 mmol, yield 46%), a white solid compound. 1H NMR(500MHz,DMSO-d6)δ9.87(s,1H),9.71(d,J=6.5Hz,1H),9.19(d,J=2.5Hz,1H),8.67(dd ,J=8.5,2.5Hz,1H),8.53(d,J=21.0Hz,2H),8.11(d,J=9.0Hz,1H),7.64(d,J=8.0Hz,1H),7. 29(d,J=8.0Hz,1H),7.21(d,J=8.5Hz,1H),7.09(d,J=9.0Hz,1H),4.95(s,2H),4.85(p,J=7. 0Hz,1H),4.69(t,J=7.0Hz,2H),4.55(t,J=6.5Hz,2H),3.21(s,3H),2.53(s,3H).LCMS[M+H] + m / z:540.3.

[0351] Example 56: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-2-methyl-N-(oxetane-3-yl)propionamide (compound 56)

[0352]

[0353] Preparation route of compound 56:

[0354]

[0355] Step 1: Synthesis of Compound 56-1

[0356] Compound C13-1 (99 mg, 0.24 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.24 mL) was added. The reaction was carried out at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was evaporated and the resulting oily compound was directly used for the next step.

[0357] The above-mentioned oily compound and compound A4 (58 mg, 0.24 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (2.0 mL), and N,N-diisopropylethylamine (156 mg, 1.20 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution (10 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 intermediate compound 56-1 (58 mg, 0.11 mmol, yield 45%). LC-MS [M+H] + m / z:532.1.

[0358] Step 2: Synthesis of Compound 56

[0359] Intermediate compound 56-1 (58 mg, 0.11 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.6 mL) was added. The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed by rotary evaporation under reduced pressure. The resulting oily compound was directly used for the next step.

[0360] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (2.0 mL), and N,N-diisopropylethylamine (57 mg, 0.44 mmol, 4.0 equivalent), HATU (84 mg, 0.22 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (16 mg, 0.22 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride aqueous solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (10 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 obtain compound 56 (28 mg, 0.05 mmol, yield 48%), a white solid. 1 H NMR (500MHz, DMSO-d6)δ

[0361] 9.58(s,1H),8.24(s,2H),8.07(dd,J=7.5,3.5Hz,3H),7.98(d,J=9.0Hz,1H),7.54(d,J=8.0Hz,1H),7.28–7.14(m,3H),6.99(d,J=9.0Hz, 1H),4.76(s,2H),4.74–4.65(m,1H),4.60(dd,J=7.5,6.0Hz,2H),4.36(t,J=6.5Hz,2H),3.02(s,3H),2.46(s,3H),1.39(s,6H).LCMS[M+H] + m / z:531.2.

[0362] Example 57: Synthesis of 2-(2-((4-((8-chloro-7-methylquinoline-2-yl)amino)benzyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acrylamide (compound 57)

[0363]

[0364] Preparation route of compound 57:

[0365]

[0366] Compound 13 (20 mg, 0.04 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (2.0 mL), and potassium carbonate (17 mg, 0.12 mmol, 3.0 equivalent) and paraformaldehyde (3 mg, 0.08 mmol, 2.0 equivalent) were added. The reaction was carried out at 30 °C for 4 hours, and the reaction was monitored by TLC. After the reaction was completed, saturated ammonium chloride aqueous solution (5.0 mL) was added to quench the reaction, and a mixed solution was obtained. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phases were combined. The organic phase was washed with saturated sodium chloride aqueous solution (10 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 obtain compound 57 (8 mg, 0.02 mmol, yield 39%), a white solid. 1HNMR (500MHz, DMSO-d6) δ9.59 (s, 1H), 8.86 (d, J = 6.5 Hz, 1H), 8.38 (s, 2H), 8.08 (d, J = 8. 5Hz,2H),7.98(d,J=9.0Hz,1H),7.54(d,J=8.0Hz,1H),7.19(d,J=5.0Hz,1H),7.17(d,J= 6.0Hz,2H),7.00(d,J=9.0Hz,1H),5.76(s,1H),5.65(s,1H),4.89–4.82(m,1H),4.80(s, 2H),4.67(t,J=6.5Hz,2H),4.48(t,J=6.5Hz,2H),3.05(s,3H),2.46(s,3H).LC-MS[M+H] + m / z:515.3.

[0367] Example 58: Synthesis of 8-chloro-7-methyl-N-(4-((methyl(5-((oxecyclobutane-3-ylamino)methyl)pyrimidin-2-yl)amino)methyl)phenyl)quinoline-2-amine (compound 58)

[0368]

[0369] Preparation route of compound 58:

[0370]

[0371] Step 1: Synthesis of Compound 58-1

[0372] Compound 13-1 (82 mg, 0.20 mmol, 1.0 equivalent) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.20 mL) was added. The reaction was carried out at room temperature for 3 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was evaporated and the resulting oily compound was directly used for the next step.

[0373] The above-mentioned oily compound and compound A5 (29 mg, 0.20 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (2.0 mL), and N,N-diisopropylethylamine (129 mg, 1.00 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution, 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 compound 58-1 (50 mg, 0.12 mmol, 60% yield). LC-MS [M+H]+ m / z:420.3.

[0374] Step 2: Synthesis of Compound 58

[0375] Under nitrogen protection, intermediate compound 58-1 (50 mg, 0.12 mmol, 1.0 equivalent) was dissolved in dichloromethane (4.0 mL), and manganese dioxide (156 mg, 1.79 mmol, 15 equivalent) was added. The reaction was carried out overnight at 40 °C, and the reaction was monitored by TLC. After the reaction was completed, the solid was collected by diatomaceous earth filtration, and the solvent was then removed by rotary evaporation. The resulting oily compound was directly used in the next step.

[0376] Under nitrogen protection, the above-mentioned oily compound was dissolved in tetrahydrofuran (3.0 mL), and sodium cyanoborohydride (15 mg, 0.24 mmol, 2.0 equivalent) was added sequentially, followed by acetic acid (7 mg, 0.12 mmol, 1.0 equivalent) and 3-oxacyclobutamine (18 mg, 0.24 mmol, 2.0 equivalent). The reaction was carried out at 40 °C for 10 hours, and the reaction was monitored by TLC. After the reaction was completed, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution (5.0 mL), yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phases were combined. The organic phase was washed with saturated sodium chloride aqueous solution (10 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 58 (22 mg, 0.05 mmol, yield 39%) as a white solid. 1 H NMR (500MHz, DMSO-d6) δ9.63 (s, 1H), 8.30 (s, 2H), 8.12 (d, J = 8.5Hz, 2H), 8.04 ( d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.20(d,J=8.5Hz, 2H),7.05(d,J=9.0Hz,1H),4.82(s,2H),4.57(t,J=6.5Hz,2H),4.29(t,J=6.0H z,2H),3.86(p,J=6.5Hz,1H),3.46(s,2H),3.07(s,3H),2.52(s,3H).LCMS[M+H] + m / z:475.2.

[0377] Example 59: Synthesis of 8-chloro-7-methyl-N-(4-((methyl(5-((4-(methanesulfonyl)piperazin-1-yl)methyl)pyrimidin-2-yl)amino)methyl)phenyl)quinoline-2-amine (compound 59)

[0378]

[0379] Following the preparation process of compound 58 in Example 58, oxetane-3-amine was replaced with 1-(methylsulfonyl)piperazine, and other operating steps were similar, resulting in compound 59 as a white solid with a yield of 47%. 1 H NMR (500MHz, DMSO-d6) δ9.63 (s, 1H), 8.29 (s, 2H), 8.13 (d, J = 8.5Hz, 2H), 8. 04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.22(d,J =8.5Hz,2H),7.06(d,J=9.0Hz,1H),4.83(s,2H),3.37(s,2H),3.13–3.08(m ,4H),3.08(s,3H),2.85(s,3H),2.52(s,3H),2.45-2.44(m,4H).LCMS[M+H] + m / z: 566.3

[0380] Example 60: Synthesis of (R)-8-chloro-N-(4-(((5-((3-(dimethylamino)pyrrolidone-1-yl)methyl)pyrimidin-2-yl)(methyl)amino)methyl)phenyl)-7-methylquinoline-2-amine (compound 60)

[0381]

[0382] Following the preparation process of compound 58 in Example 58, (R)-N,N-dimethylpyrrolidine-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, to obtain compound 60 as a white solid with a yield of 49%. 1 H NMR(500MHz,DMSO-d6)δ9.70(s,1H),8.29(s,2H),8.14(d,J=8.5Hz,2H),8.04(d,J= 9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.38-7.16(m,3H),7.08(d,J=9.0Hz,1H),4.82(s, 2H),3.40(s,2H),3.07(s,3H),3.05-2.98(m,1H),2.70-2.66(m,1H),2.55-2.49(m,5 H),2.46-2.41(m,1H),2.27(s,6H),1.98-1.88(m,1H),1.75-1.68(m,1H).LCMS[M+H] + m / z:516.3.

[0383] Example 61: Synthesis of (S)-8-chloro-7-methyl-N-(4-((methyl(5-(((tetrahydrofuran-3-yl)amino)methyl)pyrimidin-2-yl)amino)methyl)phenyl)quinoline-2-amine (compound 61)

[0384]

[0385] Following the preparation process of compound 58 in Example 58, tetrahydrofuran-3-amine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 61 as a white solid with a yield of 46%. 1 H NMR(500MHz,DMSO)δ9.64(s,1H),8.31(s,2H),8.13(d,J=8.5Hz,2H),8.04(d,J=9.0Hz,1H) ,7.60(d,J=8.0Hz,1H),7.24(d,J=8.0Hz,1H),7.21(d,J=8.5Hz,2H),7.06(d,J=9.0Hz,1H), 4.82(s,2H),3.77–3.73(m,1H),3.72–3.68(m,1H),3.65–3.61(m,1H),3.43-3.40(m,3H),3. 27–3.22(m,1H),3.07(s,3H),2.52(s,3H),1.97–1.88(m,1H),1.71–1.62(m,1H).LCMS[M+H] + m / z:489.3.

[0386] Example 62: Synthesis of 8-chloro-7-methyl-N-(4-((methyl(5-(morpholinomethyl)pyrimidin-2-yl)amino)methyl)phenyl)quinoline-2-amine (compound 62)

[0387]

[0388] Following the preparation process of compound 58 in Example 58, morpholine was used instead of oxetane-3-amine, and other operating steps were similar, resulting in compound 62 as a white solid with a yield of 44%. 1H NMR (500MHz, DMSO-d6) δ9.81(s,1H),8.28(s,2H),8.16(d,J=8.5Hz,2H),8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.25(d,J=8.0Hz,1H),7.22 (d,J=8.5Hz,2H),7.12(d,J=9.0Hz,1H),4.82(s,2H),3.55(t,J=4.5Hz,4 H),3.31(s,2H),3.07(s,3H),2.52(s,3H),2.37-2.30(m,4H).LCMS[M+H] + m / z:489.3.

[0389] Example 63: Synthesis of 8-chloro-7-methyl-N-(4-((methyl(5-(2-(oxecyclobutane-3-ylamino)ethyl)pyrimidin-2-yl)amino)methyl)phenyl)quinoline-2-amine (compound 63)

[0390]

[0391] Preparation route of compound 63:

[0392]

[0393] Step 1: Synthesis of Compound 63-1

[0394] Compound 13-2 (50 mg, 0.10 mmol, 1.0 equivalent) was added to a double-necked flask equipped with a stir bar. Under a nitrogen atmosphere, the atmosphere was purged three times, followed by the addition of tetrahydrofuran (2.0 mL). The reaction system was cooled to 0 °C in a cryogenic bath, and a 1 M lithium aluminum hydride tetrahydrofuran solution (0.30 mL, 0.30 mmol, 3.0 equivalent) was added dropwise. The reaction was carried out at 0 °C for 1 hour, and the reaction was monitored for completeness by TLC. The reaction was quenched by adding saturated ammonium chloride solution (10 mL), yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure to obtain 43 mg of crude intermediate compound 63-1 as a white solid. LC-MS [M+H] + m / z:434.2.

[0395] Step 2: Synthesis of Compound 63-2

[0396] Compound 63-1 (43 mg, 0.10 mmol, 1.0 eq) and methanesulfonic anhydride (26 mg, 0.15 mmol, 1.5 equivalence) were added to a double-necked flask equipped with a stir bar. Under a nitrogen atmosphere, the mixture was purged three times, and dichloromethane (3.0 mL) was added. The reaction system was cooled to 0 °C in a cryogenic bath, and triethylamine (20 mg, 0.20 mmol, 2.0 equivalence) was added dropwise. The reaction was carried out at 0 °C for 1 hour, and the reaction was monitored for completeness by TLC. The reaction was quenched by adding saturated ammonium chloride solution (10 mL), yielding a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), and the organic phases were combined, washed with saturated sodium chloride aqueous solution (5.0 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 obtain intermediate compound 63-2 (20 mg, 0.04 mmol, yield 39%). LC-MS [M+H] + m / z:512.3.

[0397] Step 3: Synthesis of Compound 63

[0398] Compound 63-2 (20 mg, 0.04 mmol, 1.0 equivalent) and 3-oxacyclobutane (6 mg, 0.08 mmol, 2.0 equivalent) were dissolved in dimethyl sulfoxide (2.0 mL), followed by the addition of N,N-diisopropylethylamine (26 mg, 0.20 mmol, 5.0 equivalent). The reaction 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed three times with saturated sodium chloride solution (5.0 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 obtain intermediate compound 63 (2 mg, 0.004 mmol, 10% yield). LCMS [M+H] + m / z:489.3.

[0399] Example 64: Synthesis of 2-(2-(((4-((8-methoxy-1,7-naphthidin-2-yl)amino)cyclohexyl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 64)

[0400]

[0401] Step 1: Synthesis of Compound 64-2

[0402] Compound 1-1 (194 mg, 1.00 mmol, 1.0 equivalent), compound 64-1 (228 mg, 1.00 mmol, 1.0 equivalent), BINAP (125 mg, 0.20 mmol, 0.2 equivalent), Pd2(dba)3 (92 mg, 0.10 mmol, 0.1 equivalent), and sodium tert-butoxide (192 mg, 2.00 mmol, 2.0 equivalent) were added to a 25 mL double-necked flask. Under a nitrogen atmosphere, the atmosphere was purged three times. Tetrahydrofuran (10 mL) was added, and the reaction mixture was heated and stirred overnight at 60 °C. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was quenched by adding saturated ammonium chloride aqueous solution (10 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting mixture was purified by silica gel column chromatography to give intermediate compound 64-2 (174 mg, 0.45 mmol, yield 45%). LC-MS [M+H] + m / z:387.3.

[0403] Step 2: Synthesis of Compound 64-3

[0404] Compound 64-2 (39 mg, 0.10 mmol, 1.0 equivalent) was added to a 25 mL double-necked flask. The flask was purged three times under a nitrogen atmosphere. Tetrahydrofuran (5.0 mL) was added, followed by 0.5 mL of 1 M lithium aluminum tetrahydrofuran solution. The reaction was carried out at 60 °C for 5 hours, monitored by TLC. After the reactants had reacted completely, water (2.0 mL), 15% sodium hydroxide solution (2.0 mL), and water (5.0 mL) were added dropwise to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The resulting oily compound was directly used in the next step.

[0405] The above-mentioned oily compound and compound A1 (21 mg, 0.10 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (2.0 mL), and N,N-diisopropylethylamine (65 mg, 0.50 mmol, 5.0 equivalent) was added. The reaction mixture was heated and 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 saturated ammonium chloride solution (5.0 mL) to obtain a mixed solution. The mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, and the mixture was dried over anhydrous sodium sulfate solution (10 mL). The solution was 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 compound 64-3 (28 mg, 0.06 mmol, yield 57%). LC-MS [M+H] + m / z:493.3.

[0406] Step 3: Synthesis of Compound 64

[0407] Intermediate compound 64-3 (10 mg, 0.02 mmol, 1.0 equivalent) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (0.3 mL) was added. The reaction was carried out at room temperature for 2 hours, and the reaction was monitored by TLC. After the reaction of the starting material was completed, the solvent was removed by rotary evaporation under reduced pressure. The resulting oily compound was directly used for the next step.

[0408] The above-mentioned oily compound was dissolved in N,N-dimethylformamide (1.0 mL), and N,N-diisopropylethylamine (10 mg, 0.08 mmol, 4.0 equivalent), HATU (15 mg, 0.04 mmol, 2.0 equivalent), and 3-oxacyclobutylamine (3 mg, 0.04 mmol, 2.0 equivalent) were added sequentially. The reaction solution was reacted at room temperature for 1 hour. After the reaction was completed, the reaction was quenched with saturated ammonium chloride aqueous solution (3.0 mL) to obtain a mixed solution. The above mixed solution was extracted three times with ethyl acetate (3.0 mL), the organic phases were combined, washed with saturated sodium chloride aqueous solution (5 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 obtain compound 64 (6 mg, 0.01 mmol, yield 61%), a white solid compound. 1 H NMR(500MHz,DMSO-d6)δ8.73(d,J=6.5Hz,1H),8.13(s,2H),7.83-7.59(m,2H),7.17-6 .96(m,2H),6.86(s,1H),4.74-4.66(m,1H),4.63(t,J=6.5Hz,2H),4.35(t,J=6.5Hz,2 H),3.90(s,3H),3.85-3.76(m,1H),3.43(d,J=7.0Hz,2H),3.20(s,2H),3.03(s,3H),1 .97-1.93(m,2H),1.76-1.67(m,1H),1.65-1.61(m,2H),1.13-1.01(m,4H).LC-MS[M+H] + m / z:492.3.

[0409] Example 65: Synthesis of 2-(2-(((4-(((8-chloroquinoline-2-yl)amino)cyclohexyl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 65)

[0410]

[0411] Following the preparation process of compound 64 in Example 64, 2-chloro-8-methoxy-1,7-naphthidine was replaced with 2,8-dichloroquinoline, and other operating steps were similar, resulting in compound 65 as a white solid with a yield of 34%. 1 H NMR (500MHz, DMSO-d6) δ8.73(d,J=6.5Hz,1H),8.13(s,2H),7.79(d,J=8.0Hz,1H),7.54(d,J=7.0Hz,1 H),7.50(d,J=7.5Hz,1H),7.09(s,1H),7.01(t,J=7.0Hz,1H),6.72(d,J=7.0Hz,1H),4.74-4.66(m,1H ),4.63(t,J=6.5Hz,2H),4.35(t,J=6.5Hz,2H),3.92-3.79(m,1H),3.43(d,J=7.0Hz,2H),3.20(s,2H) ,3.03(s,3H),2.07-2.03(m,2H),1.77-1.69(m,1H),1.65-1.62(m,2H),1.15-1.03(m,4H).LC-MS[M+H] + m / z:495.3.

[0412] Example 66: Synthesis of 2-(2-(((4-(((8-bromoquinoline-2-yl)amino)cyclohexyl)methyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 66)

[0413]

[0414] Following the preparation process of compound 64 in Example 64, 2-chloro-8-methoxy-1,7-naphthidine was replaced with 8-bromo-2-chloroquinoline, and other operating steps were similar, resulting in compound 66 as a white solid with a yield of 58%. 1H NMR (500MHz, CDCl3) δ8.22(s,2H),7.82(dd,J=7.5,1.0Hz,1H),7.75(d,J=9.0Hz,1H),7.51(d ,J=7.0Hz,1H),7.02(t,J=7.5Hz,1H),6.62(d,J=9.0Hz,1H),6.45(d,J=7.0Hz,1H),5.05–4.9 8(m,2H),4.88(t,J=6.5Hz,2H),4.50(t,J=6.5Hz,2H),3.89(s,1H),3.52(d,J=7.0Hz,2H),3. 35(s,2H),3.16(s,3H),2.30–2.23(m,2H),1.87–1.79(m,3H),1.29–1.20(m,4H).LC-MS[M+H] + m / z:539.3.

[0415] Example 67: Synthesis of (S)-2-(2-((1-(4-(((8-chloro-7-methylquinoline-2-yl)amino)phenyl)ethyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (compound 67)

[0416]

[0417] Following the preparation process of compound 13 in Example 13, (S)-4-(1-(methylamino)ethyl)aniline was used to replace oxetane-3-amine, and other operating steps were similar, to obtain compound 67 as a white solid with a yield of 52%. 1 H NMR (500MHz, DMSO-d6) δ9.65 (s, 1H), 8.82 (d, J = 6.5 Hz, 1H), 8.26 (s, 2H), 8.16 (d, J = 8. 5Hz,2H),8.04(d,J=9.0Hz,1H),7.60(d,J=8.0Hz,1H),7.27-7.23(m,3H),7.06(d,J=9. 0Hz,1H),6.16(q,J=7.0Hz,1H),4.79(q,J=7.0Hz,1H),4.71(t,J=6.5Hz,2H),4.43(t,J =6.5Hz,2H),3.31(s,2H),2.79(s,3H),2.52(s,3H),1.52(d,J=7.0Hz,3H).LC-MS[M+H] + 517.3.

[0418] Biological assay

[0419] 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.

[0420] Main instruments and equipment for the experiment:

[0421] Centrifuge (Beckman Avanti J-15R); Ultra-low temperature freezer (thermo FDE60086FV); Carbon dioxide incubator (ESCO CLM-170B-CN); Microplate reader (Spark Tecan); Multichannel pipette (20-200uL); Cell counter (Bio-Rad TC20).

[0422] Example 68:

[0423] G401 ELISA (H3K27me3 methylation) assay

[0424] Step 1

[0425] The compound of the present invention was diluted 3-fold each time in DMSO, for a total of 9 dilutions, to obtain a total of 10 concentrations. The compound was then added to G401 cells cultured in 96-well plates to obtain compound solutions with final concentrations of 0 nM, 0.152 nM, 0.457 nM, 1.372 nM, 4.115 nM, 12.346 nM, 37.037 nM, 111.111 nM, 333.333 nM, and 1000 nM. Before the ELISA procedure, the cells were cultured in an incubator at 37°C and 5% CO2 for 48 h.

[0426] Step 2

[0427] Wash the cells in the 96-well plate with 1×PBS buffer and add 100 μL / well of lysis buffer (0.4N HCl) for lysis. Incubate the plate on a shaker at 150 rpm and 4°C for 30 min. Add neutralization buffer (0.5M disodium hydrogen phosphate, pH 12.5, 1mM DTT; 80 μL / well) to neutralize, and pipette approximately 20 times to thoroughly mix the solution. After neutralization, centrifuge the 96-well plate at 2000 rpm for 2 min.

[0428] Step 3

[0429] 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.

[0430] Step 4

[0431] After incubation, wash five times with TBST: add 60 μL of TBST buffer to each well and wash for 5 min at 200 rpm and 25°C on a shaker. Add 50 μL of blocking buffer (TBST, 2% BSA) to each well, centrifuge at 2000 rpm for 2 min, and incubate at 200 rpm and 25°C on a shaker for 1 h.

[0432] Step 5

[0433] Remove the blocking buffer and add 30 μL of primary antibody (2% BSA, 0.1% H3K27me3; 0.03% H3) to each well. Centrifuge the plate at 2000 rpm for 2 min and incubate on a shaker at 150 rpm and 25 °C for 1 h.

[0434] Step 6

[0435] Wash five times with TBST to remove primary antibody, add 30 μL of secondary antibody (2% BSA, 0.04% anti-rabbit antibody) to each well, centrifuge at 2000 rpm for 2 min in a 384-well plate, and incubate at 150 rpm and 25°C for 1 h on a shaker.

[0436] Step 7

[0437] 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.

[0438] Step 8

[0439] After incubation at room temperature for 5 minutes, the signal was read on a microplate reader. The H3K27me3 methylation readings were normalized using the H3 signal, and the inhibition percentage was calculated for the DMSO-treated samples. The data were fitted to a dose-response curve using the GraphPad Prism program to obtain the IC50 of the test compound. 50 value.

[0440] Following the preparation methods of Novartis' patent CN114746414A and Shanghai Sailan Biotechnology Co., Ltd.'s patent CN117384153A, compound C13 in CN114746414A was used as Comparative Example 1, PA001 in CN117384153A was used as Comparative Example 2, and PA062 was used as Comparative Example 3. The characterization results are as follows:

[0441] C13: A white solid compound. 1H NMR (500MHz, CDCl3) δ8.21(s,2H),7.74(d,J=6.0Hz,1H),7.51(d,J=8.5Hz,1H),7.15(d,J=2.0Hz,1 H),7.06(d,J=6.0Hz,1H),6.98(dd,J=8.5,2.5Hz,1H),6.53(d,J=5.0Hz,1H),5.07–4.99(m,1H),4. 90(t,J=6.5Hz,2H),4.50(t,J=6.5Hz,2H),4.10(s,3H),3.41–3.38(m,1H),3.36(s,2H),3.33(t,J= 6.5Hz,2H),2.28–2.20(m,2H),1.99–1.91(m,2H),1.72–1.58(m,1H),1.25–1.12(m,4H).LC-MS[M+H] + m / z:477.0.

[0442] PA001: White solid compound. 1 H NMR(500MHz,DMSO-d6)δ9.57(s,1H),8.77(d,J=6.5Hz,1H),8.08(s,2H),7.94(d,J =9.0Hz,1H),7.87(d,J=8.5Hz,2H),7.77(d,J=5.5Hz,1H),7.47(t,J=6.5Hz,1H),7 .20-7.18(m,3H),7.15(d,J=5.5Hz,1H),4.73-4.65(m,1H),4.62(t,J=7.0Hz,2H), 4.38(d,J=6.0Hz,2H),4.34(t,J=6.0Hz,2H),3.96(s,3H),3.19(s,2H).LC-MS[M+H] + m / z:472.3.

[0443] PA062: A white solid compound. 1H NMR(500MHz, CDCl3)δ8.17(s,2H),7.88(d,J=5.5Hz,1H),7.83(d,J=9.0Hz,1H), 7.38-7.35(m,4H),7.21(d,J=9.0Hz,1H),7.07(d,J=5.5Hz,1H),6.38(d,J=7.0Hz ,1H),5.78(d,J=7.5Hz,1H),5.18-5.12(m,1H),5.03-4.95(m,1H),4.87(t,J=6. 5Hz,2H),4.44(t,J=6.5Hz,2H),4.17(s,3H),3.32(s,2H),1.56(d,J=7.0Hz,3H).

[0444] LC-MS[M+H] + m / z: 486.3.

[0445] Compound 64 is structurally similar to Comparative Example 1 (C13), except that a substituent methyl group is introduced at the N-position of the linker in Comparative Example 1. The structural comparison is as follows:

[0446]

[0447] After testing Comparative Example 1 (Compound C13 in CN114746414A) and Compound 64 according to the experimental method of Example 68, it was found that the activity of Comparative Example 1 was 58.1 nM and the activity of Compound 64 was 5.0 nM. The activity of the newly invented Compound 64 was unexpectedly increased by nearly 12 times, showing an unexpected increase in activity, as shown in Table 2.

[0448] Table 2 Comparative methylation inhibitory activities of Example 1 and Compound 64 at the cellular level

[0449] Compound numbering <![CDATA[G401-ELISA IC 50 (nM)]]> Comparative Example 1 58.1 64 5.0

[0450] The compounds of Comparative Example 2 (PA001) and Comparative Example 3 (PA062) disclosed in CN117384153A are structurally similar to Compound 1 of the present invention. Compared to Comparative Example 2, Comparative Example 3 introduces a methyl group at the benzylic position of the linker. Compound 1 of the present invention also introduces a substituent methyl group at the N-position of the linker in Comparative Example 2; the structural comparison is as follows.

[0451]

[0452] Comparative Examples 2, 3, and 1 were tested according to the experimental method of Example 68. It was found that Comparative Example 2 (PA001) had an activity greater than 1000 nM, while Comparative Example 3 (PA062) showed almost no change in activity compared to Comparative Example 2. However, similar to the unexpectedly high activity of Compound 64, the activity of Compound 1 of the present invention was significantly improved after introducing a methyl group onto the N-terminus of the Linker, reaching 1C1. 50 The value is 33 nm. Therefore, the introduction of a methyl group (PA062) at the benzylic position of the linker did not substantially improve its activity. However, the introduction of a methyl group (compound 1) at the N-position of the linker unexpectedly resulted in an activity increase of over 30 times, as shown in the data below. Figure 1 As shown.

[0453] By comparison, the introduction of specific groups (such as methyl groups) onto the nitrogen atom of this type of linker generally resulted in an activity increase of at least 12-fold. This demonstrates that the introduction of specific groups, such as methyl groups, is crucial for enhancing activity.

[0454] Table 3 shows the activity data for other similar invention compounds.

[0455] Table 3. Methylation inhibitory activity of compounds at the cellular level

[0456] Compound numbering <![CDATA[G401-ELISA IC 50 (nM)]]> Compound numbering <![CDATA[G401-ELISA IC 50 (nM)]]> 2 47.8 35 71.9 3 ≥200 36 424.3 4 ≥200 37 583.5 5 ≥200 38 73.8 6 177 39 12.7 7 26.7 40 >100 8 17.3 41 70.9 9 ≥200 42 46.7 10 52.7 43 43.9 11 50 44 50.4 12 44.2 45 48.7 13 5.3 46 ≥100 14 6.5 47 ≥100 15 9.4 48 ≥100 16 2.3 49 ≥100 17 27.2 50 77.9 18 16.2 51 ≥1000 19 ≥1000 52 26.3 20 167 53 3.6 21 ≥1000 54 43.8 22 771 55 63.9 23 2.72 56 347.6 24 63.2 57 209.5 25 3.7 58 12.9 26 12.8 59 4.7 27 16.8 60 155.6 28 9.5 61 167.4 29 ≥200 62 162.9 30 65 63 110.2 31 26.8 65 4.3 32 6.4 66 2.2 33 11 67 2.8 34 34.5

[0457] The compounds of this invention exhibit methylation inhibitory activity at the G401 cell level that is generally below 1 μM, with some compounds even showing activity below 10 nM. This demonstrates that these compounds all possess significant target activity.

[0458] Analysis of cell proliferation

[0459] Example 69:

[0460] Analysis of proliferation of suspension cells: WSU-DLCL2 / KARPAS 422 cells

[0461] 1) Culture medium:

[0462] 10% FBS (Cat: A5669401; gibco) + 89% RPMI-1640 (Cat: PM150110; Pricela) + 1% Pen Strep (Cat: 15140; gibco).

[0463] 2) Before seeding 96-well plates, treat WSU-DLCL2 / KARPAS 422 cells with 50 μL / well of poly-L-lysine for at least one hour. After treatment, wash with 100 μL / well of PBS and air dry. After air drying, seed WSU-DLCL2 / KARPAS 422 cells in 96-well plates with 100 μL / well of culture medium (approximately 2000 cells / well, two replicates for each test compound). Administer the drug the next day.

[0464] 3) On the second day, solutions of the test compounds at different concentrations were prepared using DMSO. Nine solutions of the corresponding test compounds at different concentrations were prepared by equal-fold (3-fold) dilution with DMSO. The concentrations were 185.19, 61.73, 20.58, 6.86, 2.29, 0.76, 0.25, 0.08, and 0.03 μM.

[0465] 4) After dilution, take 2 μL of DMSO to prepare different concentrations of the test compound solution and add 198 μL of culture medium, then mix thoroughly (pipe and stir at least 20 times). Then, add 25 μL / well of the above-mentioned culture medium containing different concentrations of the test compound to each of the 96 wells containing cells, and pipette and stir 10 times. Finally, culture media containing the test compound (containing 0.2% DMSO) with final concentrations of 370.37, 123.46, 41.15, 13.72, 4.57, 1.52, 0.51, 0.17, and 0.06 nM were obtained, and a DMSO control solution was prepared.

[0466] 5) Then, the cells treated with the test compound or DMSO were placed back into the 37°C incubator for 3-4 days of treatment, and then the drug was changed.

[0467] 6) During drug change: Add 80 μL of fresh culture medium to a 96-well plate that has been pre-treated with poly-L-lysine. Then, prepare different concentrations of culture medium containing the test compound, as in the first drug treatment. Add 25 μL of the test compound culture medium of different concentrations to each well and mix by pipetting 4 times with a pipette. For the WSU-DLCL2 / KARPAS 422 cells from step 5), mix the cells by pipetting 10 times with a pipette. Then, transfer 20 μL / well of the cell solution containing the test compound to the 96-well plate containing the test compound culture medium, and mix by pipetting 10 times. Perform drug change treatment every 3-4 days, for a total of 4 drug treatments.

[0468] 7) Cell viability assay was performed 14 days after treatment. First, the culture medium was pipetted 10-20 times with a pipette to fully disperse the cells. Take 20 μL of culture medium containing cells per well, add 20 μL of trypan blue, mix thoroughly, and then count the cells.

[0469] 8) Cell viability calculation: Calculation formula = 100 * drug administration group / DMSO group

[0470] By subtracting the control group from all group data and then calculating the corresponding inhibition rate based on the standard curve, the IC50 can be determined. 50 value.

[0471] Example 70:

[0472] Adherent cells: Analysis of G401 cell proliferation

[0473] 1) Culture medium:

[0474] 10% FBS (Cat: F8318; Sigma) + 89% McCoy's 5A (Cat: 16600-082; gibco) + 1% PenStrep (Cat: 15140; gibco).

[0475] 2) Inoculate 500 μL / well of G401 cells into 24-well plates (approximately 10,000 cells / well, with a single replicate for each test compound) and administer the drug the next day.

[0476] 3) On the second day, solutions of the test compounds at different concentrations were prepared using DMSO. The solutions were diluted 3-fold with DMSO to prepare nine different concentrations of the corresponding test compounds. The concentrations were 5000, 1666.67, 555.56, 185.19, 61.73, 20.58, 6.86, 2.29, and 0.76 μM.

[0477] 4) After dilution, take 2 μL of DMSO to prepare different concentrations of the test compound solution and add 198 μL of culture medium, then mix thoroughly (pipe and stir more than 20 times). Then, add 125 μL / well of the above-mentioned culture medium containing different concentrations of the test compound to each of the 24 wells with cells already seeded. Finally, culture media containing the test compound (containing 0.2% DMSO) with final concentrations of 10000, 3333.33, 1111.11, 370.37, 123.46, 41.15, 13.72, 4.57, and 1.52 nM were obtained, and a DMSO control solution was prepared.

[0478] 5) Subsequently, cells treated with different concentrations of the test compound solution or DMSO were placed back into a 37°C incubator for culture. After 3-4 days of treatment, the drug was changed.

[0479] 6) During drug change: After aspirating the culture medium from G401 cells, wash once with 500 μL PBS, then add 100 μL trypsin and digest for 4 min. Add 400 μL of culture medium to neutralize, and centrifuge at 1000 rpm for 5 min. After centrifugation, remove the supernatant, resuspend the cells in 100 μL of culture medium, and transfer 10 μL of the resuspended solution to a 24-well plate containing 490 μL of fresh culture medium. Add 125 μL of culture medium containing different concentrations of the test compound to each well and mix thoroughly. Perform drug change treatment every 3-4 days.

[0480] 7) During the fourth dressing change, after washing, digesting, and neutralizing as in step 6), resuspend the cells in 500 μL of culture medium. Then, add 20 μL of cell-containing fluid to a 96-well blackboard containing 80 μL of culture medium + 25 μL of culture medium containing different concentrations of the test compound, setting up two replicates. Hoechst staining was performed on day 14.

[0481] 8) After 14 days, collect the cells and remove most of the culture medium by tilting the plate. Invert the plate onto absorbent paper.

[0482] 9) Preparation of fixation and staining solution: Use 37% formaldehyde solution at 10×. Prepare 3.7% formaldehyde solution using PBS. Each 96-well plate requires 5 ml of 3.7% formaldehyde solution. Dilute Hoechst 33342 (1000X) in the 3.7% formaldehyde solution to a final concentration of 10 μg / ml.

[0483] 10) Add fixative staining solution in a pipette at a rate of 50 μL per well. Incubate on a horizontal shaker for 30 minutes at room temperature (RT) in the dark.

[0484] 11) Remove almost all Hoechst 33342 using the tilting method. Gently add 100 μL of PBS using a pipette for washing. Incubate on a horizontal shaker for 5 minutes at room temperature (RT) in the dark. Repeat twice. Finally, remove all PBS using the tilting method and invert the plate onto absorbent paper.

[0485] 12) The excitation light of the ELISA reader is 320 (25), and the emission light is 460 (20).

[0486] 13) The 96-well plate aluminum foil that has been read is stored sealed at -20°C.

[0487] 14) Calculation of adherent cell viability:

[0488] Cell viability (%) = [A(drug+) - A(blank)] / [A(drug-) - A(blank)] × 100%

[0489] A (Drug+): Absorbance of the pores containing cells and drug solution;

[0490] A(Drug-): Absorbance of pores containing cells but without drug solution;

[0491] A (blank): Absorbance of pores containing culture medium but without cells.

[0492] *Cell viability: Cell proliferation activity or cytotoxic activity

[0493] By subtracting the control group from all group data and then calculating the corresponding inhibition rate based on the standard curve, the IC50 can be determined. 50 value.

[0494] Karpas422, WSU-DLCL2, and G401 cells are common tumor cells induced by EZH2 mutations or overexpression, and are often used as models for evaluating antitumor activity at the cellular level. Using Tazemetostat and Comparative Example 1 as control compounds, the compounds of this invention, tested according to the above experimental methods, showed significant antitumor activity; some results are shown in Table 4.

[0495] Table 4. Inhibitory activity of compounds against the proliferation of PRC2-dependent tumor cells.

[0496]

[0497] Example 71:

[0498] Stability test of the compound in liver microsomes

[0499] The liver microsomes used in the experiment are as follows:

[0500] Table 5. Information related to liver microsomes

[0501]

[0502] Table 6. Composition of the experimental incubation system:

[0503] Types of substances to be added Final concentration Liver microsomes 0.5 mg protein / mL Test compound 1μM Acetonitrile 0.99％ dimethyl sulfoxide 0.01％ β-NADPH <![CDATA[1 mM (10 mM MgCl2 solution)]]>

[0504] Experimental steps:

[0505] (1) Preheat the empty “incubation” plates T60 and NCF60 for 10 minutes.

[0506] (2) Dilute liver microsomes to 0.56 mg / mL in 100 mM phosphate buffer to obtain working microsome solution.

[0507] (3) Transfer 445 μL of working microsome solution (0.56 mg / mL) to preheated “incubation” plates T60 and NCF60, and then continuously shake the “incubation” plates T60 and NCF60 at 37 °C for 10 minutes. Subsequently, transfer 54 μL of shaken liver microsome solution to a blank plate, then add 6 μL of NAPDH coenzyme solution to the blank plate, and then add 180 μL of quenching solution to the blank plate.

[0508] (4) Add 5 μL of the compound working solution (100 μM) to the “incubation” plates (T60 and NCF60) containing microparticles and mix thoroughly 3 times.

[0509] (5) Add 50 μL of phosphate buffer to the NCF60 plate and mix thoroughly three times. Start timing and incubate the NCF60 plate at 37°C for 60 minutes while shaking.

[0510] Add 180 μL of quenching solution to the quenching plate T0. Ensure the plate is cooled to prevent evaporation.

[0511] Mix once, and at the 60-minute time point, transfer 60 μL of the sample from NCF60 incubation to the “quench” plate T0 containing the quench solution.

[0512] (6) For the T60 plate, mix thoroughly three times and immediately remove 54 μL of the mixture, which is then spotted onto the "quench" plate and marked as 0 minutes. Then add 44 μL of NAPDH coenzyme solution to the T60 plate. Start timing and incubate the plate at 37°C for 60 minutes while shaking.

[0513] At 5, 15, 30, 45 and 60 minutes, 180 μL of quenching solution was added to the "quenching" plate, mixed once, and 60 μL of sample was continuously transferred from the T60 plate to the "quenching" plate at each time point.

[0514] (7) Shake all the “quenching” plates to be tested for 10 minutes, and then centrifuge at 4000 rpm for 20 minutes at 4°C.

[0515] (8) Transfer 80 μL of the supernatant after centrifugation to 240 μL of HPLC water and mix for 10 minutes with a shaker to obtain the sample to be analyzed.

[0516] (9) Before LC-MS / MS analysis, the samples to be analyzed were transferred to bioanalytical plates, and each bioanalytical plate was sealed and shaken for 10 minutes. After shaking, the samples were analyzed by LC-MS / MS. The half-life (t1 / 2) and clearance rate (CL) of the compounds were calculated using first-order kinetic formulas, and the results are shown in Table 7 below.

[0517] Table 7 Results of Liver Microsome Stability Test

[0518]

[0519] The results of liver microsomal stability tests on the compounds of the present invention show that the compounds of the present invention have good drug stability in the liver microsomes of humans, mice, and beagle dogs.

Claims

1. A compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of said compound, stereoisomers, or mixture, wherein the compound has a structure of Formula 1: in, A 1 Selected from N or CR 11 A 3 Selected from N or CR 13 A 5 Selected from N or CR 15 And A 1 A 3 A 5 The number of N in the text is 1 or 2; R 12 Selected from H, halogens, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl substituted with 1-3 halogens, and C1-C3 alkoxy substituted with 1-3 halogens; R 11 R 13 R 14 R 15 R 16 R 17 Each is independently selected from H, halogen, C1-C3 alkyl, C1-C3 alkoxy, and C1-C3 alkyl substituted with 1-3 halogens; R 2 Selected from H or methyl; In the group, It is a single bond or a double bond, and all of them Both can be single bonds or both can be double bonds; when In When it is a single bond, B 1 For NH or CHR 21 B 2 For NH or CHR 22 B 3 For NH or CHR 23 B 4 For NH or CHR 24 R 3 For H; when In When it is a double bond, B 1 For N or CR 21 B 2 For N or CR 22 B 3 For N or CR 23 B 4 For N or CR 24 R 3 It is H or C1-C3 alkyl; B 1 B 2 B 3 and B 4 In this context, the number of N or NH is 0 or 1. R 21 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens; R 22 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens; R 23 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens; R 24 Selected from H, halogens, C1-C3 alkyl groups or C1-C3 alkyl groups substituted with 1-3 halogens; R 4 Selected from C1-C3 alkyl groups or 1-3 deuterated C1-C3 alkyl groups; C 1 Selected from N or CH; L is selected from -C(R) 6 R 7 -, -C(=CH2)-; R 6 R 7 Each is independently selected from H, halogen, hydroxyl, C1-C3 alkyl, hydroxyl-substituted C1-C3 alkyl, C1-C3 alkyl substituted with 1-3 halogens, and C1-C3 alkyl substituted with hydroxyl and 1-3 halogens; R 5 Selected from R a -C(=O)R a -CH2R a ; R a Selected from -NHR c , In the group, For single or double bonds, R a1 R a2 R a3 Each is independently selected from -CH2-, -CH=, =CH-, -CH(CH3)-, =C(CH3)-, -C(CH3)=, or -C(=O)-; R b Selected from H, halogens, C1-C3 alkyl groups, and cyano-substituted C1-C3 alkyl groups; R c Selected from C1-C4 alkyl groups substituted with hydroxyl groups and C2-C3 alkyl groups substituted with C1-C3 alkoxy groups; D 1 -C(R) d R e )-; D 2 Selected from -O-, -SO 2 -、-CH(OH)-、-S-、-C(R f R g )-; R d R e Each is independently selected from H, halogens, and C1-C3 alkyl groups; R f R g Each is independently selected from H and halogens; E 1 Selected from -C(=O)- or -C(R) h R i )-; E 2 Selected from -NH- or -O-; E 3 Selected from -NH-, -O-, or -CH(N(CH3)2)-; R h R i Each is independently selected from H or halogen; F 1 Selected from -N(R) j - or -O-; Among them, R j Selected from H or C1-C3 alkyl groups.

2. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 1, characterized in that: In Equation 1 The group structure is selected from:

3. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 1, characterized in that: The compound has the structure of Formula 2:

4. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 1, characterized in that: The compound has the structure of Formula 3:

5. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 3 or 4, characterized in that: R 21 Selected from H, halogens, methyl groups, or CF3; R 22 Selected from H, halogens, methyl groups, or CF3; R 23 Selected from H, halogens, methyl groups, or CF3; R 24 Selected from H, halogen, methyl or CF3.

6. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 3, characterized in that: The group structure is selected from:

7. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomers, or mixtures as described in claims 3, 4, or 6, characterized in that: R 4 Selected from methyl, ethyl or -CD3.

8. The compound, its stereoisomers, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, its stereoisomers, or the mixture, as described in claim 1, characterized in that: R a Selected from 9. A compound selected from the following structures:

10. A pharmaceutical composition comprising the compound of any one of claims 1-9, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer or mixture thereof, and at least one pharmaceutically acceptable excipient.

11. A method for treating a disease or symptom mediated by at least one of PRC2, EZH1, and EZH2, characterized in that, Administer to a subject requiring the treatment a therapeutically effective amount of any one of claims 1-9, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, or mixture thereof, or a pharmaceutical composition of claim 10.

12. The use of the compound of any one of claims 1-9, its stereoisomers, mixtures of different stereoisomers of the compound, or pharmaceutically acceptable salts of said compound, stereoisomers, or mixtures; or the pharmaceutical composition of claim 10, in the preparation of a medicament for treating diseases or symptoms mediated by at least one of PRC2, EZH1, and EZH2.

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