CN118515653A - A kind of EZH2 specific inhibitor and its preparation method and use - Google Patents

Abstract

The invention provides a compound with EZH2 specific inhibitor activity, a preparation method and application thereof. The compounds of the present invention have potent inhibitory activity and suitable pharmaceutical properties and are useful in the treatment of EZH1, EZH2 and/or PRC2 mediated diseases and/or conditions.

Description

A kind of EZH2 specific inhibitor and its preparation method and use

Technical Field

The present invention relates to the field of chemical medicines, and in particular to an EZH2 specific inhibitor and a preparation method and use thereof.

Background Art

The human homolog of the Drosophila enhancer of zeste gene (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) to achieve monomethylation, dimethylation and trimethylation of H3K27 (H3K27me3), thereby inhibiting the expression of tumor suppressor genes and participating in the regulation of cell cycle, cell senescence, differentiation and cancer and other physiological or case processes. Many 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 deterioration, metastasis and poor prognosis of related cancers. In addition, there are multiple gain-of-function mutations in the active site of the EZH2 catalytic domain SET. For example, EZH2 tyrosine 641 mutations (Y641C, Y641F, Y641N, Y641S, Y641N, and Y641H) are present in more than 20% of diffuse large B-cell lymphomas and 7% of follicular lymphomas. These mutations increase the trimethylation level of H3K27, leading to the silencing of tumor suppressor genes. In addition to this mechanism of tumor suppressor gene silencing caused by EZH2-dependent histone methylation, EZH2 can also function by methylating non-histone substrates in a PRC2-independent manner, or form a transcription complex with other factors to activate the transcription of downstream target genes and increase the expression of target genes. For example, in castration-resistant prostate cancer cells (CRPC), phosphorylated EZH2 can assist the androgen receptor-associated complex to activate androgen gene expression, leading to the further development of castration-resistant prostate cancer cells. These evidences indicate that EZH2 is a very promising therapeutic target. Currently, there are several EZH2 inhibitors, such as Tazemetostat, which has been approved by the FDA for the treatment of epithelioid sarcoma and follicular lymphoma; Valemetostat, which has been approved by the MHLW for adult T-cell lymphoma. However, these SAM competitive inhibitors have their own limitations, such as insufficient inhibitory activity against EZH1/2, low selectivity, drug-resistant mutations, and high toxicity.

Therefore, it is necessary to develop a new class of EZH2 inhibitors, which should have higher catalytic activity in inhibiting EZH2, less toxic side effects, and play an important role in cancer treatment.

Summary of the invention

The present invention provides compounds that inhibit EZH2, their stereoisomers, or mixtures of different stereoisomers of the compound of formula 1, pharmaceutically acceptable salts of the above compounds, stereoisomers, and mixtures, methods for preparing the above compounds, stereoisomers, or mixtures of different stereoisomers of the compound of formula 1 and pharmaceutically acceptable salts of the above compounds, stereoisomers, and mixtures, as well as methods and pharmaceutical compositions for treating or preventing diseases or symptoms associated with EZH1, EZH2 and/or PRC2.

In the first aspect of the present invention, the present invention provides a compound having formula 1 or a stereoisomer thereof, or a mixture of different stereoisomers of the compound of formula 1, or a pharmaceutically acceptable salt of the above compound, stereoisomer, or mixture:

Wherein, A ¹ is selected from N or CR ¹¹ ;

A ² is selected from N or CR ¹² ;

A ³ is selected from N or CR ¹³ ;

^R1 is selected from H or halogen;

R ² is selected from H, halogen or C ₁ -C ₃ alkyl;

R ¹¹ is selected from H, halogen or C ₁ -C ₃ alkyl;

R ¹² is selected from H, halogen or C ₁ -C ₃ alkyl;

R ¹³ is selected from H, halogen or C ₁ -C ₃ alkyl;

L ¹ is selected from -NH- or -CH ₂ -:

^B1 is selected from N or CH:

R ³ is C ₁ -C ₃ alkyl;

^R4 is H or _{C1- C3} alkyl;

The C ₃ alkyl group is a straight chain alkyl group, a branched chain alkyl group or a cycloalkyl group;

^C1 is selected from N or CH;

^L2 is selected from ^-CHR5NH- , C( ^R5 ) _2NH- , -CHR5C(=O)NH-, -C( ^R5 ) _2C (=O)NH- ^, -CHR5C(=O) _NHCH2- or -C( ^{R5 )} _2C (=O) _NHCH2- ;

R ⁵ , at each occurrence, is independently selected from H, -OH, -CN, C ₁ -C ₂ alkyl or halogen.

In some embodiments of the present invention, the compound has the structure of Formula 2, Formula 3, Formula 4, Formula 5, Formula 6 or Formula 7:

Wherein R ¹ is H or halogen;

L ¹ is -NH-, B ¹ is CH; or L ¹ is CH ₂ , B ¹ is N.

In some embodiments of the present invention, R ¹ is halogen.

In some embodiments of the present invention, the compound has the structure of Formula 8 or Formula 9 below:

.

In some embodiments of the present invention, Select from the following structures:

.

In some embodiments of the present invention, R ³ is methyl or cyclopropyl, and R ⁴ is methyl or H.

In some embodiments of the present invention, R ³ and R ⁴ are both methyl.

In some embodiments of the present invention, ^R3 is methyl and ^R4 is H.

In some embodiments of the present invention, R ² is selected from H, halogen or methyl;

In some embodiments of the present invention, R ¹¹ is selected from H, halogen or methyl;

In some embodiments of the present invention, R ¹² is selected from H, halogen or methyl;

In some embodiments of the present invention, R ¹³ is selected from H, halogen or methyl.

In some embodiments of the invention, ^L2 is selected from _-CH2NH- , -CH(OH)C(=O)NH-, -CHF2C(=O)NH-, -CH( _CH3 )C(=O)NH-, -CH(CN ₎ C(=O)NH-, -CH2C(=O)NH- _{or -CH2C} (=O) _NHCH2- .

In some embodiments of the invention, the compound is selected from the following structures:

.

In a second aspect of the present invention, a pharmaceutical composition is provided, comprising a compound of one of Formulas 1 to 9 or one of Compounds 1 to 30, a stereoisomer or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise another active agent.

In some embodiments, the pharmaceutically acceptable carrier is selected from a pharmaceutically acceptable vehicle and a pharmaceutically acceptable adjuvant. In some embodiments, the pharmaceutically acceptable carrier is selected from a diluent, a solvent, a dispersant, an antioxidant, a preservative, a buffer, an emulsifier, a pharmaceutically acceptable filler, a disintegrant, a surfactant, a binder, a flavoring agent, a dye and a lubricant and a combination thereof.

In some embodiments, the additional active pharmaceutical agents may include one or more of anticancer agents, biologics, radiopharmaceuticals, and hormonal drugs.

The 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 to 9 or one of Compounds 1-30, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt, or a pharmaceutical composition comprising a compound of one of Formulas 1 to 9 or one of Compounds 1-30, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the above-mentioned compound, stereoisomer or mixture of different stereoisomers of the compound is administered to a subject in need of the treatment.

In some embodiments, 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 rhabdoid tumor, or oropharyngeal cancer, etc.

In a fourth aspect of the present invention, a compound of one of Formulas 1 to 9 or one of Compounds 1 to 30, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the above compound, stereoisomer or mixture of different stereoisomers of the compound, or a pharmaceutical composition containing the same is provided for use in the preparation of a medicament for treating a disease or symptom mediated by at least one of PRC2, EZH1 and EZH2.

In a fifth aspect of the present invention, a method for preparing a compound of one of Formulas 1 to 9 or one of Compounds 1 to 30 and a stereoisomer thereof is provided.

The compounds of the present invention can be prepared using the following methods or other similar methods understood by those skilled in the art of organic synthesis. Compounds of one of Formulas 1 to 9 or one of Compounds 1-30 having a chiral center can be prepared in substantially optically pure form by using substantially optically pure starting materials or by separation chromatography, recrystallization, or other separation techniques well known in the art.

The starting material Boc-protected amino para-substituted ketone and optically pure tert-butylsulfenamide are subjected to dehydration condensation to obtain tert-butylsulfenyl imide intermediate 1 (INT-1), INT-1 is then subjected to reduction reaction to obtain tert-butylsulfenamide intermediate 2 (INT-2), and INT-2 is then selectively de-tert-butylsulfenyl to obtain intermediate 3; or when R ⁴ needs further alkyl substitution, the amino group is further monoalkylated to R ⁴ is substituted to obtain intermediate 3 (INT-3), and INT3 is directly subjected to electrophilic aromatic substitution reaction or metal-catalyzed coupling reaction with heterocyclic aromatic halides after removing the Boc protecting agent to obtain the corresponding intermediate INT-4, INT-4 is subjected to metal-catalyzed coupling reaction or electrophilic aromatic substitution reaction with nitrogen-containing heterobicyclic aromatic halides, or is subjected to reductive amination reaction with nitrogen-containing heterobicyclic aromatic aldehydes to obtain intermediate INT-5, and INT-5 is subjected to acid-amine condensation reaction with corresponding oxetane-3-amine or oxetane-3-ylmethylamine after the ester group of the M portion is hydrolyzed to form a free carboxylic acid, or the ester group of the M portion is reduced to obtain an aldehyde group and then subjected to reductive amination reaction to obtain the compound of formula 1.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Docking results of compound 8, comparative example 2 (C36 in CN114746414A) and target protein molecules

DETAILED DESCRIPTION

Definition of terms

The term "a" or "an" in the present invention includes "at least one" or "at least one", and the meaning of the noun includes singular and plural forms. For example, "another agent" means one, two or more agents.

The term "PRC2" in the present invention refers to the epigenetic control factor polycombrepressive complex 2.

The term "EZH2" in the present invention is the human homolog 2 of the Drosophila enhancer of zeste gene, a core component of PRC2, which regulates the transcription of target genes by introducing 3 methylation (H3K27me3) on histone H3 lysine 27, thereby regulating their expression, differentiation and development.

The term "subject" in the present invention refers to animals including humans.

The term "therapeutically effective amount" in the present invention refers to the amount of a compound that produces the desired effect of its administration (e.g., improvement of a disease and/or disease symptom mediated by EZH1, EZH2 and/or PRC2, reduction of severity of a corresponding disease and/or disease symptom, and/or slowing down the progression of a corresponding disease and/or symptom). The exact amount of a therapeutically effective amount will depend on the purpose of the treatment, and can be determined by a person skilled in the art using known techniques (see, e.g., Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, the term "treat" and its cognates refer to slowing or stopping the progression of a disease. As used herein, "treat" and its cognates include, but are not limited to, the following: complete or partial relief, cure of diseases and/or symptoms mediated by EZH1, EZH2 and/or PRC2, reduction in the risk of corresponding diseases and/or symptoms of diseases. The improvement of any of these symptoms or reduction in severity can be evaluated according to methods and techniques known in the art.

The term "compound" in the present invention, when referring to the compounds of the present invention, means a collection of molecules having the same chemical structure, unless otherwise specified as a collection of stereoisomers (e.g., a collection of racemates, a collection of cis/trans stereoisomers, or a collection of (E) and (Z) stereoisomers), but does not exclude isotopologues contained in the atoms that make up the molecules. Therefore, it should be clear to those skilled in the art that when a deuterium atom is contained in a specific chemical structure of a compound, the compound also contains a small amount of isotopologues in which one or more deuterium atom sites are actually hydrogen atoms. In the compounds of the present invention, the relative amount of such isotopologues will depend on many factors, including, for example: the isotopic purity of the reagents used to prepare the compounds, the efficiency of isotope incorporation in the various synthetic steps used to prepare the compounds. However, as described above, the relative amount of such isotopologues will be less than 49.9% of the compound in total. In other embodiments, the relative amounts of such isotopologues will total 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.

The term "stereoisomer" in the present invention includes cis-trans isomers, enantiomers and diastereomers.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" in the present invention is formed 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.

The term "pharmaceutically acceptable" refers to a composition that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without excessive toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to any non-toxic salt that can directly or indirectly provide the compounds of the present invention after being administered to a recipient. Suitable pharmaceutically acceptable salts are those disclosed, for example, by S. M. Berge et al. in J. Pharmaceutical Sciences, 1977, 66, pp. 1 to 19.

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, and 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. Thus, such pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioic acid, 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.

Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N ⁺ (C _1-4 alkyl) ₄ salts. The present invention also contemplates the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Suitable non-limiting examples of alkali metal and alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts and magnesium salts. Further non-limiting examples of pharmaceutically acceptable salts include ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates. Other suitable non-limiting examples of pharmaceutically acceptable salts include benzenesulfonates and glucosamine salts.

The active drugs described in the present invention may include one or more of anticancer agents, biological agents, hormone drugs and radioactive drugs:

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, Hexamethasone Cyanamide, mitomycin C, Oncaspar, Hycamtin, vinblastine sulfate, Fludara, 6-TG, daunorubicin hydrochloride, Nipent, Camptosar, Neutrexin, DIC, Accutane, Tespa, Vidaza, Temodar, Matulane, Cytoxan, Adriamycin, mithracin, Alimta, 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, Prolife Prospan 20 with carbamazepine implant, Pazopanib, Sorafenib, Erlotinib, Osimertinib.

Biologics, such as interferon, Herceptin, lenalidomide, Tarceva, Velcade, BCG, Iressa, Revlimid, Zevalin, Bevacizumab, Pembrolizumab, Nimotuzumab, Bexxar, Interleukin-2, Ontak, Campath, Rituxan, TrastuzumabDeruxtecan, Erlotinib, Mylotarg, Abraxane, Ergamisol, Tykerb, Pegasys, Thalomid.

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

Radioactive drugs, such as Phosphocol, Iodotope, Samarium SM-153, Metastron.

The diluent, solvent, dispersant, antioxidant, preservative, buffer, emulsifier, pharmaceutically acceptable filler, disintegrant, surfactant, binder, flavoring, dye and lubricant in the present invention have their well-known meanings in the art.

The term "halogen" in the present invention includes F, Cl, Br and I, i.e., fluorine, chlorine, bromine and iodine, respectively. In certain embodiments, halogen is preferably F, Cl, Br.

The term "alkyl" in the present invention refers to a fully saturated branched, branched or cyclic, substituted or unsubstituted hydrocarbon chain. For example, "C ₁ -C ₃ alkyl" is an alkyl group comprising 1 to 3 carbon atoms, i.e., C ₁ , C ₂ or C ₃ , methyl, ethyl, propyl, isopropyl or cyclopropyl, respectively. In some embodiments, the alkyl group is substituted.

The term "straight chain alkyl group" in the present invention refers to an alkyl group having no side chain or branch.

The term "branched alkyl group" in the present invention refers to an alkyl group in which one or more carbon atoms on the main chain of the alkyl group are connected to a side chain.

The term "cycloalkyl group" in the present invention refers to an alkyl group in which carbon atoms form a closed ring through single bonds.

The term "substituted" in the present invention means that one or more H in the group is replaced by other atoms or groups, for example, "C ₁ -C ₃ alkyl substituted by 1-3 halogens" means that 1-3 H in the C ₁ -C ₃ alkyl is replaced by halogens.

The terms "cyano" and "nitrile" in the present invention mean a "-CN" group.

The term "hydroxyl" in the present invention means a "-OH" group.

The cancer or tumor in the present invention includes 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.

Breast tumors include, for example, breast cancer with positive hormone receptor status, breast cancer with 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.

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.

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

Gastrointestinal tumors include, for example, colorectal cancer, anal cancer, gastric cancer, pancreatic cancer, esophageal cancer, gallbladder cancer, small intestine cancer, salivary gland cancer, neuroendocrine tumors, and gastrointestinal stromal tumors.

Genitourinary tract tumors include, for example, bladder cancer, renal cell carcinoma, and cancer of the renal pelvis and urinary tract. Ocular tumors include, for example, retinoblastoma and intraocular melanoma.

Liver tumors include, for example, hepatocellular carcinoma and cholangiocarcinoma.

Skin tumors include, for example, malignant melanoma, basal carcinoma, myeloma, Kaposi's sarcoma, and Merkel cell carcinoma.

Head and neck tumors include, for example, laryngeal cancer as well as cancers of the pharynx and oral cavity.

Sarcomas include, for example, soft tissue sarcomas, synovial sarcomas, rhabdoid sarcomas, and osteosarcomas. Lymphomas include, for example, non-Hodgkin's lymphoma, Hodgkin's lymphoma, cutaneous lymphoma, central nervous system lymphoma, and AIDS-related lymphoma.

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

The abbreviations of the present invention have the common meanings in the art or the definitions in the following Table 1:

Table 1 Abbreviations

DIBAL-H Diisobutylaluminum hydride Boc tert-Butyloxycarbonyl DCM Dichloromethane THF Tetrahydrofuran DMF dimethylformamide DMSO Dimethyl sulfoxide EtOAc Ethyl acetate DIPEA N-Ethyl-N-isopropyl-2-amine NMP N-Methylpyrrolidone HATU 2-(7-Aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoroformate LiAlH ₄ Lithium aluminum hydride _MnO2 Manganese dioxide (HCHO ₎ Paraformaldehyde Pd ₂ (dba) ₃ Tris(dibenzylideneacetone)dipalladium NaO ^t Sodium tert-butoxide BINAP 2,2'-Bis(diphenylphosphino)-1,1'-binaphthyl XPhos 2-(Dicyclohexylphosphino)-2',4',6'-triisopropylbiphenyl HPLC High performance liquid chromatography LCMS Liquid chromatography-mass spectrometry HMR NMR spectroscopy ¹ H Proton s Single Peak d Double Peak t Triple Peak dd Two double peaks m Multiple peaks Hz hertz MHz Megahertz m/z Mass-to-charge ratio ELISA ELISA PBS Phosphate buffered saline TBST Tris-HCl buffer, sodium chloride and Tween-20 solution BSA BSA rpm RPM ECL Electrochemiluminescence mL ml μL microliter mg mg h Hour min minute ^o C Celsius M Mol/L nM Nanomoles/L mmol mmol nmol Namor DTT Dithiothreitol PDL Poly-D-lysine RT Room temperature

Process for preparing the compounds of the present invention

The compounds of the present invention can be prepared using the methods of Examples 1-27, or other similar methods understood by those skilled in the art.

Example 1: Synthesis of (S)-2-(2-((-1-(-4-((8-chloro-7-methylquinolin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 1)

1. Preparation of key starting material (S)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate (1-3):

Step A Synthesis of Intermediate 1-1

To a reaction flask with a stirrer, tert-butyl (trans-4-acetylcyclohexyl)carbamate (4.82 g, 20 mmol, 1.0 equivalent), (S)-tert-butylsulfenamide (2.91 g, 24 mmol, 1.2 equivalent) were added in sequence, nitrogen was replaced three times, tetrahydrofuran (100 mL), tetraethyl titanate (6.84 g, 30 mmol, 1.5 equivalent) were added, and the mixture was heated to reflux for 20 h, and the plate was monitored. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was poured into a container of saturated sodium bicarbonate (100 mL) under vigorous stirring, filtered, and the filter cake was washed with ethyl acetate (100 mL). The organic phases were combined, and the solvent was removed by vacuum rotary evaporation to obtain compound intermediate 1-1, which was directly carried out to the next step.

Step B Synthesis of Intermediate 1-2

The above oily substance 1-1 was dissolved in tetrahydrofuran (50 mL), sodium borohydride (1.51 g, 40 mmol, 2.0 equivalents) was added under stirring, and stirred at room temperature for 20 h, and the reaction was monitored by spot plate. After the reaction was completed, water (40 mL) was added to quench. The mixed solution was extracted three times with ethyl acetate (10.0 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 1-2 (4.29 g, 12.4 mmol, yield 62%) as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 4.60 (brs, 1H), 3.25-3.12 (m, 1H), 1.96-1.78 (m,4H), 1.43 (s, 9H), 1.23 (s, 9H), 1.22-1.13 (m, 7H). LCMS [M+H] ⁺ m/ z: 347.2.

Step C Synthesis of key starting materials 1-3

The solid 1-2 (4.29 g, 12.4 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (30 mL) and water (6 mL), and iodine (2.20 g, 8.68 mmol, 0.7 equivalent) was added under stirring, and the mixture was heated to 70 ° C for 4 h, and the plate was monitored. After the reaction was completed, saturated sodium thiosulfate solution (30.0 mL) and ethyl acetate (10.0 mL) were added to extract the mixed solution three times, and the organic phases were combined, washed twice with saturated sodium carbonate solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain compound 1-3 (2.57 g, 10.6 mmol, yield 86%) as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 4.41 (brs, 1H), 3.35 (brs, 1H), 2.73-2.68 (m, 1H), 2.04-2.02 (m, 2H), 1.85-1.76 (m, 4H), 1.45-1.42 (m, 9H), 1.09 -1.02 (m, 7H). LCMS [M+H] ⁺ m/z: 243.2.

2. Preparation of Compound 1:

Step 1 Synthesis of intermediate 1-4

Compound 5-bromo-2-fluoropyrimidine (35.0 g, 198 mmol, 1.0 equivalent), XPhos (9.43 g, 19.8mmol, 0.1 equivalent), Pd ₂ (dba) ₃ (9.06 g, 9.9 mmol, 0.05 equivalent) were added to a two-necked bottle equipped with a stirrer, and then THF (200 mL) and a tetrahydrofuran solution containing a zinc reagent (0.5 M, 396 mL, 198 mmol) were added under a nitrogen atmosphere. The reaction solution was stirred at 60°C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, saturated ammonium chloride solution (300 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (200 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography to obtain compound 1-4 (6.00 g, 28.1 mmol, yield 14%) as a light yellow solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.56 (d, J =1.2 Hz, 2H), 3.56 (s, 2H), 1.46 (s, 9H). LCMS [M+H] ⁺ m/z: 213.1.

Step 2 Synthesis of intermediate 1-5

Compound (S)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate 1-3 (0.46 g, 2.0 mmol, 1.0 equivalent) was dissolved in dimethyl sulfoxide (10 mL), and compound 1-4 (0.42 g, 2.0 mmol, 1.0 equivalent) and DIPEA (1.29 g, 10.0 mmol, 5.0 equivalent) were added. The resulting reaction solution was reacted at 100°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The resulting mixture was separated and purified by silica gel column chromatography to obtain intermediate 1-5 as a white solid. LCMS [M+H] ⁺ m/z: 435.3.

Step 3 Synthesis of intermediate 1-6

The intermediate 1-5 (0.43 g, 1.0 mmol, 1.0 equivalent) was dissolved in methanol (5.0 mL), and a hydrochloric acid ethyl acetate solution (4.0 M, 0.75 mL, 3.0 equivalent) was added dropwise to the reaction solution, and the plate was tested. After the reaction was completed, the white solid intermediate was obtained by rotary evaporation under reduced pressure. The white solid, 2,8-dichloro-7-methylquinoline (0.21 g, 1.0 mmol, 1.0 equivalent), BINAP (0.12 g, 0.2 mmol, 0.2 equivalent), Pd ₂ (dba) ₃ (0.09 g, 0.1 mmol, 0.1 equivalent) and sodium tert-butoxide (0.29 g, 3.0 mmol, 3.0 equivalent) were added to a 10 mL two-necked flask equipped with a stirrer, and the flask was vented three times under a nitrogen atmosphere, and then THF (5.0 mL) was added, and the reaction solution was stirred at 65 ° C for 4 h. The reaction was monitored by spot plate. After the reaction was completed, the mixture was cooled to room temperature, saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 1-6 (0.15 g, 0.3 mmol, yield 32%) as a light yellow solid. LCMS [M+H]+ m/z: 510.3.

Step 4 Synthesis of compound 1

The intermediate 1-6 (0.15 g, 0.3 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (5.0 mL) and methanol (5.0 ml), and then 2.0 M sodium hydroxide (0.75 mL, 1.5 mmol, 5.0 equivalent) was added. The reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw materials, 1.0 M dilute hydrochloric acid was used to neutralize the reaction pH to 7-8, and dichloromethane (10 mL) was extracted three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained oily compound was directly used for the next step.

The above oily compound was dissolved in N, N-dimethylformamide (5.0 mL), and compound DIPEA (0.39 g, 3 mmol, 10.0 equivalents), HATU (0.15 g, 0.6 mmol, 2.0 equivalents) and 3-oxetaneamine (0.048 g, 0.6 mmol, 2.0 equivalents) were added in sequence. The obtained reaction solution was reacted at room temperature for 1 h. After the reaction was completed, a saturated ammonium chloride solution (5 mL) was added to quench the reaction, and the above mixed solution was extracted three times with ethyl acetate (5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain the target compound 1 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.18 (s, 2H), 7.75 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 8.8 Hz, 1H), 6.43 (d, J = 6.4 Hz, 1H), 5.22 (d, J = 8.7 Hz, 1H), 5.02 (dt, J = 13.2, 6.6Hz, 1H), 4.91 (t, J = 7.0 Hz, 1H), 4.47 (t, J = 6.3 Hz, 2H), 4.05-3.98 (m, 1H), 3.78 (brs, 1H), 3.33 (s, 2H), 2.54 (s, 3H), 2.28-2.24 (m, 2H), 1.97-1.94(m, 1H), 1.88-1.84 (m, 1H), 1.50-1.48 (m, 1H), 1.36-1.23 (m, 4H ), 1.20 (d, J= 6.5 Hz, 3H). LCMS [M+H] ⁺ m/z: 509.3.

Example 2: Synthesis of Compound (R)-2-(2-((1-(4-((8-chloro-7-methylquinolin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 2)

Preparation of the key starting material (R)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate (2-2):

Step A Synthesis of Intermediate 2-1

Synthesize by referring to the preparation method of compound 1-3 in Example 1. Add intermediate 1-1 (5.0 g, 14.5 mmol, 1.0 equivalent) to a reaction bottle with a stirrer, replace nitrogen three times, add tetrahydrofuran (40.0 mL), add tri-sec-butyl lithium borohydride (1.0 M in THF, 21.8 mL, 21.8 mmol, 1.5 equivalent) dropwise at -78 ° C, return to room temperature and stir for 3 hours, and monitor with a plate. After the reaction is completed, slowly quench with water at 0 ° C, extract the above mixed solution three times with ethyl acetate (20.0 mL), combine the organic phases, wash twice with saturated sodium chloride solution (40.0 mL), dry with anhydrous sodium sulfate, filter, and remove the solvent by vacuum rotary evaporation. The obtained mixture is separated and purified by silica gel column chromatography to obtain compound 2-1 as a white solid (2.9 g, 8.4 mmol, yield 58%). ¹ H NMR (500 MHz, CD ₃ OD) δ 4.58 (brs, 1H), 3.32-3.06 (m,1H), 1.96-1.91 (m, 3H), 1.77-1.74 (m, 1H), 1.43 (s, 9H), 1.27-1.22 (m, 12H), 1.17- 1.06 (m, 4H). LCMS [M+H] ⁺ m/z: 347.2.

Step B Synthesis of key starting material 2-2

The solid 2-1 (2.9 g, 8.4 mmol, 1.0 equivalent) was dissolved in tetrahydrofuran (20 mL) and water (4 mL), and iodine (1.49 g, 5.88 mmol, 0.7 equivalent) was added under stirring, and the mixture was heated to 70°C for 4 hours and monitored by spot plate. After the reaction, saturated sodium thiosulfate solution (20 mL) and ethyl acetate (10.0 mL) were added to extract the mixed solution three times, the organic phases were combined, washed twice with saturated sodium carbonate solution (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain compound 2-2 (1.93 g, 7.14 mmol, yield 85%) as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 4.06 (brs, 1H), 3.28 (brs, 1H), 2.65-2.61 (m,1H), 1.98-1.96 (m, 2H), 1.79-1.54 (m, 4H), 1.45-1.42 (m, 9H), 1.04 -0.96 (m,7H). LCMS [M+H] ⁺ m/z: 243.2.

2. Preparation of Compound 2:

Referring to the preparation process of compound 1 in Example 1, in step 2, the key starting material compound (S)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate 1-3 was replaced with the key starting material compound (R)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate 2-2, and the other operation steps were similar to obtain compound 2 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.18 (s, 2H), 7.75 (d, J = 8.8 Hz, 1H), 7.37 (d, J = 8.0Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 8.8 Hz, 1H), 6.4 3 (d, J = 6.4Hz, 1H), 5.22 (d, J = 8.7 Hz, 1H), 5.02 (dt, J = 13.2, 6.6 Hz, 1H), 4.91 (t,J = 7.0 Hz, 1H), 4.47 (t, J = 6.3 Hz, 2H), 4.05-3.98 (m , 1H), 3.78 (brs, 1H), 3.33 (s, 2H), 2.54 (s, 3H), 2.28-2.24 (m, 2H), 1.97-1.94 (m, 1H), 1.88-1.84(m, 1H), 1.50-1.48 (m, 1H), 1.36-1.23 (m , 4H), 1.20 (d, J = 6.5 Hz, 3H). LCMS[M+H] ⁺ m/z: 509.3.

Example 3: Synthesis of (S)-2-(2-((1-(4-((8-chloro-7-methylquinolin-2-yl)amino)cyclohexyl)ethyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 3)

1. Preparation of key starting material (S)-tert-butyl (4-(1-(methylamino)ethyl)cyclohexyl)carbamate (3-2):

Step A Synthesis of Intermediate 3-1

Compound 1-3 (1.50 g, 6.19 mmol) and ethyl formate (30.0 mL) were added to the reaction flask, and the mixture was reacted at 50°C for 12 hours. The reaction was detected by LCMS. After the reaction, the solvent was dried under reduced pressure to obtain a white solid 3-1, which was directly used for the next step. LCMS [M+Na] ⁺ m/z: 293.2.

Step B Synthesis of key starting material 3-2

Under nitrogen atmosphere, the above solid (1.60 g, 5.92 mmol) was dissolved in tetrahydrofuran solution (160 mL), and BH ₃ ^. THF (1.0 M, 29.6 mL) was added to the system. The reaction was allowed to react at room temperature for 12 hours and the plate was monitored. After the reaction was completed, water (30 mL) was added dropwise at 0°C to quench the reaction. Sodium hydroxide (1.0 M, 20 mL) and ethyl acetate (100 mL) were added, the organic phase was separated, washed with saturated sodium chloride (30 mL), and dried over anhydrous sodium sulfate. After filtering, the solvent was removed by rotary evaporation under reduced pressure, and the obtained mixture was separated by silica gel column chromatography to obtain product 3-2 (460 mg, 1.80 mmol, yield 29%) as a white solid. ¹ H NMR (400MHz, CDCl ₃ ) δ 4.41 (br s, 1H), 3.81 (br s, 1H), 3.36 (br s, 1H), 2.57 (d, J =6.0 Hz, 2H), 2.36-2.47 (m, 1H), 2.00-2.11 (m, 2H), 1. 70-1.95 (m, 3H), 1.44 (s, 9H), 1.16-1.24 (m, 3H). LCMS [M-56+H] ⁺ m/z: 201.4.

2. Preparation of compound 3:

Step 1 Synthesis of intermediate 3-3

Compound 3-2 (460 mg, 1.80 mmol, 1.0 equivalent) and compound 1-4 (381 mg, 1.80 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (5.0 mL), and N, N-diisopropylethylamine (1.16 g, 9.00 mmol, 5.0 equivalent) was added and stirred at 100 °C overnight. The reaction was monitored by spot plate. After the reaction was completed, it was cooled to room temperature, saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 3-3 (420 mg, 0.97 mmol, yield 54%). LC-MS [M+H] ⁺ m/z: 449.3.

Step 2 Synthesis of intermediate 3-4

Compound 3-3 (100 mg, 0.23 mmol, 1.0 equivalent) was dissolved in methanol (1.0 mL), and a 4 M hydrochloric acid solution in 1, 4-dioxane (1.0 mL) was added. The reaction was allowed to react at room temperature for 3 hours with a plate monitor. When the reaction was complete, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step. The oily compound, 2,8-dichloro-7-methylquinoline (45 mg, 0.23 mmol, 1.0 equivalent), Pd ₂ (dba) ₃ (21 mg, 0.023 mmol, 0.1 equivalent), BINAP (29 mg, 0.046 mmol, 0.2 equivalent), sodium tert-butoxide (44 mg, 0.46 mmol, 2.0 equivalent) were dissolved in tetrahydrofuran (5.0 mL), reacted at 40 °C for 16 hours under nitrogen protection, and the reaction was completed by spot plate monitoring. The reaction was cooled to room temperature, saturated aqueous ammonium chloride solution (15 mL) was added, and ethyl acetate (15 mL) was used for extraction three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The yellow oily compound 3-4 (37 mg, 0.07 mmol, yield 32%) was obtained by separation and purification by silica gel column chromatography. LCMS [M+H] ⁺ m/z: 524.3

Step 3 Synthesis of compound 3

The intermediate 3-4 (37 mg, 0.07 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (1.0 mL) and methanol (1.0 mL), and then a 2 M aqueous sodium hydroxide solution (0.14 mL, 0.28 mmol, 4.0 equivalent) was added and reacted at room temperature. The reaction was monitored by spot plate. After the reaction of the raw material was completed, the reaction solution was neutralized with a 1 M aqueous hydrochloric acid solution to neutrality, extracted three times with dichloromethane (15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to obtain a yellow oil, which was directly used for the next step. The oil was dissolved in N, N-dimethylformamide (1.0 mL), and N, N-diisopropylethylamine (36 mg, 0.28 mmol, 4.0 equivalents), HATU (53 mg, 0.16 mmol, 2.0 equivalents) and 3-oxetaneamine (11 mg, 0.16 mmol, 2.0 equivalents) were added to the reaction solution in sequence. The reaction was carried out at room temperature. After the reaction was completed, saturated aqueous ammonium chloride solution (10 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (15 mL). The organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The white solid synthetic compound 3 (11.2 mg, 0.021 mmol, yield 30%) was obtained by separation and purification by silica gel column chromatography. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.22 (s, 2H), 7.82(d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 6.65(d, J = 8.2 Hz, 1H), 6. 08 (d, J = 7.0 Hz, 1H), 5.10 -4.97 (m, 1H), 4.91 (t, J= 7.1 Hz, 2H), 4.75 -4.64 (m, 1H), 4.47 (t, J = 6.5 Hz, 2H), 3.75-3.67 (m,2H), 3.37 (s, 2H), 2.96 (s, 3H), 2.55 (s, 3H), 2.29-2.27 (m, 1H), 2.17-2.14(m, 1H), 2.03 -1.92 (m, 2H), 1.26-1.20 (m, 7H). LCMS [M+H] ⁺ m/z: 523.3.

Example 4: Synthesis of (R)-2-(2-((1-(4-((8-chloro-7-methylquinolin-2-yl)amino)cyclohexyl)ethyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 4)

1 Preparation of key starting material (R)-tert-butyl (4-(1-(methylamino)ethyl)cyclohexyl)carbamate (4-2):

Step A Synthesis of Intermediate 4-1

Compound 2-3 (1.50 g, 6.19 mmol) and ethyl formate (30 mL) were added to the reaction flask, and the mixture was reacted at 50°C for 12 hours. The reaction was detected by LCMS. After the reaction, the solvent was removed under reduced pressure to obtain a white solid, namely intermediate 4-1, which was directly subjected to the next step. LCMS [M+Na] ⁺ m/z: 293.2.

Step B Synthesis of key starting material 4-2

Under nitrogen atmosphere, 4-1 (1.60 g, 5.92 mmol) was dissolved in tetrahydrofuran solution (160 mL), and BH ₃ ^. THF (1.0 M, 29.6 mL) was added to the system. The reaction was allowed to react at room temperature for 12 hours and monitored by spot plate. After the reaction was completed, water (30 mL) was added dropwise at 0 °C to quench the reaction. Sodium hydroxide (1.0 M, 20 mL) and ethyl acetate (100 mL) were added, the organic phase was separated, washed with saturated sodium chloride (30 mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by rotary evaporation under reduced pressure, and the obtained mixture was separated by silica gel column chromatography to obtain the product 4-2 (394 mg, 1.54 mmol, yield 26%) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 4.33 (br s, 1H), 3.77 (br s, 1H), 3.30 (br d, J = 1.2 Hz, 1H), 2.68 (br t, J = 6.0 Hz, 1H), 2.31 (d, J = 6.0 Hz, 3H), 2.0 0 (br d, J = 13.2 Hz, 2H), 1.58-1.79 (m, 3H), 1.41-1.50 (m, 2H), 1.37 (s, 9H), 1.13 (br d, J = 6.8Hz, 3H). LCMS [M-56+H] ⁺ m/z: 201.4.

2 Preparation of compound 4:

Referring to the preparation process of compound 3 in Example 3, the key starting material 3-2 was replaced with the key starting material compound 4-2 in step 1, and the other operation steps were similar to obtain compound 4 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ8.22 (s, 2H), 7.82 (d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.10 (d, J= 8.0 Hz, 1H), 6.65 (d, J = 8.2 Hz, 1H), 6.0 8 (d, J = 7.0 Hz, 1H), 5.10 -4.97 (m, 1H), 4.91 (t, J = 7.1 Hz, 2H), 4.75 -4.64 (m, 1H), 4.47 (t, J = 6.5 Hz, 2H), 3.75-3.67 (m, 2H), 3.37 (s, 2H), 2.96 LCMS [M+H] ⁺ m/z: 523.3.

Example 5: Synthesis of (S)-2-(2-((1-(4-((8-chloroquinolin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 5)

Referring to the preparation process of compound 1 in Example 1, 2,8-dichloroquinoline was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 5 as a white solid. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ8.82 (d, J = 6.5 Hz, 1H), 8.11 (s, 2H), 7.85 (d, J = 9.0 Hz, 1H), 7.60 (dd, J= 7.5, 1.5 Hz, 1H), 7.56 (dd, J = 8.0, 1.0 Hz, 1H), 7.12 (d, J = 7.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.78 (d, J = 9.0 Hz, 1H), 4.81 – 4.74 (m, 1H), 4.70 (t, J = 6.5 Hz, 2H), 4.42 (t, J = 6.5 Hz, 2H), 3.95– 3.82 (m, 2H), 3.24 (s, 2H), 2.18 – 2.10 (m, 2H), 1.88 – 1.79 (m, 2H), 1.49– 1.41 (m, 1H), 1.26 – 1.20 (m, 2H), 1.18 – 1.15 (m, 2H), 1.09 (d, J = 6.5Hz, 3H). LC-MS [M+H] ⁺ m/z: 495.3.

Example 6: Synthesis of (S)-2-(2-((1-(4-((8-bromoquinolin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 6)

Referring to the preparation process of compound 1 in Example 1, 8-bromo-2-chloroquinoline was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 6 as a white solid. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ8.75 (d, J = 6.6 Hz, 1H), 8.04 (s, 2H), 7.77 (d, J = 8.9 Hz, 1H), 7.72 (dd, J= 7.6, 1.2 Hz, 1H), 7.54 (dd, J = 7.9, 1.1 Hz, 1H), 7.08 (d, J = 6.6 Hz, 1H), 6.95 (t, J = 7.7 Hz, 1H), 6.78 (s, 1H), 6.69 (d, J = 8.9 Hz, 1H), 4.74-4.66(m, 1H), 4.64 (t, J = 6.7 Hz, 2H ), 4.35 (t, J = 6.3 Hz, 2H), 3.84-3.76 (m,2H) 3.17 (s, 2H), 2.13-2.09 (m, 2H), 1.81-1.75 (m, 2H), 1.41-1.38 (m, 1H),1.38-1.05 (m, 4H), 1.03 ( d, J = 6.5 Hz, 3H).. LCMS [M+H] ⁺ m/z: 539.2.

Example 7: Synthesis of (S)-2-(2-((1-(4-((8-bromoquinazolin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 7)

Referring to the preparation process of compound 1 in Example 1, 8-bromo-2-chloroquinazoline was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 7 as a white solid. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 9.00 (s, 1H), 8.75 (d, J = 6.5 Hz, 1H), 8.04 (s, 2H), 7.94 (d, J = 7.5Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 7.0 Hz, 1H), 7.05 (t, J = 7.5Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 4.74 – 4.66 (m, 1H), 4.64 (t, J = 6.5 Hz, 2H), 4.35 (t, J = 6.5 Hz, 2H), 3.86 – 3 .70 (m, 2H), 3.17 (s, 2H), 2.07 – 1.99 (m, 1H), 1.81 – 1.73 (m, 2H), 1.44 – 1.33 (m, 1H), 1.27 – 1.19 (m, 2H), 1.11 – 1.03 (m, 2H), 1.02 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 540.3.

Example 8: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 8)

Preparation of compound 8:

Step 1 Synthesis of intermediate 8-1

Compound 1-5 (491 mg, 1.1 mmol, 1.0 equivalent) was dissolved in 5.0 mL methanol, and 2.0 mL of 4 M hydrochloric acid in 1,4-dioxane solution was added. The reaction was carried out at room temperature for 3 h, and the plate was monitored. After the reaction of the raw material was completed, the solvent was dried by spin drying, and the obtained oily compound was directly used for the next step. Then, the above oily compound, 2-chloro-8-methoxy-1,7-naphthyridine (240 mg, 1.1 mmol, 1.0 equivalent), BINAP (140 mg, 0.23 mmol, 0.2 equivalent), Pd ₂ (dba) ₃ (110 mg, 0.12 mmol, 0.1 equivalent) and sodium tert-butoxide (434 mg, 4.5 mmol, 4.0 equivalent) were added to a 25 mL double-necked flask. Under a nitrogen atmosphere, the nitrogen was replaced three times, and then tetrahydrofuran (10 mL) was added, and the reaction solution was stirred at room temperature overnight. After the reaction was completed, saturated aqueous ammonium chloride solution (10 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (10 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 8-1 (47 mg, 0.1 mmol, yield 8%), [M+H] ⁺ m/z: 496.3.

Step 2 Synthesis of intermediate 8-2

First, the intermediate 8-1 (47 mg, 0.1 mmol, 1.0 equivalent) was dissolved in 5.0 mL of ethyl acetate, and then a 4 M hydrochloric acid ethyl acetate solution (0.1 mL, 0.4 mmol, 4.0 equivalent) was added, and the reaction was carried out at 40 °C for 20 h. Then, ethanol (2.0 mL) was added to the reaction solution, and the reaction was refluxed at 90 °C for 20 h. After the reaction of the raw materials was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step. Then, the above oily compound was dissolved in 2.0 mL of phosphorus oxychloride and reacted at 110 °C for 1.5 h. After the reaction was completed, the phosphorus oxychloride was removed under reduced pressure, and the reaction solution was diluted with ethyl acetate, and then adjusted to neutrality with a saturated aqueous solution of NaHCO ₃ at 0 °C. The above mixed solution was extracted three times with ethyl acetate (4.0 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 obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 8-2 (35 mg, 0.08 mmol, yield 75%). [M+H] ⁺ m/z: 496.3.

Step 3 Synthesis of compound 8

First, the intermediate 8-2 (35 mg, 0.08 mmol, 1.0 equivalent) was dissolved in a mixed solvent of 1.0 mL tetrahydrofuran and 1.0 mL methanol, and then 2 M sodium hydroxide aqueous solution (0.2 mL, 0.4 mmol, 5.0 equivalent) was added, and the reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw material was completed, it was neutralized with 1 M HCl aqueous solution until neutral, and the mixed solution was extracted three times with dichloromethane (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained oily compound was directly subjected to the next step of reaction. Then, the above oily compound was dissolved in 1.0 mL N, N-dimethylformamide, and compounds N, N-diisopropylethylamine (39 mg, 0.3 mmol, 4.0 equivalents), HATU (57 mg, 0.15 mmol, 2.0 equivalents) and 3-oxetaneamine (22 mg, 0.3 mmol, 2.0 equivalents) were added in sequence, and the resulting reaction solution was reacted at room temperature for 1 h. After the reaction was completed, saturated aqueous ammonium chloride solution (2.0 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, the organic phases were washed with saturated aqueous sodium chloride solution (10 mL), the organic phases were dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain 8 (6 mg, 0.01 mmol, yield 16%) as a white solid compound. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.76 (d, J = 6.5 Hz, 1H), 8.05 (s, 2H), 7.89 (d, J = 5.0 Hz, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.49 (d, J= 5.0 Hz, 1H), 7 .43 (d, J = 6.5 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.79 (d, J= 9.0 Hz, 1H), 4.75 – 4.67 (m, 1H), 4.64 (t, J = 6.5 Hz, 2H), 4.35 (t, J =6.5 Hz, 2H ), 3.87 – 3.77 (m, 2H), 3.18 (s, 2H), 2.13 – 2.02 (m, 2H), 1.83 –1.72 (m, 2H), 1.44 – 1.34 (m, 1H), 1.14 – 1.09 (m, 4H), 1.03 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 496.3.

Example 9: Synthesis of 2-(2-(((4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)(cyclopropyl)methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 9)

1. Preparation of the key starting material (tert-butyl 4-(amino(cyclopropyl)methyl)cyclohexyl)carbamate (9-5)

Step A Synthesis of Intermediate 9-1

Trans-4-((tert-butyloxycarbonyl)amino)cyclohexane-1-carboxylic acid (10 g, 41.0 mmol, 1.0 equivalent), dimethylhydroxylamine hydrochloride (4.4 g, 45.0 mmol, 1.1 equivalent), DMTMM (14.4 g, 45.0 mmol, 1.1 equivalent), NMM (8.3 g, 82.0 mmol, 2.0 equivalent) were dissolved in tetrahydrofuran (100 mL) and methanol (100 mL), and the reaction was carried out at 25 °C for 4 h. After the reaction was completed by spot plate monitoring, water (200 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 9-1 (10 g, 35.0 mmol, yield 85%), LC-MS [M+H-56] ⁺ m/z: 231.0.

Step B Synthesis of Intermediate 9-2

The intermediate 9-1 (10 g, 35.0 mmol) was added to a 500 mL three-necked flask equipped with a stirrer. Under a nitrogen atmosphere, the flask was evacuated three times, and then tetrahydrofuran (200 mL) was added. The reaction system was cooled to -10 °C in a low-temperature bath, and 3 M cyclopropane magnesium bromide (14 mL) was added dropwise with a constant pressure dropping funnel. After the addition was complete, the reaction was carried out at 0 °C for 4 hours, and the reaction was monitored by a plate. A saturated ammonium chloride solution (200 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (200 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The crude product was purified by slurrying with 50 mL of petroleum ether to obtain the intermediate 9-2 (6 g, 22.5 mmol, yield 64%), LC-MS [M+H] ⁺ m/z: 268.3.

Step C Synthesis of Intermediate 9-3

Intermediate 9-2 (4.0 g, 15.0 mmol, 1.0 equivalent), tetraethyl titanate (2.2 g, 18.0 mmol, 1.2 equivalent), S-tert-butylsulfenamide (5.1 g, 22.5 mmol, 1.5 equivalent) were added to a 100 mL three-necked flask equipped with a stirrer. The mixture was evacuated three times under a nitrogen atmosphere, and then tetrahydrofuran (60 mL) was added. The reaction was carried out at 80 °C for 12 hours, and the reaction was monitored to be complete by spot plate. A saturated ammonium chloride solution (100 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (100 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 9-3 (1.0 g, 2.7 mmol, yield 18%), LC-MS [M+H] ⁺ m/z: 371.3.

Step D Synthesis of Intermediate 9-4

Intermediate 9-3 (500 mg, 1.4 mmol, 1.0 equivalent) was added to a 20 mL three-necked flask equipped with a stirrer. Under a nitrogen atmosphere, the flask was evacuated three times, and then tetrahydrofuran (10 mL) and water (0.2 mL) were added. The reaction system was cooled to -50 °C in a low-temperature bath, and sodium borohydride (158 mg, 4.2 mmol, 3.0 equivalent) was added in batches. After the addition was complete, the reaction was carried out at -50 °C for 2 hours, and the reaction was monitored by a plate. A saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The crude product was purified by recrystallization from dichloromethane (5.0 mL) and petroleum ether (15 mL) to obtain intermediate 9-4 (300 mg, 0.81 mmol, yield 58%), LC-MS [M+H] ⁺ m/z: 373.2.

Step E Synthesis of Intermediate 9-5

Intermediate 9-4 (300 mg, 0.81 mmol, 1.0 equivalent) and I ₂ (1 M, 1.6 mL, 2.0 equivalent) were dissolved in tetrahydrofuran (5.0 mL) and methanol (1.0 mL), and the reaction was carried out at 50 °C overnight. The reaction was monitored by spot plate. 1 M sodium hydroxide aqueous solution (2.0 mL) and saturated sodium thiosulfate (2.0 mL) were added to quench the reaction, and 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 vacuum rotary evaporation to obtain intermediate 9-5 (200 mg, 0.74 mmol, yield 18%). LC-MS [M+H] ⁺ m/z: 269.3.

2 Preparation process of compound 9

Referring to the preparation process of compound 8 in Example 8, the key starting material (tert-butyl 4-(amino(cyclopropyl)methyl)cyclohexyl)carbamate was used to replace (S)-tert-butyl (4-(1-aminoethyl)cyclohexyl)carbamate, and the other operation steps were similar to obtain compound 9 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.15 (s, 2H), 8.04 (d, J= 5.0 Hz, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.34 (d, J = 5.0 Hz, 1H), 6.80 (d, J= 9.0 Hz, 1H), 6. 28 (d, J = 7.5 Hz, 1H), 5.25 (d, J = 9.0 Hz, 1H), 5.06-5.04(m Hz, 1H), 4.91 (t, J = 7.0 Hz, 2H), 4.47 (t, J = 6.5 Hz, 2H), 3.87 (brs,1H), 3.40 ( m, 1H), 3.34 (s, 2H), 2.28 (d, J = 12.0 Hz, 2H), 2.10 – 1.93 (m,3H), 1.74 – 1.61 (m, 3H), 1.51 – 1.35 (m, 2H), 0.97 – 0.89 (m, 1H), 0.60 (m,1H), 0.43 (m, 1H), 0.32 (m, 2H). LC-MS [M+H] ⁺ m/z: 522.3.

Example 10: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 10)

Preparation of the key starting material 8-chloro-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine (10-5):

Step A Synthesis of Intermediate 10-1

2-Methylmercapto-5-bromopyrimidine-4-carboxylic acid methyl ester (10.0 g, 38.00 mmol) was dissolved in a mixed solvent of 1,4-dioxane (60 mL) and water (12 mL), and potassium carbonate (15.8 g, 114.00 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.7 g, 2.32 mmol) and (E)-1-ethoxyvinyl-2-boronic acid pinacol ester (11.3 g, 57.00 mmol) were added in sequence. The reaction mixture was stirred at 90 °C for 2 hours under a nitrogen system. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, poured into water (100 mL), extracted three times with ethyl acetate (80 mL), and the organic phases were combined and washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether:ethyl acetate = 20:1 to 10:1) to give intermediate 10-1 (7.2 g, yield: 75%) as a yellow oil. LC-MS [M+H] ⁺ m/z: 255.2.

Step B Synthesis of Intermediate 10-2

10-1 (6.5 g, 25.56 mmol) was dissolved in a methanol solution of amine (20 mL, 7 M). The reaction mixture was stirred at 85 °C for 2 hours. After the reaction was completed by TLC monitoring, the reaction was concentrated under reduced pressure to obtain intermediate 10-2 (6.5 g, crude product), which was used directly in the next step without further purification.

Step C Synthesis of Intermediate 10-3

10-2 (6.5 g, 27.20 mmol) was dissolved in toluene (100 mL), and p-toluenesulfonic acid (468.0 mg, 2.72 mmol) was added to the reaction solution. The reaction mixture was stirred at 90 °C for 2 hours. After the reaction was completed as monitored by LCMS, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, dichloromethane: methanol = 30:1 to 20:1) to obtain intermediate 10-3 (4.2 g, yield: 80%) as a yellow solid. LC-MS [M+H] ⁺ m/z: 194.3.

Step D Synthesis of Intermediate 10-4

10-3 (4.1 g, 21.22 mmol) was dissolved in acetonitrile (100 mL), and phosphorus oxychloride (5.9 mL, 63.70 mmol) was added to the reaction solution. The reaction mixture was stirred at 70 °C for 2 hours. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature and poured into ice water (130 mL). The aqueous phase was adjusted to pH 7-8 with saturated sodium bicarbonate solution, extracted three times with ethyl acetate (50 mL), and the organic phases were combined and washed twice with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain intermediate 10-4 (3.6 g, yield: 80%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ )δ 9.22 (s, 1H), 8.40 (d, J = 5.4 Hz, 1H), 7.60 (d, J = 5.4 Hz, 1H), 2.75 (s,3H). LC-MS [M+H] ⁺ m/z: 212.2.

Step E Synthesis of Intermediate 10-5

10-4 (2.0 g, 9.45 mmol) was dissolved in dichloromethane (20 mL), and m-chloroperbenzoic acid (4.6 g, 22.68 mmol) was added to the reaction solution under ice bath conditions. The reaction mixture was stirred at 25 °C for 2 hours. LCMS monitored the completion of the reaction, and the reaction solution was poured into water (30 mL), extracted three times with ethyl acetate (20 mL), and the organic phases were combined and washed twice with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10:1 to 1:5) to obtain compound 10-5 (1.4 g, yield: 61%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.74 (s, 1H), 8.74 (d, J = 5.5 Hz, 1H), 7.87 (d,J = 5.5 Hz, 1H), 3.57 (s, 3H). LC-MS [M+H] ⁺ m/z: 244.0.

2 Preparation process of compound 10

Referring to the preparation process of compound 1 in Example 1, 8-chloro-2-(methylsulfonyl)pyrido[3,4-d]pyrimidine was used to replace 2,8-dichloro-7-methylquinoline in step 3, and other subsequent operation steps were similar to obtain compound 10 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 9.05-9.03 (m, 1H), 8.20 (s, 2H), 8.10 (d, J = 5.5Hz, 1H), 7.41 (d, J = 5.0 Hz, 1H), 6.27 (d, J = 6.5 Hz, 1H), 5.64-5. ( m, 1H), 3.36 (s,2H), 2.38 – 2.15 (m, 2H), 2.02 – 1.95 (m, 1H), 1.90 – 1.84 (m, 2H), 1.57 –1.48 (m, 1H), 1.31 – 1.27 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 497.3.

Example 11: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)(methyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 11)

Preparation of compound 11:

Step 1 Synthesis of intermediate 11-1

Compound 3-3 (100 mg, 0.22 mmol, 1.0 equivalent) was dissolved in methanol (1.0 mL), and a solution of hydrochloric acid in 1,4-dioxane (1.0 mL) was added. The reaction was allowed to react at room temperature for 3 hours with a spot plate for monitoring. When the reaction was complete, the solvent was dried and the obtained oily compound was directly used for the next step. The oily compound and 2-chloro-8-methoxy-1,7-naphthyridine (43 mg, 0.22 mmol, 1.0 equivalent), BINAP (14 mg, 0.044 mmol, 0.2 equivalent), Pd ₂ (dba) ₃ (20 mg, 0.022 mmol, 0.1 equivalent) and sodium tert-butoxide (42 mg, 0.44 mmol, 2.0 equivalent) were added to tetrahydrofuran (5.0 mL). Under N ₂ protection, the reaction was carried out at 40 ° C for 16 hours. The reaction was monitored by spot plate. The reaction was cooled to room temperature, saturated aqueous ammonium chloride solution (15 mL) was added, and ethyl acetate (15 mL) was used for extraction three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The yellow oily compound 11-1 (47 mg, 0.09 mmol, yield 41%) was obtained by separation and purification by silica gel column chromatography. LCMS [M+H] ⁺ m/z: 507.3.

Step 2 Synthesis of intermediate 11-2

Compound 11-1 (47 mg, 0.09 mmol, 1.0 equivalent) was dissolved in ethyl acetate (5.0 mL), and then a 4 M hydrochloric acid solution in ethyl acetate (0.36 mL, 0.36 mmol, 4.0 equivalent) was added. After reacting at 40 °C for 20 hours, ethanol (5.0 mL) was added and refluxed at 90 °C for 2 hours. The raw material was reacted after monitoring by the spot plate, and the solvent was removed under reduced pressure. The obtained oily compound was directly used for the next step. The oily compound was dissolved in phosphorus oxychloride (2.0 mL) and reacted at 110 °C for 1.5 hours. The reaction was completed by monitoring by the spot plate, and phosphorus oxychloride was removed under reduced pressure. An ice-cold saturated sodium bicarbonate aqueous solution was added to the reaction solution, and the reaction solution was adjusted to neutral, and extracted with ethyl acetate (15 mL) three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The compound 11-2 was separated and purified by silica gel column chromatography to obtain a light yellow oily compound (35 mg, 0.07 mmol, yield 78%), LCMS [M+H] ⁺ m/z: 483.3.

Step 3 Synthesis of compound 11

Compound 11-2 (35 mg, 0.07 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (1.0 mL) and methanol (1.0 mL), and a 2 M aqueous sodium hydroxide solution (0.14 mL, 0.28 mmol, 4.0 equivalent) was added. The reaction was allowed to react at room temperature for 2 hours with a spot plate monitor. After the reaction of the raw materials was completed, the reaction solution was adjusted to neutrality with a 1 M aqueous hydrochloric acid solution, extracted three times with dichloromethane (15 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained oily compound was directly used for the next step. The oily compound was dissolved in N, N-dimethylformamide (1.0 mL), and N, N-diisopropylethylamine (36 mg, 0.28 mmol, 4.0 equivalents), HATU (27 mg, 0.14 mmol, 2.0 equivalents) and 3-oxetaneamine (21 mg, 0.14 mmol, 2.0 equivalents) were added thereto in sequence. After reacting at room temperature for 1 hour, the reaction was monitored by spot plate. When the reaction was completed, saturated aqueous ammonium chloride solution (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (15 mL) three times. The organic phases were combined, washed with saturated aqueous sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The mixture was separated and purified by silica gel column chromatography to obtain a white solid 11 (7.8 mg, 0.02 mmol, yield 21%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.22 (s,2H), 8.04 (d, J = 5.2 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.34 (d, J = 5.3 Hz,1H), 6.81 (d, J = 8.9 Hz, 1H), 6.19 (d, J = 7.1 Hz, 1H), 5.09 - 5.01 (m, 1H), 4.91 (t, J = 7.1 Hz, 2H), 4.76 -4.69 (m, 1H), 4.46 (t, J = 6.4 Hz, 2H), 3.86 (s, 1H), 3.36 (s, 2H), 2.97 (s, 3H), 2.36 - 2.28 (m, 1H), 2.23 - 2.16 (m,1H), 2.00 - 1.91 (m, 1H), 1.79 - 1.65 (m, 2H), 1.59 - 1.47 (m, 1H), 1.38 -1.23 (m, 3H), 1.22 ( d, J = 6.8 Hz, 3H). LCMS [M+H] ⁺ m/z: 510.3.

Example 12: Synthesis of (S)-2-(6-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyridin-3-yl)-N-(oxetane-3-yl)acetamide (Compound 12)

Preparation of compound 12:

Step 1 Synthesis of intermediate 12-1

Compound 1-3 (265 mg, 1.1 mmol, 1.2 equivalents), methyl 2-(6-bromopyridin-3-yl)acetate (210 mg, 0.91 mmol, 1.0 equivalents), cuprous iodide (52 mg, 0.27 mmol, 0.3 equivalents), L-proline (63 mg, 0.55 mmol, 0.6 equivalents) and potassium phosphate (386 mg, 1.82 mmol, 2.0 equivalents) were added to a 10 mL double-necked flask equipped with a stirrer, and the mixture was evacuated three times under a nitrogen atmosphere, and then dimethyl sulfoxide (3.0 mL) was added, and the reaction solution was stirred at 100 °C overnight. The reaction was monitored by spot plate. After the reaction was completed, the mixture was cooled to room temperature, saturated ammonium chloride solution (10 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (20 mL), the organic phases were combined, the organic phases were washed with saturated sodium chloride aqueous solution, the organic phases were dried with anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 12-1 (74 mg, 0.19 mmol, yield 17%). LC-MS [M+H] ⁺ m/z: 392.3.

Step 2 Synthesis of intermediate 12-2

The intermediate 12-1 (98 mg, 0.25 mmol, 1.0 equivalent) was dissolved in methanol (2.0 mL), and a 4 M hydrochloric acid solution in ethyl acetate (0.25 mL) was added. The reaction was carried out at room temperature for 3 h with spot plate monitoring. After the reaction of the raw material was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step.

The above oily compound and 2-chloro-8-methoxy-1,7-naphthyridine (46 mg, 0.25 mmol, 1.0 equivalent) were dissolved in dimethyl sulfoxide (1.0 mL), potassium carbonate (104 mg, 0.75 mmol, 3.0 equivalent) was added and stirred at 100 °C overnight. The reaction was monitored by spot plate. After the reaction was completed, it was cooled to room temperature, saturated ammonium chloride solution (5.0 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 12-2 (20 mg, 0.044 mmol, yield 18%). LC-MS [M+H] ⁺ m/z: 450.3.

Step 3 Synthesis of intermediate 12-3

First, the intermediate 12-2 (20 mg, 0.044 mmol, 1.0 equivalent) was dissolved in ethyl acetate (1.0 mL), and then a 4 M hydrochloric acid ethyl acetate solution (0.044 mL, 0.18 mmol, 4.0 equivalent) was added, and the mixture was reacted at 40 °C for 20 h. Subsequently, ethanol (1.0 mL) was added to the reaction solution, and the mixture was refluxed at 90 °C for 20 h. After the reaction of the raw material was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step.

Then, the above oily compound was dissolved in phosphorus oxychloride (1.0 mL) and reacted at 110 °C for 1.5 h. The reaction was monitored by spot plate. After the reaction was completed, the phosphorus oxychloride was removed under reduced pressure, the reaction solution was diluted with ethyl acetate (5.0 mL), and then adjusted to neutral with a saturated aqueous solution of NaHCO ₃ at 0 °C. The mixed solution was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 12-3 (6 mg, 0.013 mmol, yield 30%). LC-MS [M+H] ⁺ m/z: 468.3.

Step 4 Synthesis of compound 12

First, the intermediate 12-3 (6 mg, 0.013 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (0.5 mL) and methanol (0.5 mL), and then a 2M sodium hydroxide aqueous solution (0.025 mL, 0.05 mmol, 5.0 equivalent) was added, and the reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw material was completed, the reaction was neutralized with a 1 M HCl aqueous solution until it was neutral, and the mixed solution was extracted three times with dichloromethane (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained oily compound was directly subjected to the next step of reaction.

The above oily compound was dissolved in N, N-dimethylformamide (0.5 mL), and compound N, N-diisopropylethylamine (7 mg, 0.052 mmol, 4.0 equivalents), HATU (10 mg, 0.026 mmol, 2.0 equivalents) and 3-oxetaneamine (2 mg, 0.026 mmol, 2.0 equivalents) were added in sequence, and the resulting reaction solution was reacted at room temperature for 1 h. After the reaction was completed, a saturated ammonium chloride solution (2.0 mL) was added to quench the reaction, and the above mixed solution was extracted three times with ethyl acetate (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The resulting mixture was separated and purified by silica gel column chromatography to obtain compound 12 (2 mg, 0.004 mmol, yield 31%) as a white solid compound. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.82 (d, J = 6.5 Hz, 1H), 8.01 (d, J = 5.0 Hz, 1H), 7.97 (d, J =9.0 Hz, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 5.0 Hz, 1H), 7.55 (d, J =7.0 Hz, 1H), 7.30 (dd, J = 8.5, 2.0 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H), 6.49 (d, J = 8.5 Hz, 1H), 6.34 – 6.17 (m, 1H), 4.85 – 4.78 (m, 1H), 4.75 (t, J =6.5 Hz, 2H), 4.46 (t, J = 6.0 Hz, 2H), 3.96 – 3.88 (m, 2H), 3.27 (s, 2H), 2.27 – 2.14 (m, 2H), 1.95 – 1.83 (m, 2H), 1.54 – 1.47 (m, 1H), 1.28 – 1.21 (m, 4H), 1.13 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 495.3.

Example 13: Synthesis of 2-(2-(((S)-1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)propanamide (Compound 13)

Preparation of compound 13:

Step 1 Synthesis of intermediate 13-1

Compound 1-4 (300 mg, 1.4 mmol, 1.0 equivalent) was added to a 10 mL double-necked flask equipped with a stirrer. The mixture was evacuated three times under a nitrogen atmosphere, and then N, N-dimethylformamide (3.0 mL) was added. The reaction system was cooled to -30°C in a low-temperature bath, and NaH (85 mg, 2.1 mmol, 1.5 equivalent) and iodomethane (298 mg, 2.1 mmol, 1.5 equivalent) were added to react for 1 h, and the reaction was monitored by spot plate. A saturated ammonium chloride solution (10 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (20 mL). The organic phases were combined, washed with a saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 13-1 (60 mg, 0.27 mmol, yield 19%), LC-MS [M+H] ⁺ m/z: 227.2.

Step 2 Synthesis of intermediate 13-2

Compound 13-1 (225 mg, 1.0 mmol, 1.0 equivalent) and compound 1-3 (222 mg, 1.2 mmol, 1.2 equivalent) were dissolved in DMSO (3.0 mL), and N,N-diisopropylethylamine (645 mg, 5.0 mmol, 5.0 equivalent) was added. The reaction was carried out at 100°C overnight, and the reaction was monitored by spot plate. A saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 13-2 (200 mg, 0.45 mmol, yield 45%), LC-MS [M+H] ⁺ m/z: 449.3.

Step 3 Synthesis of intermediate 13-3

Intermediate 13-2 (200 mg, 0.45 mmol, 1.0 equivalent) was dissolved in methanol (3.0 mL), and a 4M hydrochloric acid solution in ethyl acetate (0.25 mL) was added. The reaction was carried out at room temperature for 3 h with spot plate monitoring. After the reaction of the raw material was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step.

The above oily compound and 2-chloro-8-methoxy-1,7-naphthyridine (87 mg, 0.45 mmol, 1.0 equivalent), BINAP (56 mg, 0.09 mmol, 0.2 equivalent), Pd ₂ (dba) ₃ (46 mg, 0.05 mmol, 0.1 equivalent) and sodium tert-butoxide (172 mg, 1.8 mmol, 4.0 equivalent) were added to a 25 mL double-necked flask, and nitrogen was replaced three times under a nitrogen atmosphere, and then THF (4.0 mL) was added, and the reaction solution was stirred at 40°C overnight. The reaction was monitored by spot plate. After the reaction was completed, it was cooled to room temperature, and a saturated ammonium chloride solution (5.0 mL) was added to quench the reaction. The above 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 vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 13-3 (23 mg, 0.045 mmol, yield 10%), LC-MS [M+H] ⁺ m/z: 507.3.

Step 4 Synthesis of intermediate 13-4

Intermediate 13-3 (45 mg, 0.09 mmol, 1.0 equivalent) was dissolved in ethyl acetate (5.0 mL), and then a 4M hydrochloric acid ethyl acetate solution (0.088 mL, 0.36 mmol, 4.0 equivalent) was added, and the mixture was reacted at 40°C for 20 h. Then, ethanol (5.0 mL) was added to the reaction solution, and the mixture was refluxed at 90°C for 20 h. After the reaction of the raw material was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step.

The above oily compound was dissolved in phosphorus oxychloride (1.0 mL) and reacted at 110°C for 1.5 h. The reaction was monitored by spot plate. After the reaction was completed, the phosphorus oxychloride was removed under reduced pressure, the reaction solution was diluted with EA, and then adjusted to neutral with a saturated aqueous solution of NaHCO ₃ at 0°C. The mixed solution was extracted three times with ethyl acetate (3.0 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 13-4 (40 mg, 0.08 mmol, yield 92%), LC-MS [M+H] ⁺ m/z: 483.3.

Step 5 Synthesis of compound 13

The intermediate 13-4 (40 mg, 0.08 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (0.5 mL) and methanol (0.5 mL), and then a 2M sodium hydroxide aqueous solution (0.20 mL, 0.42 mmol, 5.0 equivalent) was added. The reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw material was completed, the reaction was neutralized with a 1M hydrochloric acid aqueous solution. The mixed solution was extracted three times with dichloromethane (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained oily compound was directly subjected to the next step of reaction.

The above oily compound was dissolved in N, N-dimethylformamide (1.0 mL), and N, N-diisopropylethylamine (43 mg, 0.33 mmol, 4.0 equivalents), HATU (63 mg, 0.17 mmol, 2.0 equivalents) and 3-oxetaneamine (12 mg, 0.17 mmol, 2.0 equivalents) were added in sequence, and the 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, and the mixed solution was extracted three times with ethyl acetate (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain compound 13 (24 mg, 0.05 mmol, yield 57%) as a white solid compound. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.21 (s, 2H), 8.01 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 5.0Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.54-6.49 (m, 1H), 5.28 (d, J = 9.0 Hz,1H), 5.10 (brs, 1H), 5.04-4.95 (m, 1H), 4.92-4.84 (m, 2H), 4.46 (t, J = 6.5Hz, 1H), 4.42 (t, J = 6.5 Hz, 1H), 4.06-3.96 (m, 1H), 3.87-3.85 (m, 1H), 3.36(q, J = 7.0 Hz, 1H), 2.34 – 2.13 (m, 2H), 2.03 – 1.81 ( m, 2H), 1.46 (d, J =7.0 Hz, 3H), 1.37 – 1.25 (m, 4H), 1.18 (d, J = 6.5 Hz, 3H).LC-MS [M+H] ⁺ m/z:510.3.

Example 14: Synthesis of (S)-8-chloro-N-(4-(1-((5-(oxetane-3-ylamino)methyl)pyrimidin-2-yl)amino)ethyl)cyclohexyl)-1,7-naphthyridin-2-amine (Compound 14)

Preparation of compound 14:

Step 1 Synthesis of intermediate 14-1

Compound 2-chloropyrimidine-5-carboxylic acid methyl ester (480 mg, 2.0 mmol, 1.0 equivalent) was dissolved in dimethyl sulfoxide (10 mL), and compound 1-3 (340 mg, 2.0 mmol, 1.0 equivalent) and N, N-diisopropylethylamine (1290 mg, 10.0 mmol, 5.0 equivalent) were added. The resulting reaction solution was reacted at 100 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and a saturated ammonium chloride solution (10 mL) was added to quench the reaction. 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 vacuum rotary evaporation. The resulting mixture was separated and purified by silica gel column chromatography to obtain intermediate 14-1 (414 mg, 1.1 mmol, yield 55%). LC-MS [M+H]+ m/z: 379.3.

Step 2 Synthesis of intermediate 14-2

First, the intermediate 14-1 (414 mg, 1.1 mmol, 1.0 equivalent) was dissolved in 5.0 mL of methanol, and 2.0 mL of 4 M hydrochloric acid in 1, 4-dioxane solution was added. The reaction was carried out at room temperature for 3 h and the plate was monitored. After the reaction of the raw material was completed, the solvent was dried and the obtained oily compound was directly used for the next step.

Then, the above oily compound and 2-chloro-8-methoxy-1,7-naphthyridine (212 mg, 1.09 mmol, 1.0 equivalent), BINAP (134 mg, 0.22 mmol, 0.2 equivalent), Pd ₂ (dba) ₃ (100 mg, 0.11 mmol, 0.1 equivalent) and sodium tert-butoxide (415 mg, 4.3 mmol, 4.0 equivalent) were added to a 25 mL double-necked flask, evacuated three times under a nitrogen atmosphere, and then tetrahydrofuran (10 mL) was added and stirred at room temperature overnight. After the reaction was completed, it was cooled to room temperature, saturated aqueous ammonium chloride solution (20 mL) was added to quench the reaction, and it was extracted three times with ethyl acetate (15 mL), the organic phases were combined, the organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 14-2 (186 mg, 0.43 mmol, yield 41%). LC-MS [M+H]+ m/z: 437.3.

Step 3 Synthesis of intermediate 14-3

First, the intermediate 14-2 (186 mg, 0.43 mmol, 1.0 equivalent) was dissolved in 5.0 mL of ethyl acetate, and then a 4 M hydrochloric acid ethyl acetate solution (0.43 mL, 1.72 mmol, 4.0 equivalent) was added, and the reaction was carried out at 40 °C for 20 h. Subsequently, 5.0 mL of ethanol was added to the reaction solution, and the reaction was refluxed at 90 °C for 20 h. After the reaction of the raw material was completed, the solvent was removed under reduced pressure, and the obtained oily compound was directly used for the next step.

Then, the above oily compound was dissolved in 2.0 mL of phosphorus oxychloride and reacted at 110 °C for 1.5 h. The reaction was monitored by spot plate. After the reaction was completed, the phosphorus oxychloride was removed under reduced pressure, the reaction solution was diluted with ethyl acetate (5.0 mL), and then adjusted to neutral with a saturated aqueous solution of NaHCO3 at 0 °C. 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 vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 14-3 (47 mg, 0.1 mmol, yield 24%). LC-MS [M+H]+ m/z: 455.3.

Step 4 Synthesis of compound 14

Under nitrogen atmosphere, the intermediate 14-3 (47 mg, 0.1 mmol, 1.0 equivalent) was dissolved in 1.0 mL of tetrahydrofuran, cooled to -78 °C, and then 1.5 M diisobutylaluminum hydride toluene solution (0.13 mL, 0.2 mmol, 2.0 equivalent) was slowly added. The reaction was monitored by spot plate. After the reaction was completed, 2 M sodium hydroxide solution (2.0 mL) was added to quench the reaction. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained oily compound was directly subjected to the next step of reaction. Under nitrogen protection, the above oily compound (13 mg, 0.03 mmol, 1.0 equivalent) and 3-oxetaneamine (4.4 mg, 0.06 mmol, 2.0 equivalent) were dissolved in 1.0 mL of methanol and stirred for 1 h. Then, acetic acid (1.8 mg, 0.03 mmol, 1.0 equivalent) and sodium cyanoborohydride (4 mg, 0.06 mmol, 2.0 equivalent) were added and reacted at room temperature overnight. The reaction was monitored by spot plate. After the reaction was completed, water (5.0 mL) was added to quench the reaction. The mixed solution was extracted three times with ethyl acetate (5.0 mL), and the organic phase was collected, dried over anhydrous sodium sulfate, and then the solvent was removed by rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain 14 (1 mg, 0.002 mmol, yield 7%). ¹ H NMR (500MHz, CDCl ₃ ) δ 8.22 (s, 2H), 8.05 (d, J = 5.0Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.34 (d, J = 5.5 Hz, 1H), 6.80 (d, J = 9.0 Hz, 1H), 5.08 ( d, J = 9.0 Hz, 1H), 5.02 – 4.91 (m, 1H), 4.82 (t, J = 7.0 Hz, 2H), 4.45 (t, J = 6.5 Hz, 2H), 4.08 – 3.97 (m, 2H), 3.57 (s, 2H), 2.33 – 2.26 (m, 2H), 2.00 – 1.95 (m, 1H), 1.92 – 1.85 (m, 2H), 1.37 – 1.28 (m, 4H), 1.21 (d, J = 6.5 Hz, 3H). LC-MS [M+H]+ m/z: 468.3.

Example 15: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetan-3-ylmethyl)acetamide (Compound 15)

Preparation of compound 15:

The intermediate 8-2 (6 mg, 0.013 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (0.5 mL) and methanol (0.5 mL), and then a 2 M sodium hydroxide aqueous solution (0.025 mL, 0.05 mmol, 4.0 equivalent) was added. The reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw material was completed, the reaction was neutralized with a 1 M hydrochloric acid aqueous solution until it was neutral. The mixed solution was extracted three times with dichloromethane (10 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained oily compound was directly subjected to the next step of reaction. The above oily compound was dissolved in N, N-dimethylformamide (0.5 mL), and compounds N, N-diisopropylethylamine (7 mg, 0.052 mmol, 4.0 equivalents), HATU (10 mg, 0.026 mmol, 2.0 equivalents) and 3-oxetaneamine (2.3 mg, 0.026 mmol, 2.0 equivalents) were added in sequence, and the resulting reaction solution was reacted at room temperature for 1 h. After the reaction was completed, a saturated ammonium chloride solution (1.0 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (5.0 mL), the organic phases were combined, washed with a saturated sodium chloride solution (5.0 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The resulting mixture was separated and purified by silica gel column chromatography to obtain 15 (3 mg, 0.006 mmol, yield 46%) as a white solid compound. ¹ H NMR (500 MHz, CD ₃ OD) δ 8.08 (s, 2H), 7.81 (d, J =5.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 5.5 Hz, 1H), 6.85 (d, J =9.0 Hz, 1H), 4.8 0 – 4.75 (m, 1H), 4.67 (t, J = 6.0 Hz, 2H), 4.31 (t, J = 6.0Hz, 2H), 3.89 – 3.84 (m, 1H), 3.38 (d, J = 7.0 Hz, 2H), 3.24 (s, 2H), 3.10 –3.04 (m , 1H), 2.19 LC-MS [M+H] ⁺ m/z: 510.3.

Example 16: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,6-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 16)

Referring to the preparation process of compound 1 in Example 1, 2,8-dichloro-[1,6]naphthyridine was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 16 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.71 (s, 1H), 8.56 (s, 1H), 8.19 (s, 2H), 7.94 – 7.78 (m, 1H), 6.77– 6.61 (m, 1H), 6.45 (d, J = 7.0 Hz, 1H), 5.15 (d, J = 9.0 Hz, 1H), 5.08 –5.01 (m, 1H), 4.92 (t, J = 7.0 Hz, 2H), 4.47 (t, J = 6.5 Hz, 2H), 4.08 – 3.98(m, 1H), 3.36 (s, 2H), 2.32 – 2.20 (m , 2H), 1.97 (d, J = 12.0 Hz, 1H), 1.88 (d, J = 12.5 Hz, 1H), 1.54 – 1.46 (m, 1H), 1.36 – 1.24 (m, 4H), 1.20 (d, J =6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 496.3.

Example 17: Synthesis of (S)-2-(6-((1-(4-((8-chloro-1,6-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyridin-3-yl)-N-(oxetane-3-yl)acetamide (Compound 17)

Referring to the preparation process of compound 12 in Example 12, 2,8-dichloro-[1,6]naphthyridine was used to replace 2-chloro-8-methoxy-1,7-naphthyridine in step 1, and there was no need to carry out the reaction in step 2. The other operation steps were similar to obtain compound 17 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.71 (s, 1H), 8.56 (s, 1H), 8.19 (s, 2H), 7.94 –7.78 (m, 1H), 6.77 – 6.61 (m, 1H), 6.45 (d, J = 7.0 Hz, 1H), 5.15 ( d, J = 9.0Hz, 1H), 5.08 – 5.01 (m, 1H), 4.92 (t, J = 7.0 Hz, 2H), 4.47 (t, J = 6.5 Hz, 2H), 4.08 – 3.98 (m, 1H), 3.36 (s, 2H), 2.32 – 2.20 (m, 2H), 1.97 (d, J =12.0 Hz, 1H), 1.88 (d, J = 12.5 Hz, 1H), 1.54 – 1.46 (m, 1H), 1.36 – 1.24 (m,4H), 1.20 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 496.3.

Example 18: Synthesis of (S)-2-(6-((1-(4-((8-bromo-1,6-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyridin-3-yl)-N-(oxetane-3-yl)acetamide (Compound 18)

1 Preparation of key starting material 8-bromo-2-chloro-[1,6]naphthyridine:

Step A Synthesis of Intermediate 18-1

3,5-Dibromopyridin-4-amine (5.0 g, 5.35 mmol, 1.0 equivalent) was dissolved in N,N-dimethylformamide (20 mL), and ethyl acrylate (4.0 g, 40.00 mmol, 2.0 equivalent), N,N-diisopropylethylamine (7.7 g, 60.0 mmol, 3.0 equivalent), tri(o-methylphenyl)phosphine (1.2 g, 4.0 mmol, 0.2 equivalent) and palladium acetate (448 mg, 2.0 mmol, 0.1 equivalent) were added in sequence. The reaction mixture was stirred at 100 °C for 10 hours under nitrogen system. The reaction was monitored. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted three times with ethyl acetate (10 mL), and the organic phases were combined and washed three times with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to obtain intermediate 18-1 (1.76 g, 6.5 mmol, yield: 32 %). LCMS [M+H] ⁺ m/z: 271.0.

Step B Synthesis of Intermediate 18-2

18-1 (271 mg, 1.00 mmol, 1.0 equivalent) was dissolved in acetic acid (2.0 mL), and tributylphosphine (202 mg, 1.00 mmol, 1.0 equivalent) was added to the reaction solution. The reaction mixture was stirred at 110 °C for 1 hour. After the reaction was completed as monitored by LCMS, the reaction solution was cooled to room temperature, poured into water (10 mL), filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (10 mL). The filter cake was collected and concentrated under reduced pressure to obtain intermediate 18-2 (100 mg, 0.44 mmol, 44%) as a white solid. LCMS [M+H] ⁺ m/z: 224.9.

Step C Synthesis of Intermediate 18-3

The intermediate 18-2 obtained above was dissolved in phosphorus oxychloride (1.0 mL). The reaction mixture was stirred at 110 °C for 2 hours. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, the reaction solution was concentrated under reduced pressure, and the obtained mixture was separated and purified by silica gel column chromatography to obtain compound 18-3 (95 mg, 0.39 mmol, yield: 89%). ¹ H NMR (500MHz, CDCl ₃ ) δ 9.18 (s, 1H), 9.02 (s, 1H), 8.26 (d, J = 8.5 Hz, 1H), 7.60 (d,J = 8.5 Hz, 1H), LCMS [M+H] ⁺ m/z: 242.9.

2 Preparation of compound 18:

Referring to the preparation process of compound 1 in Example 1, 8-bromo-2-chloro-[1,6]naphthyridine was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 18 as a white solid. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.88 (d, J = 6.5 Hz, 1H), 8.80 (s, 1H), 8.66 (s, 1H), 8.17 (s,2H), 7.99 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 7.0 Hz, 1H ), 6.95 – 6.84 (m, 2H), 4.87 – 4.79 (m, 1H), 4.76 (t, J = 6.5 Hz, 2H), 4.47 (t, J = 6.5 Hz, 2H), 4.04 – 3.97 (m, 1H), 3.97 – 3.90 (m, 1H), 3. 30 (s, 2H), 2.25 – 2.15 (m, 2H), 1.95 – 1.86 (m, 2H), 1.56 – 1.47 (m, 1H), 1.31 – 1.25 (m, 2H), 1.25 – 1.18 (m,2H), 1.15 (d, J = 6.5 Hz, 3H).LC-MS [M+H ] ⁺ m/z: 540.3.

Example 19: Synthesis of (S)-2-(2-((1-(4-((7-chloro-1,6-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 19)

Referring to the preparation process of compound 1 in Example 1, 2,7-dichloro-1,6-naphthyridine was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 19 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ )δ 8.59 (s, 1H), 8.20 (s, 2H), 7.79 (d, J = 8.5 Hz, 1H), 7.46 (s, 1H), 6.60 (d, J = 7.5 Hz, 1H), 6.38 (s, 1H), 5.12 (d, J = 9.0 Hz, 1H), 5.07 – 5.02 (m,1H), 4.91 (t, J = 7.0 Hz, 2H), 4.49 (t, J = 6.5 Hz, 2H), 4.06 – 3.99 (m, 1H), 3.36 (s, 2H), 2.25 – 2.17 (m, 2H) , 1.99 – 1.92 (m, 2H), 1.89 – 1.84 (m, 2H), 1.46 – 1.42 (m, 4H), 1.20 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 496.3.

Example 20: Synthesis of (S)-2-(2-((1-(4-((7-methyl-1,6-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 20)

Preparation of key starting material 2-chloro-7-methyl-1,6-naphthyridine (20-3):

Step A Synthesis of Intermediate 20-1

5-Bromo-2-methylpyridin-4-amine (1.0 g, 5.35 mmol) was dissolved in N,N-dimethylformamide (20 mL), and ethyl acrylate (1.1 g, 10.70 mmol, 2.0 equivalents), N,N-diisopropylethylamine (2.1 g, 16.0 mmol, 3.0 equivalents), tri(o-methylphenyl)phosphine (325 mg, 1.07 mmol, 0.2 equivalents) and palladium acetate (120 mg, 0.54 mmol) were added in sequence. The reaction mixture was stirred at 120 °C for 10 hours under a nitrogen system. The reaction was monitored. After the reaction was completed, the reaction solution was cooled to room temperature, poured into water (20 mL), extracted three times with ethyl acetate (10 mL), and the organic phases were combined and washed three times with saturated sodium chloride solution (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography to give intermediate 20-1 (850 mg, 4.1 mmol, yield: 77%). ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.34 (s, 1H), 7.69 (d, J = 16.0 Hz, 1H), 6.44 (s, 1H), 6.36 (d, J = 16.0 Hz, 1H), 4.49 (s, 2H), 4.27 (q, J = 7.0 Hz, 2H), 2.43 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H).

Step B Synthesis of Intermediate 20-2

20-1 (778 mg, 3.76 mmol, 1.0 equivalent) was dissolved in acetic acid (15 mL), and tributylphosphine (760 mg, 3.76 mmol, 1.0 equivalent) was added to the reaction solution. The reaction mixture was stirred at 110 °C for 1 hour. After the reaction was completed as monitored by LCMS, the reaction solution was cooled to room temperature, poured into water (10 mL), filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (20 mL). The filter cake was collected and concentrated under reduced pressure to obtain intermediate 20-2 as a white solid. LCMS [M+H] ⁺ m/z: 161.0.

Step C Synthesis of key starting material 20-3

The intermediate 20-2 obtained above was dissolved in phosphorus oxychloride (4.0 mL). The reaction mixture was stirred at 110 °C for 2 hours. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and the obtained mixture was separated and purified by silica gel column chromatography to obtain compound 20-3 (580 mg, 3.24 mmol, yield 86%). ¹ H NMR (500 MHz,CDCl ₃ ) δ 9.18 (s, 1H), 8.19 (d, J = 8.5 Hz, 1H), 7.66 (s, 1H), 7.44 (d, J =8.5 Hz, 1H).LCMS [M+H] ⁺ m/z: 179.0.

2 Preparation of compound 20:

Referring to the preparation process of compound 1 in Example 1, 2-chloro-7-methyl-1,6-naphthyridine was used to replace 2,8-dichloro-7-methylquinoline in step 3, and the other operation steps were similar to obtain compound 20 as a white solid. LC-MS [M+H] ⁺ m/z: 476.3.

Example 21: Synthesis of (S)-2-(2-((1-(4-((5-chloropyrido[4,3-d]pyrimidin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 21)

1 Preparation of the key starting material 5-chloro-2-(methylsulfonyl)pyrido[4,3-d]pyrimidine

Step A Synthesis of Intermediate 21-1

Methyl thiocarbamide (10.0 g, 53.10 mmol) was dissolved in N,N-dimethylformamide (100 mL), and sodium acetate (8.7 g, 106.00 mmol) and ethyl (Z)-2-(ethoxymethylene)-3-oxobutanoate (19.8 g, 106.00 mmol) were added in sequence. The reaction mixture was stirred at 80 °C for 4 hours. After the reaction was completed as monitored by LCMS, the reaction solution was cooled to room temperature, poured into water (50 mL), filtered with filter paper, and the filter cake was washed with water (30 mL). The filter cake was collected and concentrated under reduced pressure to obtain intermediate 21-1 (11.2 g, crude product), which was used directly in the next step without further purification. LC-MS [M+H] ⁺ m/z: 213.3.

Step B Synthesis of Intermediate 21-2

21-1 (11.2 g, 52.80 mmol) was dissolved in N,N-dimethylformamide (110 mL), and N,N-dimethylformamide dimethyl acetal (14.1 mL, 106.00 mmol) was added to the reaction mixture. The reaction mixture was stirred at 150 °C for 3 hours. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, poured into water (200 mL), extracted three times with ethyl acetate (100 mL), and the organic phases were combined and washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain intermediate 21-2 (14.0 g, crude product), which was used directly in the next step without further purification. LC-MS [M+H] ⁺ m/z: 268.3 .

Step C Synthesis of Intermediate 21-3

Dissolve 21-2 (14.0 g, 52.40 mmol) in ethanol (100 mL), and add ammonium acetate (12.1 g, 157.00 mmol) and ammonium hydroxide (14.0 mL, 360.00 mmol) in turn. The reaction mixture was stirred at 100 °C for 12 hours. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, filtered through filter paper, and the filter cake was washed with ethanol (100 mL). The solid was collected and concentrated under reduced pressure to obtain intermediate 21-3 (7.5 g, crude product), which was used directly in the next step without further purification. LC-MS [M+H] ⁺ m/z: 194.2.

Step D Synthesis of Intermediate 21-4

21-3 (4.0 g, 20.70 mmol) was dissolved in phosphorus oxychloride (19.3 mL, 207.0 mmol). The reaction mixture was stirred at 80 °C for 2 hours. After the reaction was completed as monitored by LCMS, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10:1 to 2:1) to obtain intermediate 21-4 (3.5 g, yield: 80 %) as a yellow solid. LC-MS [M+H] ⁺ m/z: 212.1.

Step E Synthesis of key starting material 21-5

21-4 (2.5 g, 11.81 mmol) was dissolved in dichloromethane (250 mL), and chloroperoxybenzoic acid (5.8 g, 28.30 mmol) was added to the reaction mixture under ice bath conditions. The reaction mixture was stirred at 25 °C for 2 hours. After the reaction was completed by LCMS monitoring, the reaction solution was poured into water (200 mL), extracted three times with dichloromethane (100 mL), the organic phases were combined, washed twice with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10:1 to 1:1) to obtain compound 21-5 (1.0 g, yield: 35%) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.00 (s, 1H), 8.86 (d, J = 5.9 Hz, 1H), 8.01 (dd, J = 5.9, 0.8 Hz, 1H), 3.49 (s, 3H). LC-MS [M+H] ⁺ m/z: 244.0.

2 Preparation of compound 21:

Referring to the preparation process of compound 1 in Example 1, 2,8-dichloro-7-methylquinoline was replaced with 5-chloro-2-(methylsulfonyl)pyrido[4,3-d]pyrimidine in step 3, and the other operation steps were similar to obtain compound 21 as a white solid. ¹ HNMR (500 MHz, CDCl ₃ ) δ 9.32 (brs, 1H), 8.30 (d, J = 6.0 Hz, 1H), 8.20 (s,2H), 7.28 (d, J = 6.0 Hz, 1H), 6.14 (d, J = 6.0 Hz, 1H), 5.61 (d, J = 8 .0 Hz,1H), 5.19 – 5.11 (m, 1H), 5.09 – 5.01 (m, 1H), 4.92 (t, J = 6.5 Hz, 2H), 4.47(t, J = 6.5 Hz, 2H), 4.07 – 4.01 (m, 1H), 3.99 – 3.89 (m, 1H), 3.36 (s, 2H), 2.28 – 2.19 (m, 2H), 1.99 – 1.94 (m, 1H), 1.91 – 1.86 (m, 1H), 1.55 – 1.49 (m, 1H), 1.37 – 1.30 (m, 2H), 1.21 (d, J = 6.5 Hz, 3H) .LC-MS [M+H] ⁺ m/z:497.3.

Example 22: Synthesis of (S)-2-(2-((1-(1-(1-(8-chloroquinolin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 22)

Preparation of compound 22:

Step 1: Preparation of intermediate 22-1

Compound (S)-4-(1-aminoethyl)piperidine-1-carboxylic acid tert-butyl ester (0.46 g, 2.0 mmol, 1.0 equivalent) was dissolved in dimethyl sulfoxide (10 mL), and compound 1-4 (0.42 g, 2.0 mmol, 1.0 equivalent) and DIPEA (1.29 g, 10.0 mmol, 5.0 equivalent) were added. The resulting reaction solution was reacted at 100°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, saturated ammonium chloride solution (10 mL) was added to quench the reaction, and 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 vacuum rotary evaporation. The resulting mixture was separated and purified by silica gel column chromatography to obtain intermediate 22-1 (0.67 g, 1.6 mmol, yield 80%) as a light yellow solid. LCMS [M+H] ⁺ m/z: 421.3.

Step 2: Preparation of intermediate 22-2

The intermediate 22-1 (0.67 g, 1.6 mmol, 1.0 equivalent) was dissolved in methanol (5.0 mL), and a hydrochloric acid ethyl acetate solution (4.0 M, 1.2 mL, 3.0 equivalent) was added dropwise to the reaction solution, and the plate was tested. After the reaction was completed, a white solid was obtained by rotary evaporation under reduced pressure. The white solid was dissolved in 1,2-dichloroethane (5.0 mL), and compound 8-chloroquinoline-2-carboxaldehyde (0.31 g, 1.6 mmol, 1.0 equivalent) and sodium triacetoxyborohydride (0.68 g, 3.2 mmol, 2.0 equivalent) were added in sequence, and the plate was tested. After the reaction was completed, a saturated ammonium chloride solution (5.0 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (10.0 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 obtained mixture was separated and purified by silica gel column chromatography to obtain intermediate 22-2 (0.46 g, 0.93 mmol, yield 58%) as a light yellow solid. LCMS [M+H] ⁺ m/z: 496.3.

Step 3: Synthesis of target compound 22

The intermediate 22-2 (40 mg, 0.08 mmol, 1.0 equivalent) was dissolved in a mixed solvent of tetrahydrofuran (1.0 mL) and methanol (1.0 mL), and a 2 M sodium hydroxide aqueous solution (0.16 mL, 0.32 mmol, 4.0 equivalent) was added. The reaction was carried out at room temperature for 2 h, and the plate was monitored. After the reaction of the raw material was completed, the reaction was neutralized with a 1 M hydrochloric acid aqueous solution until it was neutral. The mixed solution was extracted with dichloromethane (5.0 mL) three times, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained oily compound was directly subjected to the next step of reaction.

The above oily compound was dissolved in N,N-dimethylformamide (1.0 mL), and compound N,N-diisopropylethylamine (41 mg, 0.32 mmol, 4.0 equivalents), HATU (61 mg, 0.16 mmol, 2.0 equivalents) and 3-oxetaneamine (12 mg, 0.16 mmol, 2.0 equivalents) were added in sequence, and the resulting reaction solution was reacted at room temperature for 1 h. After the reaction was completed, saturated aqueous ammonium chloride solution (2.0 mL) was added to quench the reaction, and the mixed solution was extracted three times with ethyl acetate (10 mL), the organic phases were combined, and the saturated sodium chloride solution was washed three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum rotary evaporation. The obtained mixture was separated and purified by silica gel column chromatography to obtain compound 22 (8 mg, 0.016 mmol, yield 20%) as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 8.33 (d, J = 8.4 Hz, 1H), 8.09 (s, 2H), 7.82 (d, J =7.9 Hz, 2H), 7.51-7.45 (m, 2H), 4.87-4.70 (m, 5H), 4.55 (s, 2H ), 4.45 (t, J =6.2 Hz, 2H), 3.98-3.91 (m, 1H), 3.74 (d, J = 11.8 Hz, 2H), 3.11-3.02 (m, 2H), 2.04-1.96 (m, 2H), 1.82-1.72 (m, 1H), 1.70-1 .55 (m, 2H), 1.12 (d, J = 7.9 Hz, 3H). LCMS [M+H] ⁺ m/z: 495.3.

Example 23: Synthesis of (R)-2-(2-((1-(1-((8-chloroquinolin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 23)

Using compound (R)-4-(1-aminoethyl)piperidine-1-carboxylic acid tert-butyl ester instead of the enantiomer (S)-4-(1-aminoethyl)piperidine-1-carboxylic acid tert-butyl ester, refer to the procedures of Example 22 to obtain the target compound 23 as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 8.33 (d, J = 8.4 Hz, 1H), 8.09 (s, 2H), 7.82 (d, J= 7.9 Hz, 2H), 7.51-7.45 (m, 2H), 4.87-4.70 (m, 5H), 4.55 (s, 2 H), 4.45 (t, J= 6.2 Hz, 2H), 3.98-3.91 (m, 1H), 3.74 (d, J = 11.8 Hz, 2H), 3.11-3.02 (m,2H), 2.04-1.96 (m, 2H), 1.82-1.72 (m, 1H), 1.70 -1.55 (m, 2H), 1.12 (d, J =6.5 Hz, 3H). LCMS [M+H] ⁺ m/z: 495.3.

Example 24: Synthesis of (S)-2-(2-((1-(1-((8-bromoquinolin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 24)

The compound 8-bromo-2-quinolinecarboxaldehyde was used to replace the compound 8-chloroquinoline-2-carboxaldehyde, and the operation of Example 22 was referred to to obtain the target compound 24 as a white solid. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.75 (d, J = 6.5Hz, 1H), 8.37 (brs, 1H), 8.07 (dd, J = 7.5, 1.0 Hz, 1H), 8.06 (s, 2H), 7.96 (d, J = 8.9 Hz, 1H), 7.63 ( d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.9 Hz, 1H), 6.85-6.83 (m, 1H), 4.72-4.64 (m, 1H), 4.63 (t, J = 7.1 Hz, 2H), 4.34 (t, J =6.5 Hz, 2H), 4.56 (s, 2H), 4.46 (t, J = 6.2 Hz, 2H), 3.97-3.90 (m, 1H), 3.73 (d, J = 11.8 Hz, 2H), 3.10-3.00 (m, 2H), 2.05-1.94 (m, 2H), 1.83-1.73 (m,1H), 1.71-1 .53 (m, 2H), 1.02 (d, J = 6.5 Hz, 3H). LCMS [M+H] ⁺ m/z: 539.2

Example 25: Synthesis of (S)-2-(2-((1-(1-((8-chloro-1,7-naphthyridin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 25)

Preparation of the key starting material 8-chloro-1,7-naphthyridine-2-carbaldehyde (25-3):

Step A Synthesis of Intermediate 25-1

2-Chloro-8-methoxy-1,7-naphthyridine (2.5 g, 12.85 mmol) was dissolved in a mixed solvent of 1,4-dioxane (25 mL) and water (5.0 mL), and sodium carbonate (4.1 g, 38.50 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.0 g, 1.29 mmol) and potassium ethylene trifluoroborate (3.4 g, 25.70 mmol) were added in sequence. The reaction mixture was stirred at 95 °C for 2 hours under a nitrogen system. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, poured into water (100 mL), extracted three times with ethyl acetate (50 mL), and the organic phases were combined and washed twice with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 20:1 to 10:1) to give intermediate 25-1 (2.1 g, yield: 89%) as a yellow oil. LCMS [M+H] ⁺ m/z: 187.3.

Step B Synthesis of Intermediate 25-2

25-1 (2.1 g, 11.28 mmol) was dissolved in a mixed solvent of tetrahydrofuran (20 mL) and water (20 mL), and potassium osmate dihydrate (0.4 g, 1.13 mmol) and sodium periodate (7.3 g, 33.80 mmol) were added in sequence. The reaction mixture was stirred at 25 °C for 3 hours. After the reaction was completed by LCMS monitoring, the reaction solution was poured into water (100 mL), extracted three times with ethyl acetate (50 mL), the organic phases were combined, washed twice with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10:1 to 5:1) to obtain intermediate 25-2 (1.6 g, yield: 75%) as a white solid. LCMS [M+H] ⁺ m/z: 189.3.

Step C Synthesis of key starting material 25-3

25-2 (1.4 g, 7.44 mmol) was dissolved in phosphorus oxychloride (20 mL, 215.00 mmol). The reaction mixture was stirred at 110 °C for 1 hour. After the reaction was completed as monitored by LCMS, the reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 5:1 to 3:1) to obtain the key starting material 25-3 (525 mg, yield: 37%) as a yellow solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.30 (d, J = 0.8 Hz, 1H), 8.51 (d,J = 5.6 Hz, 1H), 8.39 – 8.36 (m, 1H), 8.29 (d, J = 8.5 Hz, 1H), 7.72 (d, J =5.6 Hz, 1 H). LCMS [M+H] ⁺ m/z: 193.2.

3 Preparation of compound 25:

The compound 8-chloro-1,7-naphthyridine-2-carbaldehyde was used to replace the compound 8-chloroquinoline-2-carbaldehyde, and the operation of Example 22 was referred to to obtain the target compound 25 as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 8.34 (d, J =8.5 Hz, 1H), 8.27 (d, J = 5.5 Hz, 1H), 8.12 (s, 2H), 7.92 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 5.5 Hz, 1H), 4.8 9 – 4.84 (m, 1H), 4.82 (t, J = 6.5 Hz, 2H), 4.51 (t, J = 6.5 Hz, 2H), 3.96 (s, 2H), 3.93 – 3.88 (m, 1H), 3.35 (s, 2H), 3.11 – 3.04 (m, 2H), 2. 33 – 2.24 (m, 2H), 1.85 – 1.73 (m, 2H), 1.54 – 1.48 (m, 1H), 1.48 – 1.39 (m, 2H), 1.14 (d, J = 6.5 Hz, 3H). LCMS [M+H] ⁺ m/z:496.3.

Example 26: Synthesis of (S)-2-(2-((1-(1-((8-chloro-1,6-naphthyridin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 26)

Preparation of key starting material 8-chloro-1,6-naphthyridine-2-carbaldehyde (26-5):

Step A Synthesis of Intermediate 26-1

3-Bromo-5-chloropyridin-4-amine (12.0 g, 57.8 mmol) was dissolved in N, N-dimethylformamide (120 mL), and ethyl acrylate (11.6 g, 116.0 mmol), N, N-diisopropylethylamine (30.3 mL, 174.0 mmol), tri(o-methylphenyl)phosphine (3.5 g, 11.57 mmol) and palladium acetate (1.3 g, 5.78 mmol) were added in sequence. The reaction mixture was stirred at 100 °C for 10 hours under a nitrogen system. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, poured into water (100 mL), extracted twice with ethyl acetate (100 mL), and the organic phases were combined and washed twice with saturated sodium chloride solution (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether:ethyl acetate = 10:1 to 3:1) to give Intermediate 26-1 (9.0 g, yield: 69%) as a yellow solid. LCMS [M+H] ⁺ m/z: 227.2.

Step B Synthesis of Intermediate 26-2

26-1 (6.0 g, 26.5 mmol) was dissolved in acetic acid (30 mL), and tributylphosphine (6.5 mL, 26.50 mmol) was added to the reaction solution. The reaction mixture was stirred at 110 °C for 1 hour. After the reaction was completed by LCMS monitoring, the reaction solution was cooled to room temperature, poured into water (100 mL), filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate (20 mL). The filter cake was collected and concentrated under reduced pressure to obtain 26-2 (4.0 g, yield: 84%) as a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ11.69 (s, 1H), 8.80 (s, 1H), 8.57 (s, 1H), 8.06 (d, J = 9.6 Hz, 1H), 6.68 (d,J = 9.6 Hz, 1H). LCMS [M+H] ⁺ m/z: 181.2 .

Step C Synthesis of Intermediate 26-3

26-2 (3.8 g, 21.04 mmol) was dissolved in phosphorus oxychloride (20 mL, 210.4 mmol). The reaction mixture was stirred at 120 °C for 2 hours under nitrogen. After the reaction was completed, the reaction solution was cooled to room temperature and concentrated under reduced pressure to obtain intermediate 26-3 (4.0 g, crude product), which was used directly in the next step without further purification. LCMS [M+H] ⁺ m/z: 199.1.

Step D Synthesis of Intermediate 26-4

26-3 (4.0 g, 20.10 mmol) was dissolved in a mixed solvent of 1,4-dioxane (40 mL) and water (8.0 mL), and sodium carbonate (6.4 g, 60.30 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.5 g, 2.01 mmol) and potassium ethylene trifluoroborate (5.4 g, 40.2 mmol) were added in sequence. The reaction mixture was stirred at 80 °C for 8 hours under a nitrogen system. After the reaction was completed as monitored by LCMS, the reaction solution was cooled to room temperature, poured into water (100 mL), extracted three times with ethyl acetate (100 mL), and the organic phases were combined and washed twice with a saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether:ethyl acetate = 5:1 to 3:1) to give intermediate 26-4 (2.0 g, yield: 52%) as a white solid. LCMS [M+H] ⁺ m/z: 191.1.

Step E Synthesis of key starting material 26-5

26-4 (500 mg, 2.62 mmol) was dissolved in a mixed solvent of tetrahydrofuran (10 mL) and water (10 mL), and potassium osmate dihydrate (80 mg, 0.26 mmol) and sodium periodate (1.7 g, 7.87 mmol) were added in sequence. The reaction mixture was stirred at 25 °C for 3 hours. After the reaction was completed by LCMS monitoring, the reaction solution was poured into water (20 mL), extracted three times with ethyl acetate (30 mL), the organic phases were combined, washed twice with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10:1 to 5:1) to obtain intermediate 26-5 (300 mg, yield: 59%) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.33 (d,J = 0.9 Hz, 1H), 9.29 (s, 1H), 8.96 (s, 1H), 8.53 (dd, J = 8.4, 0.9 Hz, 1H), 8.22 (d, J = 8.4 Hz, 1H).LCMS [M+H] ⁺ m/z: 193.2.

1 Preparation of compound 26:

The compound 8-chloro-1,6-naphthyridine-2-carbaldehyde was used to replace the compound 8-chloroquinoline-2-carbaldehyde, and the operation of Example 22 was referred to to obtain the target compound 26 as a white solid. ¹ H NMR (500 MHz, CD ₃ OD) δ 9.24 (s, 1H), 8.79 (s, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.20 (s, 2H), 7.96 (d, J = 8.5 Hz, 1H), 4.95 – 4.91 (m, 1H), 4. 89 (t, J = 6.5 Hz, 2H), 4.58 (t, J = 6.5 Hz, 2H), 3.98 (s, 2H), 3.98 – 3.94 (m, 1H), 3.38 (s, 2H), 3.07 (t, J = 10.0 Hz, 2H), 2.33 – 2.20 (m, 2H ), 1.91 – 1.77 (m, 2H), 1.57 – 1.42 (m, 3H), 1.21 (d, J =6.5 Hz, 3H).LCMS [M+H] ⁺ m/z: 496.3.

Example 27: Synthesis of (R)-2-(2-((1-(1-((8-chloro-1,6-naphthyridin-2-yl)methyl)piperidin-4-yl)ethyl)amino)pyrimidin-5-yl)-N-(oxetane-3-yl)acetamide (Compound 27)

Using compound (R)-4-(1-aminoethyl)piperidine-1-carboxylic acid tert-butyl ester to replace (S)-4-(1-aminoethyl)piperidine-1-carboxylic acid tert-butyl ester, refer to the procedures of Example 26 to obtain the target compound 27 as a white solid. ¹ H NMR (500MHz, CD ₃ OD) δ 9.20 (s, 1H), 8.75 (s, 1H), 8.51 (d, J = 8.5 Hz, 1H), 8.16 (s,2H), 7.91 (d, J = 8.5 Hz, 1H), 4.93 – 4.87 (m, 1H), 4. 86 (t, J = 6.5 Hz, 2H), 4.54 (t, J = 6.5 Hz, 2H), 3.95 (d, J = 4.5 Hz, 2H), 3.95 – 3.90 (m, 1H), 3.35 (s, 2H), 3.04 (t, J = 10.0 Hz, 2H), 2.31 – 2.19 (m, 2H), 1.89 – 1.74 (m, 2H),1.54 – 1.41 (m, 3H), 1.17 (d, J = 6.5 Hz, 3H).LCMS [M+H] ⁺ m/z: 496.3.

Example 28: Synthesis of 2-(2-(S)-1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-hydroxy-N-(oxetane-3-yl)acetamide (Compound 28)

The reaction was carried out according to the procedure of Example 13 to obtain the target compound 28 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.58 (s, 2H), 8.01 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 5.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.5 4-6.49 (m, 1H), 5.82(s, 1H), 5.28 (d, J = 9.0 Hz, 1H), 5.12 (br s, 1H), 5.10 (brs, 1H), 5.04-4.95(m, 1H), 4.92-4.84 (m, 2H), 4.46 (t, J = 6.5 Hz, 1H), 4.42 (t, J = 6.5 Hz, 1H), 4.06-3.96 (m, 1H), 3.36 (q, J = 7.0 Hz, 1H), 2.34-2.13 (m, 2H), 2.03-1.81 (m, 2H), 1.37-1.25 (m, 4H), 1. 18 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z:512.2.

Example 29: Synthesis of (S)-2-(2-((1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2,2-difluoro-N-(oxetane-3-yl)acetamide (Compound 29)

The reaction was carried out according to the procedure of Example 13 to obtain the target compound 29 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.58 (s, 2H), 8.01 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 5.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.5 4-6.49 (m, 1H), 5.28 (d, J = 9.0 Hz, 1H), 5.10 (brs, 1H), 5.04-4.95 (m, 1H), 4.92-4.84 (m, 2H), 4.46 (t, J = 6.5 Hz, 1H), 4.42 (t, J = 6.5 Hz, 1H), 4.06-3.96 (m, 1H), 3.36 (q, J = 7.0 Hz, 1H), 2.34-2.13 (m, 2H), 2.03-1.81 (m, 2H), 1.37-1.25 (m, 4H), 1.19 (d, J = 6.5 Hz, 3H). LC-MS [M+H ] ⁺ m/z: 532.2.

Example 30: Synthesis of 2-(2-(((S)-1-(4-((8-chloro-1,7-naphthyridin-2-yl)amino)cyclohexyl)ethyl)amino)pyrimidin-5-yl)-2-cyano-N-(oxetane-3-yl)acetamide (Compound 30)

The reaction was carried out according to the procedure of Example 13 to obtain the target compound 30 as a white solid. ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.58 (s, 2H), 8.20 (d, J = 5.0 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.33 (d, J = 5.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.5 4-6.49 (m, 1H), 5.28 (d, J = 9.0 Hz, 1H), 5.10 (brs, 1H), 5.04-4.95 (m, 1H), 4.92-4.84 (m, 2H), 4.72 (t, J = 6.5 Hz, 1H), 4.42 (t, J = 6.5 Hz, 1H), 4.06-3.96 (m, 1H), 3.87-3.85 (m, 1H), 3.36 (q, J = 7.0 Hz, 1H), 2.34 – 2.13 (m, 2H), 2.03 – 1.81 (m,2H), 1.37 – 1.25 (m, 4H), 1.18 (d, J = 6.5 Hz, 3H). LC-MS [M+H] ⁺ m/z: 521.2.

Biological assays

Compounds of the invention can be evaluated for their ability to inhibit EZH2 using the assays described below, as well as other assays known in the art.

Main experimental instruments and equipment:

Centrifuge (Beckman Avanti J-15R); ultra-low temperature refrigerator (Thermo FDE60086FV); carbon dioxide incubator (ESCO CLM-170B-CN); microplate reader (Spark tecan); slug gun (20-200 µL); cell counter (Bio-Rad TC20).

Example 31: EZH2 in vitro enzyme activity assay

Step 1

Prepare reaction buffer (4X): 80 mM Tris, pH 8.0, 0.04% BSA, 0.04% Triton-100, 2 mM DTT. Adjust pH to 8.5 and add DTT before use.

Step 2

The compound of the present invention was diluted 3 times in DMSO each time, and diluted 9 times in succession to obtain 10 concentrations. In order to make the final DMSO concentration in the reaction system 1%. In the last step, the compound was diluted 10 times with water. Each concentration of the test compound (1.92 μL each) was added to 8 rows. A 1 mg/mL wild-type PRC2 (6 components) complex (0.1056 μL), 1 mM SAM (0.16 μL), 2 mM H3[21-44] (0.08 μL) and reaction buffer (2 μL) were added to the wells of the 8 rows, mixed with a spray gun, and incubated with the test compound at 25°C for 2 h. The final composition of the reaction solution contained 40 nM wild-type PRC2, 20 nM SAM, 20 μM H3[21-44] and different concentrations of compounds (0 μM, 0.00152 μM, 0.00457 μM, 0.01372 μM, 0.04115 μM, 0.12346 μM, 0.37037 μM, 1.11111 μM, 3.33333 μM, 10 μM). The blank control consisted of 20 nM SAM and 20 μM H3[21-44]. After the reaction was completed, 4.8 μL of quenching solution (1% TFA) was added to the wells of the 8-row wells and the reaction was terminated at 25°C for 5 min.

Step 3

Add 4.8 μL of 6×MTase-GLo ^TM reagent (10×MTase-GLo ^TM reagent, 3 mM MgCl ₂ , 50 mMNaCl, 20 mM Tris, pH 8.0, 0.01% BSA, 0.01% Triton-100, 0.5 mM DTT) mixture and react at 25°C for 30 min. After the reaction is completed, add 28.8 μL/well of detection buffer to the tube, mix well, add 20 μL/well of the reaction mixture to the 384-well plate, centrifuge at 2000 rpm for 2 min, and react at 25°C for 30 min. Read the chemiluminescent signal on the microplate reader, and the detection time is 1000 ms. Subtract the corresponding data of the control group from all group data, and then calculate the corresponding inhibition rate according to the standard curve, and calculate the IC ₅₀ value.

Example 32: G401 ELISA (H3K27me3 methylation) assay

Step 1

The compounds of the present invention were diluted 3-fold each time in DMSO, serially diluted 9 times to obtain a total of 10 concentrations. The compounds were 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. Cells were cultured in an incubator at 37°C and 5% _CO2 for 48 h before the ELISA procedure.

Step 2

The cells in the 96-well plate were washed with 1× PBS buffer and lysed by adding lysis buffer (0.4N HCl) at 100 μL/well, and the plate was incubated on a shaker at 150 rpm and 4°C for 30 min. Neutralization buffer (0.5 M disodium hydrogen phosphate, pH 12.5, 1 mM DTT; 80 μL/well) was added for neutralization, and the solution was mixed thoroughly by blowing for about 20 times with a spray gun. After neutralization, the 96-well plate was centrifuged at 2000 rpm for 2 min.

Step 3

Take 10-50 μL/well of the cell lysate in the centrifuged 96-well plate into the wells of the 384-well plate, and adjust the volume in the well 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 about 16 h.

Step 4

After incubation, wash five times with TBST: add 60 μL TBST buffer to each well and wash on a shaker at 200 rpm and 25°C for 5 min.

Step 5

50 μL of blocking buffer (TBST, 2% BSA) was added to each well, and the plate was centrifuged at 2000 rpm for 2 min and incubated on a shaker at 200 rpm and 25°C for 1 h.

Step 6

The blocking buffer was removed and 30 μL of primary antibody (2% BSA, 0.1% H3K27me3; 0.03% H3) was added to each well. The plate was centrifuged at 2000 rpm for 2 min and incubated on a shaker at 150 rpm and 25°C for 1 h.

Step 7

The primary antibody was removed by washing five times with TBST, and 30 μL of secondary antibody (2% BSA, 0.04% anti-rabbit antibody) was added to each well. The 384-well plate was centrifuged at 2000 rpm for 2 min and incubated on a shaker at 150 rpm and 25°C for 1 h.

Step 8

The 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.

Step 9

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

The compounds of the present invention were tested according to the above experimental method, and some of the results are shown in Table 2. The methylation inhibitor activity of the compounds at the target level and the G401 cell level is basically below 1 uM, and has significant target activity. The present invention uses Tazemetostat (EPZ-6438), the first marketed drug targeting EZH2, with the Chinese name: Tazemetostat, as comparative example 1, and the most active compound C36 in the CN114746414A patent as comparative example 2. The compounds in some inventions show obvious advantages.

Table 2 Methylation inhibitory activity of compounds at protein and cell levels

Compound No. EZH2 IC ₅₀ (nM) G401-ELISA IC ₅₀ (nM) Comparative Example 1 - 16.2 Comparative Example 2 36.8 13.5 1 93 4.7 2 195.8 57.7 3 358.3 20.4 4 125.1 17.6 5 52.6 4.0 6 64.4 1.5 7 47.2 36.5 8 26.8 3.1 9 ≥1000 ≥1000 10 100.4 122.3 11 143.2 19.5 12 35.6 6.5 13 22.2 0.7 14 45.1 6.5 15 ≥1000 ≥1000 16 71.9 10.6 17 59.6 13.5 18 44.5 10.6 19 183.2 309.5 20 127.6 ≥1000 twenty one 150.6 67.7 twenty two 315.5 84.6 twenty three ≥1000 ≥1000 twenty four 44.8 25.4 25 131.3 292.2 26 198.5 ≥1000 27 ≥1000 ≥1000 28 20 1.3 29 23.2 2.5 30 31.2 3.1

Compared with the comparative example compounds, the compounds in this invention generally adopt the introduction of chiral methyl groups. The structures of compound 8 in the embodiment and comparative example 2 are compared as follows:

The inventors found that the introduction of a methyl structure into compound 8 surprisingly brought about an unexpected activity effect.

Molecular docking was performed using Glide software in XP mode, and the SAH molecule was retained to restore the interaction between C36 and SAH, thereby improving the reliability of the docking calculation. Comparison of the molecular docking results of C36 and compound 8. The red molecule in the figure is C36, the green molecule is compound 8, and the yellow molecule is SAH. It can be seen that the overall conformation of compound 8 and C36 is consistent (as shown in Figure 1). The additional methyl stereo configuration in compound 8 occupies the hydrophobic space formed by the benzene ring of Y658 and the side chains of C663 and T678, which improves the binding ability between the molecule and the protein. It can be seen that the introduction of methyl groups is very important for improving activity, which is highly consistent with the laboratory test results, as shown in Table 3:

Table 3 Scoring results of molecular docking of compound 8 and C36 with target protein EZH2

Compound No. Molecular docking binding energy (kcal/mol) Comparative Example 2 -7.56 8 -7.90

Analysis of cell proliferation

Embodiment 33:

Suspension cells: Analysis of WSU-DLCL2/KARPAS 422 cell proliferation 1) Culture medium:

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

2) Before plating in 96-well plates, WSU-DLCL2/KARPAS 422 cells were treated with 50 µL/well poly-lysine for more than one hour, washed with 100ul/well PBS, and dried. After drying, WSU-DLCL2/KARPAS 422 cells were added with 100 µL/well culture medium and seeded in 96-well plates (about 2000 cells/well, and two replicates were performed for each test compound), and drug administration was performed the next day.

3) On the second day, different concentrations of the test compound solutions were prepared with DMSO. 9 different concentrations of the corresponding test compound solutions were prepared by equal dilution (3 times) 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, respectively.

4) After the dilution is completed, take 2 µL of the test compound solution prepared with DMSO at different concentrations and add 198 µL of culture medium to mix thoroughly (by pipetting more than 20 times). Then add 25 µL/well of the culture medium containing different concentrations of the test compound to the 96 wells where cells have been plated, and pipette 10 times. Finally, the final concentrations of the test compound-containing culture medium (containing 0.2% DMSO) 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.

5) The cells treated with the test compound or DMSO were then placed back into the 37°C incubator for culture. After 3-4 days of treatment, the treatment was changed.

6) When changing the medicine: add 80 µL of new culture medium to the 96-well plate treated with poly-lysine in advance. Then prepare different concentrations of culture medium containing the test compound as in the first drug addition treatment, add 25 µL of different concentrations of culture medium containing the test compound to each well and mix them by blowing 4 times with a dispenser. Use a dispenser to blow 10 times with a dispenser to mix the WSU-DLCL2/KARPAS422 cells in step 5), then take 20 µL/well of the cell solution and transfer it to the 96-well plate with the culture medium of the test compound added, and then blow 10 times to mix. Change the medicine every 3-4 days, and give the medicine 4 times in total.

7) Perform cell viability test 14 days after treatment. Use a spray gun to blow the culture medium 10-20 times to fully disperse the cells, take 20 µL/well of the culture medium containing cells, add 20 µL of trypan blue, mix thoroughly, and count the cells.

8) Calculation of cell viability: Calculation formula = 100*drug administration group/DMSO group

The control group data was subtracted from all groups, and the corresponding inhibition rate was calculated based on the standard curve, and the IC ₅₀ value was calculated.

Embodiment 34:

Adherent cells: Analysis of G401 cell proliferation 1) Culture medium:

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

2) G401 cells were seeded in 24-well plates with 500 µL/well culture medium (about 10,000 cells/well, and each compound to be tested was replicated) and administered the next day.

3) On the second day, prepare different concentrations of the test compound solution with DMSO. Make equal (3-fold) dilutions with DMSO to prepare 9 different concentrations of the corresponding test compound. The concentrations are 5000, 1666.67, 555.56, 185.19, 61.73, 20.58, 6.86, 2.29, and 0.76 µM.

4) After the dilution is completed, take 2 µL of the test compound solution of different concentrations prepared with DMSO and add 198 µL of culture medium to mix thoroughly (pipette more than 20 times). Then add 125 µL/well of the above culture medium containing different concentrations of the test compound to the 24 wells where cells have been plated. Finally, the final concentrations of the test compound culture medium (containing 0.2% DMSO) are obtained, and the DMSO control solution is prepared.

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

6) When changing the dressing: After removing the culture medium of G401 cells, wash once with 500 µL PBS, then add 100 µL trypsin, digest for 4 minutes, then add 400 µL culture medium to neutralize, centrifuge at 1000rpm for 5 minutes. After centrifugation, remove the supernatant, take 100 µL culture medium to resuspend the cells, take 10 µL of the resuspended liquid and spread it in a 24-well plate with 490 µL fresh culture medium added. Add 125 µL of culture medium containing the test compound of different concentrations to each well and shake to mix. Change the dressing every 3-4 days.

7) At the fourth dressing change, wash, digest and neutralize according to step 6), and resuspend the cells in 500 µL of culture medium. Then take 20 µL of cell solution and add it to a 96-well black plate with 80 µL of culture medium + 25 µL of culture medium containing different concentrations of the compound to be tested, set up 2 replicate wells, and perform Hoechst staining on the 14th day.

8) After 14 days, harvest the cells, remove almost all the culture medium by inversion, and invert the well plate onto absorbent paper.

9) Prepare the fixative staining solution: Use 37% formaldehyde water at 10×, and use PBS to prepare 3.7% formaldehyde. Each 96-well plate needs to be prepared with 5 ml of 3.7% formaldehyde. Dilute Hoechst 33342 (1000X) in 3.7% formaldehyde solution to a final concentration of 10 ug/ml.

10) Add 50 uL of fixative staining solution to each well by pipetting. Shake horizontally for 30 min at RT, away from light.

11) Remove almost all Hoechst 33342 by tipping over. Gently add 100 µL PBS with a pipette to wash. Shake horizontally for 5 min at RT, away from light. Repeat twice, and finally remove all PBS by tipping over, and invert the plate onto absorbent paper.

12) The excitation light of the microplate reader is 320 (25) and the emission light is 460 (20).

13) The read 96-well plate was sealed with aluminum foil and stored at -20°.

14) Calculation of adherent cell viability:

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

A (drug+): absorbance of wells with cells and drug solution;

A (drug-): absorbance of wells with cells but no drug solution;

A (blank): absorbance of wells with culture medium but no cells.

*Cell viability: cell proliferation activity or cytotoxic activity

The control group data was subtracted from all groups, and the corresponding inhibition rate was calculated based on the standard curve, and the IC ₅₀ value was calculated.

Karpas422, WSU-DLCL2 and G401 cells are common tumor cells caused by EZH2 mutation or overexpression, and are usually used as models for evaluating anti-tumor activity at the cellular level. Using Tazemetostat and C36 as control compounds, the compounds of the present invention were tested according to the above experimental method and showed significant anti-tumor activity. Some of the results are shown in Table 4.

Table 4 Proliferation inhibition activity of compounds on tumor KARPAS422 cells

Compound No. Karpas 422IC ₅₀ (nM) G401 IC ₅₀ (nM) WSU-DLCL2IC ₅₀ (nM) Comparative Example 1 22.6 122.7 77.8 Comparative Example 2 15.7 117 10.9 1 5.8 - - 5 3.3 - - 6 2.9 - - 8 3.3 18.6 1.9 11 8.0 - - 12 3.2 - - 16 6.7 75.1 5.9 17 7.9 - - 18 3.7 - - twenty four 49.8 - -

Biological assays

Example 35: Pharmacokinetic properties testing in mouse models

Oral administration: The compound was mixed with 10% DMSO + 10% Solutol + 80% Saline at a dose of 10 mg/kg, and blood was collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h and 24h after administration;

Intravenous administration: The compound was mixed with 10% DMSO + 10% Solutol + 80% Saline at a dose of 5 mg/kg. Blood was collected via the submandibular vein or other appropriate methods at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h and 24 h after administration.

About 30 μL of each sample was collected per time point, anticoagulated with K2-EDTA, and placed on ice after collection. The plasma was separated by centrifugation within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). The plasma samples were stored in a -80°C refrigerator before analysis and were ready for LC-MS/MS analysis.

The pharmacokinetic parameters were calculated using WinNonlin software based on the blood drug concentration-time data obtained from the test. The compound showed good bioavailability and had excellent drugability prospects.

Some results are shown in Table 5:

Table 5 Pharmacokinetic parameters of intravenous and oral administration in ICR mice

Compound No. Intravenous AUC _last (h*ng/mL) Oral AUC _last (h*ng/mL) Bioavailability F (%) 8 3735 3105 41.6 16 268 144 26.9

Claims (10)

1. A compound, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, mixture, having the structure of formula 1:

wherein a ¹ is selected from N or CR ¹¹;

A ² is selected from N or CR ¹²;

a ³ is selected from N or CR ¹³;

R ¹ is selected from H, halogen or C ₁-C₃ alkyl;

R ² is selected from H, halogen or C ₁-C₃ alkyl;

R ¹¹ is selected from H, halogen or C ₁-C₃ alkyl;

R ¹² is selected from H, halogen or C ₁-C₃ alkyl;

R ¹³ is selected from H, halogen or C ₁-C₃ alkyl;

L ¹ is selected from-NH-or-CH ₂ -:

b ¹ is selected from N or CH:

R ³ is C ₁-C₃ alkyl, R ⁴ is H or C ₁-C₃ alkyl;

the C ₃ alkyl is a straight chain alkyl, branched alkyl or cycloalkyl;

c ¹ is selected from N or CH;

L ² is selected from -CHR⁵NH-、C(R⁵)₂NH-、-CHR⁵C(=O)NH-、-C(R⁵)₂C(=O)NH-、-CHR⁵C(=O)NHCH₂- or-C (R ⁵)₂ C(=O)NHCH₂ -;

R ⁵ at each occurrence is independently selected from H, -OH, -CN, C ₁-C₂ alkyl or halogen.

2. The compound, stereoisomer thereof, mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, mixture of the compounds, of claim 1,

In formula 1Selected from the following structures:

。

3. The compound, stereoisomer thereof, mixture of different stereoisomers of the compound, or pharmaceutically acceptable salt of the compound, stereoisomer, mixture of claims 1-2, wherein L ¹ is-NH-, and B ¹ is CH; or L ¹ is-CH ₂-, B¹ is N.

4. A compound according to any one of claims 1-2, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, mixture, wherein:

r ³ is methyl, and the chirality of the carbon atom to which the methyl is attached is S configuration.

5. The compound, stereoisomer thereof, mixture of different stereoisomers of the compound, or pharmaceutically acceptable salt of the compound, stereoisomer, mixture of the compounds according to any one of claims 1 to 2, wherein L ² is selected from -CH₂NH-、-CH(OH)C(=O)NH-、-CHF₂C(=O)NH-、-CH(CH₃)C(=O)NH-、-CH(CN)C(=O)NH-、-CH₂C(=O)NH- or-CH ₂C(=O)NHCH₂ -.

6. A compound, a stereoisomer thereof, or a mixture of different stereoisomers of the compound, wherein the compound is selected from the group consisting of the structures:

。

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer or mixture, and at least one pharmaceutically acceptable excipient.

8. A method of treating a disease or condition mediated by at least one of PRC2, EZH1, and EZH2, wherein a therapeutically effective amount of a compound, stereoisomer thereof, mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, or mixture, or a pharmaceutical composition of claim 7, is administered to a subject in need of such treatment.

9. The compound of any one of claims 1-6, a stereoisomer thereof, a mixture of different stereoisomers of the compound, or a pharmaceutically acceptable salt of the compound, stereoisomer, or mixture; or the pharmaceutical composition of claim 7, for use in the manufacture of a medicament for the treatment of a disease or condition mediated by at least one of PRC2, EZH1 and EZH 2.

10. A process for the preparation of a compound according to any one of claims 1 to 6, characterized in that:

Step 1

The para-substituted ketone with the amino protected by the initial raw material Boc and the optically pure tertiary butyl sulfinamide are subjected to dehydration condensation to obtain a tertiary butyl sulfinimide intermediate INT-1;

Step 2

The INT-1 is subjected to reduction reaction to obtain a tertiary butyl sulfinamide intermediate INT-2;

Step 3

INT-2 is selectively removed from tert-butylsulfinyl to obtain an intermediate INT-3; or when R ⁴ needs further alkyl substitution, continuing amino monoalkylation R ⁴ substitution to obtain an intermediate INT-3;

Step 4

The INT3 is directly subjected to electrophilic aryl substitution reaction or metal catalyzed coupling reaction with heterocyclic aryl halide after removing the Boc protective agent to obtain an intermediate INT-4;

Step 5

Carrying out metal-catalyzed coupling reaction or electrophilic aryl substitution reaction on INT-4 and nitrogen-containing heterobicyclic aryl halide, or carrying out reductive amination reaction on INT-4 and nitrogen-containing heterobicyclic aryl aldehyde to obtain an intermediate INT-5;

Step 6

INT-5 hydrolyzes the ester group of the M part to form free carboxylic acid, and then carries out acid-amine condensation reaction with corresponding oxetan-3-amine or oxetan-3-ylmethylamine or reduction of the ester group of the M part to obtain aldehyde group, and then carries out reductive amination reaction to obtain the compound of formula 1.