PRMT5 inhibitor as well as composition and pharmaceutical application thereof

Abstract

The invention relates to a heterocyclic compound for inhibiting PRMT5 activity and a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The heterocyclic compound for inhibiting PRMT5 activity has a structure as shown in a formula (I). The invention further provides a composition containing the compound and application of the composition as a PRMT5 inhibitor. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to a PRMT5 inhibitor compound, its stereoisomers, or its pharmaceutically acceptable salts, and also relates to a pharmaceutical composition containing the compound as an active ingredient and its pharmaceutical use in the treatment of diseases, disorders, syndromes or afflictions related to the PRMT5 enzyme. Background Art

[0002] Arginine methylation is a type of histone methylation and one of the most common post-translational modifications in mammals, mainly regulated by the PRMT gene family. Arginine methyltransferases (PRMTs) regulate arginine methylation in three different forms: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) methylation. There are a total of 9 PRMTs in mammals, which are divided into three types: type I (PRMT1, 2, 3, 4, 6, 8; mainly catalyzing the formation of MMA and ADMA), type II (PRMT5, 9; mainly catalyzing the formation of MMA and SDMA), and type III (PRMT7; mainly catalyzing the formation of MMA). PRMTs can methylate histones and a variety of non-histones. Studies have shown that changes in the enzymatic activity of PRMTs, gene mutations or deletions usually lead to abnormal development of animal individuals, and at the same time induce the occurrence, development or metastasis of cancer. Among many PRMTs, especially the role of PRMT5 in cancer has been increasingly confirmed and has received the most attention.

[0003] Protein arginine methyltransferase 5 (PRMT5) is a typical type II methyltransferase that transfers a methyl group from SAM to the two ω-guanidino nitrogen atoms of arginine, resulting in ω-NG, NG-disymmetric methylation of the protein substrate. It is located in the nucleus and cytoplasm and plays different functions by modifying histones or non-histones. As an epigenetic enzyme, the most attractive aspect of PRMT5 is the "synthetic lethality" mechanism.

[0004] The so-called synthetic lethality means that for two genes in a cell, when any one of them is mutated or does not function alone, it will not cause cell death; while when both are mutated or cannot be expressed, it will lead to cell death. Using this principle, selective killing of tumor cells can be achieved without affecting normal somatic cells.

[0005] In 2016, two articles published in *Science* first reported the "synthetic lethality" effect of inhibiting PRMT5 in MTAP-deficient tumors. The gene that forms synthetic lethality with PRMT5 is methylthioadenosine phosphorylase (MTAP), which is a tumor suppressor gene and is often deleted in tumors. Patients with MTAP deletion account for approximately 15% of all solid tumors, including approximately 15% of non-small cell lung cancer (NSCLC), 28% of esophageal cancer, 26% of bladder cancer, and 10% of esophagogastric cancer.

[0006] In addition to the synthetic lethality effect, PRMT5 also participates in DNA repair, cell cycle, and transcriptional regulation through methylation modification of the substrate arginine, and has a crucial impact on various cellular functions. Currently, the research on small molecule inhibitors of PRMT5 has become a hot topic in anti-tumor drug development.

[0007] The results of clinical trials of most first-generation PRMT5 inhibitors entering clinical trials are generally not ideal: Clinical studies have shown that GSK-3326595 has responses to various tumor types. However, 89% of the participants reported adverse events, including anemia, thrombocytopenia, neutropenia, and fatigue. The phase I clinical trial of JNJ-64619178 showed that intermittent dosing had a maintaining target inhibition effect on cancer patients. In contrast, JNJ-64619178 showed limited / no efficacy in patients with myelodysplastic syndrome. Most patients treated with JNJ-64619178 had adverse events, including thrombocytopenia, anemia, and nausea. PF-06939999, as a PRMT5 inhibitor, had an objective tumor response in patients with head and neck squamous cell carcinoma and non-small cell lung cancer. Cytopenia was observed in clinical trials, and they were dose-dependent and reversible dose modifications. The clinical trials of the above PRMT5 inhibitors seem to have been halted.

[0008] In fact, except for the project of Prelude Therapeutics, most active projects are dedicated to second-generation PRMT5 inhibitors, namely PRMT5-MTA inhibitors. In cancer cells lacking MTAP, MTA replaces SAM to bind to PRMT5, forming an inactive PRMT5-MTA complex. Second-generation PRMT5 inhibitors bind to the PRMT5-MTA complex, killing cancer cells lacking MTAP while sparing normal cells. This property allows second-generation PRMT5 inhibitors to more specifically target MTAP-deficient cancer cells and may reduce the incidence of adverse events.

[0009] Currently, the second-generation PRMT5 inhibitors that have been reported and entered the clinical phase I / II study include MRTX-1719, SKL-27969, TNG-908, TNG-462, AMG-193, etc., but no drugs have been marketed yet; documents such as WO2022132914 and WO2022115377 disclose PRMT5 inhibitors. SUMMARY OF THE INVENTION Summary of the Invention

[0011] The object of the present invention is to provide a novel PRMT5 inhibitor compound with good activity, good selectivity, good metabolic stability, good permeability, fast pharmacokinetic absorption, good pharmacokinetic exposure, and having pharmacokinetic advantages, and its use in the treatment of cancer. The heterocyclic compound provided by the present invention has a good inhibitory effect on PRMT5.

[0012] The present invention solves the above technical problems through the following technical solutions.

[0013] On the one hand, the present invention provides a compound represented by formula I, its stereoisomers, or its pharmaceutically acceptable salts:

[0014]

[0015] Wherein,

[0016] X is selected from O, -NR, -CH2;

[0017] R is selected from H, -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 haloalkyl, -C3-C6 cycloalkyl, -(CH2) r OC1-C6 alkyl, -C1-C6 hydroxyalkyl, -(CH2) n -NR a2 R a3 ;

[0018] R1, R2, and R3 are each independently selected from H, halogen, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C1-C6 haloalkyl, -CN, -OR a1 , -NR a2 R a3 ;

[0019] R a1 、R a2 、R a3 are each independently selected from H, -C1-C6 alkyl, -C3-C6 cycloalkyl;

[0020] n is 0, 1, 2, 3, 4;

[0021] m is 0, 1, 2;

[0022] r is 1, 2, 3, or 4;

[0023] R4 is selected from H, a halogen group, -C1-C6 alkyl, -C1-C6 haloalkyl, or when n is 2, two R4s together with the carbon atom to which they are attached form a bridged, fused, or spiro 3- to 10-membered cycloalkyl or 3- to 10-membered heterocyclic group;

[0024] R5 and R6 are each independently selected from H, a halogen group, -CN, -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 haloalkyl;

[0025] R7 and R8 are each independently selected from H, oxo, -C1-C6 alkyl, -C2-C6 haloalkyl, -C3-C6 cycloalkyl;

[0026] The dashed line indicates that the bond here can be a single bond or a double bond.

[0027] As a preferred technical solution, the compound is selected from:

[0028]

[0029] As a preferred technical solution, the compound is selected from:

[0030]

[0031] As a preferred technical solution, X is selected from O, -CH2, -NCH3, -NCH2CH3, -NCH(CH3)2, -NCH2CF3, -N-cyclopropyl, -NH, -N CH2CH(CH3)2, -N(CH2)2CH3, -N(CH2)2OCH3.

[0032] As a preferred technical solution, R1 is selected from H.

[0033] As a preferred technical solution, R2 and R3 are each independently selected from H, amino, methyl, ethyl, isopropyl, fluorine, dimethylamino, cyano, methoxy, trifluoromethyl, cyclopropyl.

[0034] As a preferred technical solution, R4 is selected from H, methyl, fluorine, trifluoromethyl.

[0035] As a preferred technical solution, two R4s together with the carbon atom to which they are attached form

[0036]

[0037] As a preferred technical solution, R5 and R6 are each independently selected from H, methyl, trifluoromethyl, fluorine, methoxy, -CN.

[0038] As a preferred technical solution, each of R7 and R8 is independently selected from H, oxo, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoroethyl, and cyclopropyl.

[0039] The present invention also provides a compound selected from the following formula, its stereoisomers, or a pharmaceutically acceptable salt thereof:

[0040]

[0041]

[0042]

[0043] The present invention also provides the compound or its pharmaceutically acceptable salt described in any one of the above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0044] The present invention also provides the use of the compound and its stereoisomers or its pharmaceutically acceptable salt described in any one of the above in the preparation of a drug for treating a disease, disorder, syndrome, or affliction associated with PRMT5.

[0045] As a preferred technical solution, the disease, disorder, syndrome, or affliction associated with the PRMT5 enzyme is cancer, blood disease, inflammatory disease, autoimmune disease, metabolic disease, genetic disease, hormone-related disease, immunodeficiency disease, disease related to cell death, destructive bone disease, thrombin-induced platelet aggregation, liver disease, and cardiovascular disease. The disease, disorder, syndrome, or affliction associated with the PRMT5 enzyme is preferably cancer. More preferably, the cancer is advanced solid tumor, metastatic pancreatic cancer, metastatic non-small cell lung cancer, neuro-oncology, adenocarcinoma, endometrioid carcinoma, metastatic esophageal cancer, metastatic head and neck cancer, squamous cell carcinoma, cervical tumor, myelodysplastic syndrome, non-Hodgkin lymphoma, acute myeloid leukemia, adenoid tumor, blood tumor, melanoma, pancreatic tumor, brain tumor, glioblastoma, glioma, myelofibrosis, breast tumor, chronic myelomonocytic leukemia, diffuse large B-cell lymphoma, bladder cancer, cholangiocarcinoma, mesothelioma, ovarian cancer, lung cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer, and hepatocellular carcinoma.

[0046] The present invention also provides a method for preparing the compound, its stereoisomers, or its pharmaceutically acceptable salt represented by formula (I) according to claim 1, the method comprising the steps:

[0047]

[0048] A condensation reaction occurs between a compound of general formula (Ia) or its isomer or salt and a compound of general formula (Ib) or its isomer or salt to obtain the compound shown in formula (Ⅰ) and its stereoisomers or its pharmaceutically acceptable salts.

[0049] The definitions of each group are as defined previously.

[0050] The present invention also provides a method for preparing a compound shown in formula (Ⅰa) and its stereoisomers or its pharmaceutically acceptable salts, and the method comprises the steps of:

[0051]

[0052] (1) An acylation reaction occurs between a compound of general formula (Id) or its isomer or salt and a compound of (Ie) to form a compound of general formula (Ic) or its isomer or salt;

[0053] (2) An ester hydrolysis reaction occurs on the compound of general formula (Ic) or its isomer or salt to obtain the compound shown in formula (Ⅰa) and its stereoisomers or its pharmaceutically acceptable salts,

[0054] The definitions of each group are as defined previously.

[0055] Finally, the present invention provides the compounds shown in the following formulae (Ⅰa), (Ⅰc), (Ⅰd) and their stereoisomers or their pharmaceutically acceptable salts,

[0056] The definitions of each group are as described previously.

[0057] The present invention also provides an intermediate compound and its stereoisomers or its pharmaceutically acceptable salts, which are selected from any one of the following compounds:

[0058]

[0059]

[0060] The present invention also provides an intermediate compound and its stereoisomers or its pharmaceutically acceptable salts, which are selected from any one of the following compounds:

[0061]

[0062]

[0063] Finally, the present invention provides an intermediate compound and its stereoisomers or its salts, which are selected from any one of the following compounds:

[0064]

[0065]

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] Through computer-aided design technology, the present invention modifies analogs such as TNG908 and TNG462. Through computer simulation, molecular docking analysis, research on the structure-activity relationship of drugs, etc., a variety of compounds with novel structures are screened. This series of compounds can bind to the PRMT5-MTA complex and inhibit the function of PRMT5, and have extremely strong PRMT5 inhibitory activity. In vitro and in vivo experiments further show that this series of compounds can prevent and / or treat cancers, blood diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, genetic diseases, hormone-related diseases, immunodeficiency diseases, diseases related to cell death, destructive bone diseases, thrombin-induced platelet aggregation, liver diseases, cardiovascular diseases, etc. They have good activity, good selectivity, good metabolic stability, good permeability, fast absorption, and show good pharmacokinetic exposure in mouse pharmacokinetic tests. Detailed Description of the Invention

[0069] The following further describes various aspects and characteristics of the present invention.

[0070] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed by these documents are inconsistent with those of the present invention, the description of the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to make more detailed explanations and interpretations of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings expressed by the present invention shall prevail. The following are the definitions of various terms used in the present invention, and these definitions apply to the terms used throughout the specification of the present application, unless otherwise specified in specific cases.

[0071] Compounds according to the present invention may exist in tautomeric forms, and the present invention includes all tautomeric forms.

[0072] The compounds of the present invention have asymmetric centers. Compounds containing asymmetrically substituted atoms in the present invention can be separated into optically active or racemic forms. Those skilled in the art know how to prepare optically active forms, such as by resolution of racemates or synthesis from optically active starting materials. Unless specifically indicating a particular stereochemistry or isomeric form, the present invention includes all chiral, diastereomeric, and racemic forms. The methods for preparing the compounds of the present invention and their intermediates are part of the present invention. All tautomers of the compounds of the present invention also belong to the present invention.

[0073] "Alkyl" refers to a group of straight-chain or branched-chain saturated hydrocarbon groups having 1 to 10 carbon atoms ("C1-C10 alkyl"). In some embodiments, the alkyl has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl has 1 to 6 carbon atoms ("C1-C6 alkyl"), the alkyl has 1 to 5 carbon atoms ("C1-C5 alkyl"), the alkyl has 1 to 4 carbon atoms ("C1-C4 alkyl"), the alkyl has 1 to 3 carbon atoms ("C1-C3 alkyl"), the alkyl has 1 to 2 carbon atoms ("C1-C2 alkyl"), the alkyl has 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). Additional examples of alkyl include n-heptyl (C7), n-octyl (C8), etc. Each instance of alkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted by one or more substituents; for example, substituted by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl"). In certain embodiments, the alkyl is an unsubstituted C1-C10 alkyl (e.g., -CH3). In certain embodiments, the alkyl is a substituted C1-C6 alkyl. Common alkyl abbreviations include Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0074] As described herein, the terms "halogen", "halo", "halo group", etc. denote fluorine, chlorine, bromine, or iodine, particularly denote fluorine, chlorine, bromine, and are particularly preferably fluorine, chlorine.

[0075] "Haloalkyl" refers to an alkyl as described herein (e.g., C1-C6 alkyl) in which one or more hydrogen atoms are replaced by a halogen (e.g., monohaloalkyl, dihaloalkyl, and trihaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloro-fluoroalkyl, chloro-difluoroalkyl, and 2-fluoroisobutyl.

[0076] "Alkoxy" refers to an alkyl group as described herein (e.g., C1-C6 alkyl) that is attached to a molecule through an oxygen atom. This includes moieties where the alkyl portion can be straight-chain or branched, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, and n-hexyloxy.

[0077] In some embodiments, "cycloalkyl" is a monocyclic saturated cycloalkyl having 3 to 10 ring carbon atoms ("C3-10 cycloalkyl" or "C3-C10 cycloalkyl"). In some embodiments, the cycloalkyl has 3 to 8 ring carbon atoms ("C3-8 cycloalkyl" or "C3-C8 cycloalkyl"). In some embodiments, the cycloalkyl has 3 to 6 ring carbon atoms ("C3-6 cycloalkyl" or "C3-C6 cycloalkyl"). In some embodiments, the cycloalkyl has 5 to 6 ring carbon atoms ("C5-6 cycloalkyl" or "C5-C6 cycloalkyl"). In some embodiments, the cycloalkyl has 5 to 10 ring carbon atoms ("C5-10 cycloalkyl" or "C5-C10 cycloalkyl"). Examples of C5-C6 cycloalkyl include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl include the foregoing C5-C6 cycloalkyl, as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl include the foregoing C3-C6 cycloalkyl, as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of cycloalkyl is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, the cycloalkyl is an unsubstituted C3-C10 cycloalkyl. In certain embodiments, the cycloalkyl is a substituted C3-C10 cycloalkyl.

[0078] The terms "heterocycle", "heterocyclic group" are used interchangeably and refer to substituted and unsubstituted 3- to 7-membered monocyclic groups, 7- to 11-membered bicyclic groups, and 10- to 15-membered tricyclic groups that have at least one heteroatom (O, S, or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2, or 3 heteroatoms selected from O, S, and N. Each ring of such heteroatom-containing groups can contain one or two oxygen or sulfur atoms or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. The nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen atoms can be optionally quaternized. The fused rings that complete the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or fully unsaturated. The heterocyclic group can be attached at any available nitrogen or carbon atom; the "heterocyclic group" as described herein includes heterocycloalkyl.

[0079] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrole-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, dithiolanyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azocanyl, oxocanyl, and thiooctanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, and the like. Exemplary 6-membered heterocyclic groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0080] In addition to the heteroaryl groups described below, exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolinyl, isoxazolinyl, thiazolinyl, isothiazolinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridone, 4-piperidone, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholine sulfoxide, thiomorpholine sulfone, 1,3-dioxolane, and tetrahydro-1,1-dioxothienyl, and the like. Exemplary bicyclic heterocyclic groups include quinolinyl.

[0081] As used herein, the term "pharmaceutically acceptable salt" means a salt that is not only physiologically acceptable to a subject but also refers to a synthetic substance that has utility in pharmacy, such as a salt formed as an intermediate during chiral resolution. Although such an intermediate salt cannot be directly administered to a subject, the salt can play a role in obtaining the end product of the present invention.

[0082] As described herein, the term "disease" refers to a physical state of the subject that is related to the disease described in the present invention. For example, the arterial peripheral diseases and neurodegenerative-related diseases described in the present invention.

[0083] The cancers of the present invention include standard treatments such as surgery, radiotherapy, chemotherapy, and hormone therapy.

[0084] "Cancer" or "malignant tumor" refers to any of a variety of diseases characterized by uncontrolled abnormal cell proliferation, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cells" refer to cells that have undergone early, intermediate, or late stages of multi-step tumor progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis carcinoma, and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine body cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumor, head and neck cancer, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hepatocellular carcinoma, gallbladder cancer, bronchial tumor, advanced solid tumor, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, and plasmacytoma, lymphoma, pancreatic ductal adenocarcinoma, etc.

[0085] The compounds or pharmaceutical compositions containing them in the present invention can be administered in unit dosage form, and the administration routes can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0086] The dosage forms can be liquid dosage forms, solid dosage forms, or semi-solid dosage forms. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w type, w / o type, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, aerosols (powder aerosols), sprays, etc.; semi-solid dosage forms can be ointments, gels, pastes, etc.

[0087] For the purpose of achieving the medicinal objective and enhancing the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any well-known administration method.

[0088] The compounds or compositions of the present invention can be taken alone or used in combination with other therapeutic drugs or symptomatic drugs. When there is a synergistic effect between the compounds of the present invention and other therapeutic drugs, the dosage should be adjusted according to the actual situation. Detailed implementation manners

[0089] The following listed examples are helpful for those skilled in the art to better understand the technical solutions of the present invention, but do not limit the present invention in any way.

[0090] For all the following examples, standard operations and methods known to those skilled in the art can be used. Unless otherwise specified, all temperatures are expressed in °C (degrees Celsius). The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectrometry (MS).

[0091] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) or / and liquid chromatography - mass spectrometry (LC - MS). The NMR chemical shift (δ) is in parts per million (ppm). The nuclear magnetic resonance was measured with a Bruker avance - 400 nuclear magnetic resonance spectrometer, and the solvents were deuterated dimethyl sulfoxide (DMSO - d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0092] For the LC - MS measurement of the liquid phase part, an ACQUITY UPLC ultra - high - pressure liquid chromatography was used, and for the mass spectrometry part, an Xevo G2 - S Qtof mass spectrometer was used.

[0093] The starting materials in the examples of the present invention are known and can be purchased on the market, or can also be used or synthesized according to the methods known in the art.

[0094] Example 1: Synthesis of N-(6 - amino - 5 - ethylpyridin - 3 - yl)-2-[(2R,5S)-2-(3,4 - dihydro - 1H - benzo[4,5]imidazo[2,1 - c][1,4]oxazin - 8 - yl)-5 - methylpiperidin - 1 - yl)]-2 - oxoacetamide (Compound 1)

[0095]

[0096] Step 1: Synthesis of 4-(4 - bromo - 2 - nitrophenyl)morpholine

[0097] To a solution of 1,4-dibromo-2-nitrobenzene (5.0 g, 17.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (30 mL), morpholine (4.7 g, 53.9 mmol, 3.0 equiv.) and potassium carbonate (7.4 g, 53.9 mmol, 3.0 equiv.) were added. The reaction was carried out at 120 °C for 16 h. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate, washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain 4-(4-bromo-2-nitrophenyl)morpholine (4.5 g, yield 88%). LCMS: m / z = [M+H] 287.0. 1H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J = 2.4 Hz, 1H), 7.79 (dd, J = 8.8, 2.4 Hz, 1H), 7.32 (d, J = 8.9 Hz, 1H), 3.76–3.54 (m, 4H), 3.02–2.91 (m, 4H).

[0098] Step 2: Synthesis of 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0099] In a sealed tube, to a solution of 4-(4-bromo-2-nitrophenyl)morpholine (4.5 g, 15.7 mmol, 1.0 equiv.) in formic acid (20 mL), I2 (800 mg, 3.2 mmol, 0.2 equiv.) was added. The reaction was carried out at 120 °C for 18 h. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate, washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1 g, yield 25%). LCMS: m / z = [M+H]+: 253.0. 1H NMR (400 MHz, DMSO-d6) δ 8.09 (m, J = 3.8, 1.8 Hz, 1H), 7.89–7.80 (m, 1H), 7.71 (m, J = 7.8, 4.0, 1.8 Hz, 1H), 5.17 (d, J = 4.2 Hz, 2H), 4.38 (m, J = 4.3, 3.6 Hz, 2H), 4.29–4.21 (m, 2H).

[0100] Step 3: Synthesis of 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0101] The compound 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (742 mg, 2.9 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (10 mL). Bis(pinacolato)diboron (3000 mg, 11.7 mmol, 4.0 eq), potassium acetate (866 mg, 8.7 mmol, 3.0 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (218 mg, 0.3 mmol, 0.1 eq) were added to the system. The system was purged with nitrogen three times and heated to 110 °C for reaction for 6 hours. After the reaction was completed, it was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the white solid 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (400 mg, yield 46%). LCMS: m / z = [M+H] 301.1.

[0102] Step 4: Synthesis of tert-butyl (S)-6-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0103] The compound 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (322 mg, 1.1 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (9 mL) and water (3 mL). (S)-tert-Butyl 3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (379 mg, 1.1 mmol, 1.0 equiv.), sodium carbonate (341 mg, 3.3 mmol, 3.0 equiv.) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (78 mg, 0.1 mmol, 0.1 equiv.) were added to the system. The system was purged with nitrogen three times and heated to 90 °C for reaction for 6 hours. After the reaction was completed, it was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the yellow solid tert-butyl (S)-6-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (121 mg, yield 40%). LCMS: m / z = [M+H]-Boc 270.20.

[0104] Step 5: Synthesis of (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0105] Dissolve tert-butyl (S)-6-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (121 mg, 0.45 mmol) in dichloromethane (1.5 mL), add trifluoroacetic acid (0.5 mL), stir for 30 minutes, and monitor the reaction by TLC and LCMS. After the reaction is completed, directly concentrate the reaction solution, adjust the pH to 8 - 9 with saturated sodium carbonate solution, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (88 mg). LCMS: m / z = [M + H] 270.1.

[0106] Step 6: Synthesis of 8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0107] Dissolve (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (88 mg, 0.33 mmol, 1.0 equiv.) in methanol (3 mL). Under ice bath conditions, add sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) portionwise and stir the reaction for 1 hour. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the yellow solid 8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (60 mg, yield 68%). LCMS: m / z = [M + H] 272.2.

[0108] Step 7: Synthesis of ethyl 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate

[0109] 8-[(5S)-5-Methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (60 mg, 0.22 mmol, 1.0 equiv.) was added into a reaction flask. After the system was purged with nitrogen three times, triethylamine (30 mg, 0.26 mmol, 1.2 equiv.) was added. Subsequently, under ice bath conditions, ethyl oxalyl chloride (36 mg, 0.26 mmol, 1.2 equiv.) was slowly added dropwise. The mixture was stirred at room temperature, and the reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction was quenched with water. The mixture was extracted with water and dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and loaded onto a silica gel column for chromatography to obtain ethyl 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (23 mg, yield 28%). LCMS: m / z = [M+H] 372.2.

[0110] Step 8: Synthesis of 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid

[0111] Ethyl 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (23 mg, 0.06 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (1 mL) and water (1 mL). Lithium hydroxide hydrate (6 mg, 0.12 mmol, 2 equiv.) was added to the system, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to 5 - 6 with 1 M aqueous hydrochloric acid, and the mixture was concentrated to obtain the crude product 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (20 mg). LCMS: m / z = [M+H] 344.1.

[0112] Step 9: Synthesis of 5-nitro-3-vinylpyridin-2-amine

[0113] Dissolve compound 3-bromo-5-nitropyridin-2-amine (1000 mg, 4.6 mmol, 1.0 equiv.) in 1,4-dioxane (40 mL) and water (10 mL). Add potassium vinyltrifluoroborate (741 mg, 5.5 mmol, 1.2 equiv.), potassium carbonate (3179 mg, 23.0 mmol, 5.0 equiv.) and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) (334 mg, 0.46 mmol, 0.1 equiv.) to the system. Replace the gas in the system with nitrogen 3 times and heat to 80 °C for reaction for 8 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain white solid 5-nitro-3-vinylpyridin-2-amine (388 mg, yield 44%). LCMS: m / z = [M+H] 166.1. 1H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 2.7 Hz, 1H), 8.27 (d, J = 2.6 Hz, 1H), 7.58 (s, 2H), 6.84 (dd, J = 17.2, 11.0 Hz, 1H), 5.93 (d, J = 17.2 Hz, 1H), 5.46 (d, J = 11.0 Hz, 1H).

[0114] Step 10: Synthesis of 3-ethylpyridine-2,5-diamine

[0115] Dissolve compound 5-nitro-3-vinylpyridin-2-amine (384 mg, 1.44 mmol, 1.0 equiv.) in methanol (6 mL). Add palladium on carbon (Pd 5%, containing about 55% water, 76 mg). Replace the gas in the system with hydrogen 3 times and react at room temperature for 24 hours. After the reaction is completed, filter the reaction solution and separate by column chromatography to obtain black oily liquid 3-ethylpyridine-2,5-diamine (80 mg, yield 41%). LCMS: m / z = [M+H] 138.1.

[0116] Step 11: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0117] Dissolve 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (20.0 mg, 0.05 mmol, 1.0 equiv.), 3-ethylpyridine-2,5-diamine (6.2 mg, 0.05 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (15.0 mg, 0.11 mmol, 2.5 equiv.) in N,N-dimethylformamide (1.5 mL), stir for 5 minutes, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.0 mg, 0.07 mmol, 1.5 equiv.), and stir at room temperature for 2 hours. After the reaction is completed, purify by SFC preparative chromatography to obtain the white solid N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (5 mg, yield 21%). LCMS: m / z = [M+H] 463.2379. 1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.08 (s, 1H), 7.63–7.40 (m, 3H), 7.17 (d, J = 35.0 Hz, 1H), 5.76–5.24 (m, 3H), 4.94 (s, 2H), 4.17 (m, J = 8.3, 4.3 Hz, 5H), 3.41 (d, J = 87.8 Hz, 1H), 2.47–2.23 (m, 3H), 2.12 (m, 1H), 1.82 (m, J = 44.1 Hz, 2H), 1.37 (m, 1H), 1.18–1.03 (m, 6H).

[0118] Example 2: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-{5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide (Compound 2)

[0119]

[0120]

[0121] Step 1: Synthesis of 4-(4-bromo-2-nitrophenyl)morpholine

[0122] To a solution of 1,4-dibromo-2-nitrobenzene (5.0 g, 17.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (30 mL), (S)-2-methylmorpholine (5.4 g, 53.9 mmol, 3.0 equiv.) and potassium carbonate (7.4 g, 53.9 mmol, 3.0 equiv.) were added, and the reaction was carried out at 120 °C for 16 h. After the reaction was completed, the reaction mixture was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain (S)-4-(4-bromo-2-nitrophenyl)-2-methylmorpholine (4.7 g, yield 88%). LCMS: m / z = [M+H] 301.0.

[0123] Step 2: Synthesis of (S)-8-bromo-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0124] In a sealed tube, to a solution of (S)-4-(4-bromo-2-nitrophenyl)-2-methylmorpholine (4.7 g, 15.7 mmol, 1.0 equiv.) in formic acid (20 mL), I2 (800 mg, 3.2 mmol, 0.2 equiv.) was added, and the reaction was carried out at 120 °C for 18 h. After the reaction was completed, the reaction mixture was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain (S)-8-bromo-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1.5 g, yield 35.9%). LCMS: m / z = [M+H] + : 267.0.

[0125] Step 3: Synthesis of (S)-3-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0126] Dissolve the compound 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (771.4 mg, 2.9 mmol, 1.0 eq) in N,N-dimethylformamide (15 mL). Add bis(pinacolato)diboron (3000 mg, 11.7 mmol, 4.0 eq), potassium acetate (866 mg, 8.7 mmol, 3.0 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (218 mg, 0.3 mmol, 0.1 eq) to the system. Replace the system with nitrogen three times and heat it to 110 °C for reaction for 6 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the white solid (S)-3-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (390 mg, yield 42.8%). LCMS: m / z = [M+H] 315.2.

[0127] Step 4: Synthesis of tert-butyl (S)-3-methyl-6-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3,4-dihydropyridine-1(2H)-carboxylate

[0128] The compound (S)-3-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (345.4 mg, 1.1 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (9 mL) and water (3 mL). To the system was added (S)-tert-butyl 3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (379 mg, 1.1 mmol, 1.0 equiv.), sodium carbonate (341 mg, 3.3 mmol, 3.0 equiv.), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (78 mg, 0.1 mmol, 0.1 equiv.). The system was purged with nitrogen three times and heated to 90 °C for reaction for 6 hours. After the reaction was completed, it was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the yellow solid (S)-tert-butyl 3-methyl-6-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3,4-dihydropyridine-1(2H)-carboxylate (200 mg, yield 47.5%). LCMS: m / z = [M+H]-Boc 284.2.

[0129] Step 5: Synthesis of (S)-3-methyl-8-[(S)-5-methyl-1,4,5,6-tetrahydropyridin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0130] (S)-tert-Butyl 3-methyl-6-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3,4-dihydropyridine-1(2H)-carboxylate (191.5 mg, 0.5 mmol, 1.0 eq) was dissolved in dichloromethane (1.5 mL). Trifluoroacetic acid (0.5 mL) was added. After stirring for 30 minutes, the reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction solution was directly concentrated, the pH was adjusted to 8 - 9 with saturated sodium carbonate solution, diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product (S)-3-methyl-8-[(S)-5-methyl-1,4,5,6-tetrahydropyridin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (100 mg, yield 72%). LCMS: m / z = [M+H] 284.1.

[0131] Step 6: Synthesis of (3S)-3-Methyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0132] Dissolve (S)-3-Methyl-8-[(S)-5-methyl-1,4,5,6-tetrahydropyridin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (93.72 mg, 0.33 mmol, 1.0 equiv.) in methanol (3 mL). Under ice bath conditions, add sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) portionwise and stir the reaction for 1 hour. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the yellow solid (3S)-3-Methyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (67 mg, yield 71%). LCMS: m / z = [M+H] 286.2.

[0133] Step 7: Synthesis of Ethyl 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate

[0134] Add (3S)-3-Methyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (63 mg, 0.22 mmol, 1.0 equiv.) to the reaction flask. After purging the system with nitrogen three times, add triethylamine (30 mg, 0.26 mmol, 1.2 equiv.). Subsequently, under ice bath conditions, slowly add ethyl chlorooxoacetate (36 mg, 0.26 mmol, 1.2 equiv.) dropwise and stir at room temperature while monitoring the reaction by TLC and LCMS. After the reaction is completed, quench the reaction with water, extract and separate the layers with water and dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter, mix with the sample, and separate by column chromatography to obtain the white solid Ethyl 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate (33 mg, yield 39%). LCMS: m / z = [M+H] 386.2.

[0135] Step 8: Synthesis of 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid

[0136] Dissolve ethyl 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate (23 mg, 0.06 mmol, 1.0 equiv.) in tetrahydrofuran (1 mL) and water (1 mL). Add lithium hydroxide hydrate (6 mg, 0.12 mmol, 2 equiv.) to the system and react at room temperature for 2 hours. After the reaction is completed, adjust the pH to 5 - 6 with 1 M aqueous hydrochloric acid and concentrate to obtain the crude product 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid (21 mg). LCMS: m / z = [M + H] 358.2.

[0137] Step 9: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-{(2R,5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide

[0138] Dissolve 2-{(5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid (18 mg, 0.05 mmol, 1.0 equiv.), 3-ethylpyridine-2,5-diamine (6.2 mg, 0.05 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (15.0 mg, 0.11 mmol, 2.5 equiv.) in N,N-dimethylformamide (1.5 mL), stir for 5 minutes, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.0 mg, 0.07 mmol, 1.5 equiv.), and stir at room temperature for 2 hours. After completion of the reaction, prepare and purify to obtain the yellow solid N-(6-amino-5-ethylpyridin-3-yl)-2-{(2R,5S)-5-methyl-2-[(S)-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide (8.3 mg). LCMS: m / z = [M+H]+ 477.2.

[0139] Example 3: Synthesis of N-(6-amino-5-methylpyridin-3-yl)-2-[(2R,5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 3)

[0140]

[0141] Step 1: Synthesis of N-(6-amino-5-methylpyridin-3-yl)-2-[(2R,5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0142] 2-[(5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (68.6 mg, 0.2 mmol, 1.0 equiv.), 3-methylpyridine-2,5-diamine (59 mg, 0.2 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (64.5 mg, 0.5 mmol, 2.5 equiv.) were dissolved in N,N-dimethylformamide (1.5 mL), stirred for 5 minutes, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114 mg, 0.3 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, purification by normal-phase column chromatography gave the yellow solid N-(6-amino-5-methylpyridin-3-yl)-2-[(2R,5S)-2-(3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (10.2 mg). LCMS: m / z = [M+H]+ 449.2.

[0143] Example 4: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-{(2R,5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide (Compound 4)

[0144]

[0145] Step 1: Synthesis of 4-[4-bromo-2-nitro-5-(trifluoromethyl)phenyl]morpholine

[0146] To a solution of 1-bromo-4-fluoro-5-nitro-2-(trifluoromethyl)benzene (5.11 g, 17.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (30 mL) were added morpholine (4.7 g, 53.9 mmol, 3.0 equiv.) and potassium carbonate (7.4 g, 53.9 mmol, 3.0 equiv.), and the reaction was carried out at 120 °C for 16 hours. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to give 4-[4-bromo-2-nitro-5-(trifluoromethyl)phenyl]morpholine (5.54 g, yield 87%). LCMS: m / z = [M+H] 354.9.

[0147] Step 2: Synthesis of 8-bromo-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0148] In a sealed tube, to a solution of 4-[4-bromo-2-nitro-5-(trifluoromethyl)phenyl]morpholine (5.54 g, 15.7 mmol, 1.0 equiv.) in formic acid (20 mL), add I₂ (800 mg, 3.2 mmol, 0.2 equiv.), and react at 120 °C for 18 h. After completion of the reaction, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain 8-bromo-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1.2 g, yield 23.9%). LCMS: m / z = [M+H] + : 320.9.

[0149] Step 3: Synthesis of 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0150] Dissolve 8-bromo-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (925.1 mg, 2.9 mmol, 1.0 eq) in N,N-dimethylformamide (15 mL). Add bis(pinacolato)diboron (3000 mg, 11.7 mmol, 4.0 eq), potassium acetate (866 mg, 8.7 mmol, 3.0 eq), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (218 mg, 0.3 mmol, 0.1 eq) to the system. Flush the system with nitrogen 3 times and heat to 110 °C for reaction for 6 h. After completion of the reaction, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the white solid 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (415 mg, yield 38.9%). LCMS: m / z = [M+H] 369.1.

[0151] Step 4: Synthesis of tert-butyl (S)-3-methyl-6-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3,4-dihydropyridine-1(2H)-carboxylate

[0152] Dissolve 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (404.8 mg, 1.1 mmol, 1.0 equiv.) in 1,4-dioxane (9 mL) and water (3 mL). Add (S)-tert-butyl 6-[(trifluoromethyl)sulfonyloxy]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (379 mg, 1.1 mmol, 1.0 equiv.), sodium carbonate (341 mg, 3.3 mmol, 3.0 equiv.) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (78 mg, 0.1 mmol, 0.1 equiv.) to the system. Flush the system with nitrogen three times and heat to 90 °C for reaction for 6 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the yellow solid (S)-tert-butyl 6-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (200 mg, yield 47.5%). LCMS: m / z = [M+H]-Boc 337.2.

[0153] Step 5: Synthesis of (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0154] Dissolve (S)-tert-butyl 6-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (191.5 mg, 0.5 mmol, 1.0 eq) in dichloromethane (1.5 mL), add trifluoroacetic acid (0.5 mL), stir for 30 minutes, and monitor the reaction by TLC and LCMS. After the reaction is completed, directly concentrate the reaction solution, adjust the pH to 8 - 9 with saturated sodium carbonate solution, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (120 mg, yield 71%). LCMS: m / z = [M+H] 338.1.

[0155] Step 6: Synthesis of 8-[(5S)-5-methylpiperidin-2-yl]-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0156] Dissolve (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (111.5 mg, 0.33 mmol, 1.0 equiv.) in methanol (5 mL). Under ice-bath conditions, add sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) portionwise and stir the reaction for 1 hour. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the yellow solid 8-[(5S)-5-methylpiperidin-2-yl]-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (75 mg, yield 67%). LCMS: m / z = [M+H] 340.1.

[0157] Step 7: Synthesis of ethyl 2-{(5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate

[0158] Add 8-[(5S)-5-methylpiperidin-2-yl]-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (75 mg, 0.22 mmol, 1.0 equiv.) to the reaction flask. After purging the system with nitrogen three times, add triethylamine (30 mg, 0.26 mmol, 1.2 equiv.). Subsequently, under ice-bath conditions, slowly add ethyl oxalyl chloride (36 mg, 0.26 mmol, 1.2 equiv.) dropwise and stir at room temperature. Monitor the reaction by TLC and LCMS. After the reaction is completed, quench the reaction with water, extract and separate the layers with water and dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, filter, mix with silica gel, and separate by column chromatography to obtain the white solid ethyl 2-{(5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate (33 mg, yield 34%). LCMS: m / z = [M+H] 440.2.

[0159] Step 8: Synthesis of 2-{(5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid

[0160] Dissolve ethyl 2-{(5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetate (26 mg, 0.06 mmol, 1.0 equiv.) in tetrahydrofuran (1 mL) and water (1 mL). Add lithium hydroxide hydrate (6 mg, 0.12 mmol, 2 equiv.) to the system and react at room temperature for 2 hours. After the reaction is completed, adjust the pH to 5 - 6 with 1 M aqueous hydrochloric acid and concentrate to obtain the crude product 2-{(5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid (21 mg). LCMS: m / z = [M+H] 412.1.

[0161] Step 9: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-{(2R,5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide

[0162] 2-{(5S)-5-Methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetic acid (21 mg, 0.05 mmol, 1.0 equiv.), 3-ethylpyridine-2,5-diamine (6.2 mg, 0.05 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (15.0 mg, 0.11 mmol, 2.5 equiv.) were dissolved in N,N-dimethylformamide (1.5 mL), stirred for 5 minutes, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.0 mg, 0.07 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, yellow solid N-(6-amino-5-ethylpyridin-3-yl)-2-{(2R,5S)-5-methyl-2-[7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl]piperidin-1-yl}-2-oxoacetamide (6.5 mg) was prepared by purification. LCMS: m / z = [M+H]+ 531.2.

[0163] Example 5: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide (Compound 5)

[0164]

[0165] Step 1: Synthesis of tert-butyl 4-(4-bromo-2-nitrophenyl)-3-oxopiperazine-1-carboxylate

[0166] tert-Butyl 3-oxopiperazine-1-carboxylate (5.0 g, 25 mmol, 1.1 eq) was added to a flask. After protecting with nitrogen, N,N-dimethylformamide (50 mL) was added. After stirring at room temperature for 10 minutes, sodium hydride (1.4 g, 60% dispersed in mineral oil) was added. Stirring was continued for 30 minutes. Finally, 4-bromo-1-fluoro-2-nitrobenzene (5.0 g, 22.9 mmol, 1.0 eq) was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, ethyl acetate (600 mL) and saturated ammonium chloride solution (200 mL) were added. The layers were separated, and the organic phase was washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to dryness. The crude product was purified by column chromatography (mobile phase: petroleum ether / ethyl acetate = 2 / 1 (V / V)) to obtain tert-butyl 4-(4-bromo-2-nitrophenyl)-3-oxopiperazine-1-carboxylate (3.8 g, yield 42.6%). LCMS: m / z = [M+H]: 400.0.

[0167] Step 2: Synthesis of tert-butyl 8-bromo-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0168] tert-Butyl 4-(4-bromo-2-nitrophenyl)-3-oxopiperazine-1-carboxylate (3.8 g, 9.5 mmol, 1.0 eq), reduced iron powder (2.5 g, 47.5 mmol, 5.0 eq), and acetic acid (50 mL) were added to a flask, and the mixture was refluxed for 1 hour. After the reaction was completed, the organic phase was concentrated to dryness, ethyl acetate (50 mL) was added, and the mixture was washed with saturated aqueous sodium bicarbonate solution (50 mL). The organic phase was dried, concentrated, and the crude product was purified by column chromatography (mobile phase: dichloromethane / methanol = 50 / 1 (V / V)) to obtain tert-butyl 8-bromo-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.1 g, yield 62.9%). LCMS: m / z = [M-Boc+H]: 252.0.

[0169] Step 3: Synthesis of 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0170] tert-Butyl 8-bromo-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.1 g, 5.9 mmol, 1.0 eq) was dissolved in dichloromethane (15 mL), 4N hydrochloric acid dioxane solution (10 mL) was added, and the mixture was stirred overnight. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction solution was concentrated, saturated sodium carbonate solution was added to adjust the pH to 8 - 9, diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain the crude product 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.4 g, yield 94.5%). LCMS: m / z = [M + H]: 252.0.

[0171] Step 4: Synthesis of 8-bromo-2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0172] 8-Bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.38 g, 5.5 mmol, 1.0 eq), aqueous formaldehyde solution (36%, 1.1 g), sodium triacetoxyborohydride (2.3 g, 11 mmol, 2.0 eq), acetic acid (0.5 g) and 1,2-dichloroethane (20 mL) were added to the reaction flask and stirred at room temperature overnight. After the reaction was completed, the organic phase was concentrated to dryness, neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate, and after concentration of the organic phase, the crude product was purified by column chromatography (mobile phase: dichloromethane / methanol = 20 / 1 (V / V)) to obtain 8-bromo-2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.12 g). LCMS: m / z = [M + H]: 266.0.

[0173] Step 5: Synthesis of 2-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0174] Dissolve the compound 8-bromo-2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.12 g, 4.2 mmol, 1.0 eq) in DMF (10 ml). Add bis(pinacolato)diboron (4.2 g, 17 mmol, 4.0 eq), potassium acetate (1.3 g, 12.6 mmol, 3.0 eq), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (310 mg, 0.42 mmol, 0.1 eq) to the system. Replace the gas in the system with nitrogen 3 times and heat to 110 °C for reaction for 6 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain 560 mg of a white solid. LCMS: m / z = [M+H] 314.2.

[0175] Step 6: Synthesis of tert-butyl (S)-3-methyl-6-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3,4-dihydropyridine-1(2H)-carboxylate

[0176] Dissolve the compound 2-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (560 mg, 1.8 mmol, 1.0 equiv.) in 1,4-dioxane (12 ml) and water (4 ml). Add tert-butyl 5-methyl-2-(((trifluoromethyl)sulfonyl)oxy)cyclohex-2-ene-1-carboxylate (621 mg, 1.8 mmol, 1.0 equiv.), sodium carbonate (558 mg, 5.4 mmol, 3.0 equiv.), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (150 mg, 0.18 mmol, 0.1 equiv.) to the system. Replace the gas in the system with nitrogen 3 times and heat to 90 °C for reaction for 6 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain 360 mg of a yellow solid. LCMS: m / z = [M - Boc + H] 283.2.

[0177] Step 7: Synthesis of 2-methyl-8-((2R,5S)-5-methylpiperidin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0178] Dissolve tert-butyl (S)-3-methyl-6-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3,4-dihydropyridine-1(2H)-carboxylate (360 mg, 1.3 mmol) in DCM (5 mL), add TFA (0.5 mL), stir for 0.5 h, and monitor the reaction by TLC and LCMS. After the reaction is completed, rotary evaporate the solvent, adjust the pH to 8 - 9 with saturated sodium carbonate solution, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and rotary evaporate the solvent to obtain the crude intermediate product (S)-2-methyl-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine, LCMS: m / z = [M + H] 283.1928. Then dissolve this crude product in methanol (5 mL), add sodium borohydride (100 mg, 2.6 mmol, 2.0 equiv.) portionwise under ice bath conditions, stir for 0.5 h, monitor the reaction by TLC and LCMS. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the product (195 mg) LCMS: m / z = [M + H] 285.2.

[0179] Step 8: Synthesis of 2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetic acid

[0180] 2-Methyl-8-((2R,5S)-5-methylpiperidin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (195 mg, 0.66 mmol, 1.0 equiv.) was added to a reaction flask. After protection with N2, triethylamine (90 mg, 0.78 mmol, 1.2 equiv.) was added, and monoethyl oxalyl chloride (108 mg, 0.79 mmol, 1.2 equiv.) was slowly added dropwise under ice bath conditions, and the reaction was stirred at room temperature. The reaction was monitored by TLC and LCMS. After the reaction was completed, the reaction was quenched with water, and the mixture was extracted with water and dichloromethane and separated. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and loaded onto a silica gel column for chromatography to obtain (69 mg) of ethyl 2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetate as a white solid. LCMS: m / z = [M+H] 385.1918; The obtained crude product was dissolved in tetrahydrofuran (1 mL) and water (1 mL), lithium hydroxide hydrate (18 mg, 0.36 mmol, 2 equiv.) was added to the system, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to 5-6 with 1 M aqueous hydrochloric acid, and the mixture was concentrated to obtain the crude product 2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetic acid (45 mg). LCMS: m / z = [M+H] 357.2.

[0181] Step 9: Synthesis of N-(6-Amino-5-ethylpyridin-3-yl)-2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide

[0182] 2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetic acid (20.0 mg, 0.05 mmol, 1.0 equiv.), 5-amino-3-vinylpyridin-2-amine (6.2 mg, 0.05 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (15.0 mg, 0.11 mmol, 2.5 equiv.) were dissolved in N,N-dimethylformamide (1.5 mL), stirred for 5 minutes, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (26.0 mg, 0.07 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, purification by preparative HPLC gave the white solid N-(6-amino-5-ethylpyridin-3-yl)-2-((2R,5S)-5-methyl-2-(2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide (5 mg). LCMS: m / z = [M+H] 476.3.

[0183] Example 6: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 10)

[0184]

[0185] Step 1: Synthesis of 4-(4-bromo-2-nitrophenyl)-2,2-dimethylmorpholine

[0186] 2,2-Dimethylmorpholine (2 g, 17.4 mmol, 1.0 eq), 4-bromo-1-fluoro-2-nitrobenzene (4.59 g, 20.8 mmol, 1.2 eq) and potassium carbonate (7.21 g, 52.2 mmol, 3.0 eq) were added to acetonitrile (40 mL), the system was purged with nitrogen twice, and the reaction was carried out at 60 °C for 16 hours. After completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated and mixed with silica gel, and purified by normal-phase column chromatography with a petroleum ether and ethyl acetate system. When the ethyl acetate content was 10%, orange oil 4-(4-bromo-2-nitrophenyl)-2,2-dimethylmorpholine (5.0 g, 8.0 mmol, yield 92%) was obtained.

[0187] Step 2: Synthesis of 8-bromo-3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0188] 4-(4-Bromo-2-nitrophenyl)-2,2-dimethylmorpholine (4.5 g, 14.3 mmol, 1.0 eq) and iodine (1.46 g, 5.7 mmol, 0.4 eq) were dissolved in formic acid (50 mL), and the reaction was carried out at 120 °C for 16 h. After the reaction was completed, the reaction solution was filtered, the filtrate was concentrated, and the pH value was adjusted to about 10 with sodium carbonate. Then, it was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate to remove excess water, and then rotary evaporated and mixed with silica gel. It was separated and purified by a normal-phase column. When the content of ethyl acetate in the petroleum ether and ethyl acetate system was 26%, the target product eluted. After the obtained solution was concentrated, it was slurried with a mixed solvent of petroleum ether and ethyl acetate (the volume ratio of petroleum ether to ethyl acetate was 5:1) to obtain 8-bromo-3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine as a brown solid (1.7 g, 6.1 mmol, yield 42%). LCMS (ESI) [M+H]+ = 281. 1H NMR (400 MHz, DMSO) δ 7.80 (d, J = 1.8 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.39 (dd, J = 8.5, 1.8 Hz, 1H), 4.97 (s, 2H), 4.08 (s, 2H), 1.36 (s, 6H).

[0189] Step 3: Synthesis of 3,3-dimethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0190] 8-Bromo-3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1 g, 3.8 mmol, 1.0 eq), bis(pinacolato)diboron (1.81 g, 7.1 mmol, 2.0 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (261 mg, 0.4 mmol, 0.1 eq) and potassium acetate (1.05 g, 10.7 mmol, 3.0 eq) were added to N,N-dimethylformamide (15 mL), and the gas in the system was replaced with nitrogen. The reaction was carried out at 100 °C for 4 h. After the reaction was completed, the mixture was filtered, concentrated and mixed with silica gel. It was separated and purified by a normal-phase column. When the content of ethyl acetate in the petroleum ether and ethyl acetate system was 35%, 3,3-dimethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine was obtained as a white solid (1.7 g, 6.1 mmol, yield 42%), LCMS (ESI) [M+H] + = 329.

[0191] Step 4: Synthesis of tert-butyl (S)-6-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0192] 3,3-Dimethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (236 mg, 0.72 mmol, 1.0 eq), tert-butyl (S)-3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (300 mg, 0.87 mmol, 1.2 eq), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (52 mg, 0.07 mmol, 0.1 eq) and potassium carbonate (300 mg, 2.16 mmol, 3.0 eq) were dissolved in dioxane (3 mL) and water (1 mL). Under nitrogen protection, the reaction was carried out at 90 °C for 16 hours. After the reaction was completed, the reaction solution was filtered, and the filtrate was concentrated and rotary evaporated to mix the samples. It was separated and purified by a normal-phase column. In the system of petroleum ether and ethyl acetate, when the content of ethyl acetate was 30%, brown solid tert-butyl (S)-6-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (230 mg, 0.58 mmol, yield 80%) was obtained. LCMS (ESI) [M+H]+ = 398.

[0193] Step 5: Synthesis of (S)-3,3-dimethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0194] (S)-6-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (230 mg, 0.58 mmol, 1.0 eq) was dissolved in trifluoroacetic acid (1 mL) and dichloromethane (3 mL). The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the reaction solution was directly concentrated to obtain crude white solid (S)-3,3-dimethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (103 mg, 0.35 mmol, yield 59.8%). LCMS (ESI) [M+H]+ = 298.

[0195] Step 6: Synthesis of 3,3-dimethyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0196] (S)-3,3-Dimethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (100 mg, 0.34 mmol, 1.0 eq), sodium borohydride (25 mg, 0.67 mmol, 2.0 eq) were dissolved in methanol (2 mL), and the reaction was stirred at room temperature for 30 minutes. After complete reaction, the reaction solution was directly concentrated to obtain the crude white solid 3,3-dimethyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (100 mg), which was directly used in the next step. LCMS (ESI) [M+H]+: 300.

[0197] Step 7: Synthesis of ethyl 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate

[0198] 3,3-Dimethyl-8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (100 mg, 0.334 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (2 mL), and then a solution of triethylamine (50 mg, 0.5 mmol, 1.5 eq) and ethyl oxalyl chloride (45.6 mg, 0.501 mmol, 1.5 eq) in N,N-dimethylformamide (1 mL) was added in small portions under ice bath. The reaction was carried out at room temperature for 2 hours (monitored continuously). After complete reaction, the reaction solution was concentrated to obtain the crude white solid ethyl 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (200 mg), which was directly used in the next step. LCMS (ESI) [M+H]+ = 400.

[0199] Step 8: Synthesis of 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid

[0200] Ethyl 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (100 mg, 0.25 mmol, 1.0 eq), lithium hydroxide (6 mg, 2.5 mmol, 10 eq) were dissolved in methanol (4 mL) and water (0.4 mL), and then reacted at room temperature for 1 hour. After complete reaction, the pH value was adjusted to about 4 with dilute hydrochloric acid, and extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate to remove excess water, and concentrated to obtain crude 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (100 mg) as a white solid, which was directly used for the next reaction. LCMS (ESI) [M+H]+ = 372.

[0201] Step 9: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0202] 2-[(5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (80 mg, 0.22 mmol, 1.0 eq) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (92 mg, 0.33 mmol, 1.5 eq) were dissolved in N,N-dimethylacetamide (2 mL), then N,N-diisopropylethylamine (70 mg, 0.55 mmol, 2.5 eq) was added. After reacting at room temperature for 30 minutes, 3-ethylpyridine-2,5-diamine (35 mg, 0.26 mmol, 1.2 eq) was added, and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, it was separated and purified by high performance liquid chromatography and SFC to obtain N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (5.6 mg, yield 5.4%) as a yellow powder. LCMS (ESI) [M+H]+: 491.2772. 11H NMR (400 MHz, DMSO) δ 10.57 (d, J = 15.4 Hz, 1H), 8.26 (s, 1H), 8.16–7.96 (m, 1H), 7.59–7.40 (m, 3H), 7.34–7.14 (m, 1H), 5.73–5.61 (m, 2H), 4.95 (s, 2H), 4.05 (d, J = 5.2 Hz, 2H), 3.44 (s, 2H), 2.42 (d, J = 7.5 Hz, 1H), 2.07 (s, 1H), 1.90–1.67 (m, 2H), 1.35 (s, 7H), 1.17–0.99 (m, 6H).

[0203] Example 7: Synthesis of N-(6-Amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 11)

[0204]

[0205] Step 1: Synthesis of 8-bromo-2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0206] To 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg, 0.996 mmol, 1.0 Equiv) was added acetaldehyde (131.5 mg, 2.988 mmol, 3.0 Equiv), sodium triacetoxyborohydride (1.055 g, 4.980 mmol, 5.0 Equiv), acetic acid (10 drops) and methanol (8 mL). The reaction was carried out at room temperature for 18 hours, and then sent for LCMS. The sample was stirred and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain 8-bromo-2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine as a yellow oily liquid (99.2 mg, yield 35.7%). LCMS: [M+H] = 280.0320. 1 1H NMR (400 MHz, DMSO) δ 7.75 (s, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.39–7.31 (m, 1H), 4.17–4.07 (m, 2H), 3.80 (s, 2H), 2.98 (t, J = 5.5 Hz, 2H), 2.62 (q, J = 7.1 Hz, 2H), 1.12 (t, J = 7.1 Hz, 3H).

[0207] Step 2: Synthesis of 2-Ethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0208] To 8-Bromo-2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (292.1 mg, 1.047 mmol, 1.0 Equiv) was added 4,4,4',4',5,5,5',5'-Octamethyl-2,2'-bi(1,3,2-dioxaborolane) (531.8 mg, 2.094 mmol, 2.0 Equiv), 1,1-Bis(diphenylphosphino)ferrocene dichloropalladium (76 mg, 0.105 mmol, 0.1 Equiv), Potassium acetate (307.8 mg, 3.141 mmol, 3.0 Equiv) and 1,4-Dioxane (15 mL). The nitrogen was displaced three times, and the reaction was refluxed at 100 °C for 18 hours, monitored by TLC and LCMS. Extracted with ethyl acetate and water, the organic phase was dried, concentrated, triturated, and column chromatography (D:M = 10:1 + 0.1% triethylamine) gave 2-Ethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine as a yellow oily liquid (502.3 mg, yield 146.6%). LCMS: [M+H] = 328.2277.

[0209] Step 3: Synthesis of (S)-tert-Butyl 6-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0210] To 2-ethyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (340 mg, 1.04 mmol, 1.0 Equiv), (S)-tert-butyl 3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (860.9 mg, 1.25 mmol, 1.2 Equiv), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (75.5 mg, 0.104 mmol, 0.1 Equiv), sodium carbonate (330.6 mg, 3.120 mmol, 3.0 Equiv), 1,4-dioxane (15 mL) and water (5 mL) were added. Nitrogen was displaced three times, and the reaction was refluxed at 90 °C for 18 hours, monitored by LCMS. Extracted with ethyl acetate, the organic phase was concentrated, mixed with samples, and column chromatography (D:M = 10:1 + 0.1% triethylamine) gave (S)-tert-butyl 6-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (361 mg, yield 87.6%). LCMS: [M - Boc + H] = 297.2101.

[0211] Step 4: Synthesis of (S)-2-ethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0212] (S)-tert-butyl 6-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (361 mg, 0.912 mmol, 1.0 Equiv) was dissolved in dichloromethane (5.0 mL), trifluoroacetic acid (0.8 mL, 13.674 mmol, 15.0 Equiv) was added, and the reaction was carried out at room temperature for 1 hour, monitored by LCMS and TLC. The trifluoroacetic acid and dichloromethane were evaporated to dryness, a small amount of water was added for dissolution, the pH was adjusted to alkaline with saturated sodium carbonate, and the solvent was evaporated to dryness to obtain the crude product of (S)-2-ethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine. LCMS: [M + H] = 297.2005.

[0213] Step 5: Synthesis of 2-ethyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0214] (S)-2-Ethyl-8-(5-methyl-3,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (270 mg, 0.912 mmol, 1.0 Equiv) was dissolved in methanol (5.0 mL). At 0 °C, sodium borohydride (87 mg, 2.280 mmol, 2.5 Equiv) was added portionwise. After 5 minutes, the reaction mixture was transferred to room temperature and reacted for 1 hour, monitored by LCMS and TLC. The reaction mixture was concentrated, purified by flash column chromatography (D:M = 10:1 + 0.1% triethylamine), scraped, dissolved, and evaporated to dryness to obtain 2-ethyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (129.1 mg, yield 47.5%). LCMS: [M+H] = 299.1845.

[0215] Step 6: Synthesis of ethyl 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate

[0216] 2-Ethyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (129.1 mg, 0.433 mmol, 1.0 Equiv) was dissolved in N,N-dimethylformamide (2.5 mL). Triethylamine (52.5 mg, 0.520 mmol, 1.2 Equiv) and ethyl 2-chloro-2-oxoacetate (70.7 mg, 0.520 mmol, 1.2 Equiv) were added. The reaction mixture was stirred at room temperature, monitored by LCMS. After the reaction was complete, ethanol was immediately added to quench the reaction, and the mixture was mixed with silica gel and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain ethyl 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate as a yellow oily liquid (163.8 mg, yield 95.0%). LCMS: [M+H] = 399.2373.

[0217] Step 7: Synthesis of 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid

[0218] Ethyl 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (163.8 mg, 0.412 mmol, 1.0 Equiv) was added with lithium hydroxide (29.6 mg, 1.235 mmol, 3.0 Equiv), tetrahydrofuran (2.5 mL), methanol (1.0 mL) and water (1.0 mL), and the reaction was stirred at room temperature for 0.5 h. The solvent was evaporated, a small amount of water was added, the pH was adjusted to acidic with dilute hydrochloric acid, and the solvent was evaporated to obtain the crude compound 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid. LCMS: [M+H] = 371.1800.

[0219] Step 8: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0220] 2-[(5S)-2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (152.4 mg, 0.412 mmol, 1.0 Equiv) was dissolved in N,N-dimethylformamide (4.0 mL), 3-ethylpyridine-2,5-diamine (84.7 mg, 0.618 mmol, 1.5 Equiv) and N,N-diisopropylethylamine (132.9 mg, 1.030 mmol, 2.5 Equiv) were added, and the reaction was stirred at room temperature for 5 minutes, then 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (234.8 mg, 0.618 mmol, 1.5 Equiv) was added, and the reaction was carried out at room temperature for 1 h. After the reaction was completed, the reaction solution was filtered and separated and purified by high performance liquid chromatography and SFC to obtain pink powder N-(6-amino-5-ethylpyridin-3-yl)-2-[2-(2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (46 mg, yield 22.8%). LCMS: [M+H] = 490.2928. 11H NMR (400 MHz, DMSO) δ 10.59 (s, 1H), 7.57–7.45 (m, 3H), 7.16 (d, J = 7.3 Hz, 1H), 5.67 (t, J = 19.5 Hz, 2H), 4.12 (s, 1H), 3.79 (s, 2H), 3.46 (d, J = 13.1 Hz, 1H), 3.30–3.20 (m, 1H), 2.97 (s, 2H), 2.62 (q, J = 7.1 Hz, 2H), 2.47–2.25 (m, 3H), 2.12 (d, J = 45.9 Hz, 1H), 1.94–1.66 (m, 2H), 1.37 (t, J = 15.3 Hz, 1H), 1.18–1.00 (m, 9H).

[0221] Example 8: Synthesis of N-(6-Amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 12)

[0222]

[0223] Step 1: Synthesis of 8-Bromo-2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0224] Dissolve 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (230 mg, 0.92 mmol, 1.0 Equiv.) and acetone (160 mg, 2.76 mmol, 3.0 Equiv.) in dichloromethane (10 mL), then add sodium triacetoxyborohydride (976 mg, 4.6 mg, 5.0 Equiv.). Stir the reaction at room temperature and monitor the reaction by TLC and LCMS. After the reaction is completed, purify by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain 8-bromo-2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (224 mg) as a yellow oil. LC-MS: [M+H] = 293.9969. 1 1H NMR (400 MHz, DMSO-d6) δ 7.74 (s, 1H), 7.49–7.45 (m, 1H), 7.34 (d, J = 10.3 Hz, 1H), 4.13–4.05 (m, 2H), 3.88 (s, 2H), 3.04–2.93 (m, 3H), 1.09 (d, J = 6.6 Hz, 6H).

[0225] Step 2: Synthesis of 2-Isopropyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0226] 8-Bromo-2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (220 mg, 0.75 mmol, 1.0 Equiv.), 4,4,4',4',5,5,5',5'-Octamethyl-2,2'-bi(1,3,2-dioxaborolane) (381 mg, 1.5 mmol, 2.0 Equiv.), 1,1-Bis(diphenylphosphino)ferrocene dichloropalladium(II) (54.8 mg, 0.075 mmol, 0.1 Equiv.) and potassium acetate (220 mg, 2.25 mmol, 3.0 Equiv) were added to 1,4-dioxane (5.0 mL). The nitrogen was displaced three times, and the reaction was refluxed at 100 °C for 18 hours. The reaction was monitored by LCMS and TLC. After the reaction was completed, the reaction solution was directly concentrated, and the crude product (250 mg) of brown oil was obtained by column chromatography (D:M = 10:1 + 0.1% triethylamine). LC-MS: [M+H] = 342.2458.

[0227] Step 3: Synthesis of (S)-tert-Butyl 6-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0228] 2-Isopropyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg, 0.73 mmol, 1.0 Equiv.), (S)-tert-butyl 3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (252 mg, 0.73 mmol, 1.0 Equiv.), 1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloride (51.2 mg, 0.07 mmol, 0.1 Equiv.) and sodium carbonate (221 mg, 2.2 mmol, 3.0 Equiv.) were added to water (2.0 mL) and 1,4-dioxane (6.0 mL). The mixture was purged with nitrogen three times and stirred at 90 °C for 18 h. The reaction was monitored by LCMS and TLC. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The combined organic phases were dried over anhydrous sodium sulfate, loaded onto a silica gel column, and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford (S)-tert-butyl 6-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (230 mg) as a yellow solid. LCMS: [M - Boc + H] = 311.2310.

[0229] Step 4: Synthesis of (S)-2-Isopropyl-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0230] (S)-tert-Butyl 6-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (230 mg, 0.56 mmol, 1.0 Equiv) was dissolved in dichloromethane (4.0 mL). Subsequently, trifluoroacetic acid (959 mg, 8.41 mmol, 15.0 Equiv.) was added and the reaction was carried out at room temperature. The reaction was monitored by LCMS and TLC. After completion of the reaction, the pH was adjusted to basic with saturated sodium carbonate, the solvent was evaporated under reduced pressure, loaded onto a silica gel column, and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford (S)-2-isopropyl-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (163 mg). LCMS: [M + H] = 311.2045.

[0231] Step 5: Synthesis of 2-Isopropyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0232] Dissolve (S)-2-isopropyl-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (163 mg, 0.52 mmol, 1.0 Equiv.) in methanol (4.0 mL). At 0 °C, add sodium borohydride (40 mg, 1.05 mmol, 2.0 Equiv.) portionwise. After 5 minutes, transfer the reaction mixture to room temperature and monitor by LCMS and TLC. After the reaction is completed, evaporate the solvent to dryness and directly proceed to the next step. LCMS: [M+H] = 313.2378.

[0233] Step 6: Synthesis of ethyl 2-[(5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate

[0234] Dissolve 2-isopropyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (163 mg, 0.52 mmol, 1.0 Equiv) in N,N-dimethylformamide (2.0 mL), add triethylamine (78.8 mg, 0.78 mmol, 1.5 Equiv), and slowly add ethyl 2-chloro-2-oxoacetate (141 mg, 1.04 mmol, 2.0 Equiv) under an ice bath. Stir the reaction mixture at room temperature for 0.5 h and monitor by LCMS. After the reaction is completed, quench with ethanol, concentrate the mixture, mix with silica gel, and separate by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain the product (85 mg). LCMS: [M+H] = 413.2531.

[0235] Step 7: Synthesis of 2-[(5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid

[0236] Add lithium hydroxide (48 mg, 2.0 mmol, 10.0 Equiv.), methanol (4 mL), and water (0.4 mL) to ethyl 2-[(5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetate (85 mg, 0.20 mmol, 1.0 Equiv.). Stir the mixture at room temperature and monitor by LCMS. After the reaction is completed, adjust the pH to acidic with dilute hydrochloric acid, evaporate the solvent to dryness, and directly proceed to the next step. LCMS: [M+H] = 385.1702.

[0237] Step 8: Synthesis of N-(6-Amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0238] Dissolve 2-[(5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetic acid (76 mg, 0.2 mmol, 1.0 Equiv.) in N,N-dimethylformamide (3.0 mL), then add 3-ethylpyridine-2,5-diamine (33.4 mg, 0.24 mmol, 1.2 Equiv.) and N,N-diisopropylethylamine (69.5 mg, 0.5 mmol, 2.5 Equiv.). After stirring the reaction at room temperature for 5 minutes, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114 mg, 0.3 mmol, 1.5 Equiv.) and react at room temperature. Monitor by LCMS. After the reaction is completed, purify by high performance liquid chromatography and SFC to obtain the pink powder N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (18 mg). LCMS: [M+H] = 504.2822. 1 1H NMR (400 MHz, DMSO-d6) δ 8.07 (d, J = 27.6 Hz, 1H), 7.52 (t, J = 13.2 Hz, 3H), 7.29–7.15 (m, 1H), 6.08 (s, 3H), 5.79–5.21 (m, 3H), 4.10 (d, J = 4.9 Hz, 2H), 3.89 (s, 2H), 3.49 (dd, J = 13.0, 4.3 Hz, 2H), 3.06–3.00 (m, 3H), 2.34–1.99 (m, 2H), 1.97–1.66 (m, 2H), 1.56–1.34 (m, 1H), 1.10 (d, J = 25.0 Hz, 12H).

[0239] Example 9: Synthesis of N-(6-Amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-5-methyl-2-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide (Compound 16)

[0240]

[0241] Step 1: Synthesis of 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0242] Dissolve 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (470 mg, 1.87 mmol, 1.0 Equiv.) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (867 mg, 3.74 mmol, 2.0 Equiv.) in acetonitrile (10 mL), then add potassium carbonate (387 mg, 2.8 mmol, 1.5 Equiv.). Stir the reaction at 45 °C and monitor by TLC and LCMS. After the reaction is completed, sample mixing is carried out, and column chromatography (D:M = 10:1 + 0.1% triethylamine) is used for separation to obtain the yellow solid 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (232 mg). LC-MS: [M+H] = 334.0156.

[0243] Step 2: Synthesis of 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0244] Add 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (354 mg, 1.39 mmol, 2.0 Equiv.), 1,1-bis(diphenylphosphino)ferrocene dichloropalladium (50.5 mg, 0.069 mmol, 0.1 Equiv.), potassium acetate (202.8 mg, 2.07 mmol, 3.0 Equiv) and 1,4-dioxane (4.0 mL) to 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (232 mg, 0.69 mmol, 1.0 Equiv.). Replace N2 three times and reflux the reaction at 100 °C for 18 hours. Monitor by LCMS and TLC. After the reaction is completed, rotary evaporate the solvent, sample mixing is carried out, and column chromatography (D:M = 10:1 + 0.1% triethylamine) is used for separation to obtain the brown oily crude product 8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg). LCMS: [M+H] = 382.1480.

[0245] Step 3: Synthesis of tert-butyl (S)-3-methyl-6-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3,4-dihydropyridine-1(2H)-carboxylate

[0246] 8-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg, 0.656 mmol, 1.0 Equiv.), tert-butyl (S)-3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (226 mg, 0.656 mmol, 1.0 Equiv.), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (48.3 mg, 0.066 mmol, 0.1 Equiv.), sodium carbonate (209 mg, 1.97 mmol, 3.0 Equiv.), water (2.0 mL) and 1,4-dioxane (6.0 mL) were taken. Nitrogen was displaced three times immediately, and the reaction was stirred at 90 °C for 18 hours, monitored by LCMS and TLC. After the reaction was completed, it was extracted with ethyl acetate and water. The organic phases were combined, dried over anhydrous sodium sulfate, mixed with samples, and column chromatography (D:M = 10:1 + 0.1% triethylamine) was carried out to obtain a brown oil, tert-butyl (S)-3-methyl-6-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3,4-dihydropyridine-1(2H)-carboxylate (204 mg). LCMS: [M - Boc + H] = 351.1422.

[0247] Step 4: Synthesis of (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0248] Dissolve tert-butyl (S)-3-methyl-6-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-3,4-dihydropyridine-1(2H)-carboxylate (200 mg, 0.44 mmol, 1.0 Equiv) in dichloromethane (4.0 mL), add trifluoroacetic acid (752 mg, 6.6 mmol, 15.0 Equiv.), react at room temperature, and monitor by LCMS and TLC. After the reaction is completed, adjust the pH to alkaline with saturated sodium carbonate, rotary evaporate the solvent, mix the sample, and separate by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg). LCMS: [M+H] = 351.1743.

[0249] Step 5: Synthesis of 8-[(5S)-5-methylpiperidin-2-yl]-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0250] Dissolve (S)-8-(5-methyl-1,4,5,6-tetrahydropyridin-2-yl)-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (250 mg, 0.71 mmol, 1.0 Equiv.) in methanol (4.0 mL), add sodium borohydride (67 mg, 1.77 mmol, 2.5 Equiv.) portionwise at 0 °C. After 5 minutes, transfer to room temperature for reaction and monitor by LCMS and TLC. After the reaction is completed, rotary evaporate, mix the sample, and separate by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain 8-[(5S)-5-methylpiperidin-2-yl]-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (59 mg). LCMS: [M+H] = 353.1751.

[0251] Step 6: Synthesis of ethyl 2-{(5S)-5-methyl-2-[2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]piperidin-1-yl}-2-oxoacetate

[0252] 8-[(5S)-5-Methylpiperidin-2-yl]-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (59 mg, 0.167 mmol, 1.0 Equiv) was dissolved in N,N-dimethylformamide (2.0 mL). Triethylamine (25.3 mg, 0.25 mmol, 1.5 Equiv) was added. Under an ice bath, ethyl 2-chloro-2-oxoacetate (45.9 mg, 0.334 mmol, 2.0 Equiv) was slowly added, and the reaction was stirred at room temperature for 0.5 h. Monitored by LCMS. After the reaction was completed, it was quenched with ethanol, concentrated, mixed with silica gel, and separated by a chromatography column (D:M = 10:1 + 0.1% triethylamine) to obtain ethyl 2-{(5S)-5-methyl-2-[2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]piperidin-1-yl}-2-oxoacetate (17 mg). LCMS: [M+H] = 453.1863.

[0253] Step 7: Synthesis of 2-{(5S)-5-methyl-2-[2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]piperidin-1-yl}-2-oxoacetic acid

[0254] Lithium hydroxide (8.8 mg, 0.37 mmol, 10.0 Equiv.), methanol (2 mL) and water (0.2 mL) were added to ethyl 2-{(5S)-5-methyl-2-[2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]piperidin-1-yl}-2-oxoacetate (17 mg, 0.037 mmol, 1.0 Equiv.), and the mixture was stirred at room temperature. Monitored by LCMS. After the reaction was completed, the pH was adjusted to acidic with dilute hydrochloric acid, and the solvent was evaporated to dryness, and then directly subjected to the next reaction. LCMS: [M+H] = 425.1618.

[0255] Step 8: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-5-methyl-2-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide

[0256] 2-{(5S)-5-Methyl-2-[2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]piperidin-1-yl}-2-oxoacetic acid (15.7 mg, 0.037 mmol, 1.0 Equiv.) was dissolved in N,N-dimethylformamide (2.0 mL). 3-Ethylpyridine-2,5-diamine (7.7 mg, 0.056 mmol, 1.5 Equiv.) and N,N-diisopropylethylamine (13 mg, 0.093 mmol, 2.5 Equiv.) were added. After stirring at room temperature for 5 minutes, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.3 mg, 0.056 mmol, 1.5 Equiv.) was added and the reaction was carried out at room temperature. Monitored by LCMS. After the reaction was completed, the product was separated and purified by Prep-HPLC and SFC to obtain white powder N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-5-methyl-2-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)piperidin-1-yl)-2-oxoacetamide (10 mg). LCMS: [M+H] = 544.2555. 1H NMR (400 MHz, DMSO-d6) δ 10.58 (d, J = 18.8 Hz, 1H), 8.06 (d, J = 25.9 Hz, 1H), 7.61–7.44 (m, 3H), 7.22 (dd, J = 32.4, 8.0 Hz, 1H), 5.85–5.21 (m, 3H), 4.14 (d, J = 23.8 Hz, 4H), 3.60–3.41 (m, 3H), 3.27 (s, 3H), 2.46–2.28 (m, 3H), 2.14 (d, J = 46.0 Hz, 1H), 1.95–1.65 (m, 2H), 1.38 (t, J = 15.0 Hz, 1H), 1.19–0.99 (m, 6H).

[0257] Example 10: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 40)

[0258]

[0259] Step 1: Synthesis of tert-butyl (S)-2-methyl-3-oxopiperazine-1-carboxylate

[0260] To a solution of compound (S)-3-methylpiperazin-2-one (4.0 g, 35 mmol, 1.0 equiv.) in dichloromethane (30 mL), di-tert-butyl dicarbonate (7.7 g, 35 mmol, 1.0 equiv.) was added, and the reaction was carried out at room temperature for 16 hours. After the reaction was completed, the sample was mixed and separated by column chromatography to obtain (S)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate (7.12 g).

[0261] Step 2: Synthesis of (S)-tert-butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate

[0262] Weigh (S)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate (3.5 g, 16 mmol, 1.0 eq.), displace the system with nitrogen three times, add N,N-dimethylformamide solution (30 mL), then under an ice bath at 0 °C, add sodium hydride (770 mg, 33 mmol, 2.0 equiv.) in batches. After stirring for 30 minutes, slowly add a solution of 4-bromo-1-fluoro-2-nitrobenzene (4.0 g, 18 mmol, 1.1 equiv.) in N,N-dimethylformamide solution (8 mL), and stir at room temperature for 3 hours. After the reaction was complete, the reaction was quenched with ammonium chloride solution, and then extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the compound (S)-tert-butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.9 g) was obtained by column chromatography. LCMS: m / z = [M - Boc + H] 314.1.

[0263] Step 3: Synthesis of (S)-tert-butyl 8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0264] Compound (S)-tert-butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.9 g, 9.4 mmol, 1.0 equiv.) and iron (2.6 g, 47.2 mmol, 5.0 equiv.) were added to acetic acid (40 mL), and the reaction was carried out at 100 °C for 1.5 hours. After the reaction was complete, the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated and then separated by column chromatography to obtain yellow (S)-tert-butyl 8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.5 g). LCMS: m / z = [M - Boc + H] 266.0.

[0265] Step 4: Synthesis of (S)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0266] Weigh (S)-tert-butyl 8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.5 g, 6.85 mmol, 1.0 equiv.), add trifluoroacetic acid (3 mL) and dichloromethane (10 mL) to dissolve it, and stir the reaction at room temperature for 1 hour. After the reaction is complete, concentrate the reaction solution to remove the solvent, adjust the pH value to 8 - 9 with saturated sodium carbonate solution, add ethyl acetate for extraction, and rotary evaporate the organic phase to obtain the crude product (S)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1 g), which is directly used for the next step. LCMS: m / z = [M - Boc + H] 266.0.

[0267] Step 5: Synthesis of (S)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0268] Add acetaldehyde (646.8 mg, 14.7 mmol, 3.0 equiv.), sodium triacetoxyborohydride (3.10 g, 14.7 mmol, 3 equiv.), acetic acid (20 drops) and methanol (15 mL) to the compound (S)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.3 g, 4.9 mmol, 0.2 equiv.), and react at room temperature for 16 hours. After the reaction is completed, rotary evaporate the solvent, mix the sample, and separate it by column chromatography to obtain (S)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.21 g). LCMS: m / z = [M + H]. 294.1.

[0269] Step 6: Synthesis of (S)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0270] The compound (S)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.14 g, 3.9 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide solution (40 mL). Bis(pinacolato)diboron (3 g, 11.8 mmol, 3.0 eq), potassium acetate (1.2 g, 11.8 mmol, 3.0 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (286 mg, 0.4 mmol, 0.1 eq) were added to the system. The system was purged with nitrogen three times and heated to 100 °C for reaction for 6 hours. After the reaction was completed, it was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the white solid (S)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.27 g). LCMS: m / z = [M+H] 342.2.

[0271] Step 7: Synthesis of tert-butyl (S)-6-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0272] The compound (S)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.26 g, 3.7 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (30 mL) and water (10 mL). (S)-tert-Butyl 5-methyl-2-{[(trifluoromethyl)sulfonyl]oxy}cyclohex-2-ene-1-carboxylate (1.2 g, 3.7 mmol, 1.0 equiv.), sodium carbonate (1.2 g, 11.0 mmol, 3.0 equiv.), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (266 mg, 0.4 mmol, 0.1 equiv.) were added to the system. The system was purged with nitrogen three times and heated to 90 °C for reaction for 6 hours. After the reaction was completed, it was diluted with water, extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the compound tert-butyl (S)-6-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (1.2 g). LCMS: m / z = [M+H]-Boc 411.2.

[0273] Step 8: Synthesis of (S)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0274] Dissolve tert-butyl (S)-6-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (1.23 g, 3 mmol, 1.0 eq) in dichloromethane (3 mL), add trifluoroacetic acid (10 mL), stir for 30 minutes, and monitor the reaction by TLC and LCMS. After the reaction is completed, rotary evaporate the solvent, adjust the pH to 8 - 9 with saturated sodium carbonate solution, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and rotary evaporate the solvent to obtain the crude product (S)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.2 g). LCMS: m / z = [M + H] 311.2.

[0275] Step 9: Synthesis of (1S)-2-ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0276] Dissolve (S)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (964 mg, 3.1 mmol, 1.0 equiv.) in methanol (10 mL). Under an ice bath, add sodium borohydride (233 mg, 6.1 mmol, 2.0 equiv.) portionwise, stir for 30 minutes, and monitor the reaction by TLC and LCMS. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain (1S)-2-ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (460 mg). LCMS: m / z = [M + H] 313.2.

[0277] Step 10: Synthesis of ethyl 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate

[0278] (1S)-2-Ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (146 mg, 0.47 mmol, 1.0 equiv.) was added to a reaction flask. After protection with nitrogen, triethylamine (71 mg, 0.7 mmol, 1.5 equiv.) was added, and monoethyl oxalyl chloride (97 mg, 0.7 mmol, 1.5 equiv.) was slowly added dropwise under ice bath conditions. The reaction was stirred at room temperature, and the reaction was monitored by TLC and LCMS. After the reaction was completed, the solvent was evaporated to dryness, triturated, and separated by column chromatography to obtain ethyl 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate (215 mg). LCMS: m / z = [M+H] 413.2.

[0279] Step 11: Synthesis of 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid

[0280] Ethyl 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate (206.5 mg, 0.5 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (4 mL) and water (4 mL). Lithium hydroxide (24 mg, 1.0 mmol, 2 equiv.) was added to the system, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to 5 - 6 with 1 M aqueous hydrochloric acid, and the solvent was evaporated to dryness to obtain the crude product 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid (200 mg). LCMS: m / z = [M+H] 385.2.

[0281] Step 12: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide

[0282] Dissolve 2-{(5S)-2-[(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid (172.8 mg, 0.45 mmol, 1.0 equiv.), 5-nitro-3-ethenylpyridin-2-amine (93 mg, 0.68 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (157 mg, 1.13 mmol, 2.5 equiv.) in N,N-dimethylformamide (4 mL), stir for 5 minutes, add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (258 mg, 0.68 mmol, 1.5 equiv.), and stir at room temperature for 0.5 hour. After the reaction is completed, separate and purify by preparative HPLC and SFC to obtain the compound N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (7.0 mg). LCMS: m / z = [M+H] 504.3.

[0283] Example 11: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-(S)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl)-5-methylpiperidin-1-yl]-2-oxoacetamide (Compound 39)

[0284]

[0285] Step 1: Synthesis of tert-butyl (R)-2-methyl-3-oxopiperazine-1-carboxylate

[0286] To a solution of compound (S)-3-methylpiperazin-2-one (4.0 g, 35 mmol, 1.0 equiv.) in dichloromethane (30 mL), add di-tert-butyl dicarbonate (7.7 g, 35 mmol, 1.0 equiv.), and react at room temperature for 16 hours. After the reaction is completed, sample and separate by column chromatography to obtain tert-butyl (R)-2-methyl-3-oxopiperazine-1-carboxylate (6.84 g).

[0287] Step 2: Synthesis of tert-butyl (R)-4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate

[0288] Weigh (S)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate (3.50 g, 16.34 mmol, 1.0 eq.). Flush the system with nitrogen three times. Add N,N-dimethylformamide solution (30 mL). Then, under an ice bath at 0 °C, add sodium hydride (770 mg, 33 mmol, 2.0 equiv.) portionwise. After stirring for 30 minutes, slowly add a solution of 4-bromo-1-fluoro-2-nitrobenzene (4.0 g, 18 mmol, 1.1 equiv.) in N,N-dimethylformamide solution (8 mL). Stir the reaction at room temperature for 3 hours. After the reaction is complete, quench the reaction with ammonium chloride solution, then extract with ethyl acetate and water. Dry the organic phase with anhydrous sodium sulfate and concentrate it. Obtain the compound tert-butyl (R)-4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.96 g) by column chromatography. LCMS: m / z = [M - Boc + H] 314.1.

[0289] Step 3: Synthesis of tert-butyl (R)-8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0290] Add the compound tert-butyl (R)-4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.9 g, 9.4 mmol, 1.0 eq.) and iron (2.6 g, 47.2 mmol, 5.0 eq.) to acetic acid (40 mL). React at 100 °C for 1.5 hours. After the reaction is complete, filter the reaction solution through diatomaceous earth, concentrate the filtrate, and obtain the yellow tert-butyl (R)-8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.56 g) by column chromatography. LCMS: m / z = [M - Boc + H] 266.0.

[0291] Step 4: Synthesis of (R)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0292] Weigh (R)-tert-butyl 8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.5 g, 6.85 mmol, 1.0 equiv.), add trifluoroacetic acid (3 mL) and dichloromethane (10 mL) to dissolve it, and stir the reaction at room temperature for 1 hour. After the reaction is complete, concentrate the reaction solution to remove the solvent, adjust the pH value to 8 - 9 with saturated sodium carbonate solution, add ethyl acetate for extraction, and rotary evaporate the organic phase to obtain the crude product (R)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.09 g), which is directly used for the next step. LCMS: m / z = [M - Boc + H] 266.0.

[0293] Step 5: Synthesis of (R)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0294] Add acetaldehyde (646.8 mg, 14.7 mmol, 3.0 equiv.), sodium triacetoxyborohydride (3.10 g, 14.7 mmol, 3 equiv.), acetic acid (20 drops) and methanol (15 mL) to the compound (R)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.3 g, 4.9 mmol, 0.2 equiv.), and react at room temperature for 16 hours. After the reaction is completed, rotary evaporate the solvent, mix the sample, and separate by column chromatography to obtain (R)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.27 g). LCMS: m / z = [M + H]. 294.1.

[0295] Step 6: Synthesis of (R)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0296] Dissolve the compound (R)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.14 g, 3.9 mmol, 1.0 eq) in N,N-dimethylformamide solution (40 mL). Add bis(pinacolato)diboron (3 g, 11.8 mmol, 3.0 eq), potassium acetate (1.2 g, 11.8 mmol, 3.0 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (286 mg, 0.4 mmol, 0.1 eq) to the system. Replace the gas in the system with nitrogen 3 times and heat to 100 °C for reaction for 6 hours. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain the white solid (R)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.29 g). LCMS: m / z = [M+H] 342.2.

[0297] Step 7: Synthesis of tert-butyl (S)-6-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate

[0298] The compound (S)-2-ethyl-1-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.26 g, 3.7 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (30 mL) and water (10 mL). To the system were added (S)-tert-butyl 5-methyl-2-{[(trifluoromethyl)sulfonyl]oxy}cyclohex-2-ene-1-carboxylate (1.2 g, 3.7 mmol, 1.0 equiv.), sodium carbonate (1.2 g, 11.0 mmol, 3.0 equiv.), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (266 mg, 0.4 mmol, 0.1 equiv.). The system was purged with nitrogen three times and heated to 90 °C for reaction for 6 hours. After the reaction was completed, water was added for dilution, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain the compound (S)-tert-butyl 6-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (1.27 g). LCMS: m / z = [M+H] - Boc 411.2.

[0299] Step 8: Synthesis of (R)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0300] (S)-tert-butyl 6-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-3-methyl-3,4-dihydropyridine-1(2H)-carboxylate (1.23 g, 3 mmol, 1.0 eq) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (10 mL) was added, and after stirring for 30 minutes, the reaction was monitored by TLC and LCMS. After the reaction was completed, the solvent was evaporated, saturated sodium carbonate solution was added to adjust the pH to 8 - 9, water was added for dilution, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain the crude product (R)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.05 g). LCMS: m / z = [M+H] 311.2.

[0301] Step 9: Synthesis of (1R)-2-ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0302] Dissolve (R)-2-ethyl-1-methyl-8-[(S)-5-methyl-3,4,5,6-tetrahydropyridin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (964 mg, 3.1 mmol, 1.0 equiv.) in methanol (10 mL). Under an ice bath, add sodium borohydride (233 mg, 6.1 mmol, 2.0 equiv.) portionwise. After stirring for 30 minutes, monitor the reaction by TLC and LCMS. After the reaction is completed, dilute with water, extract with ethyl acetate, wash with saturated sodium chloride, combine the organic phases, dry over anhydrous sodium sulfate, filter, and separate by column chromatography to obtain (1R)-2-ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (425 mg). LCMS: m / z = [M+H] 313.2.

[0303] Step 10: Synthesis of ethyl 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate

[0304] Add (1R)-2-ethyl-1-methyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (146 mg, 0.47 mmol, 1.0 equiv.) to a reaction flask. After protecting with nitrogen, add triethylamine (71 mg, 0.7 mmol, 1.5 equiv.). Slowly add ethyl oxalyl chloride (97 mg, 0.7 mmol, 1.5 equiv.) dropwise under an ice bath. Stir the reaction at room temperature and monitor the reaction by TLC and LCMS. After the reaction is completed, rotary evaporate the solvent, mix the sample, and separate by column chromatography to obtain ethyl 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate (213 mg). LCMS: m / z = [M+H] 413.2.

[0305] Step 11: Synthesis of 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid

[0306] The ethyl 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate (206.5 mg, 0.5 mmol, 1.0 equiv.) was dissolved in tetrahydrofuran (4 mL) and water (4 mL). Lithium hydroxide (24 mg, 1.0 mmol, 2 equiv.) was added to the system, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the pH was adjusted to 5 - 6 with 1 M aqueous hydrochloric acid, and the solvent was evaporated to dryness to obtain the crude product 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid (187 mg). LCMS: m / z = [M+H] 385.2.

[0307] Step 12: Synthesis of N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl]-2-oxoacetamide

[0308] 2-{(5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetic acid (172.8 mg, 0.45 mmol, 1.0 equiv.), 5-nitro-3-ethenylpyridin-2-amine (93 mg, 0.68 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (157 mg, 1.13 mmol, 2.5 equiv.) were dissolved in N,N-dimethylformamide (4 mL), stirred for 5 minutes, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (258 mg, 0.68 mmol, 1.5 equiv.) was added, and the mixture was stirred at room temperature for 0.5 hour. After the reaction was completed, the compound N-(6-amino-5-ethylpyridin-3-yl)-2-[(2R,5S)-2-[(R)-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazin-8-yl]-5-methylpiperidin-1-yl]-2-oxoacetamide (4.0 mg) was obtained by preparative HPLC and SFC separation and purification. LCMS: m / z = [M+H] 504.3.

[0309] The compounds listed in Table 1 below were prepared by a method similar to that described in the examples, with appropriate variations in the reactants, amounts of reagents, protection and deprotection, solvents, and reaction conditions. The characterization data of the compounds are summarized in Table 1 below.

[0310] Table 1: Structures and Characterizations of Some Compounds

[0311]

[0312]

[0313]

[0314]

[0315] Experimental Example 1: Test for the Binding Ability of the Compounds of the Present Invention to PRMT5 Protein

[0316] Experimental Purpose: To detect the binding ability of the compound to PRMT5 protein by the CETSA experimental method

[0317] Background Principle: The CETSA experiment is a molecular detection method for measuring the affinity of a drug for a target protein. The principle is that after the drug binds to the target protein, its structure becomes more stable. When using a cell or tissue sample treated with a candidate drug, if the candidate drug is an inhibitor of PRMT5, then the candidate drug can bind to PRMAT5, making the PRMT5 protein more stable. After heating the sample, the PRMT5 protein in the sample will be more easily detected by a specific antibody, and the PRMT5 protein will be more easily detected by Western blot; conversely, after heating, the stability of the PRMT5 protein will be worse, and the amount of protein detected will be lower. Thus, the binding ability of the drug to the target protein is evaluated for the screening of PRMT5 protein inhibitors.

[0318] Specific experimental procedures: HCT116 cells in the logarithmic growth phase (cell viability > 90%) were taken, washed 3 times with PBS, and centrifuged at 2000g for 2 min; lysed on ice for 30 min with cell lysis buffer containing protease inhibitor PMSF; the protein concentration of the protein sample was measured using a BCA kit. The samples were incubated with the candidate drug, control drug, and control reagent for 30 min respectively, and heated at about 10 set temperature points for each group of samples; restored to room temperature, centrifuged the samples at 20000g, and collected the supernatant; the protein samples were heated and denatured at 100 °C for 10 min using sample buffer. After the samples were restored to room temperature, western blot detection was performed on the samples; the protein loading amount was controlled at 20 μg. After determining the mutation temperature, the compound was set with a concentration gradient of generally 9 points, the samples were incubated, and the above operations were performed for western blot detection. Protein electrophoresis: The voltage of the stacking gel was set at 60 v, and the voltage of the separating gel was set at 120 v; after electrophoresis, electrotransfer was started. The electrotransfer conditions were set at 250 mA for 2 h; blocked with 5% BSA for 1 h; added with specific primary antibody and incubated overnight on a shaker at 4 °C; washed 4 times with TBST, 2.5 min each time; incubated with secondary antibody on a shaker at room temperature for 1 h; washed 4 times with TBST, 2.5 min each time; developed using ECL to detect the PRMT5 protein expression levels at different groups and each temperature point. The western blot bands were processed by image J and GraphPad software to calculate the EC 50 value.

[0319] EC50, the half-maximal effective concentration, refers to the drug concentration that can cause 50% of individuals to be effective. The PRMT5 inhibitors TNG908 and TNG462 were used as positive reference compounds, and the preparation method was referred to the examples in the patent document WO2022026892.

[0320]

[0321] Experimental Example 2: Determination of the effect of the compound of the present invention on the proliferation of HCT116 MTAP- / - cells

[0322] Experimental purpose: The purpose of this test example is to test the effect of the compound on the proliferation of HCT116 MTAP- / - cells.

[0323] Background principle: Protein arginine methyltransferase 5 (PRMT5) can methylate a variety of proteins and plays an important role in biological processes such as gene expression, splicing, and DNA damage repair. Methionine adenosylphosphate phosphorylase (MTAP) is often co-deleted with the common tumor suppressor gene CDKN2A in the body, and the proportion of this co-deletion in tumors can reach 9% - 15%. It has been found that inhibiting PRMT5 has a synthetic lethal effect in MTAP-deficient tumors. Therefore, by detecting the inhibition rate of the compound on the proliferation of HCT116 MTAP- / - cells, it can be used for the screening of PRMT5 protein inhibitors.

[0324] Specific experimental procedure:

[0325] Construct HCT116 MTAP knockout cells and screen monoclonal cells. HCT116 MTAP - / - and WT cells in the logarithmic growth phase were inoculated into 96-well plates, 90 μL per well, 1000 cells per well, and left standing overnight in a 37°C incubator. The next day, 10 μL of compounds with different concentrations were added, with the highest concentration being 100 μM, diluted 10-fold, with 9 gradients (the final concentration of DMSO was 1%), and incubated in a 37°C incubator for 7 days. On the 7th day, the old culture medium was aspirated, and 110 μL of culture medium (the ratio of culture medium to CCK8 was 100:10) was added and incubated at 37°C for 1 - 4 h. The absorbance was measured at 450 nM, and the IC50 was calculated by processing with GraphPad software. By comparing with the positive drug, the compound was screened.

[0326] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It can indicate the half amount of a certain drug or substance (inhibitor) in inhibiting certain biological procedures (or certain substances included in this procedure, such as enzymes, cell receptors, or microorganisms). The compounds TNG908 and TNG462 described above were used as positive reference compounds. The test results are shown in Table 2:

[0327] Table 2

[0328]

[0329] The test results show that the representative compounds of the present invention have good inhibitory effects on HCT116 MTAP - / - cells, comparable to or better than the positive control, while having a weak inhibitory effect on HCT116 WT cells and having excellent selectivity.

[0330] Experimental example 3: Test of the inhibitory effect of the compounds of the present invention on the function of PRMT5 enzyme

[0331] Experimental purpose: To detect the inhibitory effect of compounds on the function of PRMT5 enzyme by MTase-Glo method

[0332] Background principle: The gene that constitutes synthetic lethality with PRMT5 is MTAP (methylthioadenosine phosphorylase). MTAP is involved in the metabolism of 2-methylthioadenosine (MTA) and regenerates methionine required for the synthesis of SAM. The deletion of MTAP gene causes the accumulation of MTA in cells, and MTA competes with the substrate S-adenosyl-L-methionine (SAM) of PRMT5, resulting in a decrease in PRMT5 activity. Based on the MTase-Glo method, taking advantage of the property that the methyltransferase (PRMT5) provides a methyl group from SAM (S-adenosylmethionine) to the substrate to generate SAH, the PRMT5 methyltransferase activity test experiment is carried out in the presence or absence of MTA to screen for selective PRMT5 inhibitors

[0333] Specific experimental procedure:

[0334] The MTase-Glo methyltransferase fluorescence method was used to monitor the conversion of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) by the recombinant PRMT5:MEP50 enzyme complex in the presence or absence of 5'-methylthioadenosine (MTA). The enzymatic reaction was carried out in a white 96-well plate. The reaction buffer contained 20 mM bicine (pH 7.60), 25 mM NaCl, 1 mM DTT, and 0.1% (w / v) CHAPS. The test compounds and positive control were prepared, diluted 10-fold, with 9 concentration gradients and 3 replicates, and the highest concentration was 100 μM. In the absence of MTA, the compounds were incubated with the reaction mixture containing 1 nM recombinant PRMT5:MEP50 enzyme complex, 2.5 μM H41-21 histone peptide, and 2.5 μM S-adenosylmethionine (SAM) for the experiment. Similarly, in the presence of MTA, the compounds were incubated with the mixture composed of 2 nM enzyme complex, 1.5 μM MTA, 2.5 μM H41-21 histone peptide, and 2.5 μM SAM. After incubation at room temperature for 5 h, the reaction was terminated by adding TFA to each well. Subsequently, 5X MTase-GloTM reagent was added to each well and incubated at room temperature to convert SAH generated in the reaction into ADP. After 10 min, MTase-Glo detection solution was added to each well and incubated at room temperature for 30 min, and then detected by a microplate reader

[0335] IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It can indicate the half amount of a certain drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances included in this process, such as enzymes, cell receptors, or microorganisms). Using the compounds TNG908 and MRTX1719 described above as positive reference compounds, the test results are shown in Table 3:

[0336] Table 3

[0337]

[0338] The results show that the compounds of the present invention have a very strong inhibitory effect on the function of PRMT5 protease, and the IC50 values all reach the nM level, equivalent to or less than the positive drug. This strong inhibitory effect has important therapeutic significance for the treatment of diseases or disorders related to PRMT5 inhibition.

[0339] Experimental Example 4: Determination of the in vitro metabolic stability of the compounds of the present invention

[0340] 1. Experimental principle: Liver microsomes contain most of the phase I enzymes, and the most important one is the microsomal mixed function oxidase system mainly composed of CYP450. When using liver microsomes for research, adding the corresponding cofactor NADPH can reconstruct the in vitro metabolic system, and thus conduct phase I metabolic stability research through the in vitro incubation method.

[0341] 2. Experimental method:

[0342] 2.1 Thaw each component of the kit in an ice bath and place it on ice for later use;

[0343] 2.2 Prepare pre-incubation solution A (1X), 10 ul of solution A, 2 ul of solution B, and 28 ul of 0.1M PBS buffer, mix well, incubate at 37 °C for 5 min, and then dispense 40 ul into each tube for later use;

[0344] 2.3 Prepare pre-incubation solution B (1X), 154 ul of 0.1M PBS buffer, 1 ul of the test substance (or positive substrate), and 5 ul of liver microsomes, and mix well;

[0345] 2.4 Add 160 ul of pre-incubation solution B to the centrifuge tube containing 40 ul of pre-incubation solution A, pipette 3 times to mix well, and immediately place it at 37 °C for incubation timing;

[0346] 2.5 At the set time point, add pre-cooled acetonitrile to terminate the reaction, centrifuge at 20000 rpm, 4 °C for 10 min, take the supernatant for detection.

[0347] Set up control groups: 1) Positive control group: Replace the test substance with a positive substrate; 2) Negative control group: Do not add Solution A and Solution B; 3) Blank control group: Only contain the substrate and PBS buffer solution.

[0348] Experimental Example 5: In vitro permeability Caco-2 test of the compound of the present invention

[0349] 1. Caco-2 experimental method

[0350] 1.1 Caco-2 cell culture:

[0351] (1) Add 50 μL and 25 mL of cell culture medium to each well of the Transwell insert and the reservoir, respectively.

[0352] (2) Before cell inoculation, pre-incubate the Transwell plate at 37 °C and 5% CO2 for 1 hour.

[0353] (3) Inoculate 50 μL of cell suspension (2×105 cells / mL) into the insert and culture at 37 °C, 5% CO2, and 95% relative humidity for 21 - 28 days.

[0354] (4) Replace the cell culture medium every other day within 7 days and daily after 7 days.

[0355] (5) Measure the transepithelial electrical resistance (TEER) of the monolayer using EVOM3.

[0356] 1.2 ABBA experimental procedure:

[0357] (1) Wash the Transwell plate twice with pre-warmed HBSS (10 mM HEPES, pH 7.4) and then incubate at 37 °C for 30 minutes.

[0358] (2) Prepare a 1 mM test compound in DMSO and dilute it 200-fold to 5 μM with HBSS (10 mM HEPES, pH 7.4).

[0359] (3) From apical to basolateral direction: Add 75 μL of 5 μM working solution (10 mM HEPES, pH 7.4) to the apical chamber and 235 μL of HBSS (10 mM HEPES, pH 7.4) to the basolateral chamber.

[0360] (4) From basolateral to apical direction: Add 235 μL of 5 μM working solution (10 mM HEPES, pH

[0361] 7.4) to the basolateral chamber and 75 μL of HBSS (10 mM HEPES, pH 7.4) to the apical chamber.

[0362] (5) Transfer 50 μL of 5 μM working solution (10 mM HEPES, pH 7.4) to a sample plate containing 200 μL of cold methanol (IS* as CO).

[0363] (6) Incubate the cell plate at 37 °C, 5% CO2, and 95% relative humidity for 2 hours.

[0364] (7) After 2 hours of incubation, transfer 50 μL of the solution from the apical chamber and the basolateral chamber to a sample plate containing 200 μL of cold methanol and IS*.

[0365] (8) Centrifuge the sample plate at 3220 g for 40 minutes.

[0366] (9) Transfer 100 μL of the supernatant to an analytical plate containing an appropriate volume of H2O for LC-MS / MS analysis.

[0367] 1.3 Lucifer Yellow Fluorescence Permeation Detection

[0368] (1) Add 100 μL of 100 μM Lucifer Yellow solution (10 mM HEPES, pH 7.4) to the apical chamber and add 300 μL of 10 mM HEPES (pH 7.4) to the outside of the basolateral chamber.

[0369] (2) Incubate the cell plate at 37 °C, 5% CO2, and 95% relative humidity for 0.5 hour.

[0370] (3) Transfer 100 μL from the apical chamber and the basolateral chamber to a plate. After 0.5 hour of incubation, read the values of the 427 nM excitation wavelength and the 536 nM emission wavelength of the Lucifer Yellow permeation detector.

[0371] 1.4 Data Analysis

[0372] (1) Use Microsoft Excel to calculate the apparent permeability coefficient (Papp, cm / s) and the efflux ratio.

[0373]

[0374]

[0375] Vacceptor = 0.235 mL for A→B, and 0.075 mL for B→A

[0376] Vdonor = 0.075 mL for A→B, and 0.235 mL for B→A

[0377] Area = the surface area of the membrane, cm2 (0.143 for Corning 3391)

[0378] Time = the total transport time, s

[0379] (2) Calculate Lucifer Yellow fluorescence penetration using the following formula:

[0380]

[0381] I = the fluorescence intensity

[0382] Vacceptor = 0.3 mL and Vdonor = 0.1 mL for Lucifer Yellow leakage % test

[0383] *IS: 50 nM alprazolam, 50 nM labetalol and 100 nM ketoprofen

[0384] Experimental Example 6: In vivo pharmacokinetics test of the compounds of the present invention

[0385] 1. Test purpose: After single intravenous injection or oral gavage of different compounds to ICR mice, blood samples were collected at different time points, and the concentration of the test substance in the plasma of the mice after administration of the test substance was measured by LC-MS / MS and related parameters were calculated.

[0386] 2. Test protocol:

[0387] 2.1 Test animals: ICR male mice, SPF grade

[0388] 2.2 Test design:

[0389]

[0390] Note: *, The animals were fasted overnight (10 - 14 hours) before drug administration and fed 4 hours after drug administration.

[0391] 2.3 Blood sampling time points

[0392] IV after administration: 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h. PO: 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h. Blood was collected via the jugular vein, about 0.2 mL was collected for each sample, anticoagulated with K2-EDTA, and placed on ice after collection.

[0393] 2.4 Plasma sample processing: After blood samples were collected, they were placed on ice and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6800 g, 6 minutes, 2 - 8 °C). Plasma samples were stored in a -80 °C refrigerator before analysis.

[0394] 2.5 Result analysis: Based on the blood drug concentration data at different time points, pharmacokinetic parameters were calculated using Phoenix WinNonlin 7.0, providing parameters such as AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2, as well as their mean values and standard deviations.

[0395]

[0396] The results showed that the compound of the present invention exhibited good pharmacokinetic exposure in the mouse pharmacokinetic test, superior to the positive control.

Claims

1. A compound of formula (I), its stereoisomers, or a pharmaceutically acceptable salt thereof: Wherein, X is selected from O, -NR, -CH2; R is selected from H, -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 haloalkyl, -C3-C6 cycloalkyl, -(CH2) r OC1-C6 alkyl, -C1-C6 hydroxyalkyl, -(CH2) n -NR a2 R a3 ; R1, R2, and R3 are each independently selected from H, a halogen group, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C1-C6 haloalkyl, -CN, -OR a1 , -NR a2 R a3 ; R a1 、R a2 、R a3 each independently selected from H, -C1-C6 alkyl, -C3-C6 cycloalkyl; n is 0, 1, 2, 3, 4; m is 0, 1, 2; r is 1, 2, 3, 4; R4 is selected from H, halogen, -C1-C6 alkyl, -C1-C6 haloalkyl, or when n is 2, two R4 together with the carbon atom to which they are attached form a bridged, fused or spiro 3-10 membered cycloalkyl or 3-10 membered heterocyclic group; R5 and R6 are each independently selected from H, halogen, -CN, -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 haloalkyl; R7 and R8 are each independently selected from H, oxo, -C1-C6 alkyl, -C2-C6 haloalkyl, -C3-C6 cycloalkyl.

2. The compound according to claim 1, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, The compound is selected from:

3. The compound according to claim 1 or 2, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, The compound is selected from:

4. The compound according to claim 1, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, X is selected from O, -CH2, -NCH3, -NCH2CH3, -NCH(CH3)2, -NCH2CF3, -NCH2CHF2, -N-cyclopropyl, -NH, -NCH2CH(CH3)2, -N(CH2)2CH3, -N(CH2)2OCH3, -N(CH2)2N(CH3)2, -NCH2C(CH3)2OH.

5. The compound according to claim 1-2, 4 and its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, R1 is selected from H.

6. The compound according to claims 1-2, 4-5, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, R2 and R3 are each independently selected from H, amino, methyl, ethyl, isopropyl, fluorine, dimethylamino, cyano, methoxy, trifluoromethyl, cyclopropyl.

7. The compound according to claim 1, 4 - 6, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, R4 is selected from H, methyl, fluorine, trifluoromethyl.

8. The compound according to claim 1, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, Two R4s together with the carbon atom to which they are attached form 9. The compound according to any one of claims 1-7, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that, R5 and R6 are each independently selected from H, methyl, trifluoromethyl, fluorine, methoxy, -CN.

10. The compound according to claims 1-9, its stereoisomers, or its pharmaceutically acceptable salts, characterized in that, R7 and R8 are each independently selected from H, oxo, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoroethyl, cyclopropyl.

11. A compound selected from the following formula, its stereoisomers, or a pharmaceutically acceptable salt thereof:

12. A pharmaceutical composition comprising the compound according to any one of claims 1 to 11, its stereoisomers or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

13. Use of the compound according to any one of claims 1 to 11, its stereoisomers, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 12 in the preparation of a drug for treating a disease, disorder, syndrome or affliction associated with PRMT5.

14. The use of the drug according to claim 13, wherein the diseases, disorders, syndromes or afflictions related to the PRMT5 enzyme are cancer, blood diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, genetic diseases, hormone-related diseases, immunodeficiency diseases, diseases related to cell death, destructive bone diseases, thrombin-induced platelet aggregation, liver diseases and cardiovascular diseases. The diseases, disorders, syndromes or afflictions related to the PRMT5 enzyme are preferably cancer. More preferably, the cancer is advanced solid tumors, metastatic pancreatic cancer, metastatic non-small cell lung cancer, neuro-oncology, adenocarcinoma, endometrioid carcinoma, metastatic esophageal cancer, metastatic head and neck cancer, squamous cell carcinoma, cervical tumor, myelodysplastic syndrome, non-Hodgkin lymphoma, acute myeloid leukemia, adenoid tumor, blood tumor, melanoma, pancreatic tumor, brain tumor, glioblastoma, glioma, myelofibrosis, breast tumor, chronic myelomonocytic leukemia, diffuse large B-cell lymphoma, bladder cancer, cholangiocarcinoma, mesothelioma, ovarian cancer, lung cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer and hepatocellular carcinoma.

15. A method for preparing the compound represented by formula (I) according to claim 1, its stereoisomers or its pharmaceutically acceptable salts, the method comprising the steps of: The compound of general formula (Ia) or its isomers or salts reacts with the compound of general formula (Ib) or its isomers or salts to undergo a condensation reaction to obtain the compound represented by formula (I), its stereoisomers or its pharmaceutically acceptable salts, The definitions of each group are as defined in claim 1.

16. A method for preparing the compound represented by formula (Ia) and its stereoisomers or its pharmaceutically acceptable salts, the method comprising the steps of: (1) The compound of general formula (Id) or its isomers or salts reacts with the compound of general formula (Ie) to undergo an acylation reaction to generate the compound of general formula (Ic) or its isomers or salts; (2) The compound of general formula (Ic) or its isomers or salts undergoes an ester hydrolysis reaction to obtain the compound represented by formula (Ia), its stereoisomers or its pharmaceutically acceptable salts, X is a halogen, and the definitions of the other groups are as defined in claim 1.

17. Compounds represented by the following formulae (Ia), (Ic), (Id) and their stereoisomers or their pharmaceutically acceptable salts, The definitions of each group are as defined in claim 1.

18. Compounds and their stereoisomers or their pharmaceutically acceptable salts, which are selected from any one of the following compounds:

19. A compound, its stereoisomers or its pharmaceutically acceptable salts, which are selected from any of the following compounds:

20. Compounds and their stereoisomers or their pharmaceutically acceptable salts, which are selected from any one of the following compounds:

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