PRMT5 inhibitor, and composition and pharmaceutical use thereof

Abstract

The present 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 represented by formula (I). Further provided in the present invention are a composition containing the compound and the use thereof as a PRMT5 inhibitor.

Description

A PRMT5 inhibitor, its composition and pharmaceutical use Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a PRMT5 inhibitor compound and its stereoisomers, or pharmaceutically acceptable salts thereof, as well as to a pharmaceutical composition comprising the compound as an active ingredient and its pharmaceutical use in treating diseases, disorders, syndromes or conditions associated with the PRMT5 enzyme. Background Art

[0002] Arginine methylation, a type of histone methylation, is one of the most common post-translational modifications in mammals and is primarily regulated by the PRMT gene family. Arginine methyltransferases (PRMTs) regulate arginine methylation in three distinct forms: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) methylation. Nine PRMTs exist in mammals, divided into three types: type I (PRMT1, 2, 3, 4, 6, and 8; they primarily catalyze the production of MMA and ADMA), type II (PRMT5 and 9; they primarily catalyze the production of MMA and SDMA), and type III (PRMT7; they primarily catalyze the production of MMA). PRMTs can methylate histones and various non-histone proteins. Research has shown that altered PRMT activity, mutations, or deletions often lead to developmental abnormalities in animals and can also induce the initiation, progression, or metastasis of cancer. Among the numerous PRMTs, PRMT5 has garnered the most attention due to its increasingly well-established role in cancer.

[0003] Protein arginine methyltransferase 5 (PRMT5) is a typical type II methyltransferase that transfers methyl groups from SAM to the two ω-guanidino nitrogen atoms of arginine, resulting in ω-NG, NG-disymmetrical methylation of protein substrates. It is located in the cell nucleus and cytoplasm and exerts different functions by modifying histones or non-histone proteins. As an epigenetic enzyme, PRMT5 is most notable for its "synthetic lethality" mechanism.

[0004] The so-called synthetic lethality means that for two genes in a cell, if any one of them mutates or fails to function alone, it will not cause cell death; but if both of them mutate or cannot be expressed at the same time, it will cause cell death. This principle can be used to selectively kill tumor cells without affecting normal somatic cells.

[0005] In 2016, two papers published in Science first reported the "synthetic lethality" effect of PRMT5 inhibition in MTAP-deficient tumors. The gene that causes synthetic lethality with PRMT5 is methylthioadenosine phosphorylase (MTAP), a tumor suppressor gene that is frequently lost in tumors. MTAP-deficient patients account for approximately 15% of all solid tumors, including approximately 15% of non-small cell lung cancers (NSCLCs), 28% of esophageal cancers, 26% of bladder cancers, and 10% of esophageal and gastric cancers.

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

[0007] Most first-generation PRMT5 inhibitors entering clinical trials have generally yielded less than promising results. Clinical studies have shown that GSK-3326595 responded to various tumor types. However, 89% of participants reported adverse events, including anemia, thrombocytopenia, neutropenia, and fatigue. A Phase I clinical trial of JNJ-64619178 demonstrated sustained target inhibition in cancer patients with intermittent dosing. In contrast, JNJ-64619178 demonstrated limited or no efficacy in patients with myelodysplastic syndromes. Most patients treated with JNJ-64619178 experienced adverse events, including thrombocytopenia, anemia, and nausea. PF-06939999, a PRMT5 inhibitor, demonstrated objective tumor responses in patients with squamous cell carcinoma of the neck and non-small cell lung cancer. Cytoreductions were observed in clinical trials, which were dose-dependent and reversible with dose modification. Clinical trials of these PRMT5 inhibitors appear to have been halted.

[0008] In fact, with the exception of Prelude Therapeutics' program, most active programs are focused on second-generation PRMT5 inhibitors, known as PRMT5-MTA inhibitors. In cancer cells lacking MTAP, MTA replaces SAM in binding to PRMT5, forming an inactive PRMT5-MTA complex. Second-generation PRMT5 inhibitors bind to the PRMT5-MTA complex, killing MTAP-deficient cancer cells while sparing normal cells. This property allows second-generation PRMT5 inhibitors to more specifically target MTAP-deficient cancer cells and potentially reduce the incidence of adverse events.

[0009] Currently, the second-generation PRMT5 inhibitors that have been reported to have entered the clinical phase I / II research stage include MRTX-1719, SKL-27969, TNG-908, TNG-462 and AMG-193, but no drugs are still on the market; PRMT5 inhibitors are disclosed in documents such as WO2022132914 and WO2022115377. Summary of the Invention

[0010] SUMMARY OF THE INVENTION

[0011] The present invention aims to provide a novel PRMT5 inhibitor compound with good activity, good selectivity, good metabolic stability, good permeability, rapid pharmacokinetic absorption, good pharmacokinetic exposure, and 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] In one aspect, the present invention provides a compound as shown in Formula I and its stereoisomers, or a pharmaceutically acceptable salt thereof:

[0014] in,

[0015] X is selected from O, -NR, -CH2;

[0016] 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 ;

[0017] R1, R2, R3 are each independently selected from H, halogen, -C1-C6 alkyl, -C3-C6 cycloalkyl, -C1-C6 haloalkyl, -CN, -OR a1 、-NR a2 R a3 ;

[0018] R a1 、R a2 、R a3 Each is independently selected from H, -C1-C6 alkyl, -C3-C6 cycloalkyl;

[0019] n is 0, 1, 2, 3, or 4;

[0020] m is 0, 1, or 2;

[0021] r is 1, 2, 3, or 4;

[0022] R4 is selected from H, halo, -C1-C6 alkyl, -C1-C6 haloalkyl, or when n is 2, two R4 together with the carbon atoms to which they are attached form a bridged, fused or spiro 3-10 membered cycloalkyl or 3-10 membered heterocyclyl;

[0023] R5 and R6 are each independently selected from H, halo, -CN, -C1-C6 alkyl, -C1-C6 alkoxy, -C1-C6 haloalkyl;

[0024] R7 and R8 are each independently selected from H, oxo, -C1-C6 alkyl, -C2-C6 haloalkyl, -C3-C6 cycloalkyl;

[0025] The dotted line indicates that the bond here can be a single bond or a double bond.

[0026] As a preferred technical solution, the compound is selected from:

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

[0028] 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.

[0029] As a preferred technical solution, the R1 is selected from H.

[0030] As a preferred technical solution, R2 and R3 are each independently selected from H, amino, methyl, ethyl, isopropyl, fluorine, dimethylamino, cyano, methoxy, trifluoromethyl, and cyclopropyl.

[0031] As a preferred technical solution, the R4 is selected from H, methyl, fluorine, and trifluoromethyl.

[0032] As a preferred technical solution, two R4 together with the carbon atoms to which they are connected form

[0033] As a preferred technical solution, R5 and R6 are each independently selected from H, methyl, trifluoromethyl, fluorine, methoxy, and -CN.

[0034] As a preferred technical solution, R7 and R8 are each independently selected from H, oxo, methyl, ethyl, propyl, isopropyl, isobutyl, trifluoroethyl, and cyclopropyl.

[0035] The present invention also provides a compound selected from the following formula and its stereoisomers, or a pharmaceutically acceptable salt thereof:

[0036] The present invention also provides any one of the above compounds or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0037] The present invention also provides use of any of the above compounds and stereoisomers thereof, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating a disease, condition, syndrome or disorder associated with PRMT5.

[0038] As a preferred technical solution, the disease, disorder, syndrome or condition associated with the PRMT5 enzyme is cancer, blood disease, inflammatory disease, autoimmune disease, metabolic disease, genetic disease, hormone-related disease, immunodeficiency disease, disease associated with cell death, destructive bone disease, thrombin-induced platelet aggregation, liver disease and cardiovascular disease, and the disease, disorder, syndrome or condition 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, Lung cancer, neurological tumors, adenocarcinoma, endometrioid carcinoma, metastatic esophageal cancer, metastatic head and neck cancer, squamous cell carcinoma, cervical cancer, myelodysplastic syndrome, non-Hodgkin's lymphoma, acute myeloid leukemia, adenoid tumors, hematological cancers, melanoma, pancreatic cancer, brain tumors, glioblastoma, glioma, myelofibrosis, breast cancer, chronic myelomonocytic leukemia, diffuse large B-cell lymphoma, bladder cancer, bile duct cancer, mesothelioma, ovarian cancer, lung cancer, prostate cancer, colon cancer, stomach cancer, esophageal cancer and hepatocellular carcinoma.

[0039] The present invention also provides a method for preparing the compound represented by formula (I) according to claim 1 and its stereoisomers or pharmaceutically acceptable salts thereof, the method comprising the steps of:

[0040] The compound of general formula (Ia) or its isomer or salt undergoes a condensation reaction with the compound of general formula (Ib) or its isomer or salt to obtain the compound represented by formula (I) and its stereoisomer or pharmaceutically acceptable salt.

[0041] The definitions of the groups are as above.

[0042] The present invention also provides a method for preparing the compound represented by formula (Ia) and its stereoisomers or pharmaceutically acceptable salts thereof, the method comprising the steps of:

[0043] (1) A compound of formula (Id) or its isomer or salt undergoes an acylation reaction with a compound of formula (Ie) to produce a compound of formula (Ic) or its isomer or salt;

[0044] (2) the compound represented by the general formula (Ic) or its isomer or salt undergoes ester hydrolysis reaction to obtain the compound represented by the general formula (Ia) and its stereoisomer or its pharmaceutically acceptable salt,

[0045] The definitions of the groups are as above.

[0046] Finally, the present invention also provides compounds represented by the following formulas (Ia), (Ic), (Id) and their stereoisomers or pharmaceutically acceptable salts thereof:

[0047] The definitions of the groups are as described above.

[0048] The present invention also provides an intermediate compound and its stereoisomers or pharmaceutically acceptable salts thereof, which are selected from any one of the following compounds:

[0049] The present invention also provides an intermediate compound and a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is selected from any one of the following compounds:

[0050] Finally, the present invention also provides an intermediate compound and its stereoisomer or salt thereof, which is selected from any one of the following compounds:

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention utilizes computer-aided design technology to modify analogs such as TNG908 and TNG462. Through computer simulation, molecular docking analysis, and drug structure-activity relationship studies, a variety of novel compounds have been screened. This series of compounds can bind to the PRMT5-MTA complex and inhibit PRMT5 function, exhibiting extremely strong PRMT5 inhibitory activity. In vitro and in vivo experiments further demonstrate that this series of compounds can prevent and / or treat cancer, blood disorders, inflammatory diseases, autoimmune diseases, metabolic diseases, genetic diseases, hormone-related diseases, immunodeficiency diseases, diseases associated with cell death, destructive bone diseases, thrombin-induced platelet aggregation, liver disease, and cardiovascular diseases. They exhibit excellent activity, selectivity, metabolic stability, permeability, and rapid absorption, and exhibit favorable pharmacokinetic exposure in mouse pharmacokinetic tests.

[0053] Detailed Description of the Invention

[0054] Various aspects and features of the present invention are further described below.

[0055] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the 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 provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail. The following are definitions of various terms used in the present invention, which apply to the terms used throughout the specification of this application, unless otherwise specified in the specific case.

[0056] The compounds according to the present invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

[0057] The compounds of the present invention possess asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art will appreciate how to prepare optically active forms, such as by racemic resolution or synthesis from optically active starting materials. Unless otherwise specified, the present invention encompasses all chiral, non-isomerized, and racemic forms. Methods for preparing the compounds of the present invention and intermediates thereto are also intended to be included in the present invention. All tautomers of the compounds of the present invention are also intended to be included in the present invention.

[0058] "Alkyl" refers to a group of straight or branched saturated hydrocarbon groups having 1 to 10 carbon atoms ("C1-C10 alkyl"). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C1-C8 alkyl"). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C1-C6 alkyl"), the alkyl group has 1 to 5 carbon atoms ("C1-C5 alkyl"), the alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"), the alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"), the alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"), and the alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups 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 groups include n-heptyl (C7), n-octyl (C8), and the like. Each instance of an alkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents; such as, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl"). In certain embodiments, the alkyl group is an unsubstituted C1-C10 alkyl group (e.g., -CH3). In certain embodiments, the alkyl group is a substituted C1-C6 alkyl group. Common alkyl abbreviations include Me(–CH3), Et(–CH2CH3), iPr(–CH(CH3)2), nPr(–CH2CH2CH3), n–Bu(–CH2CH2CH2CH3), or i–Bu(–CH2CH(CH3)2).

[0059] As used herein, the terms "halogen", "halo", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine, bromine, and particularly preferably fluorine and chlorine.

[0060] "Haloalkyl" refers to an alkyl group 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.

[0061] "Alkoxy" refers to an alkyl group as described herein (e.g., C1-C6 alkyl) attached to the molecule through an oxygen atom. This includes groups in which the alkyl portion can be straight or branched, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, and n-hexoxy.

[0062] In some embodiments, a “cycloalkyl” is a monocyclic saturated cycloalkyl group having 3 to 10 ring carbon atoms (“C3-10 cycloalkyl” or “C3-C10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl” or “C3-C8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyl” or “C3-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6 cycloalkyl” or “C5-C6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl” or “C5-C10 cycloalkyl”). Examples of C5-C6 cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-C6 cycloalkyl groups include the aforementioned C5-C6 cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-C8 cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each example of a cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted ("substituted cycloalkyl") with one or more substituents. In certain embodiments, a cycloalkyl group is an unsubstituted C3-C10 cycloalkyl group. In certain embodiments, a cycloalkyl group is a substituted C3-C10 cycloalkyl group.

[0063] The terms "heterocycle" and "heterocyclyl" 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 having at least one heteroatom (O, S or N) in at least one ring, preferably 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of such heteroatom-containing groups may 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 may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be saturated, partially saturated or fully unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom; "heterocyclyl" as described herein includes heterocycloalkyl.

[0064] Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridine, oxirane, and thiirane. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thitanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-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, dihydropyridinyl, and thiohexyl. 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, oxepanyl, and thiecanyl. 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.

[0065] In addition to the heteroaryl groups described below, exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane, and tetrahydro-1,1-dioxythienyl, etc. Exemplary bicyclic heterocyclic groups include quinoline ring groups.

[0066] As used herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate during chiral resolution. Although the salt of this intermediate cannot be directly administered to a subject, the salt can play a role in obtaining the final product of the present invention.

[0067] As described herein, the term "disease" refers to a physical condition of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegenerative diseases described in the present invention.

[0068] Cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy, and hormone therapy.

[0069] "Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other sites in the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cell" refers to a cell that is undergoing an early, intermediate, or advanced stage of multistep neoplastic progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, larynx cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, stomach cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumors, 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 tumors, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma, lymphoma, pancreatic ductal adenocarcinoma, etc.

[0070] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0071] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0072] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known method of administration.

[0073] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation. DETAILED DESCRIPTION

[0074] The following embodiments are intended to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the present invention in any way.

[0075] For all of the following examples, standard procedures and methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectroscopy (MS).

[0076] The structures of the compounds of the present invention were confirmed by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are expressed in parts per million (ppm). NMR measurements were performed using a Bruker Avance-400 NMR spectrometer. The solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0077] Liquid phase mass spectrometry LC-MS measurement The liquid phase part used ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part used Xevo G2-S Qtof mass spectrometer.

[0078] The starting materials used in the examples of the present invention are known and can be purchased commercially, or can be used or synthesized according to methods known in the art.

[0079] 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)

[0080] Step 1: Synthesis of 4-(4-bromo-2-nitrophenyl)morpholine

[0081] To a solution of 1,4-dibromo-2-nitrobenzene (5.0 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 mixture was allowed to react at 120°C for 16 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 4-(4-bromo-2-nitrophenyl)morpholine (4.5 g, 88% yield). LCMS: m / z=[M+H]287.0.1H NMR (400MHz, DMSO-d6) δ8.07(d,J=2.4Hz,1H),7.79(dd,J=8.8,2.4Hz,1H),7.32(d,J=8.9Hz,1H),3.76–3.54(m,4H),3.02–2.91(m,4H).

[0082] Step 2: Synthesis of 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0083] To a solution of 4-(4-bromo-2-nitrophenyl)morpholine (4.5 g, 15.7 mmol, 1.0 equiv.) in formic acid (20 mL) in a sealed tube was added I2 (800 mg, 3.2 mmol, 0.2 equiv.) and allowed to react at 120°C for 18 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride, combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 8-bromo-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1 g, 25% yield). LCMS:m / z=[M+H]+:253.0.1H NMR(400MHz, DMSO-d6)δ8.09(m,J=3.8,1.8Hz,1H),7.89–7.80(m,1H),7.71(m,J=7.8, 4.0, 1.8Hz, 1H), 5.17 (d, J = 4.2Hz, 2H), 4.38 (m, J = 4.3, 3.6Hz, 2H), 4.29–4.21 (m, 2H).

[0084] 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

[0085] 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), and 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]palladium dichloride (218 mg, 0.3 mmol, 0.1 eq) were added to the system. The system was replaced with nitrogen three times and the temperature was raised to 110°C for 6 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 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, 46% yield) as a white solid. LCMS: m / z = [M+H] 301.1.

[0086] Step 4: Synthesis of (S)-tert-butyl 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

[0087] 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), and tert-butyl (S)-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 6 hours. After completion, the reaction was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (S)-tert-butyl 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, 40% yield) as a yellow solid. LCMS: m / z = [M+H]-Boc 270.20.

[0088] 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

[0089] (S)-tert-Butyl 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) was dissolved in dichloromethane (1.5 mL). Trifluoroacetic acid (0.5 mL) was added and stirred for 30 minutes. The reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction solution was concentrated and the pH was adjusted to 8-9 by adding saturated sodium carbonate solution. The solution was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give 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.

[0090] Step 6: Synthesis of 8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0091] (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.) was dissolved in methanol (3 mL). Sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) was added portionwise on ice and stirred for 1 hour. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 8-[(5S)-5-methylpiperidin-2-yl]-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (60 mg, 68% yield) as a yellow solid. LCMS: m / z = [M+H] 272.2.

[0092] 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

[0093] 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 to the reaction flask. After nitrogen was replaced three times, triethylamine (30 mg, 0.26 mmol, 1.2 equiv.) was added. Subsequently, ethyl oxalyl chloride (36 mg, 0.26 mmol, 1.2 equiv.) was slowly added dropwise in an ice bath. The mixture was stirred at room temperature and the reaction was monitored by TLC and LCMS. After the reaction, water was added to quench the reaction, and the liquid phases were extracted with water and dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate, filtered, mixed, and separated by column 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, 28% yield) as a white solid. LCMS: m / z = [M+H] 372.2.

[0094] 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

[0095] The compound 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 allowed to react at room temperature for 2 hours. After completion of the reaction, the pH was adjusted to 5-6 with 1 M aqueous hydrogen chloride solution and 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.

[0096] Step 9: Synthesis of 5-nitro-3-vinylpyridin-2-amine

[0097] 3-Bromo-5-nitropyridin-2-amine (1000 mg, 4.6 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (40 mL) and water (10 mL). Potassium vinyl trifluoroborate (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 (334 mg, 0.46 mmol, 0.1 equiv.) were added. The system was purged with nitrogen three times and heated to 80°C for 8 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 5-nitro-3-vinylpyridin-2-amine (388 mg, 44% yield) as a white solid. LCMS: m / z=[M+H]166.1.1H NMR (400MHz, DMSO-d6) δ8.83(d,J=2.7Hz,1H),8.27(d,J=2.6Hz,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).

[0098] Step 10: Synthesis of 3-ethylpyridine-2,5-diamine

[0099] The compound 5-nitro-3-vinylpyridin-2-amine (384 mg, 1.44 mmol, 1.0 equiv.) was dissolved in methanol (6 mL). Palladium on carbon (5% Pd, approximately 55% water content, 76 mg) was added, and the system was purged with hydrogen three times. The reaction was allowed to react at room temperature for 24 hours. After completion of the reaction, the reaction solution was filtered and separated by column chromatography to obtain 3-ethylpyridine-2,5-diamine (80 mg, 41% yield) as a black oily liquid. LCMS: m / z = [M+H] 138.1.

[0100] 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

[0101] 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.) were dissolved in N,N-dimethylformamide (1.5 mL) and stirred for 5 minutes. 2-(7-Azobenzotriazole)-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 the reaction was completed, the product was purified by SFC preparative chromatography to give a 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 (400MHz, DMSO-d6) δ10.46(s,1H),8.08(s,1H),7.63–7.40(m,3H),7.17(d,J=35.0Hz,1H),5.76–5.24(m,3H),4.94(s,2H),4.17( m,J=8.3,4.3Hz,5H),3.41(d,J=87.8Hz,1H),2.47–2.23(m,3H),2.12(m,1H),1.82(m,J=44.1Hz,2H),1.37(m,1H),1.18–1.03(m,6H).

[0102] 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)

[0103] Step 1: Synthesis of 4-(4-bromo-2-nitrophenyl)morpholine

[0104] To a solution of 1,4-dibromo-2-nitrobenzene (5.0 g, 17.9 mmol, 1.0 equiv.) in N,N-dimethylformamide (30 mL) were added (S)-2-methylmorpholine (5.4 g, 53.9 mmol, 3.0 equiv.) and potassium carbonate (7.4 g, 53.9 mmol, 3.0 equiv.), and the mixture was allowed to react at 120°C for 16 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (S)-4-(4-bromo-2-nitrophenyl)-2-methylmorpholine (4.7 g, 88% yield). LCMS: m / z = [M+H] 301.0.

[0105] Step 2: Synthesis of (S)-8-bromo-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0106] 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) in a sealed tube was added I2 (800 mg, 3.2 mmol, 0.2 equiv.) and allowed to react at 120°C for 18 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride and combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (S)-8-bromo-3-methyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1.5 g, 35.9% yield). LCMS: m / z = [M+H] + :267.0.

[0107] 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

[0108] 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) was dissolved in N,N-dimethylformamide (15 mL), and 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]palladium dichloride (218 mg, 0.3 mmol, 0.1 eq) were added to the system. The system was replaced with nitrogen three times and the temperature was raised to 110°C for 6 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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, 42.8% yield) as a white solid. LCMS: m / z = [M+H] 315.2.

[0109] 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

[0110] 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), and to the system were added tert-butyl (S)-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 the temperature was raised to 90°C for 6 hours. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 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 (200 mg, 47.5% yield) as a yellow solid. LCMS: m / z = [M+H]-Boc 284.2.

[0111] 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

[0112] (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, and the mixture was stirred for 30 minutes. The reaction was monitored by TLC and LCMS. After the reaction, the reaction solution was concentrated, saturated sodium carbonate solution was added to adjust the pH to 8-9, the mixture was diluted with water, and the mixture was extracted with ethyl acetate and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 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, 72% yield). LCMS: m / z = [M+H] 284.1.

[0113] 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

[0114] (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.) was dissolved in methanol (3 mL). Sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) was added portionwise on ice and stirred for 1 hour. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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, 71% yield) as a yellow solid. LCMS: m / z = [M+H] 286.2.

[0115] 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

[0116] (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.) was added to the reaction flask. After nitrogen was replaced three times, triethylamine (30 mg, 0.26 mmol, 1.2 equiv.) was added. Subsequently, ethyl oxalyl chloride (36 mg, 0.26 mmol, 1.2 equiv.) was slowly added dropwise in an ice bath. The mixture was stirred at room temperature and the reaction was monitored by TLC and LCMS. After completion of the reaction, water was added to quench the reaction, and the liquid phases were extracted with water and dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, mixed, and separated by column chromatography to afford 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, 39% yield) as a white solid. LCMS: m / z = [M+H] 386.2.

[0117] 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

[0118] 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.) was dissolved in tetrahydrofuran (1 mL) and water (1 mL). Lithium hydroxide hydrate (6 mg, 0.12 mmol, 2 equiv.) was added to the mixture and allowed to react at room temperature for 2 hours. After completion, the pH was adjusted to 5-6 with 1 M aqueous hydrogen chloride solution and concentrated to afford 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.

[0119] 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

[0120] 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.) were dissolved in N,N-dimethylformamide (1.5 mL) and stirred for 5 minutes. 2-(7-Azobenzotriazole)-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, preparative purification was performed to obtain a 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.

[0121] 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)

[0122] 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

[0123] 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) and stirred for 5 minutes. 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114 mg, 0.3 mmol, 1.5 equiv.) was added and stirred at room temperature for 2 hours. After completion of the reaction, purification by normal phase column afforded a 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.

[0124] 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)

[0125] Step 1: Synthesis of 4-[4-bromo-2-nitro-5-(trifluoromethyl)phenyl]morpholine

[0126] 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 mixture was allowed to react at 120°C for 16 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 4-[4-bromo-2-nitro-5-(trifluoromethyl)phenyl]morpholine (5.54 g, 87% yield). LCMS: m / z = [M+H] 354.9.

[0127] Step 2: Synthesis of 8-bromo-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0128] 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) was added I2 (800 mg, 3.2 mmol, 0.2 equiv.) in a sealed tube and allowed to react at 120°C for 18 hours. After completion, the reaction was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride and combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 8-bromo-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1.2 g, 23.9% yield). LCMS: m / z = [M+H] + :320.9.

[0129] 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

[0130] The compound 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) was dissolved in N,N-dimethylformamide (15 mL), and 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]palladium dichloride (218 mg, 0.3 mmol, 0.1 eq) were added to the system. The system was replaced with nitrogen three times and the temperature was raised to 110°C for 6 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 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, 38.9% yield) as a white solid. LCMS: m / z = [M+H] 369.1.

[0131] Step 4: Synthesis of (S)-tert-butyl 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

[0132] The compound 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.) was dissolved in 1,4-dioxane (9 mL) and water (3 mL), and tert-butyl (S)-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]palladium dichloride (78 mg, 0.1 mmol, 0.1 equiv.) were added to the system. The system was purged with nitrogen three times and the temperature was raised to 90°C for 6 hours. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (S)-tert-butyl 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 (200 mg, 47.5% yield) as a yellow solid. LCMS: m / z = [M+H]-Boc 337.2.

[0133] 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

[0134] (S)-tert-Butyl 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 (191.5 mg, 0.5 mmol, 1.0 eq) was dissolved in dichloromethane (1.5 mL), trifluoroacetic acid (0.5 mL) was added, and the mixture was stirred for 30 minutes. The reaction was monitored by TLC and LCMS. After the reaction, the reaction solution was concentrated, saturated sodium carbonate solution was added to adjust the pH to 8-9, the mixture was diluted with water, and the mixture was extracted with ethyl acetate and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 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, 71% yield). LCMS: m / z = [M+H] 338.1.

[0135] 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

[0136] (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.) was dissolved in methanol (5 mL). Sodium borohydride (25 mg, 0.66 mmol, 2.0 equiv.) was added portionwise on ice and stirred for 1 hour. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford 8-[(5S)-5-methylpiperidin-2-yl)-7-(trifluoromethyl)-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (75 mg, 67% yield) as a yellow solid. LCMS: m / z = [M+H] 340.1.

[0137] 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

[0138] 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.) was added to the reaction flask. After nitrogen was replaced three times, triethylamine (30 mg, 0.26 mmol, 1.2 equiv.) was added, and then ethyl oxalyl chloride (36 mg, 0.26 mmol, 1.2 equiv.) was slowly added dropwise in an ice bath. The mixture was stirred at room temperature and the reaction was monitored by TLC and LCMS. After the reaction, water was added to quench the reaction, and the liquid phases were extracted with water and dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, mixed, and separated by column chromatography to afford 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, 34% yield) as a white solid. LCMS: m / z = [M+H] 440.2.

[0139] 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

[0140] 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.) was dissolved in tetrahydrofuran (1 mL) and water (1 mL). Lithium hydroxide hydrate (6 mg, 0.12 mmol, 2 equiv.) was added to the mixture and allowed to react at room temperature for 2 hours. After completion of the reaction, the pH was adjusted to 5-6 with 1 M aqueous hydrogen chloride solution and concentrated to afford 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.

[0141] 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

[0142] 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) and stirred for 5 minutes. 2-(7-Azobenzotriazole)-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, preparative purification was performed to obtain a 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). LCMS: m / z = [M+H]+ 531.2.

[0143] 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)

[0144] Step 1: Synthesis of tert-butyl 4-(4-bromo-2-nitrophenyl)-3-oxopiperazine-1-carboxylate

[0145] tert-Butyl 3-oxapiperazine-1-carboxylate (5.0 g, 25 mmol, 1.1 eq) was added to a flask. N,N-dimethylformamide (50 mL) was added under nitrogen. After stirring at room temperature for 10 minutes, sodium hydride (1.4 g, 60% dispersion in mineral oil) was added and stirring continued for 30 minutes. Finally, 4-bromo-1-fluoro-2-nitrobenzene (5.0 g, 22.9 mmol, 1.0 eq) was added and stirred at room temperature for 16 hours. After the reaction, ethyl acetate (600 mL) and saturated ammonium chloride solution (200 mL) were added. The organic phase was separated, and the organic phase was washed sequentially 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.

[0146] Step 2: Synthesis of tert-butyl 8-bromo-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0147] 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 refluxed for 1 hour. After completion of the reaction, the organic phase was concentrated to dryness, ethyl acetate (50 mL) was added, and the mixture was washed with saturated sodium bicarbonate (50 mL). The organic phase was dried and concentrated, and the crude product was purified by column chromatography (mobile phase: dichloromethane / methanol = 50 / 1 (v / v)) to afford tert-butyl 8-bromo-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.1 g, 62.9% yield). LCMS: m / z = [M-Boc+H]: 252.0.

[0148] Step 3: Synthesis of 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0149] Dissolve 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) in dichloromethane (15 mL). Add 4N hydrochloric acid in dioxane (10 mL) and stir overnight. Monitor the reaction by TLC and LCMS. After completion, the reaction solution was concentrated, adjusted to pH 8-9 by adding saturated sodium carbonate solution, diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to give the crude product 8-bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.4 g, 94.5% yield). LCMS: m / z = [M+H]: 252.0.

[0150] Step 4: Synthesis of 8-bromo-2-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0151] 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 a reaction flask and stirred overnight at room temperature. After completion of the reaction, the organic phase was concentrated to dryness, neutralized with saturated sodium bicarbonate solution, and extracted with ethyl acetate. The organic phase was concentrated, and the crude product was purified by column chromatography (mobile phase: dichloromethane / methanol = 20 / 1 (v / v)) to afford 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.

[0152] Step 5: Synthesis of 2-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0153] 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) was dissolved in DMF (10 ml). 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]palladium dichloride (310 mg, 0.42 mmol, 0.1 eq) were added. The system was purged with nitrogen three times and heated to 110°C for 6 hours. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The organic phases were washed with saturated sodium chloride. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to yield 560 mg of a white solid. LCMS: m / z = [M+H] 314.2.

[0154] Step 6: Synthesis of (S)-tert-butyl 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

[0155] The compound 2-methyl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (560 mg, 1.8 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (12 ml) and water (4 ml). 5-methyl-2-(((trifluoromethyl)sulfonyl)oxy)cyclohex-2-ene-1-carboxylic acid tert-butyl ester (621 mg, 1.8 mmol, 1.0 equiv.), sodium carbonate (558 mg, 5.4 mmol, 3.0 equiv.), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (150 mg, 0.18 mmol, 0.1 equiv.) were added to the system. The system was purged with nitrogen three times and the temperature was raised to 90°C for 6 hours. After the reaction was completed, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to obtain 360 mg of a yellow solid. LCMS: m / z = [M-Boc+H] 283.2.

[0156] 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

[0157] (S)-tert-Butyl 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) was dissolved in DCM (5 mL), TFA (0.5 mL) was added, and the reaction was stirred for 0.5 h and monitored by TLC and LCMS. After the reaction, the solvent was dried by spin-drying, saturated sodium carbonate solution was added to adjust the pH to 8-9, 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 the solvent was dried by spin-drying to obtain the intermediate crude 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. The crude product was then dissolved in methanol (5 mL), and sodium borohydride (100 mg, 2.6 mmol, 2.0 equiv.) was added portionwise in an ice bath. After stirring for 0.5 h, the reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to give the product (195 mg) (LCMS: m / z = [M+H] 285.2).

[0158] 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

[0159] 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 the reaction flask. After N2 protection, triethylamine (90 mg, 0.78 mmol, 1.2 equiv.) was added. Ethyl oxalyl chloride (108 mg, 0.79 mmol, 1.2 equiv.) was slowly added dropwise under ice bath conditions and stirred at room temperature. The reaction was monitored by TLC and LCMS. After completion, water was added to quench the reaction, and the fractions were extracted with water and dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, mixed, and separated by column chromatography to afford 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 (69 mg) as a white solid. LCMS: m / z = [M+H] 385.1918. The crude product was dissolved in tetrahydrofuran (1 mL) and water (1 mL). Lithium hydroxide hydrate (18 mg, 0.36 mmol, 2 equiv.) was added and the mixture reacted at room temperature for 2 hours. After the reaction was complete, the pH was adjusted to 5-6 with a 1 M aqueous solution of hydrogen chloride, and the reaction mixture was concentrated to afford crude 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.

[0160] 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

[0161] 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) and stirred for 5 minutes. 2-(7-Azobenzotriazole)-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 was performed by preparative high-performance liquid chromatography to yield 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) as a white solid. LCMS: m / z = [M+H] 476.3.

[0162] 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)

[0163] Step 1: Synthesis of 4-(4-bromo-2-nitrophenyl)-2,2-dimethylmorpholine

[0164] 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 weighed and added to acetonitrile (40 mL). The atmosphere was purged with nitrogen twice and the reaction was allowed to proceed at 60°C for 16 hours. After completion of the reaction, the reaction solution was filtered, the filtrate was concentrated, and the sample was purified by normal phase column separation using petroleum ether and ethyl acetate. When the ethyl acetate content was 10%, 4-(4-bromo-2-nitrophenyl)-2,2-dimethylmorpholine (5.0 g, 8.0 mmol, 92% yield) was obtained as an orange oil.

[0165] Step 2: Synthesis of 8-bromo-3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine

[0166] 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 reacted at 120°C for 16 hours. After the reaction is completed, the reaction solution is filtered, the filtrate is concentrated and the pH value is adjusted to about 10 with sodium carbonate, and then extracted with ethyl acetate and water. The organic phase is used to remove excess water with anhydrous sodium sulfate, and the sample is spin-dried and purified by normal phase column separation. When the ethyl acetate content is 26% in a petroleum ether and ethyl acetate system, the target product flows out. The resulting solution is concentrated and then slurried with a mixed solvent of petroleum ether and ethyl acetate (the volume ratio of petroleum ether and ethyl acetate is 5:1) to obtain a brown solid 8-bromo-3,3-dimethyl-3,4-dihydro-1H-benzo[4,5]imidazo[2,1-c][1,4]oxazine (1.7 g, 6.1 mmol, yield 42%). LCMS(ESI)[M+H]+=281.1H NMR (400MHz, DMSO) δ7.80 (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).

[0167] 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

[0168] 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), pinacol diboron (1.81 g, 7.1 mmol, 2.0 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (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), the system gas was replaced with nitrogen, and the reaction was carried out at 100°C for 4 hours. After the reaction, the mixed solution was filtered, concentrated and mixed, and separated and purified by normal phase column. When the ethyl acetate content was 35%, a white solid 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 (1.7 g, 6.1 mmol, yield 42%) was obtained. LCMS (ESI) [M+H] + =329.

[0169] Step 4: Synthesis of (S)-tert-butyl 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

[0170] 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), (S)-tert-butyl 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]palladium dichloride (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) and reacted at 90°C under nitrogen for 16 hours. After completion of the reaction, the reaction solution was filtered, the filtrate was concentrated and the sample was dried by spin drying. The product was separated and purified by normal phase column chromatography using a petroleum ether and ethyl acetate system with an ethyl acetate content of 30% to obtain (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)-carboxylic acid tert-butyl ester (230 mg, 0.58 mmol, 80% yield) as a brown solid. LCMS (ESI) [M+H]+ = 398.

[0171] 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

[0172] 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, 1.0 eq) was dissolved in trifluoroacetic acid (1 mL) and dichloromethane (3 mL) and stirred at room temperature for 1 hour. After the reaction was complete, the reaction solution was concentrated to give the crude product (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, 59.8% yield) as a white solid. LCMS (ESI) [M+H]+ = 298.

[0173] 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

[0174] (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) and sodium borohydride (25 mg, 0.67 mmol, 2.0 eq) were dissolved in methanol (2 mL) and stirred at room temperature for 30 minutes. After the reaction was complete, the reaction solution was concentrated to obtain a 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 used directly in the next step. LCMS (ESI) [M+H]+: 300.

[0175] 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

[0176] 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). Then, 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) were added in small amounts in an ice bath and the mixture was reacted at room temperature for 2 hours (continuous monitoring). After the reaction was complete, the reaction mixture was concentrated to afford crude 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) as a white solid, which was used directly in the next step. LCMS (ESI) [M+H]+ = 400.

[0177] 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

[0178] 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) and 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 the reaction was complete, the pH was adjusted to approximately 4 with dilute hydrochloric acid, and the product was extracted with ethyl acetate and water. The organic phase was freed from excess water using anhydrous sodium sulfate, and concentrated to afford 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 used directly in the next reaction. LCMS (ESI) [M+H]+ = 372.

[0179] 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

[0180] 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), and 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 stirred at room temperature for 1 hour. After completion of the reaction, HPLC and SFC separation and purification were performed to obtain a yellow powder, 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, 5.4% yield). LCMS (ESI) [M+H]+: 491.2772. 1HNMR(400MHz,DMSO)δ10.57(d,J=15.4Hz,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=5.2Hz,2H),3.44(s,2H),2.42(d,J=7.5Hz,1H),2.07(s,1H),1.90–1.67(m,2H),1.35(s,7H),1.17–0.99(m,6H).

[0181] 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)

[0182] Step 1: Synthesis of 8-bromo-2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0183] 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 mixture was allowed to react at room temperature for 18 hours, and the mixture was sampled for LCMS. The sample was mixed and column chromatography (D:M = 10:1 + 0.1% triethylamine) afforded 8-bromo-2-ethyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (99.2 mg, 35.7% yield) as a yellow oil. LCMS: [M+H] = 280.0320. 1 H NMR (400MHz, DMSO) δ7.75 (s, 1H), 7.48 (d, J = 8.5Hz, 1H), 7.39–7.31 (m, 1H), 4.17–4.07 ( m, 2H), 3.80 (s, 2H), 2.98 (t, J = 5.5Hz, 2H), 2.62 (q, J = 7.1Hz, 2H), 1.12 (t, J = 7.1Hz, 3H).

[0184] 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

[0185] 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) were 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)diboron palladium chloride (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 atmosphere was replaced with nitrogen three times and the reaction was refluxed at 100°C for 18 hours. The reaction was monitored by TLC and LCMS. The product was extracted with ethyl acetate and water, and the organic phase was dried, concentrated, mixed, and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford 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 (502.3 mg, 146.6% yield) as a yellow oily liquid. LCMS: [M+H] = 328.2277.

[0186] Step 3: Synthesis of tert-butyl (S)-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

[0187] 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) was added (S)-3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (860. The reaction mixture was stirred for 1 h at 4 °C for 2 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 1 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. The mixture was stirred for 3 h. The mixture was stirred for 2 h. LCMS: [M-Boc+H]=297.2101.

[0188] 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

[0189] Tert-butyl (S)-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), and trifluoroacetic acid (0.8 mL, 13.674 mmol, 15.0 Equiv) was added. The mixture was reacted at room temperature for 1 hour, monitored by LCMS and TLC. The trifluoroacetic acid and dichloromethane were evaporated, and the mixture was dissolved in a small amount of water. 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.

[0190] Step 5: Synthesis of 2-ethyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0191] (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). Sodium borohydride (87 mg, 2.280 mmol, 2.5 Equiv) was added portionwise at 0°C. After 5 minutes, the mixture was transferred to room temperature and reacted for 1 hour. LCMS and TLC monitoring were performed. The solution was concentrated, washed with a plate (D:M = 10:1 + 0.1% triethylamine), scraped, dissolved, and dried to give 2-ethyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (129.1 mg, 47.5% yield). LCMS: [M+H]=299.1845.

[0192] 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

[0193] 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), and 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 was stirred at room temperature and monitored by LCMS. Upon completion, ethanol was added to quench the reaction, the sample was mixed, and column chromatography (D:M = 10:1 + 0.1% triethylamine) was performed 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 (163.8 mg, 95.0% yield) as a yellow oily liquid. LCMS: [M+H] = 399.2373.

[0194] 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

[0195] To 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 lithium hydroxide (29.6 mg, 1.235 mmol, 3.0 Equiv), tetrahydrofuran (2.5 mL), methanol (1.0 mL), and water (1.0 mL). The mixture was stirred at room temperature for 0.5 hours. The solvent was evaporated, a small amount of water was added, and the pH was adjusted to acidity with dilute hydrochloric acid. 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.

[0196] 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

[0197] 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), and 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. The mixture was stirred at room temperature for 5 minutes, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (234.8 mg, 0.618 mmol, 1.5 Equiv) was added and the mixture was reacted at room temperature for 1 hour. After the reaction, the reaction solution was filtered and separated and purified by HPLC and SFC to obtain a pink powder of 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, 22.8% yield). LCMS: [M+H] = 490.2928. 1HNMR(400MHz,DMSO)δ10.59(s,1H),7.57–7.45(m,3H),7.16(d,J=7.3Hz,1H), 5.67(t,J=19.5Hz,2H),4.12(s,1H),3.79(s,2H),3.46(d,J=13.1Hz,1H),3.3 0–3.20(m,1H),2.97(s,2H),2.62(q,J=7.1Hz,2H),2.47–2.25(m,3H),2.12(d ,J=45.9Hz,1H),1.94–1.66(m,2H),1.37(t,J=15.3Hz,1H),1.18–1.00(m,9H).

[0198] 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)

[0199] Step 1: Synthesis of 8-bromo-2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0200] 8-Bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (230 mg, 0.92 mmol, 1.0 eq.) and acetone (160 mg, 2.76 mmol, 3.0 eq.) were dissolved in dichloromethane (10 mL). Sodium acetate borohydride (976 mg, 4.6 mg, 5.0 eq.) was then added. The reaction was stirred at room temperature and monitored by TLC and LCMS. After completion of the reaction, 8-bromo-2-isopropyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (224 mg) was isolated and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain a yellow oil. LC-MS: [M+H] = 293.9969. 1 H NMR(400MHz,DMSO-d6)δ7.74(s,1H),7.49–7.45(m,1H),7.34(d,J=10.3Hz,1 H), 4.13–4.05 (m, 2H), 3.88 (s, 2H), 3.04–2.93 (m, 3H), 1.09 (d, J = 6.6Hz, 6H).

[0201] 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

[0202] 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)diboron palladium chloride (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 atmosphere was replaced with nitrogen three times, and the mixture 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 concentrated and separated by column chromatography (D:M=10:1+0.1% triethylamine) to obtain a crude brown oil (250 mg). LC-MS: [M+H]=342.2458.

[0203] Step 3: Synthesis of tert-butyl (S)-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

[0204] 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)-3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (252 mg, 0.7 3mmol, 1.0Equiv.), 1,1-bis(diphenylphosphino)diphenylferric palladium dichloride (51.2mg, 0.07mmol, 0.1Equiv.) and sodium carbonate (221mg, 2.2mmol, 3.0Equiv.) were added to water (2.0mL) and 1,4-dioxane (6.0mL), and the nitrogen atmosphere was replaced three times. The reaction was stirred at 90°C for 18 hours and monitored by LCMS and TLC. After completion of the reaction, the mixture was extracted with ethyl acetate and water. The organic phases were combined, dried over anhydrous sodium sulfate, mixed, and separated and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to give (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.

[0205] 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

[0206] (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), followed by the addition of trifluoroacetic acid (959 mg, 8.41 mmol, 15.0 Equiv.). The reaction was carried out at room temperature and monitored by LCMS and TLC. After the reaction, the pH was adjusted to alkaline with saturated sodium carbonate, the solvent was dried, the sample was mixed, and the product was separated by column chromatography (D:M = 10:1 + 0.1% triethylamine) to obtain (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.

[0207] Step 5: Synthesis of 2-isopropyl-8-[(5S)-5-methylpiperidin-2-yl]-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0208] (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 eq.) was dissolved in methanol (4.0 mL). Sodium borohydride (40 mg, 1.05 mmol, 2.0 eq.) was added portionwise at 0°C. After 5 minutes, the mixture was transferred to room temperature for reaction, monitored by LCMS and TLC. After completion of the reaction, the mixture was spin-dried and directly carried to the next step. LCMS: [M+H] = 313.2378.

[0209] 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

[0210] 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) was dissolved in N,N-dimethylformamide (2.0 mL). Triethylamine (78.8 mg, 0.78 mmol, 1.5 equiv) was added. Ethyl 2-chloro-2-oxoacetate (141 mg, 1.04 mmol, 2.0 equiv) was slowly added under ice-cooling. The mixture was stirred at room temperature for 0.5 hour. LCMS monitoring was used. After completion of the reaction, the mixture was quenched with ethanol, concentrated, and the sample was mixed. The product (85 mg) was isolated by column chromatography (D:M = 10:1 + 0.1% triethylamine). LCMS: [M+H] = 413.2531.

[0211] 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

[0212] 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 eq.) was added lithium hydroxide (48 mg, 2.0 mmol, 10.0 eq.), methanol (4 mL), and water (0.4 mL), and stirred at room temperature. LCMS monitoring was used. After completion of the reaction, the pH was adjusted to acidic with dilute hydrochloric acid. The solvent was evaporated and the reaction proceeded directly to the next step. LCMS: [M+H] = 385.1702.

[0213] 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

[0214] 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.) was dissolved in N,N-dimethylformamide (3.0 mL), followed by the addition of 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.). The mixture was stirred at room temperature for 5 minutes, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114 mg, 0.3 mmol, 1.5 Equiv.), and the reaction was continued at room temperature. After completion of the reaction, the product was separated and purified by HPLC and SFC to give a 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 H NMR(400MHz, DMSO-d6)δ8.07(d,J=27.6Hz,1H),7.52(t,J=13.2Hz,3H),7.29–7.15(m,1H),6.08(s,3H),5.79–5.21(m,3H),4.10(d,J=4.9Hz,2H) ,3.89(s,2H),3.49(dd,J=13.0,4.3Hz,2H),3.06–3.00(m,3H),2.34–1.9 9(m,2H),1.97–1.66(m,2H),1.56–1.34(m,1H),1.10(d,J=25.0Hz,12H).

[0215] 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)

[0216] Step 1: Synthesis of 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0217] 8-Bromo-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (470 mg, 1.87 mmol, 1.0 eq.) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (867 mg, 3.74 mmol, 2.0 eq.) were dissolved in acetonitrile (10 mL). Potassium carbonate (387 mg, 2.8 mmol, 1.5 eq.) was added and the reaction was stirred at 45°C. TLC and LCMS monitoring was performed. After completion of the reaction, the sample was mixed and separated by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford 8-bromo-2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (232 mg) as a yellow solid. LC-MS: [M+H] = 334.0156.

[0218] 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

[0219] 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 eq.) was added 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (354 mg, 1.39 mmol, 2.0 eq.), 1,1-bis(diphenylphosphino)diboron palladium chloride (50.5 mg, 0.069 mmol, 0.1 eq.), potassium acetate (202.8 mg, 2.07 mmol, 3.0 eq.), and 1,4-dioxane (4.0 mL). The atmosphere was replaced with nitrogen three times, and the reaction was refluxed at 100°C for 18 hours. LCMS and TLC monitoring was performed. After the reaction, the solvent was dried, the sample was mixed, and the mixture was separated by column chromatography (D:M = 10:1 + 0.1% triethylamine) to give a brown oily crude product 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 (250 mg). LCMS: [M+H] = 382.1480.

[0220] Step 3: Synthesis of (S)-tert-butyl 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

[0221] 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.), (S)-3-methyl-6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (226 mg, 0.656 mmol, 1.0 Equiv.) g, 0.656mmol, 1.0 Equiv.), 1,1-bis(diphenylphosphino)diphenylferric palladium dichloride (48.3mg, 0.066mmol, 0.1 Equiv.), sodium carbonate (209mg, 1.97mmol, 3.0 Equiv.), water (2.0mL) and 1,4-dioxane (6.0mL), immediately replace nitrogen three times, stir the reaction at 90°C for 18 hours, and monitor by LCMS and TLC. After completion of the reaction, the product was extracted with ethyl acetate and water. The organic phases were combined, dried over anhydrous sodium sulfate, mixed, and purified by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford (S)-tert-butyl 3-methyl-6-(2-(2,2,2-trifluoroethyl)-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]oxazin-8-yl)-3,4-hydropyridine-1(2H)-carboxylate (204 mg) as a brown oil. LCMS: [M-Boc+H] = 351.1422.

[0222] 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

[0223] (S)-tert-Butyl 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.) was dissolved in dichloromethane (4.0 mL), and trifluoroacetic acid (752 mg, 6.6 mmol, 15.0 Equiv.) was added. The reaction was carried out at room temperature and monitored by LCMS and TLC. After the reaction, the pH was adjusted to alkaline with saturated sodium carbonate, the solvent was dried, the sample was mixed, and the product was separated 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.

[0224] 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

[0225] (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.) was dissolved in methanol (4.0 mL). Sodium borohydride (67 mg, 1.77 mmol, 2.5 Equiv.) was added portionwise at 0°C. After 5 minutes, the reaction was transferred to room temperature and monitored by LCMS and TLC. After the reaction, the mixture was dried in spun-drying mode, mixed, and separated by column chromatography (D:M = 10:1 + 0.1% triethylamine) to give 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.

[0226] 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

[0227] 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), and triethylamine (25.3 mg, 0.25 mmol, 1.5 Equiv) was added. In 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 hour. After completion of the reaction, the product was quenched with ethanol, concentrated, mixed, and separated by column chromatography (D:M = 10:1 + 0.1% triethylamine) to afford 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.

[0228] 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

[0229] 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 eq.) was added lithium hydroxide (8.8 mg, 0.37 mmol, 10.0 eq.), methanol (2 mL), and water (0.2 mL), and stirred at room temperature. LCMS monitoring was used. After completion of the reaction, the pH was adjusted to acidic with dilute hydrochloric acid. The solvent was evaporated and the reaction proceeded directly to the next step. LCMS: [M+H] = 425.1618.

[0230] 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

[0231] 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) and 3-ethylpyridine-2,5-diamine was added. (7.7 mg, 0.056 mmol, 1.5 equiv.) and N,N-diisopropylethylamine (13 mg, 0.093 mmol, 2.5 equiv.) were stirred at room temperature for 5 minutes, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.3 mg, 0.056 mmol, 1.5 equiv.) and the reaction was continued at room temperature. LCMS monitoring was performed. After completion of the reaction, Prep-HPLC and SFC separation and purification were performed to obtain a 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 (400MHz, DMSO-d6) δ10.58(d,J=18.8Hz,1H),8.06(d,J=25.9Hz,1H),7. 61–7.44(m,3H),7.22(dd,J=32.4,8.0Hz,1H),5.85–5.21(m,3H),4.14(d,J =23.8Hz,4H),3.60–3.41(m,3H),3.27(s,3H),2.46–2.28(m,3H),2.14(d,J =46.0Hz,1H),1.95–1.65(m,2H),1.38(t,J=15.0Hz,1H),1.19–0.99(m,6H).

[0232] 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)

[0233] Step 1: Synthesis of (S)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate

[0234] To a solution of (S)-3-methylpiperazin-2-one (4.0 g, 35 mmol, 1.0 equiv.) in dichloromethane (30 mL) was added di-tert-butyl dicarbonate (7.7 g, 35 mmol, 1.0 equiv.), and the mixture was allowed to react at room temperature for 16 hours. After the reaction was complete, the sample was stirred and separated by column chromatography to afford tert-butyl (S)-2-methyl-3-oxopiperazine-1-carboxylate (7.12 g).

[0235] Step 2: Synthesis of (S)-tert-butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate

[0236] (S)-tert-Butyl 2-methyl-3-oxopiperazine-1-carboxylate (3.5 g, 16 mmol, 1.0 eq.) was weighed and the system was purged with nitrogen three times. N,N-dimethylformamide solution (30 mL) was added, and sodium hydroxide solution (770 mg, 33 mmol, 2.0 equiv.) was added portionwise under an ice bath at 0°C. After stirring for 30 minutes, a solution of 4-bromo-1-fluoro-2-nitrobenzene (4.0 g, 18 mmol, 1.1 equiv.) in N,N-dimethylformamide (8 mL) was slowly added dropwise. The reaction was stirred at room temperature for 3 hours. After completion of the reaction, the reaction was quenched with ammonium chloride solution, followed by extraction with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography afforded (S)-tert-Butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.9 g). LCMS: m / z = [M-Boc+H] 314.1.

[0237] Step 3: Synthesis of tert-butyl (S)-8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0238] (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 reacted at 100°C for 1.5 hours. After the reaction was complete, the filtrate was filtered through celite and concentrated by column chromatography to afford 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.

[0239] Step 4: Synthesis of (S)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0240] (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.) was weighed and dissolved in trifluoroacetic acid (3 mL) and dichloromethane (10 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed and the pH was adjusted to 8-9 with saturated sodium carbonate solution. Ethyl acetate was added for extraction. The organic phase was dried to give the crude product (S)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1 g), which was used directly in the next step. LCMS: m / z = [M-Boc+H] 266.0.

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

[0242] To (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.) was added 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). The mixture was allowed to react at room temperature for 16 hours. After completion of the reaction, the solvent was evaporated, the sample was mixed, and column chromatography was performed 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.

[0243] 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

[0244] 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 (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]palladium dichloride (286 mg, 0.4 mmol, 0.1 eq) were added to the system. The system was purged with nitrogen three times and the temperature was raised to 100°C for 6 hours. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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) as a white solid. LCMS: m / z = [M+H] 342.2.

[0245] 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

[0246] 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 tert-butyl (S)-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 the temperature was raised to 90°C for 6 hours. After the reaction was complete, the mixture was diluted with water, extracted with ethyl acetate, and 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)-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)-carboxylic acid tert-butyl ester (1.2 g). LCMS: m / z = [M+H]-Boc 411.2.

[0247] 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

[0248] (S)-tert-Butyl 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) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (10 mL) was added, and the reaction was stirred for 30 minutes and monitored by TLC and LCMS. After the reaction, the solvent was dried by rotary evaporation, saturated sodium carbonate solution was added to adjust the pH to 8-9, the mixture was diluted with water, and the mixture was extracted with ethyl acetate and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was then dried by rotary evaporation 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.

[0249] 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

[0250] (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.) was dissolved in methanol (10 mL). Sodium borohydride (233 mg, 6.1 mmol, 2.0 equiv.) was added portionwise in an ice bath. The mixture was stirred for 30 minutes and the reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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.

[0251] 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

[0252] (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 the reaction flask. Under nitrogen protection, triethylamine (71 mg, 0.7 mmol, 1.5 equiv.) was added. Under ice bath conditions, ethyl oxalyl chloride (97 mg, 0.7 mmol, 1.5 equiv.) was slowly added dropwise. The reaction was stirred at room temperature and monitored by TLC and LCMS. After the reaction, the solvent was dried, the sample was stirred, and then 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]pyrazin-8-yl]-5-methylpiperidin-1-yl}-2-oxoacetate (215 mg). LCMS: m / z = [M+H] 413.2.

[0253] Step 11: Synthesis of 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

[0254] 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 (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 mixture and allowed to react at room temperature for 2 hours. After completion of the reaction, the pH was adjusted to 5-6 with 1 M aqueous hydrogen chloride solution and the mixture was dried in a rotary evaporation chamber to obtain the crude product 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 (200 mg). LCMS: m / z = [M+H] 385.2.

[0255] 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

[0256] 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-vinylpyridin-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) and stirred for 5 minutes. 2-(7-Azabenzotriazole)-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 hours. After completion of the reaction, the product was separated and purified by HPLC preparative chromatography and SFC to give 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.

[0257] 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)

[0258] Step 1: Synthesis of (R)-tert-butyl 2-methyl-3-oxopiperazine-1-carboxylate

[0259] To a solution of (S)-3-methylpiperazin-2-one (4.0 g, 35 mmol, 1.0 equiv.) in dichloromethane (30 mL) was added di-tert-butyl dicarbonate (7.7 g, 35 mmol, 1.0 equiv.), and the mixture was allowed to react at room temperature for 16 hours. After the reaction was complete, the sample was stirred and separated by column chromatography to afford tert-butyl (R)-2-methyl-3-oxopiperazine-1-carboxylate (6.84 g).

[0260] Step 2: Synthesis of tert-butyl (R)-4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate

[0261] (S)-tert-Butyl 2-methyl-3-oxopiperazine-1-carboxylate (3.50 g, 16.34 mmol, 1.0 eq.) was weighed and the system was purged with nitrogen three times. N,N-dimethylformamide solution (30 mL) was added, and sodium hydroxide solution (770 mg, 33 mmol, 2.0 equiv.) was added portionwise under an ice bath at 0°C. After stirring for 30 minutes, a solution of 4-bromo-1-fluoro-2-nitrobenzene (4.0 g, 18 mmol, 1.1 equiv.) in N,N-dimethylformamide (8 mL) was slowly added dropwise. The reaction was stirred at room temperature for 3 hours. After completion of the reaction, the reaction was quenched with ammonium chloride solution, followed by extraction with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate and concentrated. Column chromatography afforded (R)-tert-Butyl 4-(4-bromo-2-nitrophenyl)-2-methyl-3-oxopiperazine-1-carboxylate (3.96 g). LCMS: m / z = [M-Boc+H] 314.1.

[0262] Step 3: Synthesis of tert-butyl (R)-8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate

[0263] (R)-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 reacted at 100°C for 1.5 hours. After the reaction was complete, the filtrate was filtered through celite and concentrated by column chromatography to afford yellow (R)-tert-butyl 8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylate (2.56 g). LCMS: m / z = [M-Boc+H] 266.0.

[0264] Step 4: Synthesis of (R)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0265] (R)-8-bromo-1-methyl-3,4-dihydrobenzo[4,5]imidazo[1,2-a]pyrazine-2(1H)-carboxylic acid tert-butyl ester (2.5 g, 6.85 mmol, 1.0 equiv.) was weighed and dissolved in trifluoroacetic acid (3 mL) and dichloromethane (10 mL). The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed by concentration. The pH was adjusted to 8-9 by adding saturated sodium carbonate solution. Ethyl acetate was added for extraction, and the organic phase was dried to give the crude product (R)-8-bromo-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine (1.09 g), which was used directly in the next step. LCMS: m / z = [M-Boc+H] 266.0.

[0266] Step 5: Synthesis of (R)-8-bromo-2-ethyl-1-methyl-1,2,3,4-tetrahydrobenzo[4,5]imidazo[1,2-a]pyrazine

[0267] To (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.) was added 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). The mixture was allowed to react at room temperature for 16 hours. After completion of the reaction, the solvent was evaporated, the sample was mixed, and column chromatography was performed 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.

[0268] 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

[0269] 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) was dissolved in N,N-dimethylformamide (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]palladium dichloride (286 mg, 0.4 mmol, 0.1 eq) were added to the system. The system was purged with nitrogen three times and the temperature was raised to 100°C for 6 hours. After the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The mixture was washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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) as a white solid. LCMS: m / z = [M+H] 342.2.

[0270] 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

[0271] 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 tert-butyl (S)-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 the temperature was raised to 90°C for 6 hours. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and 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)-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)-carboxylic acid tert-butyl ester (1.27 g). LCMS: m / z = [M+H]-Boc 411.2.

[0272] 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

[0273] (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 the reaction was stirred for 30 minutes and monitored by TLC and LCMS. After the reaction, the solvent was dried by rotary evaporation, saturated sodium carbonate solution was added to adjust the pH to 8-9, the mixture was diluted with water, and the mixture was extracted with ethyl acetate and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was then dried by rotary evaporation 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.

[0274] 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

[0275] (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.) was dissolved in methanol (10 mL). Sodium borohydride (233 mg, 6.1 mmol, 2.0 equiv.) was added portionwise in an ice bath. The mixture was stirred for 30 minutes and the reaction was monitored by TLC and LCMS. After completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, and washed with saturated sodium chloride. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and separated by column chromatography to afford (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.

[0276] 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

[0277] (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.) was added to the reaction flask. Under nitrogen protection, triethylamine (71 mg, 0.7 mmol, 1.5 equiv.) was added. Under ice bath conditions, ethyl oxalyl chloride (97 mg, 0.7 mmol, 1.5 equiv.) was slowly added dropwise. The reaction was stirred at room temperature and monitored by TLC and LCMS. After the reaction, the solvent was dried, the sample was stirred, and then separated 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.

[0278] 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

[0279] 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 mixture and allowed to react at room temperature for 2 hours. After completion of the reaction, the pH was adjusted to 5-6 with 1 M aqueous hydrogen chloride solution and the mixture was dried in a rotary evaporation chamber 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.

[0280] 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

[0281] 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-vinylpyridin-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) and stirred for 5 minutes. 2-(7-azabenzotriazole)-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 hours. After completion of the reaction, the product was separated and purified by HPLC preparative chromatography and SFC to give 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). LCMS: m / z = [M+H] 504.3.

[0282] The compounds listed in Table 1 below were prepared by methods similar to those described in the Examples, with appropriate variations in reactants, amounts of reagents, protection and deprotection, solvents, and reaction conditions. Characterization data for the compounds are summarized in Table 1 below.

[0283] Table 1: Structure and characterization of some compounds

[0284] Experimental Example 1: Binding ability test of the compound of the present invention to PRMT5 protein

[0285] Experimental purpose: To detect the binding ability of compounds to PRMT5 protein using CETSA experimental method

[0286] Background and Principle: The CETSA assay is a molecular assay that measures the affinity of a drug for its target protein. The principle is that drug binding to the target protein stabilizes its structure. When a candidate drug is used to treat a cell or tissue sample, if it is a PRMT5 inhibitor, it will bind to PRMT5, stabilizing the PRMT5 protein. After heating the sample, the PRMT5 protein in the sample will be more easily detected by specific antibodies and by Western blot. Conversely, heating will destabilize the PRMT5 protein, resulting in a lower amount of protein detected. This method can thus assess the binding ability of the drug to the target protein and is used to screen for PRMT5 inhibitors.

[0287] Experimental Procedure: HCT116 cells in logarithmic growth phase (cell viability >90%) were washed three times with PBS and centrifuged at 2000g for 2 minutes. Cells were lysed on ice for 30 minutes using cell lysis buffer containing the protease inhibitor PMSF. Protein concentrations were determined using a BCA assay. Samples were incubated with the candidate drug, control drug, and control reagent for 30 minutes. Each sample was heated at approximately 10 pre-set temperature points. After returning to room temperature, the samples were centrifuged at 20,000g and the supernatant was collected. Protein samples were denatured by heating at 100°C for 10 minutes using sample buffer. After returning to room temperature, samples were analyzed by western blot. The protein loading was controlled at 20 μg. After determining the transition temperature, a compound concentration gradient was established, typically 9 points, and samples were incubated. Western blot analysis was performed as above. Protein electrophoresis was performed with the stacking gel voltage set to 60V and the separating gel voltage set to 120V. After electrophoresis, electrotransfer was initiated. Electroporation conditions were set at 250 mA for 2 h; 5% BSA blocking was performed for 1 h; specific primary antibody was added and incubated overnight on a shaker at 4°C; TBST was washed four times for 2.5 min each; secondary antibody was incubated on a shaker at room temperature for 1 h; TBST was washed four times for 2.5 min each; ECL was used for development to detect PRMT5 protein expression in different groups and at various temperature points. Western blot bands were converted and processed using Image J and GraphPad software, and EC values ​​were calculated. 50 value.

[0288] EC50 is the half-maximal effect concentration, which refers to the drug concentration that causes 50% of individuals to be effective. PRMT5 inhibitors TNG908 and TNG462 were used as positive reference compounds, and the preparation method was based on the examples in patent document WO2022026892.

[0289] Experimental Example 2: Determination of the effect of the compounds of the present invention on the proliferation of HCT116 MTAP- / - cells

[0290] Experimental purpose: The purpose of this test example is to test the effect of compounds on the proliferation of HCT116 MTAP- / - cells.

[0291] Background and Principle: Protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins and plays a crucial role in biological processes such as gene expression, splicing, and DNA damage repair. Methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with the common tumor suppressor gene CDKN2A, with this co-deletion occurring in up to 9% to 15% of tumors. Studies have shown that inhibiting PRMT5 is synthetically lethal in MTAP-deficient tumors. Therefore, compounds can be screened for PRMT5 inhibitors by measuring their inhibitory effect on the proliferation of HCT116MTAP- / - cells.

[0292] Specific experimental process:

[0293] Construct HCT116 MTAP knockout cells and screen single clones. - / - WT cells were seeded into 96-well plates, with 90 μL per well (1,000 cells / well) and incubated overnight at 37°C. The next day, 10 μL of compound at various concentrations was added, with a maximum concentration of 100 μM. Nine 10-fold dilutions were performed (final DMSO concentration was 1%), and the cells were incubated at 37°C for 7 days. On day 7, the old medium was aspirated and 110 μL of medium (at a 100:10 ratio of medium to CCK8) was added. The cells were incubated at 37°C for 1-4 hours. The absorbance was measured at 450 nM, and the IC50 value was calculated using GraphPad software. Compounds were screened by comparing the results with those of active drugs.

[0294] IC50 (half maximal inhibitory concentration) refers to the half-maximal inhibitory concentration of the antagonist being measured. It indicates the half-maximum inhibitory concentration of a drug or substance (inhibitor) in inhibiting a biological process (or substances involved in that process, 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:

[0295] Table 2

[0296] The test results show that the representative compounds of the present invention have an effect on HCT116 MTAP - / - The cells have good inhibitory effect, which is comparable to or better than the positive control, while the inhibitory effect on HCT116 WT cells is weak, with excellent selectivity.

[0297] Experimental Example 3: Testing of the Inhibitory Effect of the Compounds of the Invention on PRMT5 Enzyme Function

[0298] Experimental purpose: To detect the inhibitory effect of compounds on PRMT5 enzyme function using the MTase-Glo method

[0299] Background principle: The gene that constitutes the synthetic lethality of PRMT5 is MTAP (methylthioadenosine phosphorylase). MTAP participates in the metabolism of 2-methylthioadenosine (MTA) and regenerates the methionine required for the synthesis of SAM. The loss of the MTAP gene causes the accumulation of MTA in cells, and MTA competes with the substrate of PRMT5, S-adenosyl-L-methionine (SAM), resulting in a decrease in PRMT5 activity. Based on the MTase-Glo method, the methyltransferase (PRMT5) is used to provide a methyl group to the substrate by transferring SAM (S-adenosylmethionine) 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.

[0300] Specific experimental process:

[0301] The MTase-Glo methyltransferase fluorescence assay monitors 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 performed 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. Test compounds and positive controls were prepared using a 10-fold dilution series of nine concentrations in triplicate, with a maximum concentration of 100 μM. Compounds were incubated in the absence of MTA with a reaction mixture containing 1 nM recombinant PRMT5:MEP50 enzyme complex, 2.5 μM H41-21 histone peptide, and 2.5 μM S-adenosylmethionine (SAM). Similarly, in the presence of MTA, the compounds were incubated with a mixture consisting 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 hours, the reaction was terminated by the addition of TFA to each well. 5X MTase-Glo™ reagent was then added to each well and incubated at room temperature to convert the SAH produced in the reaction to ADP. After 10 minutes, MTase-Glo detection solution was added to each well and incubated at room temperature for 30 minutes before detection using a microplate reader.

[0302] The IC50 (half maximal inhibitory concentration) is the half-maximal inhibitory concentration of the antagonist being measured. It indicates the half-maximum inhibitory concentration of a drug or substance (inhibitor) in inhibiting a biological process (or substances involved in that process, such as enzymes, cell receptors, or microorganisms). Using the aforementioned compounds TNG908 and MRTX1719 as positive reference compounds, the test results are shown in Table 3:

[0303] Table 3

[0304] The results showed that the compounds of the present invention have very strong inhibitory effects on PRMT5 protease function, with IC50 values ​​reaching the nM level, equivalent to or lower than those of positive drugs. This strong inhibitory effect has important therapeutic significance for the treatment of conditions or diseases related to PRMT5 inhibition.

[0305] Experimental Example 4: In vitro metabolic stability determination of the compounds of the present invention

[0306] 1. Experimental Principle: Liver microsomes contain most phase I enzymes, the most important of which is the microsomal mixed function oxidase system with CYP450 as the main component. When conducting research with liver microsomes, adding the corresponding cofactor NADPH can reconstitute the in vitro metabolic system, thereby conducting phase I metabolic stability studies through in vitro incubation.

[0307] 2. Experimental methods:

[0308] 2.1 Melt the components of the kit in an ice bath and place on ice until ready to use;

[0309] 2.2 Prepare pre-incubation solution A (1X): 10 μl of solution A, 2 μl of solution B, and 28 μl of 0.1M PBS buffer. Mix well, incubate at 37°C for 5 minutes, and aliquot 40 μl into each tube for use.

[0310] 2.3 Prepare pre-incubation solution B (1X), 154 μl of 0.1 M PBS buffer, 1 μl of test substance (or positive substrate), and 5 μl of liver microsomes, and mix well.

[0311] 2.4 Add 160 μl of Pre-incubation Solution B to the centrifuge tube containing 40 μl of Pre-incubation Solution A, pipette three times to mix thoroughly, and immediately incubate at 37°C for the desired time.

[0312] 2.5 At the set time point, pre-cooled acetonitrile was added to terminate the reaction, and the mixture was centrifuged at 20,000 rpm and 4°C for 10 min. The supernatant was collected for detection.

[0313] 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 or solution B; 3) Blank control group: only contains substrate and PBS buffer.

[0314] Experimental Example 5: In vitro permeability Caco-2 test of the compounds of the present invention

[0315] 1. Caco-2 assay method

[0316] 1.1 Caco-2 cell culture:

[0317] (1) Add 50 μL and 25 mL of cell culture medium to each well of the Transwell insert and reservoir, respectively.

[0318] (2) Before cell inoculation, pre-incubate the Transwell plate at 37°C, 5% CO2 for 1 hour.

[0319] (3) 50 μL of cell suspension (2х10 5 cells / mL) was inoculated into the insert and cultured at 37° C., 5% CO 2 , and 95% relative humidity for 21-28 days.

[0320] (4) Change the cell culture medium every other day for 7 days, and change it every day after 7 days.

[0321] (5) Measure the transepithelial electrical resistance (TEER) of the monolayer using EVOM3.

[0322] 1.2 ABBA experimental steps:

[0323] (1) Wash the Transwell plate twice with preheated HBSS (10 mM HEPES, pH 7.4) and then incubate at 37°C for 30 minutes.

[0324] (2) Test compounds were prepared at 1 mM in DMSO and diluted 200-fold to 5 μM with HBSS (10 mM HEPES, pH 7.4).

[0325] (3) Apical to basolateral direction: add 75 μL of 5 μM working solution (10 mM HEPES, pH 7.4) to the apical chamber and add 235 μL of HBSS (10 mM HEPES, pH 7.4) to the basolateral chamber.

[0326] (4) Basolateral to apical direction: add 235 μL of 5 μM working solution (10 mM HEPES, pH 7.4) to the basolateral chamber, and add 75 μL of HBSS (10 mM HEPES, pH 7.4) to the apical chamber.

[0327] (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).

[0328] (6) Incubate the cell plate at 37°C, 5% CO2, and 95% relative humidity for 2 hours.

[0329] (7) After 2 hours of incubation, 50 μL of solution was transferred from the apical and basolateral chambers to a sample plate containing 200 μL of cold methanol and IS*.

[0330] (8) Centrifuge the sample plate at 3220 g for 40 minutes.

[0331] (9) Transfer 100 μL of the supernatant to an assay plate containing an appropriate volume of H 2 O for LC-MS / MS analysis.

[0332] 1.3 Lucifer Yellow Fluorescent Penetrant Testing

[0333] (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 basolateral chamber.

[0334] (2) Incubate the cell plate at 37°C, 5% CO2, and 95% relative humidity for 0.5 hours.

[0335] (3) 100 μL was transferred from the apical chamber and the basolateral chamber to the plate. After incubation for 0.5 h, the excitation wavelength value of 427 nM and the emission wavelength value of 536 nM of the Lucifer Yellow permeabilization detector were read.

[0336] 1.4 Data Analysis

[0337] (1) The apparent permeability coefficient (Papp, cm / s) and efflux ratio were calculated using Microsoft Excel.

[0338] Vacceptor=0.235mL for A→B,and 0.075mL for B→A

[0339] Vdonor=0.075mL for A→B,and 0.235mL for B→A

[0340] Area=the surface area of ​​the membrane,cm2(0.143for Corning 3391)

[0341] Time=the total transport time,s

[0342] (2) Calculate the Lucifer Yellow fluorescence penetration using the following formula:

[0343] I=the fluorescence intensity

[0344] Vacceptor=0.3mL and Vdonor=0.1mL for Lucifer Yellow leakage%test

[0345] *IS:50nM alprazolam,50nM labetalol and 100nM ketoprofen

[0346] Experimental Example 6: In vivo pharmacokinetic study of the compounds of the present invention

[0347] 1. Experimental Purpose: After a single intravenous injection or oral administration of different compounds to ICR mice, blood samples were collected at different time points. The concentration of the test compound in mouse plasma after administration was determined by LC-MS / MS and related parameters were calculated.

[0348] 2. Experimental plan:

[0349] 2.1 Experimental animals: ICR male mice, SPF grade

[0350] 2.2 Experimental Design

[0351] Note: *, animals were fasted overnight (10-14 hours) before administration and fed 4 hours after administration.

[0352] 2.3 Blood collection time

[0353] IV administration: 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after administration. PO administration: 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and 24 hours after administration. Blood was collected from the jugular vein, approximately 0.2 mL per sample, anticoagulated with K2-EDTA, and placed on ice.

[0354] 2.4 Plasma Sample Processing: After blood collection, place the blood samples on ice and centrifuge them within 1 hour to separate the plasma (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C freezer before analysis.

[0355] 2.5 Analysis of results: Phoenix WinNonlin7.0 was used to calculate the pharmacokinetic parameters using the plasma drug concentration data at different time points, providing parameters such as AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2, as well as their mean and standard deviations.

[0356] The results showed that the compound of the present invention exhibited good pharmacokinetic exposure in the mouse pharmacokinetic test, which was better than the positive control.

Claims

1. A compound represented by 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 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; 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 compounds are 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 claims 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 its pharmaceutically acceptable salts, characterized in that, R5 and R6 are each independently selected from H, methyl, trifluoromethyl, fluorine, methoxy, -CN.

10. The compound according to any one of claims 1-9, its stereoisomers, or pharmaceutically acceptable salts thereof, 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 and its stereoisomers or its pharmaceutically acceptable salts, and one or more pharmaceutically acceptable carriers, diluents or excipients.

13. Use of the compound according to any one of claims 1 to 11 and its stereoisomers, or its pharmaceutically acceptable salts or the pharmaceutical composition according to claim 12 in the manufacture of a medicament for the treatment of a disease, disorder, syndrome or affliction associated with PRMT5.

14. The use of a drug according to claim 13, wherein the disease, disorder, syndrome or affliction related to 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, and the disease, disorder, syndrome or affliction related to the PRMT5 enzyme is preferably cancer, and 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, hematological 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 of formula (I) as defined in claim 1, and its stereoisomers or pharmaceutically acceptable salts thereof, the method comprising the steps: The compound of general formula (Ia) or its isomer or salt undergoes a condensation reaction with the compound of general formula (Ib) or its isomer or salt to obtain the compound of formula (Ⅰ) and its stereoisomers or its pharmaceutically acceptable salts. The definitions of the respective groups are as defined in claim 1.

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

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

18. A compound, its stereoisomer or its pharmaceutically acceptable salt, which is selected from any of the following compounds:

19. A compound, its stereoisomer or its pharmaceutically acceptable salt, which is selected from any one of the following compounds:

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

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