PRMT5 inhibitor and application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a PRMT5 inhibitor compound and its isomers, or pharmaceutically acceptable salts thereof, and 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 is a type of histone methylation and one of the most common post-translational modifications in mammals. It is mainly regulated by the PRMT gene family. Arginine methyltransferases (PRMTs) regulate arginine methylation in three different forms: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) methylation. There are 9 PRMTs in mammals, which are divided into three types: type I (PRMT1, 2, 3, 4, 6, 8; mainly catalyze the production of MMA and ADMA), type II (PRMT5, 9; mainly catalyze the production of MMA and SDMA) and type III (PRMT7; mainly catalyze the production of MMA). PRMTs can methylate histones and a variety of non-histone proteins. Studies have shown that changes in PRMTs enzyme activity, gene mutations or deletions usually lead to abnormal development of animal individuals, and also induce the occurrence, development or metastasis of cancer. Among the many PRMTs, the role of PRMT5 in cancer is increasingly confirmed and has received the most attention.

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

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

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

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

[0007] The results of most clinical trials of first-generation PRMT5 inhibitors that have entered clinical trials are generally not ideal: clinical studies have shown that GSK-3326595 responds to a variety of tumor types. However, 89% of participants reported adverse events, including anemia, thrombocytopenia, neutropenia, and fatigue. A Phase I clinical trial of JNJ-64619178 showed that intermittent dosing had a maintenance target inhibition effect on tumor patients. In contrast, JNJ-64619178 showed limited / no efficacy in patients with myelodysplastic syndrome. Most patients treated with JNJ-64619178 experienced adverse events, including thrombocytopenia, anemia, and nausea. PF-06939999, as a PRMT5 inhibitor, had objective tumor responses in patients with squamous cell carcinoma of the neck and non-small cell lung cancer. Cytoreductions were observed in clinical trials, and they were dose-dependent and reversible with dose modifications. The clinical trials of the above-mentioned PRMT5 inhibitors appear to have been stopped.

[0008] In fact, with the exception of Prelude Therapeutics' project, most active projects are dedicated to second-generation PRMT5 inhibitors, namely PRMT5-MTA inhibitors. In cancer cells lacking MTAP, MTA replaces SAM to bind to PRMT5, forming an inactive PRMT5-MTA complex. Second-generation PRMT5 inhibitors bind to the PRMT5-MTA complex and kill cancer cells lacking MTAP 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 Phase I / II clinical research stage include MRTX-1719, SKL-27969, TNG-908, TNG-462 and AMG-193, but no drugs are still on the market; WO2022132914, WO2022115377 and other documents disclose PRMT5 inhibitors, but their selectivity, metabolic stability, permeability and absorbability are poor. Therefore, it is still necessary to develop more effective, active, selective, metabolically stable, permeable, fast-absorbing and pharmacokinetics-friendly PRMT5 inhibitors. Summary of the invention SUMMARY OF THE INVENTION

[0011] The purpose of the present invention is to provide a novel PRMT5 inhibitor compound with good activity, good selectivity, good metabolic stability, good permeability, fast pharmacokinetic absorption, 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 isomers, or a pharmaceutically acceptable salt thereof:

[0014]

[0015] in,

[0016] R1 is selected from -(CH2) n -R4, -CH(CH3)-R4;

[0017] R2 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may optionally be further substituted, -CH(CH3)-R3;

[0018] R1 and R2 are different;

[0019] The R3 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 The haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups may optionally be further substituted;

[0020] The R4 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 The haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups may optionally be further substituted;

[0021] n = 0, 1, 2, 3, 4;

[0022] Or R1, R2 and the connected N form a saturated or unsaturated five-membered, six-membered or seven-membered heterocyclic ring, and contain 1 to 3 heteroatoms selected from N, O, S, wherein the S atom is optionally substituted by one or two oxo groups, and the five-membered, six-membered or seven-membered heterocyclic ring is substituted or spiro-connected with one or more R6;

[0023] The R6 is selected from C 1-6 Alkyl, halogen, C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, C 3-6 Cycloalkyl, -C(O)C 1-6 Alkyl, -C(O)C 1-6 Haloalkyl, -C(O)OC 1-6 Alkyl, -C(O)OC 1-6Haloalkyl, a saturated or partially saturated or aromatic 5-10 membered heterocyclic ring containing 1 to 3 heteroatoms selected from N, O, S, which may be optionally substituted by one or more Ra;

[0024] The Ra is selected from hydrogen, deuterium, nitrogen methyl bridged cycloheptyl, C 1-6 Alkyl, C 1-6 Alkoxy, halogen, amino, thiol, nitro, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, cyano, hydroxy, hydroxyalkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 3-6 Cycloalkyl, C 3-6 Halogenated cycloalkyl, C 3-6 Halogenated heterocycloalkyl, C 1-6 Alkylamino, formyl and optionally substituted with R b -(CH2) m -heterocycloalkyl, -(CH2) m - heterocycloalkenyl, aryl, heteroaryl, heterocyclyl;

[0025] The R b For halogen, CN, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 1-6 Haloalkyl, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy, formyl, methylamino, oxetanyl;

[0026] m = 0, 1, 2, 3, 4;

[0027] R is selected from the following structures:

[0028]

[0029] X1 is selected from N or CR7, and R7 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl;

[0030] R5 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl;

[0031] R8 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl.

[0032] As a preferred technical solution, the R8 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, isopropyl, deuterated methyl, halomethyl, and methoxy; further preferably, the R8 is selected from hydrogen, methyl, ethyl, isopropyl, and deuterated methyl.

[0033] As a preferred technical solution, the R is selected from the following structures:

[0034]

[0035] wherein R5 is as defined above.

[0036] As a preferred technical solution, the R5 is selected from hydrogen, hydroxyl, cyano, C 1-6 Alkyl, halogen, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 deuterated alkyl; further preferably, R5 is selected from hydrogen, hydroxy, cyano, methoxy, methyl, ethyl, bromine, chlorine, fluorine, deuterated methyl, -CH2F, -CHF2, -CF3, -CH2Cl, -CHCl2, -CCl3, -CD3, cyclopropyl; more preferably, R5 is selected from hydrogen, chlorine, fluorine, methyl.

[0037] As a preferred technical solution, wherein R2 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, C1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 3-6 Cycloalkyl, -CH(CH3)-six-membered heterocyclyl, -CH(CH3)-six-membered heteroaryl; further preferably, R2 is selected from methyl, isopropyl, cyclopropyl, -CH(CH3)-six-membered heteroaryl containing 1 to 3 N, O, S heteroatoms.

[0038] As a preferred technical solution, R4 is selected from any substituted 5-10 membered heteroaryl; further preferably, R4 is selected from unsubstituted, halogen, C 1-6 Alkyl, C 1-6 Alkoxy-substituted benzopyran ring, benzofuran ring, benzothiazole ring, benzopyrazole ring, benzopyridine ring, benzopyrrole ring, benzopyrimidine ring, benzothiophene ring, benzoxazole ring, pyridothiazole ring, pyridothiphene ring, pyridopyrrole ring; more preferably, R4 is selected from Most preferably, R4 is selected from

[0039]

[0040] As a preferred technical solution, the ring formed by R1, R2 and the connected N is selected from pyrrole ring, piperidine ring, methyl piperidine ring, imidazole ring, pyrazole ring, morpholine ring, azepane, azetidine, azepane, azocyclohexane, azopentane substituted or spiro-coupled with one or more R6; further preferably, the ring formed by R1, R2 and the connected N is selected from

[0041]

[0042] As a preferred technical solution, the R6 is selected from methyl, ethyl, halogen, C 1-6 haloalkyl, methoxy, halomethoxy, -C(O)methyl, cyclopropyl, a saturated or partially saturated or aromatic 9-membered heterocycle containing 1 to 3 heteroatoms selected from N, O, S, which may be optionally substituted by one or more Ra; further preferably, R6 is selected from methyl, cyclopropyl, halogen and

[0043] As a preferred technical solution, the R a is selected from hydrogen, deuterium, nitrogen, methyl, methoxy, halogen, amino, mercapto, nitro, alkenyl, alkynyl, deuterated methyl, cyano, hydroxy, cyclopropyl, methylamino, formyl, trifluoromethyl, difluoromethyl and optionally substituted with R b -(CH2) m -heterocycloalkyl, aryl, heteroaryl, heterocyclic group, said R b It is methyl, cyclopropyl, formyl, cyclobutyl, and oxetane.

[0044] As a preferred technical solution, the aryl, heteroaryl and heterocyclic group are selected from piperidinyl, piperazinyl, pyranyl, morpholinyl, pyrazolyl, pyridinyl, pyrazinyl, furanyl, pyrimidinyl, thiazolyl and oxazolyl; further preferably, the aryl, heteroaryl and heterocyclic group are selected from piperidinyl, piperazinyl, pyranyl and morpholinyl.

[0045] As a preferred technical solution, the compound is selected from:

[0046]

[0047] The present invention also provides a compound selected from the following formula or a pharmaceutically acceptable salt or isomer thereof:

[0048]

[0049]

[0050] The present invention also provides a compound selected from the following formula or a pharmaceutically acceptable salt or isomer thereof:

[0051]

[0052] The present invention also provides use of any of the above compounds and isomers thereof, or pharmaceutically acceptable salts thereof, in the preparation of a medicament for treating a disease, condition, syndrome or disorder associated with PRMT5.

[0053] 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, blood cancers, melanoma, pancreatic tumors, brain tumors, glioblastoma, glioma, myelofibrosis, breast tumors, 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.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present invention uses computer-aided design technology to modify TNG908 and other analogs, and screens a variety of novel structural compounds through computer simulation, molecular docking analysis, drug structure-activity relationship research, etc. This series of compounds can bind to the PRMT5-MTA complex and inhibit the function of PRMT5, and has extremely strong PRMT5 inhibitory activity. In vivo and in vitro experiments further show that this series of compounds can prevent and / or treat cancer, blood diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, hereditary diseases, hormone-related diseases, immunodeficiency diseases, diseases related to cell death, destructive bone diseases, thrombin-induced platelet aggregation, liver diseases and cardiovascular diseases, and have good activity, good selectivity, good metabolic stability, good permeability, and fast absorption. DETAILED DESCRIPTION OF THE INVENTION

[0057] Various aspects and features of the present invention are further described below.

[0058] 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 known meanings, the meanings described in the present invention shall prevail. Below are the definitions of various terms used in the present invention, which apply to the terms used in the entire specification of this application, unless otherwise specified in specific circumstances.

[0059] The compounds according to the invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

[0060] The compounds of the present invention have asymmetric centers. The compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art know how to prepare optically active forms, such as by racemic separation or synthesis from optically active starting materials. Unless the specific stereochemistry or isomeric form is specifically stated, the present invention includes all chiral, diastereoisomers and racemates. The methods for preparing the compounds of the present invention and the intermediates thereof are part of the present invention. All tautomers of the compounds of the present invention are also part of the present invention.

[0061] The term "substituted" means that any one or more hydrogen atoms on a specified group are replaced with a substituent as long as the valence of the specified atom is normal and the resulting compound after the substitution is stable.

[0062] The terms "alkoxy" and "alkylamino" are customary expressions and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or an amino group, respectively, wherein the alkyl group is as described herein.

[0063] As used herein, the terms "halogen", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine and bromine, with fluorine and chlorine being particularly preferred.

[0064] As described herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include its sub-groups. For example, when referring to "C1-C6 alkyl", it may also include sub-range groups represented by C1-C4 alkyl, C1-C3 alkyl, C2-C6 alkyl, C2-C4 alkyl, etc., as well as specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[0065] The terms "heterocycle", "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, which have at least one heteroatom (O, S or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of this heteroatom-containing group can contain one or two oxygen or sulfur atoms or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. Nitrogen and sulfur atoms may be optionally oxidized, and nitrogen atoms 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 completely unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom. As used herein, the terms "heterocycloalkyl," "heterocyclo," and "heterocyclyl" include "heteroaryl" groups, as defined below.

[0066] 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.

[0067] The term "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 9- or 10-membered bicyclic groups, and 11- to 14-membered tricyclic groups, which have at least one heteroatom (O, S or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of the heteroaryl containing heteroatoms can contain one or two oxygen or sulfur atoms or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated or unsaturated. Nitrogen and sulfur atoms can be optionally oxidized, and nitrogen atoms can be optionally quaternized. As a bicyclic or tricyclic heteroaryl, it must include at least one completely aromatic ring, but the other one or more fused rings can be aromatic or non-aromatic. Heteroaryl can be attached to any available nitrogen or carbon atom of any ring. If the other ring is cycloalkyl or heterocyclic it is additionally optionally substituted with =0 (oxo) where valence permits.

[0068] Unless otherwise indicated, when referring to a specifically named aryl (e.g., phenyl), heterocyclyl (e.g., pyrrolidinyl, piperidinyl, and morpholinyl), or heteroaryl (e.g., tetrazolyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, and furanyl) group, the reference is intended to include rings optionally having 0 to 3, preferably 0 to 2, substituents selected from the substituents listed above for arylheterocyclyl and / or heteroaryl groups.

[0069] As described herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate when performing chiral separation. Although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention.

[0070] As described herein, the term "disease" refers to a physical state of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegeneration-related diseases described in the present invention.

[0071] The cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy and hormone therapy.

[0072] "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 via 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 cancer and hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine 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 lymphocytic 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 plasma cell tumor, lymphoma, pancreatic ductal adenocarcinoma.

[0073] The compounds of the present invention or pharmaceutical compositions containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0074] 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 types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0075] In order 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 administration method.

[0076] 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

[0077] 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.

[0078] For all 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 ° C (Celsius). The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectroscopy (MS).

[0079] The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or / and liquid-mass spectrometry (LC-MS). The NMR chemical shift (δ) is in parts per million (ppm). The nuclear magnetic resonance is measured using a Bruker avance-400 nuclear magnetic spectrometer, the solvents are deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD) and deuterated chloroform (CDCl3), and the internal standard is tetramethylsilane (TMS).

[0080] Liquid phase mass spectrometry LC-MS measurement The liquid phase part uses ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part uses Xevo G2-S Qtof mass spectrometer.

[0081] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0082] Example SLG-1: (4-amino-1,3-dihydrofuran[3,4-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (Compound SLG-1)

[0083]

[0084] Synthesis of Intermediate 4-Cyano-2,5-dihydrofuran-3-yl Trifluoromethanesulfonate

[0085]

[0086] 4-Oxotetrahydrofuran-3-carbonitrile (0.5 g, 4.5 mmol, 1.0 equiv.) was dissolved in dichloromethane (5 mL), and after replacing nitrogen, N,N-diisopropylisohexylamine (0.94 mL) was added to the solution, and the temperature was lowered to -78 ° C. Then, trifluoromethanesulfonic anhydride (0.76 mL) was added. After reacting for 15 minutes, water was added and the liquid was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain crude 4-cyano-2,5-dihydrofuran-3-yl trifluoromethanesulfonate, which was directly used in the next step.

[0087] Intermediate 1 Synthesis of 4-amino-1,3-dihydrofuran[3,4-c]quinoline-8-carboxylic acid

[0088]

[0089] Step 1: Synthesis of methyl 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate

[0090] To a solution of methyl 4-amino-3-bromobenzoate (2.3 g, 10.0 mmol, 1.0 equiv.) in 1.4-dioxane (40 mL) were added pinacol borate (7.6 g, 30 mmol, 3.0 equiv.), dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium (726 mg, 1.0 mmol, 0.1 equiv.) and potassium acetate (3.0 g, 30 mmol, 3.0 equiv.), and the mixture was reacted at 100 °C for 6 hours under nitrogen protection. After the reaction is completed, the solvent is removed by concentration, ethyl acetate (100 mL) and water (100 mL) are added, and the mixture is filtered through diatomaceous earth to remove the black suspended matter, and the organic phase is separated. The aqueous phase is extracted three times with ethyl acetate (50 mL*3), and the organic phases are combined and dried over sodium sulfate. After concentration, a small amount of dichloromethane is added for pulping, and the mixture is washed to obtain a white solid 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid methyl ester (about 1.4 g). LCMS: [M+H]+=278.1641.

[0091] Step 2: Synthesis of methyl 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylate

[0092] To a solution of methyl 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (500 mg, 1.8 mmol, 1.0 equiv.) in 1,4-dioxane (11 mL) were added 4-cyano-25-dihydrofuran-3-yl trifluoromethanesulfonate (550 mg, 2.26 mmol, 1.25 equiv.), tetrakis(triphenylphosphine)palladium (260 mg, 0.22 mmol, 0.13equiv.), potassium carbonate (925mg, 6.70mmol, 3.7equiv.) and water (1.1mL), under nitrogen protection, react at 90℃ for 16 hours. After the reaction is completed, the reaction solution is directly filtered, and the filter cake is first washed with a small amount of ethyl acetate (2.0mL), then washed with water (1.0mL), and dried to obtain solid 4-amino-1,3-dihydrofurano[3,4-C]quinoline-8-carboxylic acid methyl ester (220mg, yield 50%). LCMS [M+H] + :245.0929; 1 H NMR (400MHz, DMSO-d6): δ8.12(d,J=2.0Hz,1H),8.01(dd,J=8.8Hz,2.0Hz,1H),7.61(d ,J=8.9Hz,1H),6.95(s,2H),5.41(t,J=3.5Hz,2H),5.04(t,J=3.5Hz,2H),3.88(s,3H).

[0093] Step 3: Synthesis of 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylic acid

[0094] Dissolve methyl 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylate (100 mg, 0.41 mmol, 1.0 equiv.) in a mixed solution of tetrahydrofuran (1.0 mL) and methanol (1.0 mL), then add a solution of lithium hydroxide (68.9 mg, 1.64 mmol, 4.0 equiv.) in water (1.0 mL) to the above solution and react at 50°C for 16 hours. After the reaction is completed, the reaction solution is directly concentrated, adjusted to pH 6.0, and filtered to obtain 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylic acid (76 mg, yield 80.5%) as a white solid. LCMS: [M+H] + =231.0957.

[0095] Intermediate 2: Synthesis of 5-(piperidin-2-yl)benzo[d]thiazole

[0096]

[0097] Step 1: Synthesis of tert-butyl 6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate

[0098] At -78°C and under N2 protection, add 1.0 mol / L lithium bis(trimethylsilyl)amide (7.53 mL, 7.53 mmol, 1.50 equiv.) to a tetrahydrofuran (20 mL) solution of the compound N-tert-butyl carbonate-piperidin-2-one (1.0 g, 5.02 mmol, 1.0 equiv.). After reacting at this temperature for 1.5 hours, slowly add a tetrahydrofuran (12 mL) solution of N-phenylbis(trifluoromethanesulfonyl)imide (2.33 g, 6.53 mmol, 1.30 equiv.), and after the addition is complete, move to room temperature and react for 2 hours. After the reaction was completed, water (10 mL) was added for dilution, and the mixture was extracted with ethyl acetate (15 mL*3), washed with saturated sodium chloride (20 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The light yellow solid 6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (1.37 g, yield: 83%) was separated by column chromatography. LCMS: [M+H] + =332.07. 1 HNMR (400MHz, CDCl3): δ5.32 (m, J = Hz, 1H), 3.63 (m, J = Hz, 2H), 2.30 (m, J = Hz, 2H), 1.82–1.71 (m, 2H), 1.58–1.49 (m, 9H).

[0099] Step 2: Synthesis of tert-butyl 6-(benzo[d]thiazol-5-yl)-3,4-dihydropyridine-1(2H)-carboxylate

[0100] Tert-butyl 6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylate (333 mg, 1.0 mmol, 1.0 equiv.), 1,4-dioxane (9 mL) and water (3 mL) were added to a round-bottom flask, followed by benzothiazole-5-boronic acid pinacol ester (287 mg, 1.10 mmol, 1.10 equiv.), sodium carbonate (318 mg, 3.0 mmol, 3.0 equiv.) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (72.5 mg, 0.10 mmol, 0.10 equiv.) were added to the system. The reaction was carried out at 90°C for 12 hours under nitrogen protection. After the reaction is completed, water (10 mL) is added to dilute, and the mixture is extracted with ethyl acetate (10 mL*3). The organic phases are combined, washed with saturated sodium chloride (10 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain a white solid 6-(benzo[d]thiazol-5-yl)-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (217 mg, yield: 69%). 1 H NMR (400MHz, CDCl3): δ9.15 (s, 1H), 8.76 (d, J = 1.5Hz, 1H), 8.15–8.06 (m, 2H), 4.64 (s, 1H), 3.16 (t, J = 7.2Hz, 2H), 1.87 (p, J = 7.3Hz, 2H), 1.66 (p, J = 7.2Hz, 2H), 1.48 (s, 9H).

[0101] Step 3: Synthesis of 5-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole

[0102] 6-(Benzo[d]thiazol-5-yl)-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (30 mg, 2.3 mmol, 1.0 equiv.) and trifluoroacetic acid (2.6 mL, 34.7 mmol, 15.0 equiv.) were added to a round-bottom flask and stirred at room temperature for 2 hours. After the reaction was complete, trifluoroacetic acid was removed by distillation under reduced pressure, then diluted with water, the pH value was adjusted to 8-9 with saturated sodium carbonate solution, and extracted with ethyl acetate three times (15 mL*3). The organic phases were combined, washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product 5-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole, which was directly used in the next step. LCMS: [M+H] + =217.07.

[0103] Step 4: Synthesis of 5-(piperidin-2-yl)benzo[d]thiazole

[0104] The crude product (50 mg) of the compound 5-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole was dissolved in methanol (1 mL), and sodium borohydride (18 mg, 0.46 mmol, 2.0 equiv.) was added in batches at 0°C, and the mixture was reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated to remove methanol, and then diluted with water (1 mL), and extracted with ethyl acetate (1 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. Column chromatography was used to separate the yellow solid 5-(piperidin-2-yl)benzo[d]thiazole (34.0 mg, yield: 41%). LCMS: [M+H] + =219.09. 1 H NMR (400MHz, CDCl3): δ9.02(s,1H),8.20(s,1H),7.91(d,J=8.3Hz,1H),7.61(d,J=8.4Hz,1H),3.90(dd,J=11.3Hz,2.6H z,1H),3.25(d,J=12.1Hz,1H),2.87(m,1H),1.96(t,J=13.6Hz,2H),1.84–1.69(m,3H),1.65–1.55(m,1H),1.28(s,1H).

[0105] Synthesis of (4-amino-1,3-dihydrofuran[3,4-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone

[0106]

[0107] 4-Amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylic acid (40 mg, 0.17 mmol, 1.0 equiv.), 5-(piperidin-2-yl)benzothiazole (45.6 mg, 0.21 mmol, 1.2 equiv.), N,N-diisopropylethylamine (67.2 mg, 0.51 mmol, 3.0 equiv.) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (132 mg, 0.34 mmol, 2.0 equiv.) were added sequentially to N,N-dimethylacetamide (6 mL) and reacted at room temperature. After the reaction is completed, the reaction solution is filtered, and the solid is the target product mixture, which is then completely dissolved with N,N-dimethylformamide and trifluoroacetic acid to obtain a clear solution, which is purified by high-performance liquid preparative chromatography to obtain a white solid (4-amino-1,3-dihydrofuran [3,4-c] quinolin-8-yl) (2- (benzo [d] thiazol-5-yl) piperidin-1-yl) ketone (42 mg, yield 57.4%). LCMS [M+H]+ :431.1938. 1 H NMR (400MHz, DMSO-d6): δ9.42(s,1H),8.56(s,1H),8.20(d,J=8.4Hz,1H),8.02(s,1H),7.82(d,J=32.8Hz,3H),7.48(d,J=8.3Hz,1H),5 .43(s,2H),5.07(s,2H),3.56(d,J=11.0Hz,2H),2.93(s,1H),2.54(s,1H),2.08–1.94(m,1H),1.69(d,J=9.5Hz,1H),1.62–1.32(m,3H).

[0108] Example SLG-2: (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[3-(benzo[d]thiazol-5-yl)morpholinyl]methanone (Compound SLG-2)

[0109]

[0110] 4-Amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (30.0 mg, 0.09 mmol, 1.0 equiv.) prepared according to the preparation method of Example 4, 3-(Benzo[d]thiazol-5-yl)morpholine (20.0 mg, 0.09 mmol, 1.0 equiv.) prepared according to the preparation method of Intermediate 2, N,N-diisopropylethylamine (30 uL, 1.2 equiv.) and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (30 mg, 0.11 mmol, 1.2 equiv.) were added to N,N-dimethylacetamide (0.5 mL) and stirred at room temperature for 2-3 hours. After the reaction, water (1 mL) was added to dilute, extracted with ethyl acetate, and the organic phases were combined and concentrated to obtain a crude product, which was separated and purified by preparative chromatography to obtain a white solid (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[3-(benzo[d]thiazol-5-yl)morpholinyl]methanone (21 mg). LCMS [M+H] + :445.1513. 1HNMR (400M, DMSO-d6): δ9.43(s,1H),8.26-8.20(m,5H),7.62(s,2H),7.60(s,1H),7.16(s,2H),4.57(d,J=8 .6Hz, 1H), 4.20 (s, 3H), 4.01 (dd, J = 8.6Hz, 3.8Hz, 2H), 3.87 (d, J = 8.5Hz, 2H), 3.69 (dt, J = 8.6Hz, 3.8Hz, 2H).

[0111] Example SLG-4: (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[3-(benzo[d]thiazol-5-yl)morpholinyl]methanone (Compound SLG-4)

[0112]

[0113] Step 1: Synthesis of methyl 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylate

[0114] Methyl 4-amino-3-(pinacol boro)benzoate (554 mg, 2.0 mmol, 1.0 equiv.), 5-bromo-1-methyl-1H-pyrazole-4-carbonitrile (410 mg, 2.2 mmol, 1.1 equiv.), potassium phosphate (1.7 g, 6.0 mmol, 3.0 equiv.) and dichlorobis[di-tert-butyl-(4-dimethylaminophenyl)phosphine]palladium(II) were added to a mixed solution of 1,4-dioxane (15 mL) and water (5 mL), replaced with nitrogen three times, and reacted at 100°C for 16 hours. After the reaction was completed, the reaction solution was cooled to room temperature, diluted with water (20 mL), allowed to stand and filtered, and the filter cake was washed with water (3 mL) and methanol (2 mL) to obtain a crude product of methyl 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylate. LCMS [M+H]+: 257.1049. 1 HNMR (400M, DMSO-d6): δ 3.91 (s, 3H), 4.44 (s, 3H), 7.40 (s, 2H), 7.63 (d, J = Hz, 1H), 8.04 (d, J = Hz, 1H), 8.30 (s, 1H), 8.83 (s, 1H).

[0115] Step 2 Synthesis of 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid

[0116] 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (100 mg, 0.4 mmol, 1.0 equiv.), lithium hydroxide (32 mg, 0.8 mmol, 2.0 equiv.), methanol (1.0 mL), water (1.0 mL) and tetrahydrofuran (1.0 mL) were added to the reaction bottle in sequence and reacted at 90°C for 2-3 hours. After the reaction, the pH was adjusted to 7 with HCl aqueous solution (1.0 M), most of the solvent was removed by rotary evaporation, and the filter cake was filtered. The filter cake was washed with water (0.5 mL) and methanol (0.5 mL) respectively, and then the filter cake was dried to obtain a crude product of 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (about 70.0 mg). LCMS [M+H] + :243.10. 1 HNMR (400M, DMSO-d6): δ4.50 (s, 3H), 7.83 (d, J = Hz, 1H), 8.21 (d, J = Hz, 1H), 8.58 (s, 1H), 8.86 (s, 1H), 13.0 (s, 1H).

[0117] Step 3 Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone

[0118] 4-Amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (30 mg, 0.09 mmol, 1.0 equiv.), 5-(piperidin-2-yl)benzothiazole (20.0 mg, 0.09 mmol, 1.0 equiv.), N,N-diisopropylethylamine (30 uL, 1.2 equiv.) and O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (30 mg, 0.11 mmol, 1.2 equiv.) were added to N,N-dimethylacetamide (0.5 mL) and stirred at room temperature for 2-3 hours. After the reaction, water (1 mL) was added to dilute, and ethyl acetate (2 mL*3) was used for extraction. The organic phases were combined and concentrated to obtain a crude product, which was separated and purified by preparative chromatography to obtain a white solid (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[2-(benzo[d]thiazol-5-yl)piperidin-1-yl]methanone (21 mg). LCMS [M+H] + :443.1649; 1H NMR (400MHz, DMSO-d6): δ9.45(s,1H),8.34(s,1H),8.30(s,1H),8.23(d,J=8.4Hz,1H),8.09(s,1H),7.97(s,2H),7.77–7.64(m,2H),7.58–7.48(m ,1H),4.21(s,3H),3.50(d,J=10.6Hz,2H),2.97(t,J=8.4Hz,1H),2.70–2 .52(m,1H),2.02(d,J=14.6Hz,1H),1.82–1.57(m,3H),1.51-1.45(m,1H).

[0119] Example SLG-6: (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[2-(benzo[d]thiazol-6-yl)piperidin-1-yl]methanone (Compound SLG-6)

[0120]

[0121] Step 1: Synthesis of tert-butyl 6-(benzo[d]thiazol-6-yl)-3,4-dihydropyridine-1(2H)-carboxylate

[0122] The compound 6-{[(trifluoromethyl)sulfonyl]oxy}-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (899 mg, 2.72 mmol, 1.0 equiv.) was dissolved in a mixed solvent of 1,4-dioxane (18 mL) and water (6 mL), and then benzothiazole-6-boronic acid pinacol ester (780 mg, 2.99 mmol, 1.1 equiv.), sodium carbonate (864 mg, 8.16 mmol, 3.0 equiv.) and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (197 mg, 0.27 mmol, 0.1 equiv.) were added to the system. The reaction was carried out at 90°C for 12 hours under nitrogen protection. After the reaction was completed, water (10 mL) was added to dilute it, and it was extracted with ethyl acetate (10 mL*3). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain white solid 6-(benzo[d]thiazol-6-yl)-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (610 mg, yield: 71%). LCMS [M+H] + :317.1707; 1H NMR (400MHz, DMSO-d6): δ9.33 (s, 1H), 8.05–7.99 (m, 2H), 7.42 (dd, J = 8.5Hz, 1.8Hz, 1H), 5.46 (t,J=3.8Hz,1H),3.73–3.60(m,2H),2.27(td,J=6.8Hz,3.7Hz,2H),1.78(m,2H),0.93(s,9H).

[0123] Step 2: Synthesis of 6-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole

[0124] The compound 6-(benzo[d]thiazol-6-yl)-3,4-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (610 mg, 1.93 mmol, 1.0 equiv.) was dissolved in trifluoroacetic acid (2.2 mL, 28.96 mmol, 15.0 eq.) and stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, diluted with water (5 mL), and the pH value was adjusted to 8-9 with saturated sodium carbonate solution, followed by extraction with ethyl acetate (5 mL*3). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product 6-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole, which was used directly in the next step.

[0125] Step 3: Synthesis of 6-(piperidin-2-yl)benzo[d]thiazole

[0126] The obtained compound 6-(1,4,5,6-tetrahydropyridin-2-yl)benzo[d]thiazole was dissolved in methanol (5 mL), and sodium borohydride (147 mg, 3.86 mmol, 2.0 equiv.) was added in batches at 0°C. After the addition, the mixture was transferred to room temperature for reaction for 2 hours. After the reaction was completed by TLC monitoring, the reaction solution was directly concentrated, diluted with water (2 mL), and extracted with ethyl acetate (5 mL*3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain a yellow liquid 6-(piperidin-2-yl)benzo[d]thiazole (395 mg, yield 94%). LCMS[M+H] +:219.1254;1H NMR (400MHz, CDCl3): δ8.99(s,1H),8.16–7.98(m,2H),7.55(dd,J=8.5Hz,1.7Hz,1H),3.81(dd,J=10.8,2.5Hz,1H),3.5 0(s,1H),3.22(dp,J=11.8,2.0Hz,1H),2.85(td,J=11.6,3.1Hz,1H),2.53(s,2H),2.04–1.81(m,2H),1.63–1.51(m,2H).

[0127] Step 4: Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[2-(benzo[d]thiazol-6-yl)piperidin-1-yl]methanone

[0128] 6-(Piperidin-2-yl)benzo[d]thiazole (15 mg, 0.07 mmol, 1.0 equiv.), 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (17 mg, 0.07 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (22 mg, 0.17 mmol, 2.5 equiv.) were dissolved in N,N-dimethylformamide (1 mL). After stirring at room temperature for five minutes, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (39 mg, 0.10 mmol, 1.5 equiv.) was added and reacted at room temperature for 16 hours. After the reaction, the mixture was separated and purified by preparative chromatography to obtain a white solid (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)[2-(benzo[d]thiazol-6-yl)piperidin-1-yl]methanone (21 mg, yield: 67.7%). LCMS [M+H] + :443.1915. 1 H NMR (400MHz, DMSO-d6): δ9.37(s,1H),8.60(s,1H),8.37(s,1H),8.25(s,1H),8.11(d,J=8.5Hz,1H),7.92(d,J=8.5Hz,1H),7.84(d ,J=8.5Hz,1H),7.55(d,J=8.5Hz,1H),4.29(s,8H),2.98(d,J=18.1Hz,1H),2.03(t,J=11.3Hz,1H),1.76–1.58(m,3H),1.49(s,1H).

[0129] Example SLG-8: 4-amino-N-(1-(benzo[d]thiazol-5-yl)ethyl)-N,1-dimethyl-1H-pyrazolo[4,3-c]quinoline-8-carboxamide (Compound SLG-8)

[0130]

[0131] Step 1: Synthesis of 1-(Benzo[d]thiazol-5-yl)-N-methylethylamine

[0132] Benzo[d]thiazol-5-yl ethyl ketone (43 mg, 0.25 mmol, 1.0 equiv.), methylamine hydrochloride (17 mg, 0.25 mmol, 1.0 equiv.), triethylamine (50 uL) and tetraethyl titanate (127 mg, 0.5 mmol, 2.0 equiv.) were added to methanol (1 mL) and reacted at 50°C for 16 hours. The reaction was monitored by TLC and LCMS, LCMS [M+H] +: 191.0652. After the reaction was completed, the reaction solution was cooled to zero degrees, and then sodium borohydride (38 mg, 4.0 equiv.) was added in batches. After the reaction, the reaction solution was concentrated directly, and water (1 mL) and ethyl acetate (2 mL) were added respectively, and then the solid was filtered out and separated. The aqueous phase was extracted with ethyl acetate (2 mL*3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was separated by column to obtain an oily product 1-(benzo[d]thiazol-5-yl)-N-methylethylamine (about 20 mg), LCMS [M+H] +: 193.0983, which was directly used in the next step reaction.

[0133] Step 2: Synthesis of 4-amino-N-(1-(benzo[d]thiazol-5-yl)ethyl)-N,1-dimethyl-1H-pyrazolo[4,3-c]quinoline-8-carboxamide

[0134] 4-Amino-1-methylpyrazolo[4,3-c]quinoline-8-carboxylic acid (30 mg, 0.13 mmol, 1.2 equiv.), 1-(benzothiazol-5-yl)-N-methyl-1-amine (20.0 mg, 0.10 mmol, 1.0 equiv.), N,N-diisopropylethylamine (40.3 mg, 0.312 mmol, 3.0 equiv.) and N,N-dimethylacetamide (1 mL) were added to the flask in sequence to dissolve, and finally 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol, 2.0 equiv.) was added and reacted at room temperature for 2-3 h. After the reaction was completed, trifluoroacetic acid was added to the reaction solution, the reaction solution was clarified, and purified by high performance liquid chromatography to obtain a white solid 4-amino-N-(1-(benzo[d]thiazol-5-yl)ethyl)-N,1-dimethyl-1H-pyrazolo[4,3-c]quinoline-8-carboxamide (20 mg, yield 47.6%). LCMS [M+H] + :417.1949. 1 H NMR (400MHz, DMSO): δ9.40(s,1H),9.12(s,1H),8.58(s,1H),8.37(s,1H),8.17(d,J=8.3Hz,1H) ,8.05(s,1H),7.92–7.77(m,2H),7.49(s,1H),4.34(s,3H),2.79(s,3H),1.72(d,J=6.8Hz,3H).

[0135] Example SLG-14: (4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (Compound SLG-14)

[0136]

[0137] Step 1: Synthesis of methyl 4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylate

[0138] Methyl 4-amino-2-chloro-3-(pinacol borate)benzoate (295 mg, 1.0 mmol, 1.0 equiv.), 5-bromo-1-methyl-1H-pyrazole-4-carbonitrile (205 mg, 1.1 mmol, 1.1 equiv.), potassium phosphate (0.85 g, 3.0 mmol, 3.0 equiv.) and dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (II) (70 mg, 0.1 mmol, 0.1 equiv.) were added to a mixed solvent of 1,4-dioxane (10 mL) and water (3 mL) in sequence. The mixture was reacted at 100°C for 16 hours under nitrogen protection. After the reaction was completed, the reaction solution was cooled to room temperature, diluted with water (10 mL), allowed to stand and filtered, and the filter cake was washed with water (2 mL) and methanol (1 mL) to obtain a crude product, which was separated and purified by liquid phase preparation to obtain solid 4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (45 mg). LCMS: [M+H] + :291.0663.

[0139] Step 2: Synthesis of 4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid

[0140] 4-Amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (45 mg, 0.15 mmol, 1.0 equiv.), lithium hydroxide (12 mg, 0.3 mmol, 2.0 equiv.), methanol (1.0 mL), water (1.0 mL) and tetrahydrofuran (1.0 mL) were added to the reaction bottle in sequence, and the reaction was carried out at 70°C for 16 hours. After the reaction was completed, the pH was adjusted to 7 with HCl aqueous solution (1.0 M), and the solid was concentrated to obtain a solid, which was then dried in vacuo to obtain a crude product (about 50 mg). LCMS [M+H] + :277.0498.

[0141] Step 3: Synthesis of (4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone

[0142] Crude 4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (30 mg, 0.09 mmol, 1.0 equiv.), 5-(piperidin-2-yl)benzo[d]thiazole (20.0 mg, 0.09 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (30 uL, 1.2 equiv.) were added to N,N-dimethylformamide (0.5 mL) solvent, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30 mg, 1.2 equiv.) and stirred at room temperature for 2-3 hours. The reaction was monitored by TLC and LCMS. After the reaction was completed, the white solid (4-amino-7-chloro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (7 mg) was obtained by separation and purification by preparative chromatography. LCMS: [M+H] + =477.1364; 1 HNMR (400M, DMSO-d6): δ9.43(s,1H),8.62(s,1H),8.46-8.45(m,1H),8.24(d,J=8.1Hz,1H),8.18(s,2H),7.99-7.89(m,1H),7.60(dd,J=8.1,3 .8Hz,1H),6.13(t,J=5.1Hz,1H),4.71-4.54(m,1H),3.93(s,3H),3.48- 3.25(m,2H),2.65-2.62(m,1H),2.20--2.01(m,2H),1.73-1.43(m,3H).

[0143] Example SLG-15: (4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (Compound SLG-15)

[0144]

[0145] Step 1: Synthesis of methyl 4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylate

[0146] Methyl 4-amino-2-fluoro-3-(pinacol borate)benzoate (295 mg, 1.0 mmol, 1.0 equiv.), 5-bromo-1-methyl-1H-pyrazole-4-carbonitrile (205 mg, 1.1 mmol, 1.1 equiv.), potassium phosphate (0.85 g, 3.0 mmol, 3.0 equiv.) and dichlorobis[di-tert-butyl-(4-dimethylaminophenyl)phosphine]palladium(II) (70 mg, 0.1 mmol, 0.1 equiv.) were added to a mixed solvent of 1,4-dioxane (10 mL) and water (3 mL), and the reaction was carried out at 100 °C for 16 hours under nitrogen protection. After the reaction was completed as monitored by HPLC and LCMS, the reaction solution was cooled to room temperature, diluted with water (10 mL), allowed to stand and then filtered, and the filter cake was washed with water (2 mL) and methanol (1 mL) to obtain a crude product of 4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (about 150 mg). 1 H NMR (400MHz, DMSOd6): δ8.75 (d, J = 7.9 Hz, 1H), 8.30 (s, 1H), 7.47 (s, 2H), 7.31 (d, J = 13.3 Hz, 1H), 4.41 (s, 3H), 3.92 (s, 3H).

[0147] Step 2: Synthesis of 4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid

[0148] 4-Amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (150 mg, 0.6 mmol, 1.0 equiv.), lithium hydroxide (50 mg, 1.2 mmol, 2.0 equiv.), methanol (1.0 mL), water (1.0 mL) and tetrahydrofuran (1.0 mL) were added into the reaction bottle in sequence, and the reaction was carried out at 90°C for 2-3 hours. After the reaction was completed, the pH was adjusted to 7 with HCl aqueous solution (1.0 M), and the solid was concentrated to obtain a solid, which was then dried in vacuo to obtain a crude product of 4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (180 mg). LCMS: [M+H] + =261.1034.

[0149] Step 3: Synthesis of (4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone

[0150] The crude 4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (35 mg, 0.09 mmol, 1.0 equiv.), 5-(piperidin-2-yl)benzo[d]thiazole (20.0 mg, 0.09 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (30 uL, 1.2 equiv.) were added to N,N-dimethylformamide (0.5 mL) solvent, followed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30 mg, 1.2 equiv.) and reacted at room temperature for 2-3 hours. The reaction was monitored by TLC and LCMS. After the reaction was completed, the white solid (4-amino-7-fluoro-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (7 mg) was obtained by separation and purification by preparative chromatography. LCMS: [M+H] + =461.1555. 1 H NMR (400MHz, DMSO-d6): δ9.41(s,1H),8.61(s,1H),8.48(s,1H),8.21(s,1H),8.07(s,1H),7.71(s,1H),7.51(s,1H) ,4.41-4.66(m,3H),3.27-3.56(m,1H),2.83-3.20(s,1H),2.50-2.67(m,1H),1.91-2.14(m,1H),1.31-1.76(m,5H).

[0151] Example SLG-18: (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone (Compound SLG-18)

[0152]

[0153] Step 1: Synthesis of methyl 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridine-8-carboxylate

[0154] 4-Amino-5-(pinacol boroyl)nicotinate methyl ester (277 mg, 1.0 mmol, 1.0 equiv.), 5-bromo-1-methyl-1H-pyrazole-4-carbonitrile (205 mg, 1.1 mmol, 1.1 equiv.), potassium phosphate (0.85 g, 3.0 mmol, 3.0 equiv.), dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (II) (70 mg, 0.1 mmol, 0.1 equiv.), 1,4-dioxane (10 mL) and water (3 mL) were added to the reaction flask in sequence. After nitrogen replacement three times, the reaction was heated at 100 degrees overnight. After the reaction was completed, the reaction was monitored by HPLC and LCMS. The reaction was cooled, water (10 mL) was added, and the reaction mixture was filtered after standing. The filter cake was washed with water (2 mL) and methanol (1 mL) to obtain a crude product of about 150 mg. LCMS [M+H] + =258.09.

[0155] Step 2: Synthesis of 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridine-8-carboxylic acid

[0156] The intermediate 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridine-8-carboxylic acid methyl ester (150 mg, 0.6 mmol, 1.0 equiv.), LiOH (50 mg, 1.2 mmol, 2.0 equiv.), methanol (1.0 mL), water (1.0 mL) and THF (1.0 mL) were added to the reaction flask in sequence, and reacted at 90 degrees for 2-3 hours. After the reaction was completed, HCl (1.0 M) was used to adjust the pH to 7, and most of the solvent was removed by rotary evaporation. The crude product was dried and directly used for the next step. LCMS [M+H] + =244.08.

[0157] Step 3: Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridin-8-yl)(2-(benzo[d]thiazol-5-yl)piperidin-1-yl)methanone

[0158] The crude product 4-amino-1-methyl-1H-pyrazolo[4,3-c][1,8]naphthyridine-8-carboxylic acid (50 mg, 0.09 mmol, 1.0 equiv.), 5-(piperidin-2-yl)benzo[d]thiazole (20.0 mg, 0.09 mmol, 1.0 equiv.), N,N-diisopropylethylamine (DIPEA) (30 uL, 1.2 equiv.) and DMF (0.5 mL) were added to a 4 mL reaction bottle, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30 mg, 1.2 equiv.) was added and stirred at room temperature for 2-3 hours. The reaction was monitored by TLC and LCMS. After the reaction was completed, a white solid (2 mg) was obtained by separation and purification by preparative chromatography. LCMS [M+H] + =441.15. 1 HNMR (400MHz, DMSO-d6): δ9.33(s,1H),8.83(d,J=1.6Hz,1H),8.75(d,J=1 .6Hz,1H),8.26(s,1H),7.85–7.79(m,1H),7.69(d,J=8.6Hz,1H),7.45–7.3 8(m,1H),7.13(s,2H),4.82–4.75(m,1H),3.89(s,3H),3.81–3.73(m,1H),3 .67–3.58(m,1H),2.25–2.14(m,1H),1.98–1.81(m,2H),1.78–1.63(m,4H).

[0159] The compounds listed in Table 1 below were prepared by methods similar to those described in the Examples, wherein the reactants, amounts of reagents, protection and deprotection, solvents and reaction conditions were appropriately varied. Characterization data of the compounds are summarized in Table 1 below.

[0160] Table 1: Structure and characterization of some compounds

[0161]

[0162]

[0163]

[0164]

[0165] Experimental Example 1: Test of the binding ability of the compound of the present invention to PRMT5 protein

[0166] Experimental purpose: To detect the binding ability of compounds to PRMT5 protein by CETSA experimental method

[0167] Background principle: CETSA experiment is a molecular detection method to measure the affinity between drugs and target proteins. The principle is that after the drug binds to the target protein, its structure becomes more stable. If the candidate drug is an inhibitor of PRMT5, the candidate drug can bind to PRMAT5, making the PRMT5 protein more stable. After heating the sample, the PRMT5 protein in the sample will be more easily detected by specific antibodies, and the PRMT5 protein will be more easily detected by Western blot experiments; conversely, the stability of the PRMT5 protein will be worse after heating, and the amount of protein detected will be lower. In this way, the binding ability of the drug to the target protein is evaluated, which is used for the screening of PRMT5 protein inhibitors.

[0168] Specific experimental process:

[0169] Take HCT116 cells in the logarithmic growth phase (cell viability>90%), wash the cells 3 times with PBS, centrifuge at 2000g for 2min; lyse on ice for 30min with cell lysis solution containing protease inhibitor PMSF; use BCA kit to measure the concentration of protein samples. Incubate the samples with candidate drugs, control drugs and control reagents for 30min, heat each group of samples at about 10 set temperature points; restore room temperature, centrifuge the samples at 20000g, collect the supernatant; use sample buffer to heat and denature the protein samples at 100℃ for 10min. After the samples return to room temperature, perform western blot detection on the samples; the protein loading amount is controlled at 20ug. After determining the mutation temperature, the compound concentration gradient is generally set to 9 points, the samples are incubated, and the same operation is performed as above for western blot detection. Protein electrophoresis: the voltage of the concentrated gel is set to 60v, and the voltage of the separation gel is set to 120v; after the electrophoresis is completed, start electrotransfer. The conditions for electroporation were set at 250 mA for 2 h; 5% BSA was blocked for 1 h; specific antibody 1 was added and incubated overnight on a shaker at 4 °C; TBST was washed 4 times, 2.5 min each time; 2 antibodies were incubated on a shaker at room temperature for 1 h; TBST was washed 4 times, 2.5 min each time; ECL was used for development to detect the expression of PRMT5 protein in different groups and at various temperature points. Western blot bands were converted and processed by image J and GraphPad software, and EC was calculated. 50 value.

[0170] EC50 is the half-maximal effect concentration, which refers to the drug concentration that can cause 50% of individuals to be effective. The PRMT5 inhibitor TNG908 was used as a positive reference compound, and the preparation method was referred to Example 616 in patent document WO2022026892.

[0171] The structure of TNG908 is as follows:

[0172]

[0173] Table 1: IC50 results

[0174]

[0175]

[0176] The results show that the compounds of the present invention can inhibit the activity of the PRMT5 target, and the IC50 value reaches the nM level, which is equivalent to or less than the positive control drug TNG908, and the selectivity is better than TNG908. This strong inhibitory effect has important application prospects and therapeutic significance for the treatment of symptoms or diseases related to PRMT5 inhibition.

[0177] Experimental Example 2: Determination of the effect of the compounds of the present invention on the proliferation of HCT116 MTAP- / - cells

[0178] Experimental purpose: The purpose of this test example is to test the effect of compounds on the proliferation of HCT116 MTAP- / - cells.

[0179] Background principle: Protein arginine methyltransferase 5 (PRMT5) can methylate a variety of proteins and play an important role in biological processes such as gene expression, splicing and DNA damage repair. Methylthioadenosine phosphorylase (MTAP) often co-deletes with the common tumor suppressor gene CDKN2A in the body, and the proportion of this co-deletion in tumors can reach 9% to 15%. Studies have found that inhibiting PRMT5 has a synthetic lethal effect in MTAP-deficient tumors. Therefore, by detecting the inhibition rate of compounds on the proliferation of HCT116 MTAP- / - cells, it can be used to screen PRMT5 protein inhibitors.

[0180] Specific experimental process:

[0181] Construct HCT116 MTAP knockout cells and screen monoclones. - / - and WT cells, inoculated in 96-well plates, 90uL per well, 1000 cells / well, and incubated overnight at 37°C incubator. On the second day, 10μl of different concentrations of compounds were added, with the highest concentration being 100uM, 10-fold dilution, 9 gradients (DMSO final concentration was 1%), and incubated at 37°C incubator for 7 days. On the 7th day, the old culture medium was aspirated, 110ul of culture medium (the ratio of culture medium to CCK8 was 100:10) was added, and incubated at 37°C for 1-4h. The absorbance was detected at 450nM, and the IC50 was calculated by GraphPad software. The compounds were screened by comparing with positive drugs.

[0182] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the antagonist being measured. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). The compound TNG908 described above was used as a positive reference compound.

[0183] The test results are shown in Table 2 below:

[0184] Table 2: IC50 results

[0185]

[0186]

[0187] The results showed that the compounds of the present invention can significantly inhibit HCT116 MTAP - / - The cell viability and IC50 values ​​reached nM level, which was equivalent to or less than the positive control drug TNG908, and the selectivity was better than TNG908. This strong inhibitory effect has important application prospects and therapeutic significance for the treatment of diseases or conditions related to PRMT5 inhibition.

[0188] Experimental Example 3: Testing of the inhibitory effect of the compounds of the present invention on PRMT5 enzyme function

[0189] Experimental purpose: To detect the inhibitory effect of compounds on PRMT5 enzyme function by MTase-Glo method

[0190] Background principle: The gene that constitutes the synthetic lethality of PRMT5 is MTAP (methylthioadenosine phosphorylase). MTAP is involved 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 SAM (S-adenosylmethionine) with a methyl group 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.

[0191] Specific experimental process:

[0192] The MTase-Glo methyltransferase fluorescence method was used to monitor the conversion of s-adenosylmethionine (SAM) to s-adenosylhomocysteine ​​(SAH) by the recombinant PRMT5:MEP50 enzyme complex in the presence or absence of 5'-methylthioadenosine (MTA). The enzymatic reaction was performed in a white 96-well plate. The reaction buffer contained 20mM bicine (pH 7.60), 25mM NaCl, 1mM DTT and 0.1% (w / v) CHAPS. The test compounds and positive controls were configured according to 10-fold dilutions, 9 concentration gradients, 3 replicates, and the highest concentration was 100uM. In the absence of MTA, the compounds were incubated with a reaction mixture containing 1nM recombinant PRMT5:MEP50 enzyme complex, 2.5uM H41-21 histone peptide and 2.5uM s-adenosylmethionine (SAM) for the experiment. Similarly, in the presence of MTA, the compound was incubated with a mixture of 2nM enzyme complex, 1.5uM MTA, 2.5uM H41-21 histone peptide and 2.5uMSAM. After incubation at room temperature for 5h, TFA was added to each well to terminate the reaction. Subsequently, 5X MTase-GloTM reagent was added to each well and incubated at room temperature to convert the SAH produced in the reaction into ADP. After 10min, MTase-Glo detection solution was added to each well, incubated at room temperature for 30min, and detected by microplate reader.

[0193] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the antagonist being measured. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). The compound TNG908 described above was used as a positive reference compound.

[0194] Some test results are shown in Table 3:

[0195] Table 3: IC50 results

[0196] Compound No. PRMT5.MTA IC50(nM) PRMT5 IC50(nM) SLG-2 5.28 50.32 SLG-15 3.61 11.74 TNG908 14.21 76.69

[0197] The results showed that the compounds of the present invention have a very strong enzyme function inhibitory effect on the PRMT5.MTA protein complex, with an IC50 value reaching the nM level, which is lower than the positive control drug TNG908, and at the same time has a selectivity higher than TNG908, achieving an unexpected technical effect.

[0198] Experimental Example 4: In vitro metabolic stability determination of the compounds of the present invention

[0199] 1. Experimental principle: Liver microsomes contain most of the phase I enzymes, the most important of which is the microsomal mixed function oxidase system with CYP450 as the main component. When using liver microsomes for research, adding the corresponding auxiliary factor NADPH can reconstruct the in vitro metabolic system, thereby conducting phase I metabolic stability research through in vitro incubation method.

[0200] 2. Experimental methods:

[0201] 2.1 Melt the components of the kit in an ice bath and place on ice for later use;

[0202] 2.2 Prepare pre-incubation solution A (1X), 10ul of solution A, 2ul of solution B, and 28ul of 0.1M PBS buffer, mix well, incubate at 37°C for 5min, and dispense 40ul into each tube for use;

[0203] 2.3 Prepare pre-incubation solution B (1X), 0.1M PBS buffer 154ul, test substance (or positive substrate) 1ul, liver microsomes 5ul, mix well;

[0204] 2.4 Add 160ul of pre-incubation solution B to the centrifuge tube containing 40ul of pre-incubation solution A, pipette 3 times to mix, and immediately place at 37℃ for incubation time;

[0205] 2.5 At the set time point, add pre-cooled acetonitrile to terminate the reaction, centrifuge at 20,000 rpm, 4°C, 10 min, take the supernatant and detect.

[0206] 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.

[0207] Experimental results:

[0208] The results of the rat & human liver microsome incubation phase I metabolism experiment showed that the relative content of the compounds of the present invention was greater than 90% after 2h MS detection, and the relative content of the compounds of some compounds such as SLG-15 was greater than 95% after 2h MS detection, and the phase I metabolism was very stable, which achieved an unexpected metabolic stability effect compared with the positive control drug TNG908. The specific values ​​are shown in Table 4.

[0209] Table 4. Metabolism report of rat & human liver microsome incubation-phase I of the compounds of the present invention

[0210]

[0211] Experimental Example 5: In vitro permeability Caco-2 test of the compounds of the present invention

[0212] 1. Caco-2 experimental method

[0213] 1.1 Caco-2 cell culture:

[0214] (1) Add 50 μL and 25 mL of cell culture medium to each well of the Transwell insert and reservoir, respectively.

[0215] (2) Before cell inoculation, the Transwell plate was pre-incubated at 37°C and 5% CO2 for 1 hour.

[0216] (3) 50 μL of cell suspension (2х105 cells / mL) was inoculated into the insert and cultured at 37°C, 5% CO2, and 95% relative humidity for 21-28 days.

[0217] (4) The cell culture medium was replaced every other day for 7 days and then every day thereafter.

[0218] (5) The transepithelial electrical resistance (TEER) of the monolayer was measured using EVOM3.

[0219] 1.2 ABBA experimental steps:

[0220] (1) Wash the Transwell plate twice with pre-warmed HBSS (10 mM HEPES, pH 7.4) and then incubate at 37°C

[0221] Incubate for 30 min.

[0222] (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).

[0223] (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.

[0224] (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.

[0225] (5) Transfer 50 μL of 5 μM working solution (10 mM HEPES, pH 7.4) to a flask containing 200 μL of cold methanol.

[0226] (IS* as CO) in the sample plate.

[0227] (6) Incubate the cell plate at 37°C, 5% CO2, and 95% relative humidity for 2 hours.

[0228] (7) After 2 h 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*.

[0229] (8) Centrifuge the sample plate at 3220 g for 40 minutes.

[0230] (9) Transfer 100 μL of the supernatant to an assay plate containing an appropriate volume of H 2 O for LC-MS / MS analysis.

[0231] 1.3 Lucifer Yellow fluorescent penetrant testing

[0232] (1) Add 100 μL of 100 μM Lucifer Yellow solution (10 mM HEPES, pH 7.4) to the apical chamber and 300 μL of 10 mM HEPES (pH 7.4) to the basolateral chamber.

[0233] (2) Incubate the cell plate at 37°C, 5% CO2, and 95% relative humidity for 0.5 h.

[0234] (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.

[0235] 1.4 Data Analysis

[0236] (1) The apparent permeability coefficient (Papp, cm / s) and efflux ratio were calculated using Microsoft Excel.

[0237]

[0238] Vacceptor=0.235 mL for A→B,and 0.075 mL for B→A

[0239] Vdonor=0.075 mL for A→B,and 0.235 mL for B→A

[0240] Area=the surface area of ​​the membrane,cm2(0.143 for Corning 3391)

[0241] Time=the total transport time,s

[0242] (2) Calculate Lucifer Yellow fluorescence penetration using the following formula:

[0243]

[0244] I=the fluorescence intensity

[0245] Vacceptor=0.3 mL and Vdonor=0.1 mL for Lucifer Yellow leakage%test

[0246] *IS:50 nM alprazolam,50 nM labetalol and 100 nM ketoprofen

[0247] 2. Experimental results

[0248] The results of the Caco-2 cell permeation experiment showed that the permeability (Papp A→B (1E-6 cm / s) of compound SLG-15 was significantly better than that of MRTX1719, and the efflux rate was less than 2%, which basically ruled out the possibility that the compound was a substrate for efflux transporters, and the oral absorption was good. The specific values ​​are shown in Table 5.

[0249] Table 5. Caco-2 test results of the compounds of the present invention

[0250]

[0251] Experimental Example 6: In vivo pharmacokinetic study of the compounds of the present invention

[0252] 1. Experimental purpose: After a single intravenous injection or oral gavage of different compounds to ICR mice, blood samples were collected at different time points. LC-MS / MS was used to determine the concentration of the test compound in the mouse plasma after administration and calculate related parameters.

[0253] 2. Experimental plan:

[0254] 2.1 Experimental animals: ICR male mice, SPF grade

[0255] 2.2 Experimental design:

[0256]

[0257] Note: *, animals were fasted overnight (10-14 hours) before administration and were fed 4 hours after administration.

[0258] 2.3 Blood collection time

[0259] IV after administration: 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, 24h.

[0260] PO:0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h

[0261] Blood was collected from the jugular vein, approximately 0.2 mL of blood was collected for each sample, anticoagulated with K2-EDTA, and placed on ice after collection.

[0262] 2.4 Plasma sample processing: After blood samples were collected, they were placed on ice and centrifuged to separate plasma within 1 hour (centrifugation conditions: 6800 g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C refrigerator before analysis.

[0263] 2.5 Result analysis: Phoenix WinNonlin7.0 was used to calculate the pharmacokinetic parameters based on the blood drug concentration data at different time points, providing parameters such as AUC0-t, AUC0-∞, MRT0-∞, Cmax, Tmax, and T1 / 2 and their mean and standard deviations.

[0264] The above examples are only representative. It can be seen from the above examples that the compounds of the present invention are ideal highly active PRMT5 inhibitors and can be expected to be used for treating or preventing disorders or diseases associated with PRMT5 inhibition.

[0265] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, and they should be covered by the protection scope of the present invention.

Claims

1. A compound represented by formula (I) and its isomers, or a pharmaceutically acceptable salt thereof: in, R1 is selected from -(CH2) n -R4, -CH(CH3)-R4; R2 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may optionally be further substituted, -CH(CH3)-R3; R1 and R2 are different; The R3 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 The haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups may optionally be further substituted; The R4 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, the amino, thiol, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl may be optionally further substituted; n = 0, 1, 2, 3, 4; Or R1, R2 and the connected N form a saturated or unsaturated five-membered, six-membered or seven-membered heterocyclic ring, and contain 1 to 3 heteroatoms selected from N, O, S, wherein the S atom is optionally substituted by one or two oxo groups, and the five-membered, six-membered or seven-membered heterocyclic ring is substituted or spiro-connected with one or more R6; The R6 is selected from C 1-6 Alkyl, halogen, C 1-6 Haloalkyl, -OC 1-6 Alkyl, -OC 1-6 Haloalkyl, C 3-6 Cycloalkyl, - C(O)C 1-6 Alkyl, -C(O)C 1-6 Haloalkyl, -C(O)OC 1-6 Alkyl, -C(O)OC 1-6 Haloalkyl, a saturated or partially saturated or aromatic 5-10 membered heterocyclic ring containing 1 to 3 heteroatoms selected from N, O, S, which may be optionally substituted by one or more Ra; The Ra is selected from hydrogen, deuterium, nitrogen methyl bridged cycloheptyl, C 1-6 Alkyl, C 1-6 Alkoxy, halogen, amino, thiol, nitro, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, cyano, hydroxy, hydroxyalkyl, C 1-6 Haloalkyl, C 1-6 Halogenated alkoxy, C 3-6 Cycloalkyl, C 3-6 Halogenated cycloalkyl, C 3-6 Halogenated heterocycloalkyl, C 1-6 Alkylamino, formyl and optionally substituted with R b -(CH2) m -heterocycloalkyl, -(CH2) m - heterocycloalkenyl, aryl, heteroaryl, heterocyclyl; The R b For halogen, CN, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 3-6 Cycloalkyl, C 3-6 Heterocycloalkyl, C 1-6 Haloalkyl, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy, formyl, methylamino, oxetane; m＝0、1、2、3、4； R is selected from the following structures: X1 is selected from N or CR7, and R7 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Deuterated alkoxy, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl; R5 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl; R8 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, C 1-6 Alkyl, alkenyl, alkynyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, hydroxyalkyl, C 3-7 Cycloalkyl, C 3-7 Halogenated cycloalkyl.

2. The compound according to claim 1, its isomer, or its pharmaceutically acceptable salt, characterized in that: The R8 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, mercapto, cyano, nitro, methyl, ethyl, isopropyl, deuterated methyl, halomethyl, and methoxy; more preferably, the R8 is selected from hydrogen, methyl, ethyl, isopropyl, and deuterated methyl.

3. The compound according to claim 1-2 and its isomer, or its pharmaceutically acceptable salt, characterized in that: The R5 is selected from hydrogen, hydroxyl, cyano, C 1-6 Alkyl, halogen, C 1-6 Haloalkyl, C 3-6 Cycloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy, C 1-6 deuterated alkyl; further preferably, R5 is selected from hydrogen, hydroxy, cyano, methoxy, methyl, ethyl, bromine, chlorine, fluorine, deuterated methyl, -CH2F, -CHF2, -CF3, -CH2Cl, -CHCl2, -CCl3, -CD3, cyclopropyl; more preferably, R5 is selected from hydrogen, chlorine, fluorine, methyl.

4. The compound according to claim 1-3 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: wherein R2 is selected from hydrogen, deuterium, halogen, amino, hydroxyl, thiol, cyano, nitro, C 1-6 Alkyl, C 1-6 Alkoxy, halogenated C 1-6 Alkoxy, C 3-6 Cycloalkyl, -CH(CH3)-six-membered heterocyclyl, -CH(CH3)-six-membered heteroaryl; further preferably, R2 is selected from methyl, isopropyl, cyclopropyl, -CH(CH3)-six-membered heteroaryl containing 1 to 3 N, O, S heteroatoms.

5. The compound according to claim 1-4 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The R4 is selected from any substituted 5-10 membered heteroaryl; further preferably, R4 is selected from unsubstituted, halogen, C 1-6 Alkyl, C 1-6 Alkoxy-substituted benzopyran ring, benzofuran ring, benzothiazole ring, benzopyrazole ring, benzopyridine ring, benzopyrrole ring, benzopyrimidine ring, benzothiophene ring, benzoxazole ring, pyridothiazole ring, pyridothiphene ring, pyridopyrrole ring; more preferably, R4 is selected from ； Most preferably, R4 is selected from 6. The compound according to claim 1-5 and its isomers, or pharmaceutically acceptable salts thereof, characterized in that: The ring formed by R1, R2 and the connected N is selected from pyrrole ring, piperidine ring, methyl piperidine ring, imidazole ring, pyrazole ring, morpholine ring, azepane, azetidine, azetidine, azohexane, azopentane substituted or spiro-coupled with one or more R6; further preferably, the ring formed by R1, R2 and the connected N is selected from 7. The compound according to claim 6, its isomer, or its pharmaceutically acceptable salt, characterized in that: The R6 is selected from methyl, ethyl, halogen, C 1-6 haloalkyl, methoxy, halomethoxy, -C(O)methyl, cyclopropyl, a saturated or partially saturated or aromatic 9-membered heterocycle containing 1 to 3 heteroatoms selected from N, O, S, which may be optionally substituted by one or more Ra; further preferably, R6 is selected from methyl, cyclopropyl, halogen and 8. The compound according to claim 7, its isomer, or its pharmaceutically acceptable salt, characterized in that: The R a is selected from hydrogen, deuterium, nitrogen, methyl, methoxy, halogen, amino, mercapto, nitro, alkenyl, alkynyl, deuterated methyl, cyano, hydroxy, cyclopropyl, methylamino, formyl, trifluoromethyl, difluoromethyl and optionally substituted with R b -(CH2) m -heterocycloalkyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, said R b It is methyl, cyclopropyl, formyl, cyclobutyl, and oxetane.

9. The compound according to claim 1-8 and its isomer, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

10. A compound selected from the following formula and its isomers, or a pharmaceutically acceptable salt thereof: 。 11. Use of a compound according to any one of claims 1 to 10 and its isomers, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a disease, disorder, syndrome or condition associated with the PRMT5 enzyme; preferably, 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 more preferably cancer disease; most preferably, the cancer is an advanced solid tumor, metastatic pancreatic cancer, metastatic non-small cell lung cancer, a neural tumor, adenocarcinoma, endometrioid carcinoma, metastatic esophageal cancer, metastatic head and neck cancer, squamous cell carcinoma, cervical tumor, myelodysplastic syndrome, non-Hodgkin's 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, bile duct cancer, mesothelioma, ovarian cancer, lung cancer, prostate cancer, colon cancer, gastric cancer, esophageal cancer and hepatocellular carcinoma.

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