Polycyclic compounds as MAT2A inhibitors

Abstract

The present invention provides a compound as shown in Formula I and its isomers, or a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition and use thereof as a MAT2A inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a polycyclic compound and its isomers, or a pharmaceutically acceptable salt thereof, as a MAT2A inhibitor, a preparation method thereof, a pharmaceutical composition, and use thereof in preparing a compound for treating the cancer in a subject suffering from the cancer. Background Art

[0002] Methionine adenosyltransferase (MAT) (also known as S-adenosylmethionine synthetase) is a cellular enzyme that catalyzes the synthesis of S-adenosylmethionine (SAM or AdoMet) from methionine and ATP; this catalysis is considered to be the rate-limiting step of the methionine cycle. SAM is an propylamine donor in polyamine biosynthesis and the major methyl donor for DNA methylation, and is involved in gene transcription and cell proliferation as well as the generation of secondary metabolites.

[0003] The three isoforms of human MAT include MAT1 and MAT3, which are expressed in liver tissue, while MAT2A is ubiquitously expressed in human cell types and is the predominant form in human tumors. Crystal structure and mechanistic studies have shown that despite their 85% identity in amino acid sequence, their mechanisms of action differ significantly. MAT2A forms functional homodimers in its purified active form and binds to the regulatory protein MAT2B. MAT2B regulates the activity of MAT2A by increasing the sensitivity of MAT2A to inhibition by the ADOMet product, but does not provide significant rate enhancement. Cellular localization studies have shown that MAT2A is present in both the cytoplasm and the nucleus. Nuclear condensation of MAT2A has been reported to occur during replication and the subsequent G2 phase, satisfying the high methylation requirements of DNA and histone methylation processes in the nucleus during the S phase. The activity of ADOMet in transmethylation reactions has been identified as a rate-limiting factor in the development of lung cancer stem cells, making MAT2A and related enzymes of the methionine cycle targets for anticancer drugs. Methionine for ADOMet production by MAT2A comes from dietary sources or the polyamine cycle, where 5-methylthio-D-ribose 1-phosphate produced by S-methyl-5'-thioadenosine phosphorylase is recycled to methionine. MAT2A protein expression has been reported to be increased in cancers such as colon cancer, hepatocellular carcinoma, gastric cancer, blood and liver cancer. Approximately 15% of human cancers show deletion of the MTAP (S-methyl-5'-thioadenosine phosphorylase) gene and therefore lack the methionine recycling pathway for polyamine synthesis. Loss of MTAP in chr9p21 often includes loss of the CDKN2a tumor suppressor gene locus. Comprehensive genetic lethality studies of MATP- / - cancer cells have shown that these cancer cells have increased sensitivity to inhibition of MAT2A, PRMT5 and PRMT1.

[0004] In hepatocellular carcinoma (HCC), downregulation of MAT1A and upregulation of MAT2A occur, which is called MAT1A:MAT2A switch. The switch accompanied by upregulation of MAT2B leads to lower SAM content, which provides a growth advantage for liver cancer cells. Since MAT2A plays a vital role in promoting the growth of liver cancer cells, it is a target for anti-tumor therapy. Recent studies have shown that silencing by using small interfering RNA substantially inhibits the growth of liver cancer cells and induces apoptosis. See, for example, T. Li et al., J. Cancer 7 (10) (2016) 1317-1327.

[0005] Some MTAP-deficient cancer cell lines are particularly sensitive to inhibition of MAT2A, Marjon et al. (Cell Reports 15(3)(2016) 574–587). MTAP (methylthioadenosine phosphorylase) is an enzyme widely expressed in normal tissues that catalyzes the conversion of methylthioadenosine (MTA) to adenine and 5-methylthioribose-1-phosphate. Adenine is salvaged to produce adenosine monophosphate, and 5-methylthioribose-1-phosphate is converted to methionine and formate. Due to this salvage pathway, MTA can serve as an alternative purine source when de novo purine synthesis is blocked, for example, by antimetabolites such as L-alanosine.

[0006] In other cancers lacking MTAP deletion, including hepatocellular carcinoma and leukemia, MAT2A is dysregulated. J. Cai et al., Cancer Res. 58 (1998) 1444-1450; TS Jani et al., Cell. Res. 19 (2009) 358-369. Silencing MAT2A expression by RNA interference can produce antiproliferative effects in various cancer models, H. Chen et al., Gastroenterology 133 (2007)

[0007] 207-218; Q. Liu et al. Hepatol. Res. 37 (2007) 376-388.

[0008] Many human and mouse malignant cells lack MTAP activity. MTAP deficiency is not only present in tissue culture cells, but also in primary leukemias, gliomas, melanomas, pancreatic cancer, non-small cell lung cancer (NSCLC), bladder cancer, astrocytomas, osteosarcomas, head and neck cancer, myxoid chondrosarcomas, ovarian cancer, endometrial cancer, breast cancer, soft tissue sarcomas, non-Hodgkin lymphomas and mesothelioma. The gene encoding human MTAP is located in region 9p21 on human chromosome 9p. This region also contains the tumor suppressor genes p16INK4A (also known as CDKN2A) and p15INK4B. These genes encode p16 and p15, which are inhibitors of cyclin D-dependent kinases cdk4 and cdk6, respectively.

[0009] Alternatively, the p16INK4A transcript can be an alternative reading frame (ARF) spliced ​​into a transcript encoding p14ARF. p14ARF binds to MDM2 and prevents degradation of p53 (Pomerantz et al. (1998) Cell 92: 713-723). The 9p21 chromosome region is of interest because it is often homozygous deleted in a variety of cancers, including leukemia, NSLC, pancreatic cancer, glioma, melanoma, and mesothelioma. Deletions usually inactivate more than one gene. For example, Cairns et al. ((1995) Nat. Gen. 11: 210-212) reported that after studying more than 500 primary tumors, almost all of the deletions identified in these tumors involved a 170kb region containing MTAP, p14ARF, and P16INK4A. Carson et al. (WO99 / 67634) reported that there is a correlation between the stage of tumor development and the homozygosity loss of the gene encoding MTAP and the gene encoding p16. For example, it is reported that the loss of the MTAP gene rather than p16INK4A indicates cancer in the early stages of development, while the loss of the genes encoding p16 and MTAP indicates that cancer is in a more advanced stage of cancer development. In some osteosarcoma patients, the MTAP gene is present at diagnosis, but is missing at a later time point (Garcia-Castellano et al., Clin. Cancer Res. 8 (3) 2002 782-787).

[0010] WO2018039972 discloses MAT2A enzyme inhibitors for treating cancer; WO2021158792 mentions MAT2A inhibitors for treating autoimmune diseases or inflammatory diseases; WO2018045071 describes MAT2A enzyme inhibitors for treating diseases or conditions mediated by overexpression of MAT2A; CN202080014106.0 also discloses the compound 3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-(pyridin-3-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one for treating MTAP-deficient non-small cell lung cancer (NSCLC) in patients in need; CN114874207A discloses MAT2A inhibitors with a 6-in-6 ring core; CN113999232A discloses polycyclic compounds that inhibit MTAP-deficient cancer cells.

[0011] Finding effective MAT2A inhibitors is an important direction for the current development of tumor targeted drugs. Summary of the invention SUMMARY OF THE INVENTION

[0013] The present invention meets the significant demand for safe and effective compounds and methods for treating, preventing and controlling cancer while reducing or avoiding the toxicity and / or side effects associated with conventional therapies. To solve the above technical problems, the present invention adopts the following technical solutions:

[0014] In one aspect, the present invention provides a compound represented by Formula I and its isomers, or a pharmaceutically acceptable salt thereof,

[0015]

[0016] in:

[0017] R 1 Select from non-existent, C 6 -C 10 Aryl, C 6 -C 10 Aryl is optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 、-CN、C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0018] R 2 Selected from hydrogen, hydroxyl, halogen, -NH 2 、-CN、C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0019] R 3 Select from non-existent, C 6 -C 10 Aryl, optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 、-CN、C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0020] R 4 Selected from hydroxyl, C 6 -C 12 Aryl, C 6 -C 12 Heteroaryl, C 6 -C 12 heterocyclic groups, each of which is optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 , carboxyl, -CN, C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0021] R 5 Selected from C 2 -C 6 -Alkenyl, C 2 -C 6 -cycloalkenyl, C 6 -C 12 Aryl, C 6 -C 12 Heteroaryl, C 6 -C 12 heterocyclic groups, each of which is optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 , carboxyl, -CN, C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0022] R 6 Selected from hydrogen, NR 1a R 1b , NR 1a R 1b Optionally substituted with one or more substituents selected from the group consisting of hydroxy, halogen, -NH 2 , carboxyl, -CN, C 1 -C 6 -alkyl, C 1 -C 6 - alkoxy;

[0023] R 1a , R 1b Each independently selected from hydrogen, C 5 -C 12 Aryl, C 5 -C 12 Heteroaryl, C 5 -C 12 Heterocyclic group.

[0024] Furthermore, the R 1 Selected from absent, methoxyphenyl.

[0025] Furthermore, the R 2 is selected from hydrogen, hydroxyl; R 3 Selected from absent, methoxyphenyl.

[0026] Furthermore, the R 4 Selected from hydroxyl and the following structures:

[0027]

[0028] The group is optionally substituted by one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

[0029] Furthermore, the R 5 Selected from cyclohexene, cyclohexadiene and the following structures:

[0030] The group is optionally substituted by one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

[0031] Furthermore, the R 6 Selected from hydrogen, , the group is optionally substituted by one or more hydroxyl groups, halogen groups, -NH 2 , carboxyl, -CN, C 1 -C 6 -alkyl, C 1 -C 6 -Alkoxy substitution.

[0032] The present invention also provides compounds represented by the following formulas (Ia), (Ib), (Ic) and their isomers, or pharmaceutically acceptable salts thereof:

[0033]

[0034] R 4 , R 5 The definition of is as above.

[0035] The present invention also provides a compound and an isomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is:

[0036]

[0037]

[0038] The present invention also provides a pharmaceutical composition comprising the compounds of formula (I), (Ia), (Ib), (Ic), etc. and their isomers, or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable excipients.

[0039] The present invention also provides the use of the compounds of formula (I), (Ia), (Ib), (Ic), etc. and their isomers, or their pharmaceutically acceptable salts, or the compositions in the preparation of a medicament for treating a disease or condition in a subject suffering from the disease or condition, wherein the disease or condition is mediated by overexpression of MAT2A, the disease or condition is preferably cancer, and the cancer is preferably characterized by reduced or absent expression of the methylthioadenosine phosphorylase (MTAP) gene, absent MTAP gene, or reduced MTAP protein function.

[0040] The inventors have found that this class of compounds is a highly effective MAT2A inhibitor, has extremely strong MAT2A inhibitory activity, and can be used to prepare a method for preventing and / or treating MAT2A inhibition-related indications, including cancers caused by reduced or absent MTAP expression, absent MTAP genes, and reduced MTAP protein function. The present invention is completed based on the above findings. DETAILED DESCRIPTION OF THE INVENTION

[0042] Various aspects and features of the present invention are further described below.

[0043] 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.

[0044] The compounds according to the invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

[0045] 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.

[0046] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring of said event or circumstance.

[0047] The term "substituted" means that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, as long as the valence state of the particular atom is normal and the resulting compound is stable after the substitution. When the substituent is =0, it means that two hydrogen atoms are replaced. When multiple hydrogen atoms are replaced by substituents, each substituent is independent of each other and can be the same substituent or different substituents. Unless otherwise specified, the type and number of substituents can be arbitrary on the basis of chemical realization.

[0048] 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.

[0049] 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.

[0050] As used herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a straight chain or branched chain alkyl group, and which may include its subgroups, for example, referring to "C 1 -C 6 When "alkyl" is present, it may also include C 1 -C 4 Alkyl, C 1 -C 3 Alkyl, C 2 -C 6 Alkyl, C 2 -C 4 Alkyl and the like represent sub-range groups, and specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc. It may be divalent, such as methylene, ethylene.

[0051] Represents a single bond or a double bond.

[0052] The terms "heterocycle", "heterocyclic" or "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, the heteroatom-containing ring preferably having 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 and / 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 fully unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom. As used herein, the terms "heterocycle," "heterocycloalkyl," "heterocyclo," "heterocyclic," and "heterocyclyl" include "heteroaryl" groups, as defined below.

[0053] "Aryl" when used alone or as part of another term refers to a carbocyclic aromatic radical, whether or not fused, having the number of carbon atoms specified, or if no number of carbon atoms is specified, up to 14 carbon atoms, such as C 6 -C 14 -Aryl. Specific aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacene, and the like.

[0054] 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 quinuclidinyl.

[0055] 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 and / 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 condensed 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 condensed 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.

[0056] 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.

[0057] 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 with 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. Specifically including acid (organic acid and inorganic acid) addition salts or base addition salts (including organic bases and inorganic bases).

[0058] 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.

[0059] The cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy and hormone therapy.

[0060] "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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The solvents used in this application can be obtained commercially. The abbreviations used in this application are as follows:

[0066] Table 1: Meaning of abbreviations

[0067]

[0068] Beneficial technical effects

[0069] The inventors found that the compounds of the present invention have good MAT2A inhibitory activity, and the IC50 is less than that of the positive control drug AG270. The present invention provides a class of MAT2A inhibitor compounds with novel structure and strong activity, which have good application prospects in preventing and / or treating indications related to MAT2A inhibition, such as reduction or loss of MTAP expression, loss of MTAP gene, reduction of MTAP protein function, etc. DETAILED DESCRIPTION

[0070] The following embodiments are helpful for those skilled in the art to better understand the technical solution of the present invention, but the protection scope of the present invention includes but is not limited to them.

[0071] 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).

[0072] 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 Brukeravance-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).

[0073] 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 XevoG2-SQtof mass spectrometer.

[0074] 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.

[0075] Example 1: 7-(4-methoxyphenyl)-9-(3-oxoisoindol-5-yl)-7,9-dihydro-8H-purin-8-one (Compound 1)

[0076]

[0077] Step 1: Synthesis of compound 1-1

[0078] Add 2-methyl-5-nitrobenzoic acid methyl ester (40.00g, 205.12mmol, 1.0eq.), N-bromosuccinimide (91.28g, 512.82mmol, 2.50eq) and azobisisobutyronitrile (6.72g, 41.02mmol, 0.2eq) to carbon tetrachloride (200ml) solvent and react at 80°C for 16 hours. After the reaction, add water and dichloromethane to extract, dry the organic phase with anhydrous sodium sulfate, separate and purify with normal phase column, petroleum ether and ethyl acetate system, when the polarity is 12%, the product flows out, and concentrate to obtain yellow solid 2-(bromomethyl)-5-nitrobenzoic acid methyl ester (30.00g, 53.57% yield). LCMS (TOF MS ES + )m / z[M+H] + :274 / 276.

[0079] 1H NMR (400MHz, CDCl3) δ9.00(d,J=2.2Hz,1H),8.34(d,J=2.3Hz,1H),7.96(s,1H),4.97(s,2H),1.57(s,3H).

[0080] Step 2: Synthesis of compound 1-2

[0081] Add 2-(bromomethyl)-5-nitrobenzoic acid methyl ester (27.3g, 100.0mmol, 1.0eq) to ammonia-methanol (500mL, 7mol / L) solution and react at room temperature for 16 hours. After the reaction is completed, the reaction solution is concentrated, ethyl acetate (100mL) is added and stirred for 10 minutes, filtered, and the filter cake is washed twice with ethyl acetate (50mL). The solid is collected to obtain a yellow solid 6-nitroisosindoline-1-one (13.0g, 73.00% yield). LCMS (TOF MS ES + )m / z[M+H] + :179.04.

[0082] Step 3: Synthesis of Compound 1-3

[0083] Compound 6-nitroisosindoline-1-one (13.0 g, 73.03 mmol, 1.0 eq.), di-tert-butyl dicarbonate (31.80 g, 146.06 mmol, 2.0 eq.) and triethylamine (22.17 g, 219.12 mmol, 3.0 eq.) were added to a dichloromethane (250 ml) solution. Under ice bath conditions, 4-dimethylaminopyridine (1.78 g, 14.60 mmol, 0.2 eq.) was slowly added and reacted at room temperature for 16 hours. After the reaction was completed, the reaction solution was added to water, extracted three times with dichloromethane (100 ml), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The product was separated and purified by normal phase column, petroleum ether and ethyl acetate system, and the polarity was 25%, and the product flowed out. The product was concentrated to obtain a yellow solid 6-nitro-1-oxoisoindole-2-carboxylic acid tert-butyl ester (9.0 g, 44.34%). LCMS (TOF MS ES + )m / z[M+H] + :279.04. 1 H NMR (400MHz, DMSO) δ8.55(dd,J=8.4,2.2Hz,1H),8.42(d,J=2.1Hz,1H),7.94(d,J=8.4Hz,1H),4.93(s,2H),1.53(s,9H).

[0084] Step 4: Synthesis of Compound 1-4

[0085] The compound 6-nitro-1-oxoisoindole-2-carboxylic acid tert-butyl ester (9.0 g, 32.37 mmol, 1.0 eq.) was added to a methanol solution (200 mL), followed by the addition of Pd / C (1.10 g), and the reaction was carried out at room temperature under hydrogen protection for 16 hours. After the reaction was completed, diatomaceous earth was quickly filtered, and the filter cake was washed with methanol. The filtrate was concentrated to obtain a crude product 6-amino-1-oxoisoindole-2-carboxylic acid tert-butyl ester (7.80 g, 97.0% yield), which was directly used for the next step reaction. LCMS (TOF MS ES + )m / z[M+H] + :249.12.

[0086] Step 5: Synthesis of Compound 1-5

[0087] Add tert-butyl 6-amino-1-oxoisoindole-2-carboxylate (7.8 g, 31.40 mmol, 1.0 eq.), 4,6-dichloro-5-nitropyrimidine (9.15 g, 47.17 mmol, 1.50 eq.) and triethylamine (9.54 g, 94.35 mmol, 3.00 eq.) to tetrahydrofuran (120 mL) solution and react at room temperature for 16 hours. After the reaction was completed, the reaction solution was added to water, extracted three times with dichloromethane (100 ml), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The product was separated and purified by a normal phase column. When the polarity of the dichloromethane and ethyl acetate system was 10%, the product flowed out and concentrated to obtain a yellow solid 6-[(6-chloro-5-nitropyrimidin-4-yl)amino]-1-oxoisoindole-2-carboxylic acid tert-butyl ester (7.8 g, 61.33% yield). LCMS (TOF MS ES + )m / z[M+H] + :406.08. 1 H NMR (400 MHz, DMSO-d 6 )δ10.38(s,1H),8.60(s,1H),7.84(s,1H),7.74(d,J=9.3Hz,1H),7.67(d,J=8.5Hz,1H),4.76(s,2H),1.49(s,9H).

[0088] Step 6: Synthesis of Compound 1-6

[0089] 6-[(6-chloro-5-nitropyrimidin-4-yl)amino]-1-oxoisoindole-2-carboxylic acid tert-butyl ester (7.8 g, 19.26 mmol, 1.0 eq.) was added to a methanol solution (200 mL), followed by the addition of Pd / C (1.10 g), and the reaction was carried out at room temperature under hydrogen protection for 40 hours. After the reaction was completed, diatomaceous earth was quickly filtered, and the filter cake was washed with dichloromethane / methanol (10:1). After the filtrate was concentrated, it was separated and purified by a normal phase column. When the polarity of the dichloromethane and dichloromethane / methanol (10:1) system was 80%, the product flowed out and was concentrated to obtain a brown solid 6-[(5-aminopyrimidin-4-yl)amino]-1-oxoisoindole-2-carboxylic acid tert-butyl ester (1.2 g, 18.27% yield). LCMS (TOF MS ES + )m / z[M+H] + :342.15. 1 H NMR (400 MHz, DMSO-d 6)δ8.74(s,1H),8.19(d,J=1.8Hz,1H),8.16(s,1H),7.93(s,1H),7.80(dd,J=8 .5,1.9Hz,1H),7.71(d,J=8.5Hz,1H),5.34(s,2H),4.79(s,2H),1.54(s,9H).

[0090] Step 7: Synthesis of Compound 1-7

[0091] 6-[(5-aminopyrimidin-4-yl)amino]-1-oxoisoindole-2-carboxylic acid tert-butyl ester (200 mg, 0.58 mmol, 1.0 eq.) was added to a dichloromethane (5 mL) solution, and triethylamine (120.0 mg, 1.17 mmol, 2.0 eq) and BTC (260 mg, 0.87 mmol, 1.50 eq) in dichloromethane were added in sequence at 0°C. The mixture was reacted at 0°C for 1 hour under nitrogen protection. After the reaction was completed, the mixture was purified by column to obtain a white solid 1-oxo-6-(8-oxo-7,8-dihydro-9H-purine-9-yl)isoindole-2-carboxylic acid tert-butyl ester (280 mg, crude product). It was used directly in the next step. LCMS (TOF MS ES+) m / z[M+H]+: 368.13 / 312.13.

[0092] Step 8: Synthesis of compound 1

[0093] 1-Oxo-6-(8-oxo-7,8-dihydro-9H-purine-9-yl)isoindole-2-carboxylic acid tert-butyl ester (280 mg, 0.76 mmol, 1.0 eq.), 4-methoxyphenylboronic acid (695.4 mg, 4.58 mmol, 6.0 eq.), anhydrous copper acetate (206.4 mg, 1.14 mmol, 1.5 eq.) and anhydrous pyridine (180.4 mg, 2.28 mmol, 3.0 eq.) were added to anhydrous N,N-dimethylformamide (8 mL) solution and reacted at room temperature for 16 hours under oxygen protection. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with methanol, and ethyl acetate (50 mL) was added after the filtrate was concentrated, and then washed with water and saturated brine, respectively, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated. Prep-HPLC prepared white solid 7-(4-methoxyphenyl)-9-(3-oxoisoindol-5-yl)-7,9-dihydro-8H-purin-8-one (13.4 mg, 10.17%). LCMS (TOF MS ES + )m / z[M+H] + :374.13. 1 H NMR (400 MHz, DMSO-d 6)δ8.71(d,J=5.3Hz,1H),8.36(s,1H),7.95(s,1H),7.92–7.81(m,2H),7.65–7.56(m,2H),7.22–7.11(m,2H),4.49(s,2H),3.85(s,3H).

[0094] Example 2: 9-(cyclohexa-1,5-dien-1-yl)-1-(4-methoxyphenyl)-8-phenyl-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (Compound 2)

[0095]

[0096] Step 1: Synthesis of compound 2-1

[0097]

[0098] 2,6-dichloropurine (21.77g, 115.0mmol, 1.0eq), cyclohexene-1-boric acid (17.28g, 138mmol, 1.2eq), anhydrous copper acetate (25.0g, 138.0mmol, 1.2eq), triethylamine (58.3g, 576.0mmol, 5.0eq) were dissolved in 500mL dichloromethane solution, stirred at room temperature for 2 hours under air protection. After the reaction was complete, diatomaceous earth was filtered and the filtrate was concentrated. Normal phase column separation, petroleum ether and ethyl acetate system, polarity of 40% to obtain 2,6-dichloro-9-cyclohexyl-9H-purine (3.3g, 10.71% yield), 1 H NMR (400 MHz, DMSO-d 6 )δ8.79(s,1H),6.28(s,1H),2.56(s,2H),2.23(m,J=6.6Hz,2H),1.82–1.74(m,2H),1.64(m,J=6.3Hz,2H).

[0099] Step 2: Synthesis of compound 2-2

[0100]

[0101] 2,6-dichloro-9-cyclohexyl-9H-purine (2.7 g, 10.0 mmol, 1.0 eq) was dissolved in tetrahydrofuran (10 mL), and then sodium hydroxide aqueous solution (1.0 g, 25.0 mmol, 12.5 eq, 30 wt.%) was added, and the reaction was carried out at room temperature for 16 hours. After the reaction was complete, 20 mL of ethyl acetate was added, the organic layer was separated, filtered, washed with ethyl acetate, and the solid was collected to obtain 2-chloro-9-cyclohexyl-1,-dihydropurine-6-one (1.46 g, 58.20% yield). 1 H NMR (400 MHz, DMSO-d 6 )δ7.74(s,1H),6.20–6.13(m,1H),2.55(t,J=3.4Hz,2),2.20(m,J=6.6,3.4Hz,2H),1.81–1.75(m,2H),1.65–1.61(m,2H).

[0102] Step 3: Synthesis of compound 2-3

[0103]

[0104] 2-Chloro-9-cyclohexyl-1,-dihydropurine-6-one (1.46 g, 5.82 mmol, 1.0 eq), 2-aminopyridine (1.10 g, 11.68 mmol, 2.0 eq), Xant-Phos (1.01 g, 1.75 mmol, 0.3 eq), palladium acetate (260.7 mg, 1.16 mmol, 0.2 eq), and cesium carbonate (3.77 g, 11.60 mmol, 2.0 eq) were dissolved in 1,4-dioxane solution (50 mL) and reacted at 120 ° C under nitrogen protection for 16 hours. After the reaction was complete, the mixture was filtered through diatomaceous earth, the filtrate was concentrated, and the mixture was separated by a normal phase column (dichloromethane / (dichloromethane:methanol (10:1)) system. When the polarity was 60%, 9-cyclohexyl-1-en-1-yl-2-pyridine-2-amino-1,9-dihydro-6H-purine-6-one (840 mg, 47.42% yield) was obtained. LCMS (ESI) [M+H]+: 309.14. 1 H NMR (400 MHz, DMSO-d 6)δ13.39(d,J=6.9Hz,1H),10.69(d,J=3.2Hz,1H),8.36(dd,J=5.3,1.7Hz,1H),7.93(s,1H),7.87–7.79(m,1H),7.28(d,J=8.5Hz,1H),7.10(dd, J=7.2,5.2Hz,1H),6.18(td,J=3.9,1.9Hz,1H),2.57(td,J=6.0,2.3Hz,2H),2.23(dq,J=6.6,3.5Hz,2H),1.85–1.79(m,2H),1.68–1.62(m,2H).

[0105] Step 4: Synthesis of compound 2-4

[0106]

[0107] 9-cyclohexyl-1-en-1-yl-2-pyridin-2-amino-1,9-dihydro-6H-purine-6-one (840 mg, 2.76 mmol, 1.0 eq), p-methoxyphenylboronic acid (4.2 g, 27.6 mmol, 10.0 eq), anhydrous copper acetate (749.3 mg, 4.14 mmol, 1.5 eq) were dissolved in anhydrous DMF (25 ml), followed by the addition of anhydrous pyridine (872.0 mg, 11.04 mmol, 4.0 eq), and reacted at room temperature for 16 hours under oxygen protection. After the reaction was complete, the mixture was filtered, the filter cake was washed with methanol, and the solid was collected to obtain 9-cyclohexyl-1-en-1-yl-1-(4-methoxyphenyl)-2-pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (660 mg, 57.97% yield). 1 H NMR (400 MHz, DMSO-d 6 )δ9.50(s,1H),8.28(s,1H),8.21(s,1H),7.71(s,1H),7.35(d,J=8.3Hz,1H),7.29–7.21(m,2H),7.09–6.99(m,2H),6.89(s ,1H),6.37(s,1H),3.82(d,J=2.2Hz,2H),2.66(d,J=7.2Hz,2H),2.26(s,2H),1.81(q,J=6.0Hz,2H),1.66(t,J=6.1Hz,2H).

[0108] Step 5: Synthesis of Compound 2-5

[0109]

[0110] N-bromosuccinimide (859.9 mg, 4.83 mmol, 4.0 eq) was added to a dichloromethane (5 mL) solution of 9-cyclohexyl-1-en-1-yl-1-(4-methoxyphenyl)-2-pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (500.0 mg, 1.21 mmol, 1.0 eq) and reacted at room temperature for 16 hours. After the reaction was complete, the reaction solution was concentrated and separated by a normal phase column. The petroleum ether and ethyl acetate system was used. When the polarity was 35%, 8-bromo-9-(3-bromocyclohexyl-1-en-1-yl)-1-(4-methoxyphenyl)-2-pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (250 mg, 36.12% yield) was obtained. LCMS (ESI) [M+H] +: 573.0. 1 H NMR (400 MHz, DMSO-d 6 )δ9.97(s,1H),8.35–8.24(m,2H),7.60(d,J=9.0Hz,1H),7.48(dd,J=8.8,2.6Hz,1H),7.29(d,J=8.7Hz,2H),7.08(d,J =8.4Hz,2H),6.46(t,J=4.1Hz,1H),6.02(s,1H),3.83(s,3H),2.34(m,J=10.8Hz,2H),2.56-2.70(m,2H),1.85(m,2H).

[0111] Step 6: Synthesis of compound 2

[0112]

[0113] 8-Bromo-9-(3-bromocyclohexyl-1-en-1-yl)-1-(4-methoxyphenyl)-2-pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (220.0 mg, 0.43 mmol, 1.0 eq), phenylboronic acid (93.8 mg, 0.77 mmol, 2.0 eq), Pd(dppf)Cl 2 (124.1mg, 0.15mmol, 0.4eq), potassium phosphate (161.0mg, 0.77mmol, 2.0eq) were added to a mixed solvent of 1,4-dioxane (3.0mL) and water (0.3mL), and reacted at 100°C under nitrogen protection for 2 hours. After the reaction was complete, diatomaceous earth was filtered, the filtrate was concentrated, and ethyl acetate was added for dilution, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and finally the filtrate was concentrated. Normal phase column separation, petroleum ether and ethyl acetate system, when the polarity was 60%, a crude product (80mg) was obtained, and a white solid (5mg) was obtained by preparative separation. LCMS (ESI) [M+H] +: 489.20.1 H NMR (400 MHz, DMSO-d 6 )δ9.69(s,1H),8.53(d,J=2.5Hz,1H),8.36(s,1H),7.86(d,J=8.8Hz,1H),7.70–7.62(m,3H),7.49(t,J=7.6Hz,2H),7.38(s,1H),7.31( d,J=9.0Hz,1H),7.10(d,J=9.0Hz,2H),6.16-6.22(m,1H),6.35-6.40(t,1H),6.43-6.49(dd,1H),2.35–2.26(m,2H),2.43-2.48(m,2H).

[0114] The compounds in Table 2 were obtained by referring to the method of Example 1:

[0115] Table 2: Structures and characterization of compounds 3 to 7

[0116]

[0117]

[0118] The compounds in Table 3 were obtained by referring to the method of Example 2:

[0119] Table 3: Structures and characterization of compounds 8-29, 32-35

[0120]

[0121]

[0122]

[0123]

[0124] Example 30: 9-(Cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (Compound 30)

[0125]

[0126] Step 1: Synthesis of compound 30-1

[0127] 1-Bromo-2-fluoro-3-nitrobenzene (5 g, 23.5 mmol, 1.0 eq), sodium carbonate (4.982 g, 47 mmol, 2.0 eq) and methylamine hydrochloride (1.904 g, 28.2 mmol, 1.7 eq) were added to 15 mL of dimethylformamide solvent and reacted at room temperature for 16 h. After the reaction was completed, the reaction solution was concentrated and chromatographed by normal phase column (ethyl acetate 5%) to obtain red liquid 2-bromo-N-methyl-6-nitroaniline (5.09 g, yield 97.4%). LCMS (ESI) [M+H] + :231 1 H NMR (400MHz, DMSO) δ7.76 (ddd, J=9.8, 8.0, 1.5Hz, 2H), 6.70 (t, J=8.0Hz, 1H), 6.33 (s, 1H), 2.70 (d, J=3.0Hz, 3H).

[0128] Step 2: Synthesis of compound 30-2

[0129] The compound 2-bromo-N-methyl-6-nitroaniline (2.0 g, 8.70 mmol, 1.0 eq) was added to a glacial acetic acid (20 mL) solution, the temperature was raised to 50°C, reduced iron powder (1.46 g, 26.1 mmol, 3 eq) was added in batches, and the reaction was maintained at 50°C for 1 h. After the reaction was completed, the solid residue was filtered off and reverse phase column chromatography (25% acetonitrile) was performed to obtain a yellow oily substance 6-bromo-N-methylbenzene-1,2-diamine (510.0 mg, yield 14.1%). LCMS (ESI) [M+H] + :201

[0130] Step 3: Synthesis of compound 30-3

[0131] 6-Bromo-N-methylbenzene-1,2-diamine (510 mg, 2.55 mmol, 1.0 eq) and p-toluenesulfonic acid monohydrate (51.0 mg, 0.296 mmol, 0.12 eq) were dissolved in triethyl orthoformate (1.5 mL) and reacted at 85°C for 1 h. After the reaction was complete, the reaction solution was concentrated and subjected to reverse phase column chromatography (acetonitrile 15%) to obtain a white solid 7-bromo-1-methyl-1H-benzo[d]imidazole (480.0 mg, yield 89.6%). LCMS (ESI) [M+H] + :211 1 H NMR (400MHz, DMSO) δ8.28 (s, 1H), 7.68 (d, J = 8.1Hz, 1H), 7.46 (d, J = 7.7Hz, 1H), 7.15 (td, J = 8.0, 1.4Hz, 1H), 4.10 (d, J = 1.4Hz, 3H).

[0132] Step 4: Synthesis of compound 30-4

[0133] 7-Bromo-1-methyl-1H-benzo[d]imidazole (480.0 mg, 2.29 mmol, 1.0 eq), 4,4,4',4',5,5',5',2'-octamethyl-2'-bis(1,3,2-dioxaborolane) (1.74 g, 6.87 mmol, 3.0 eq), Pd(dppf)Cl 2 (84.1 mg, 0.115 mmol, 0.05 eq) and potassium acetate (673.3 mg, 6.87 mmol, 3 eq) were dissolved in 1,4-dioxane solution (10 mL) and reacted at 100 ° C for 4 hours under nitrogen protection. After the reaction was complete, the filtrate was filtered and concentrated, and the filtrate was purified by normal phase column chromatography (ethyl acetate 25%) to obtain a brown solid 1-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (375 mg, yield 63.6%) LCMS (ESI) [M+H] + :259.

[0134] Step 5: Synthesis of compound 30-5

[0135] Compound (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (375 mg, 1.45 mmol, 1.0 eq), 8-bromo-2-chloro-9-(cyclohex-1-en-1-yl)-9H-purin-6-ol (477.1 mg, 1.45 mmol, 1.0 eq), Pd(dppf)Cl 2 (53.0 mg, 0.0725 mmol, 0.05 eq.) and Cs 2 CO 3 (1.43g, 4.35mmol, 3eq) was added to a mixed solution of 1,4-dioxane (4mL) and water (1mL), and stirred at 80°C for 2h under nitrogen protection. After the reaction was completed, reverse phase column chromatography (50% acetonitrile) was performed to obtain a brown solid 2-chloro-9-(cyclohex-1-en-1-yl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-1,9-dihydro-6H-purine-6-one (213mg, yield 38.7%). LCMS (ESI) [M+H] + :381.

[0136] Step 6: Synthesis of compound 30-6

[0137] Compound 2-chloro-9-(cyclohex-1-en-1-yl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-1,9-dihydro-6H-purin-6-one (213 mg, 0.56 mmol, 1.0 eq), 2-aminopyridine (79.0 mg, 0.84 mmol, 1.5 eq), Pd 2 (dba) 3 (25.6 mg, 0.028 mmol, 0.05 eq.) and Cs 2 CO 3 (551.0 mg, 1.68 mmol, 3 eq) was added to a 1,4-dioxane (5 mL) solution and stirred at 80°C for 4 h under nitrogen protection. After the reaction was completed, reverse phase column chromatography (60% acetonitrile) was performed to obtain a yellow solid 9-(cyclohex-1-en-1-yl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (122 mg, yield 49.7%)

[0138] Step 7: Synthesis of compound 30

[0139] Compound 9-(cyclohex-1-en-1-yl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (122.0 mg, 0.28 mmol, 1.0 eq), 4-methoxyphenylboronic acid (85.1 mg, 0.56 mmol, 2 eq), copper acetate (76.4 mg, 0.42 mmol, 1.5 eq.) and pyridine (66.4 mg, 0.84 mmol, 3 eq) were added to a DMF (5 mL) solution and the reaction was stirred at room temperature under oxygen protection for 16 h. After the reaction was completed, reverse phase column chromatography (60% acetonitrile) was performed to obtain a white solid 9-(cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-8-(1-methyl-1H-benzo[d]imidazol-7-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (18.5 mg, yield 12.2%). LCMS (ESI) [M+H] + :545.23.

[0140] Example 31: 8-(6-aminopyridin-3-yl)-9-(cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (Compound 31)

[0141]

[0142] Step 1: Synthesis of compound 31-1

[0143] Add 2,6-dichloro-9H-urea (60g, 317.46mmol, 1.0eq) and p-toluenesulfonic acid monohydrate (6.039g, 476.19mmol, 1.5eq) to 500mL of ultra-dry dichloromethane solvent, then slowly add 3,4-dihydro-2H-pyran (40.05g, 476.19mmol, 1.5eq) dissolved in 100ml of ultra-dry dichloromethane solvent while stirring, and react at room temperature for 4h. After the reaction is completed, filter the reaction liquid, wash the filter cake with dichloromethane, add 300ml of 10% sodium carbonate solution for extraction, collect the organic phase, add water for extraction, dry the extracted organic layer with anhydrous sodium sulfate, and concentrate the organic phase to a solid after drying. The mixture was slurried with a solvent of ethyl acetate: petroleum ether = 1:12 to obtain a white solid 2,6-dichloro-9-(tetrahydro-2H-pyran-2-ene)-9H-purine (57.4 g, yield 86.8%). LCMS (ESI) [M+H] +: 273 1 H NMR(400MHz, DMSO)δ8.95(s,1H),5.74(dd,J=10.8,2.3Hz,1H),4.06–3.97(m,1H),3.81–3.69(m,1H),2.26(tdd ,J=13.3,10.8,4.4Hz,1H),1.99(tt,J=13.2,2.8Hz,2H),1.84–1.69(m,1H),1.59(ddt,J=12.2,8.2,3.7Hz,2H).

[0144] Step 2: Synthesis of compound 31-2

[0145] Under nitrogen protection, 2,6-dichloro-9-(tetrahydro-2H-pyran-2-ene)-9H-purine (5g, 18.38mmol, 1.0eq) was dissolved in 50ml of ultra-dry tetrahydrofuran solvent and cooled to -78°C under dry ice ethanol conditions, then 2mol / L lithium diisopropylamide (13.78ml, 27.57mmol, 1.5eq) was slowly added dropwise, and stirred for 30min after addition. Then 1,2-dibromo-1,1,2,2-tetrachloroethane (8.98g, 27.57mmol, 1.5eq) dissolved in 15ml of ultra-dry tetrahydrofuran was slowly added dropwise, and the reaction was continued at -78°C for 1.5h after addition. After the reaction was complete, 10ml of saturated ammonium chloride solution was slowly added to quench the reaction solution, and the temperature did not exceed -30°C. The reaction solution was concentrated and purified by normal phase column chromatography (ethyl acetate 10%) to obtain a yellow solid 8-bromo-2,6-dichloro-9-(tetrahydro-2H-pyran-2-ene)-9H-purine (2.2554 g, yield 35.1%). LCMS (ESI) [M+H] +: 351 1H NMR (400MHz, DMSO) δ5.78–5.67(m,1H),4.66(dd,J=6.5,2.6Hz,1H),3.91–3.68(m,1H),3.36(ddd,J=11.4,7 .9,3.6Hz,1H),2.06–1.90(m,1H),1.81–1.69(m,1H),1.60(ddp,J=15.8,6.7,3.5Hz,1H),1.49–1.25(m,2H).

[0146] Step 3: Synthesis of compound 31-3

[0147] Dissolve 8-bromo-2,6-dichloro-9-(tetrahydro-2H-pyran-2-ene)-9H-purine (1g, 2.9mmol, 1.0eq) in dichloromethane solution (10mL), then add 2.5ml trifluoroacetic acid under ice bath condition, and react for 30min at room temperature. After the reaction is complete, concentrate the reaction solution, dissolve it with methanol, and add triethylamine to clarify the solution. Reverse phase column chromatography gives a light yellow solid 8-bromo-2,6-dichloro-9H-urea (616mg, yield 81.1%). LCMS (ESI) [M+H] + :267

[0148] Step 4: Synthesis of compound 31-4

[0149] 8-Bromo-2,6-dichloro-9H-urea (616 mg, 2.31 mmol, 1.0 eq), cyclohexene-1-ylboronic acid (873.2 mg, 6.93 mmol, 3.0 eq), pyridine (547.5 mg, 6.93 mmol, 3 eq), and copper acetate (627.2 mg, 3.47 mmol, 1.5 eq) were dissolved in 1,4-dioxane solution (10 mL) and reacted at room temperature for 16 hours under oxygen atmosphere. After the reaction was complete, the mixture was filtered and the mother liquor was chromatographed by reverse phase column (acetonitrile 40%) to obtain yellow solid 8-bromo-2-chloro-9-(cyclohex-1-en-1-yl)-9H-purin-6-ol (260 mg, yield 34.2%) LCMS (ESI) [M+H]+: 329.

[0150] Step 5: Synthesis of compound 2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)pyridine

[0151] 5-Bromo-2-nitropyridine (1 g, 4.95 mmol, 1.0 eq.), 4,4,4',4',5,5',5',2'-octamethyl-2'-bis(1,3,2-dioxaborolane) (1.56 g, 6.14 mmol, 1.24 eq.), Pd(dppf)Cl 2(182 mg, 0.25 mmol, 0.05 eq.) and potassium acetate (1.44 g, 24.8 mmol, 5.0 eq.) were dissolved in dioxane (20 mL), N 2 After the reaction was complete, the reaction solution was filtered, washed with ethyl acetate, and the mother liquor was retained. After being spin-dried, it was purified by reverse phase column, and water (1‰ formic acid solution) and acetonitrile system were passed through the column; 50%; the product was discharged after 15 minutes to obtain the target product of yellow solid 2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)pyridine (707mg, yield 56%). LCMS (ESI) [M+H] + :169. 1 H NMR (400MHz, DMSO-d6) δ8.81(s,1H),8.42(d,J=7.9Hz,1H),8.30(d,J=7.9Hz,1H),1.34(d,J=1.5Hz,12H)

[0152] Step 6: Synthesis of compound 31-5

[0153] Compound 8-bromo-2-chloro-9-(cyclohex-1-en-1-yl)-9H-purin-6-ol (260 mg, 0.79 mmol, 1.0 eq), 2-nitro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborol-2-yl)pyridine (200.2 mg, 1.19 mmol, 1.5 eq), Pd(dppf)Cl 2 (28.9 mg, 0.0395 mmol, 0.05 eq.) and Cs 2 CO 3 (777.4 mg, 2.37 mmol, 3 eq) was added to a mixed solution of 1,4-dioxane (4 mL) and water (1 mL), and stirred at 80°C for 2 h under nitrogen protection. After the reaction was completed, reverse phase column chromatography (50% acetonitrile) was performed to obtain a brown solid 2-chloro-9-(cyclohex-1-ene-1-yl)-8-(6-nitropyridin-3-yl)-9H-purin-6-ol (190 mg, yield 64.4%). LCMS (ESI) [M+H] +: 373

[0154] Step 7: Synthesis of compound 31-6

[0155] Compound 2-chloro-9-(cyclohex-1-en-1-yl)-8-(6-nitropyridin-3-yl)-9H-purin-6-ol (190 mg, 0.51 mmol, 1.0 eq), 2-aminopyridine (71.9 mg, 0.77 mmol, 1.5 eq), Pd 2 (dba) 3(23.3 mg, 0.0225 mmol, 0.05 eq.) and Cs 2 CO 3 (501.8 mg, 1.53 mmol, 3 eq) was added to a 1,4-dioxane (5 mL) solution and stirred at 80°C for 4 h under nitrogen protection. After the reaction was completed, reverse phase column chromatography (60% acetonitrile) was performed to obtain a yellow solid 9-(cyclohex-1-ene-1-yl)-8-(6-nitropyridin-3-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (155 mg, yield 70.5%). LCMS (ESI) [M+H]+: 431

[0156] Step 8: Synthesis of compound 31-7

[0157] Compound 9-(cyclohex-1-en-1-yl)-8-(6-nitropyridin-3-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (155 mg, 0.36 mmol, 1.0 eq), 4-methoxyphenylboronic acid (109.4 mg, 0.72 mmol, 2 eq), copper acetate (98.3 mg, 0.54 mmol, 1.5 eq.) and pyridine (85.3 mg, 1.08 mmol, 3 eq) were added to DMF (5 mL) solution and the reaction was stirred at room temperature for 16 h under oxygen protection. After the reaction was completed, reverse phase column chromatography (60% acetonitrile) was performed to obtain a yellow solid 9-(cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-8-(6-nitropyridin-3-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (90.0 mg, yield 46.6%). LCMS (ESI) [M+H]+: 537.

[0158] Step 9: Synthesis of compound 31

[0159] The compound 9-(cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-8-(6-nitropyridin-3-yl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purine-6-one (90.0 mg, 0.168 mmol, 1.0 eq) was added to a glacial acetic acid (5 mL) solution, the temperature was raised to 50°C, reduced iron powder (47.1 mg, 0.84 mmol, 5 eq) was added in batches, and the reaction was maintained at 50°C for 1 h. After the reaction was completed, the solid residue was filtered off and subjected to reverse phase column chromatography (60% acetonitrile) to obtain a white solid 8-(6-aminopyridin-3-yl)-9-(cyclohex-1-en-1-yl)-1-(4-methoxyphenyl)-2-(pyridin-2-ylamino)-1,9-dihydro-6H-purin-6-one (12.0 mg, yield 14.1%). LCMS (ESI) [M+H]+: 507.2.

[0160] The compounds in Table 4 were obtained by referring to the method of Example 31:

[0161] Table 4: Structure and characterization of compounds 36 and 37

[0162]

[0163] The preparation method of compound 36 is the same as that of Example 31, with an additional halogenation reaction step.

[0164] Experimental Example 1: Binding ability test of the compound of the present invention to mat2a protein

[0165] Experimental purpose: To detect the binding ability of the compound to MAT2A protein by CETSA experimental method

[0166] Background principle: The 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. Use candidate drugs to treat cell or tissue samples. If the candidate drug is an inhibitor of MAT2A, then the candidate drug can bind to MAT2A, making the MAT2A protein more stable. After heating the sample, the MAT2A protein in the sample will be more easily detected by specific antibodies, and the MAT2A protein will be easier to detect through Western blot experiments; conversely, the stability of the MAT2A 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 MAT2A protein inhibitors.

[0167] Specific experimental process:

[0168] 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 buffer 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 of electroporation were set at 250 mA for 2 hours; 5% BSA was blocked for 1 hour; specific antibody 1 was added and incubated overnight on a shaker at 4°C; TBST was washed 4 times, 2.5 minutes each time; 2 antibodies were incubated on a shaker at room temperature for 1 hour; TBST was washed 4 times, 2.5 minutes each time; ECL was used for development to detect the expression of TYK2 protein in different groups and at various temperature points. Western blot bands were converted and processed by image J and GraphPad software, and EC50 was calculated.

[0169] EC50 is the concentration for 50% of maximal effect (EC50), which refers to the drug concentration that can cause 50% of individuals to be effective. AG270 (Compound 153 reported in CN201780066270.4) of Angios Pharmaceuticals Co., Ltd. was used as a positive reference compound.

[0170] The EC50 values ​​of each compound are classified according to the following description:

[0171] “+” indicates that the EC50 value is greater than 100 μM;

[0172] “++” indicates that the EC50 value is less than 100 μM and greater than 10 μM;

[0173] "+++" indicates that the EC50 value is less than 10 μM.

[0174] Table 5: EC50 results

[0175]

[0176]

[0177] EC50 experimental data show that the compounds of the present invention have good binding ability with MAT2A protein. Compared with the positive control drug AG270 (prepared according to the preparation method of compound 153 reported in CN201780066270.4), the compounds of the present invention have comparable or even stronger binding ability with MAT2A protein.

[0178] Experimental Example 2: Effect of the compounds of the present invention on HCT116 MTAP - / - Cell proliferation assay

[0179] Experimental purpose: The purpose of this test case is to test the effect of compounds on HCT116 MTAP - / - Cell proliferation effect.

[0180] Background: Methionine adenosyltransferase 2A (MAT2A) is considered a synthetic lethal target in cancers that lack the methylthioadenosine phosphorylase (MTAP) gene. The MTAP gene is adjacent to the CDKN2A tumor suppressor and is co-deleted with CDKN2A in approximately 15% of cancers. Therefore, the effects of compounds on HCT116 MTAP were tested. - / - The inhibition rate of cell proliferation is used for screening of MAT2A protein inhibitors.

[0181] Specific experimental process:

[0182] 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. On the second day, 10μl of compounds of different concentrations (DMSO final concentration was 1%) were added and incubated at 37°C for 10 days. On the 10th day, the old culture medium was removed, 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.

[0183] IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It indicates the half amount of a drug or substance (inhibitor) in inhibiting a certain biological process (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). AG270 from Angios Pharmaceuticals Co., Ltd. was used as a positive reference compound.

[0184] The test results are shown in Table 6 below, where the IC50 values ​​of each compound are classified according to the following description:

[0185] +++ less than 100 nM, ++ between 100 nM and 10 μM, + greater than 10 μM.

[0186] The test results are shown in Table 6 below:

[0187] Table 6: IC50 experimental data

[0188]

[0189]

[0190] The results show that the compounds of the present invention can inhibit HCT116 MTAP-null cells, and the IC50 value reaches nM level, which is equivalent to or less than the positive control drug AG270. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to MAT2A inhibition.

[0191] Experimental Example 3: Determination of the effect of the compounds of the present invention on the function of MAT2A protease

[0192] Experimental purpose: The purpose of this test case is to test the ability of the compound to inhibit the function of MAT2A protease.

[0193] Background Principle: The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) plays an important role in metabolism and epigenetics because it is the main producer of the universal methyl donor s-adenosylmethionine (SAM). ATP and L-Met generate SAM and phosphate groups under the action of MAT2A. Therefore, after compound incubation, the ability of the compound to inhibit the enzyme function of MAT2A is evaluated by detecting the amount of SAM generated, which is used for the screening of MAT2A protein inhibitors.

[0194] Specific experimental process:

[0195] MAT2A protein expression: Full-length MAT2A was cloned into the pET24N vector with an n-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site. The constructed vector was transformed into Escherichia coli BL21 (DE3), and the cells were shaken to an OD of 0.6, and 1mM IPTG was added and cultured at 18°C ​​for 16h. The cells were collected, ultrasonically disrupted, and the supernatant was taken by centrifugation. The protein was purified by Ni-NTA, and the protein concentration and purity were determined after dialysis. SAM assay: Reaction system: add 91-x ul of 50mM TrisHCl pH 7.5, 1.5uL (10 / 3M) of KCl, 1.5uL (1M) of MgCl2, 1uL (100mM) of ATP, 1uL (80mM) of L-Met, 1uL (30Mm PH7.67) of EDTA, 1uL 5% BSA, 2uL of the above DMSO-dissolved drugs, x uL of MAT2A protein. The experimental groups were prepared with different drug concentrations, 2uL (100x) was taken out and added to the reaction system, reacted at 37°C for 18h, and solvent control group and blank control group were set up respectively. The reaction was terminated, 40uL of the system was taken out, and 4uL 10% SDS was added to quench the reaction. IC50 was calculated by GraphPad software, and the compounds were screened by comparing with positive drugs.

[0196] The test results are shown in Table 7 below, where the IC50 values ​​of each compound are classified according to the following description:

[0197] +++ is less than 10 nM, ++ is between 10 nM and 1 μM, and + is greater than 1 μM.

[0198] The test results are shown in Table 7 below:

[0199] Table 7: IC50 experimental data

[0200]

[0201]

[0202] The results show that the compounds of the present invention have a very strong inhibitory effect on the protease function of MAT2A, and the IC50 values ​​all reach the nM level, which is lower than the positive control drug AG270. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to MAT2A inhibition.

[0203] The above examples are only representative. It can be seen from the above examples that the compounds of the present invention are ideal and highly effective MAT2A inhibitors, and can be expected to be used for treating or preventing symptoms or diseases related to MAT2A inhibition.

[0204] 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 the following formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof, The R4 is selected from the following groups: The group is optionally substituted by one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

2. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is:

3. A pharmaceutical composition comprising the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

4. Use of the compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, or the composition according to claim 3, in the preparation of a medicament for treating MAT2A-mediated HCT116 human colon cancer in a subject.

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