CN116425751B - Polycyclic compounds as MAT2A inhibitors - Google Patents

Abstract

The invention provides a compound shown in a formula I and an isomer or pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition and application of the compound as an MAT2A inhibitor.

Description

Polycyclic compounds as MAT2A inhibitors

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a polycyclic compound serving as an MAT2A inhibitor and an isomer thereof, or pharmaceutically acceptable salts thereof, a preparation method, a pharmaceutical composition and application thereof in preparation of drugs for treating cancers in subjects suffering from the cancers.

Background

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 in methionine recycling. SAM is the propylamino donor in polyamine biosynthesis and is the primary methyl donor for DNA methylation and is involved in gene transcription and cell proliferation and production of secondary metabolites.

Three subtypes of human MAT include MAT1 and MAT3 expressed in liver tissue, while MAT2A is ubiquitously expressed in human cell types, the predominant form in human tumors. Crystal structure and mechanism studies showed that despite their 85% identity in amino acid sequence, there was a significant difference in their mechanism of action. MAT2A forms a functional homodimer in its purified active form and binds to the regulatory protein MAT 2B. MAT2B modulated MAT2A activity by increasing the sensitivity of MAT2A to ADOMet product inhibition, but did not provide significant rate enhancement. Cell localization studies showed that MAT2A was present in both the cytoplasm and nucleus. Nuclear concentration of MAT2A was reported to occur during replication and subsequent G2 phase, meeting the highly methylated requirements of DNA and histone methylation processes within the S phase nuclei. The activity of ADOMet in the transmethylation reaction has been identified as a rate limiting factor in the development of lung cancer stem cells, making MAT2A and the enzymes involved in methionine circulation targets for anticancer drugs. Methionine production of ADOMet by MAT2A comes from a dietary source or polyamine cycle in which 5-methylthio-D-ribose 1-phosphate produced by S-methyl-5' -thioadenylate phosphorylase is cycled to methionine. MAT2A protein has been reported to have increased expression in cancers such as colon cancer, liver cancer, stomach cancer, blood and liver. About 15% of human cancers show a deletion of the MTAP (S-methyl-5' -thioadenosine phosphorylase) gene and thus lack the methionine recovery pathway for polyamine synthesis. The deletion of MTAP in chr9p21 typically includes a deletion of the CDKN2a oncogene locus. Comprehensive genetic lethality studies of MATP-/-cancer cells indicate that inhibition of MAT2A, PRMT5 and PRMT1 by these cancer cells increases sensitivity.

In hepatocellular carcinoma (HCC), down-regulation of MAT1A and up-regulation of MAT2A occur, which is called MAT1A: MAT2A conversion. The shift accompanied by the up-regulation of MAT2B results in lower SAM content, which provides a growth advantage for hepatoma cells. MAT2A is a target of anti-tumor therapy because it plays a vital role in promoting liver cancer cell growth. Recent studies have shown that silencing by using small interfering RNAs substantially inhibits the growth of hepatoma cells and induces apoptosis. See, e.g., T.Li et al, J.cancer 7 (10) (2016) 1317-1327.

Some MTAP-deficient cancer Cell lines are particularly sensitive to inhibition of MAT2A, marjon et al (Cell Reports15 (3) (2016) 574-587). MTAP (methylthioadenosine phosphorylase) is an enzyme widely expressed in normal tissues, which catalyzes the conversion of Methylthioadenosine (MTA) to adenine and 5-methylthioribose-1-phosphate. Adenine is remedied to produce adenosine monophosphate, and 5-methylthioribose-1-phosphate is converted to methionine and formate. Because of this salvage pathway, MTA can be an alternative source of purine when de novo synthesis of purine is blocked by, for example, antimetabolites such as L-alacinone.

In other cancers lacking MTAP deletions (including hepatocellular carcinoma and leukemia), MAT2A is deregulated. Cai et al, cancer Res.58 (1998) 1444-1450; T.S. Jani et al, cell.Res.19 (2009) 358-369. Silencing MAT2A expression by RNA interference can produce antiproliferative effects in a variety of cancer models, H.Chen et al, gastroenterology133 (2007)

207-218; liu et al hepatol Res.37 (2007) 376-388.

Many human and murine malignant cells lack MTAP activity. MTAP deficiency is present not only in tissue culture cells, but also in primary leukemias, gliomas, melanomas, pancreatic cancers, non-small cell lung cancer (NSCLC), bladder cancers, astrocytomas, osteosarcomas, head and neck cancers, myxochondrosarcoma, ovarian cancers, endometrial cancers, breast cancers, soft tissue sarcomas, non-hodgkin lymphomas and mesothelioma. The gene encoding human MTAP is located in region 9p21 on human chromosome 9 p. This region also contains the tumor suppressor genes p16.sup.INK4A (also known as CDKN 2A) and p15.sup.INK4B. These genes encode p16 and p15, which are inhibitors of cyclin D-dependent kinases cdk4 and cdk6, respectively.

Alternatively, the p16.sup.INK4A transcript may be an open reading frame (ARF) spliced into a transcript encoding pl4 ARF. p14ARF binds to MDM2 and prevents degradation of p53 (Pomerantz et al (1998) Cell 92:713-723). The 9p21 chromosomal region is of interest because it is often homozygous for deletion in a variety of cancers, including leukemia, NSLC, pancreatic cancer, glioma, melanoma, and mesothelioma. Deletions typically inactivate more than one gene. For example, cairns et al ((1995) Nat. Gen.11:210-212) reported that after more than 500 primary tumors were studied, almost all deletions identified in these tumors involved a 170kb region containing MTAP, p14ARF and Pl6INK 4A. Carson et al (WO 99/67134) reported a correlation between the stage of tumor development and loss of homozygosity of the gene encoding MTAP and the gene encoding p 16. For example, the deletion of the MTAP gene, not p16.sup.INK4a, is reported to be predictive of cancer in the early stages of development, whereas the deletion of the genes encoding p16 and MTAP is reported to be predictive of cancer in the more advanced stages of cancer development. In some osteosarcoma patients, the MTAP gene is present at the time of diagnosis, but is deleted at a later point in time (Garcia-Castellano et al, clin.cancer Res.8 (3) 2002 782-787).

MAT2A enzyme inhibitors for the treatment of cancer are disclosed in WO 2018039972; WO2021158792 mentions MAT2A inhibitors for the treatment of autoimmune or inflammatory diseases; MAT2A enzyme inhibitors for the treatment of diseases or conditions mediated by overexpression of MAT2A are described in WO 2018045071; also disclosed in CN202080014106.0 is 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 the treatment of MTAP-deficient non-small cell lung cancer (NSCLC) in a patient in need thereof; CN114874207a discloses MAT2A inhibitors of 6-and 6-ring parent nuclei; CN113999232a discloses polycyclic compounds that inhibit MTAP-deleted cancer cells.

Finding effective MAT2A inhibitors is an important direction in the development of current tumor-targeted drugs. .

Disclosure of Invention

Summary of The Invention

The present invention meets the significant need for safe and effective compounds and methods for treating, preventing, and managing cancer while reducing or avoiding the toxicity and/or side effects associated with conventional therapies. In order to solve the technical problems, the invention adopts the following technical scheme:

in one aspect, the present invention provides compounds of formula I and isomers thereof, or pharmaceutically acceptable salts thereof,

Wherein:

R ₁ selected from the group consisting of absence, 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 -an alkoxy group;

R ₂ selected from hydrogen, hydroxy, halogen, -NH ₂ 、-CN、C ₁ -C ₆ -alkyl, C ₁ -C ₆ -an alkoxy group;

R ₃ selected from the group consisting of absence, 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 -an alkoxy group;

R ₄ selected from hydroxy, C ₆ -C ₁₂ Aryl, C ₆ -C ₁₂ Heteroaryl, C ₆ -C ₁₂ Heterocyclyl, each 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 -an alkoxy group;

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 Heterocyclyl, each 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 -an alkoxy group;

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 -an alkoxy group;

R _1a 、R _1b each independently selected from hydrogen, C ₅ -C ₁₂ Aryl, C ₅ -C ₁₂ Heteroaryl, C ₅ -C ₁₂ A heterocyclic group.

Further, the R ₁ Selected from the group consisting of absent, methoxyphenyl.

Further, the R ₂ Selected from the group consisting ofHydrogen, hydroxy; r is R ₃ Selected from the group consisting of absent, methoxyphenyl.

Further, the R ₄ Selected from hydroxyl groups and the following structures:

the groups are optionally substituted with one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

Further, the R ₅ Selected from cyclohexene, cyclohexadiene and the following structures:

the groups are optionally substituted with one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

Further, the R ₆ Selected from hydrogen,The radicals optionally being substituted by one or more hydroxy, halogen, -NH ₂ Carboxyl, -CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy substitution.

The invention also provides compounds represented by the following formulas (I-a), (I-b) and (I-c) and isomers thereof, or pharmaceutically acceptable salts thereof,

R ₄ 、R ₅ is defined as above.

The present invention also provides a compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, which is:

the invention also provides a pharmaceutical composition, which comprises the compounds shown in the formula (I), (I-a), (I-b), (I-c) and the like, isomers thereof or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable auxiliary materials.

The invention also provides the use of a compound comprising formula (I), (I-a), (I-b), (I-c), etc., or an isomer thereof, or a pharmaceutically acceptable salt thereof, or the composition, in the manufacture of a medicament for treating a disease or disorder in a subject suffering from said disease or disorder, wherein said disease or disorder is mediated by overexpression of MAT2A, preferably a cancer, characterized by a reduction or absence of expression of the methylthioadenosine phosphorylase (MTAP) gene, a deletion of the MTAP gene, or a reduction of MTAP protein function.

The inventor discovers that the compound is a high-efficiency MAT2A inhibitor, has extremely strong MAT2A inhibition activity, and can be used for preparing the drugs for preventing and/or treating the indications related to MAT2A inhibition, including cancers caused by reduction or deletion of MTAP expression, deletion of MTAP genes and reduction of MTAP protein functions. The present invention has been completed based on the above findings.

Detailed Description

Various aspects and features of the invention are described further below.

All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention. The following are definitions of various terms used in the present invention, which are applicable to terms used throughout the specification of the present application, unless otherwise specified in the specific context.

The compounds according to the invention may exist in tautomeric forms, the invention then embraces all tautomeric forms.

The compounds of the present invention have asymmetric centers and the compounds of the present invention containing an asymmetrically substituted atom can be isolated in optically active or racemic forms, and one skilled in the art knows how to prepare optically active forms, such as by racemate resolution or synthesis from optically active starting materials. Unless a specific stereochemistry or isomeric form is specifically indicated, the present invention includes all chiral, diastereomeric and racemates. Methods for preparing the compounds of the invention and intermediates thereof are part of the present invention. All tautomers of the compounds of the invention are also part of the invention.

The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "substituted" means that any one or more hydrogen atoms on a particular atom or group is substituted with a substituent, provided that the valence of the particular atom is normal and the resulting compound is stable after substitution. When the substituent is =o, it means that two hydrogen atoms are substituted. When a plurality of hydrogen atoms are substituted with substituents, each substituent may be the same substituent or different substituents, independently of the other. The kind and number of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.

The terms "alkoxy" and "alkylamino" are used in conventional sense to refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or amino group, respectively, wherein alkyl is as described herein.

As used herein, the term "halogen", "halo", and the like, means fluorine, chlorine, bromine, or iodine, particularly fluorine, chlorine, bromine, and particularly preferably fluorine, chlorine.

As used herein, the term "alkyl" refers to an alkyl group having the indicated number of carbon atoms, which is a straight or branched chain alkyl group, and which may include its sub-groups, e.g., reference to "C ₁ -C ₆ Alkyl "when it may also include C ₁ -C ₄ Alkyl, C ₁ -C ₃ Alkyl, C ₂ -C ₆ Alkyl, C ₂ -C ₄ Alkyl groups, etc., and specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc. It may be divalent, e.g. methylene, ethylene.

Representing a single bond or a double bond.

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, said 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. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be 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", "heterocycle", and "heterocyclyl" include "heteroaryl" groups, as defined below.

"aryl" when used alone or as part of another termRefers to carbocyclic aromatic groups, whether fused or not, having the indicated number of carbon atoms, or up to 14 carbon atoms if the number of carbon atoms is not indicated, e.g. C ₆ -C ₁₄ -aryl. Specific aryl groups are phenyl, naphthyl, biphenyl, phenanthryl, tetracenyl, and the like.

In addition to 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 sulfoxide, thiomorpholinyl sulfone, 1, 3-dioxolane, and tetrahydro-1, 1-dioxothienyl, and the like. Exemplary bicyclic heterocyclic groups include quinuclidinyl.

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 having at least one heteroatom (O, S or N) in at least one ring, said heteroatom-containing ring preferably having 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of the heteroaryl group containing a heteroatom 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 each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms and may be saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atoms may optionally be quaternized. Heteroaryl groups as bicyclic or tricyclic must include at least one fully aromatic ring, but the other fused ring or rings may be aromatic or non-aromatic. Heteroaryl groups may be attached to any available nitrogen or carbon atom of any ring. Where valence allows, if the other ring is cycloalkyl or heterocycle, it is additionally optionally substituted with =o (oxo).

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), the reference is intended to include rings optionally having 0 to 3, preferably 0 to 2 substituents selected from the substituents listed above for aryl heterocyclyl and/or heteroaryl.

As used herein, the term "pharmaceutically acceptable salt" means a salt which is not only physiologically acceptable to the subject, but also refers to a synthetic substance of pharmaceutical use, such as a salt formed as an intermediate in the preparation of chiral resolution, which salt may play a role in obtaining the end product of the present invention, although it is not directly administered to the subject. Specifically included are acid (organic and inorganic) addition salts or base addition salts (including organic and inorganic bases).

As used herein, the term "disease" refers to a physical state of the subject that is associated with the disease of the present invention. For example, the arterial peripheral disease and the neurodegenerative related disease according to the present invention.

The cancers of the invention include standard treatments such as surgery, radiation therapy, chemotherapy, hormonal therapy, and the like.

"cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled cellular abnormal proliferation, the ability of affected cells to spread locally or through the blood stream and lymphatic system to other sites, the body (i.e., metastasis), and any of a number of characteristic structures and/or molecular features. "cancer cells" refers to cells that undergo early, mid, or late stages of multistep tumor progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary system cancer, melanoma, brain tumor, head and neck cancer, acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, advanced solid tumors, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell tumor, teratocarcinoma, retinoblastoma, choriocarcinoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, sarcomas, fibrosarcoma, sarcomas, and lymphomas of the pancreas, and sarcomas.

The compound of the present invention or a pharmaceutical composition containing the same may be administered in unit dosage form by the enteral or parenteral route such as oral, intravenous, intramuscular, intravenous, subcutaneous, nasal, oral mucosal, ocular, pulmonary and respiratory tract, skin, vaginal, rectal, etc.

The dosage form may be a liquid, solid or semi-solid dosage form. The liquid preparation can be solution (including true solution and colloid solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including injection solution, powder injection and transfusion), eye drop, nasal drop, lotion, liniment, etc.; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like.

For the purpose of administration, the drug or the pharmaceutical composition of the present invention can be administered by any known administration method to enhance the therapeutic effect.

The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents. When the compound of the present invention has a synergistic effect with other therapeutic agents, its dosage should be adjusted according to the actual circumstances.

The solvents used in the present application are commercially available. The abbreviations used in this application are as follows:

table 1: abbreviation meaning

Beneficial technical effects

The inventor discovers that the compound has good MAT2A inhibition activity, and the IC50 is smaller than that of a positive control medicine AG270. The invention provides MAT2A inhibitor compounds with novel structures and strong activity, and the compounds have good application prospects in prevention and/or treatment of MAT2A inhibition related indications such as MTAP expression reduction or deletion, MTAP gene deletion, MTAP protein function reduction and the like.

Detailed Description

The following examples are presented to aid one skilled in the art in better understanding the present invention, but the scope of the present invention is not limited thereto.

For all of the following examples, standard procedures and methods known to those skilled in the art may be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structure of the compounds is determined by nuclear magnetic resonance spectroscopy (NMR) and/or Mass Spectrometry (MS).

The structure of the compounds of the invention is determined by Nuclear Magnetic Resonance (NMR) or/and liquid-mass spectrometry (LC-MS). NMR chemical shift (δ) is in parts per million (ppm). Nuclear magnetic resonance was measured using a Bruker avance-400 nuclear magnetic resonance apparatus with deuterated dimethyl sulfoxide (DMSO-d 6), deuterated methanol (CD 3 OD) and deuterated chloroform (CDCl 3) as solvents, and Tetramethylsilane (TMS) as internal standard.

Liquid phase mass spectrometry LC-MS the liquid phase fraction was measured using an ACQUITY UPLC ultra high pressure liquid chromatograph and the mass spectrum fraction was measured using a Xex og2-Sqtof mass spectrometer.

The starting materials in the examples of the present invention are known and commercially available, and may also be used or synthesized according to methods known in the art.

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

Step 1: synthesis of Compound 1-1

Methyl 2-methyl-5-nitrobenzoate (40.00 g,205.12mmol,1.0 eq.) N-bromosuccinimide (91.28 g,512.82mmol,2.50 eq.) and azobisisobutyronitrile (6.72 g,41.02mmol,0.2 eq.) were added to carbon tetrachloride (200 ml) solvent and reacted at 80℃for 16 hours. After the reaction, water and methylene chloride were added to extract, the organic phase was dried over anhydrous sodium sulfate, and the mixture was separated and purified by a normal phase column, and when the polarity was 12%, the product was eluted and concentrated to give methyl 2- (bromomethyl) -5-nitrobenzoate as a yellow solid (30.00 g,53.57% yield). LCMS (TOF MS ES) ⁺ )m/z[M+H] ⁺ :274/276。

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).

Step 2: synthesis of Compounds 1-2

Methyl 2- (bromomethyl) -5-nitrobenzoate (27.3 g,100.0mmol,1.0 eq) was added to a solution of ammonia-methanol (500 mL,7 mol/L) and reacted at room temperature for 16 hours. After completion of the reaction, the reaction mixture was concentrated, ethyl acetate (100 mL) was added and stirred for 10 minutes, and then, the mixture was filtered, and the cake was washed twice with ethyl acetate (50 mL), and the solid was collected to give 6-nitroiso Xin Duolin-1-one (13.0 g,73.00% yield) as a yellow solid. LCMS (TOF MS ES) ⁺ )m/z[M+H] ⁺ :179.04。

Step 3: synthesis of Compounds 1-3

The compound 6-nitroiso Xin Duolin-1-one (13.0 g,73.03mmol,1.0 eq.) was added to a solution of di-tert-butyl dicarbonate (31.80 g,146.06mmol,2.0 eq.) and triethylamine (22.17 g,219.12mmol,3.0 eq.) in dichloromethane (250 ml) and 4-dimethylaminopyridine (1.78 g,14.60mmol,0.2 eq.) was slowly added under ice-bath conditions at room temperature for 16 hours. After completion of the reaction, the reaction mixture was added to water, extracted three times with dichloromethane (100 ml), the organic layers were combined and washed with saturated brine, respectivelyWashing, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate, separating and purifying by normal phase column, petroleum ether and ethyl acetate system, eluting with 25% polarity, and concentrating to obtain yellow solid tert-butyl 6-nitro-1-oxo-isoindole-2-carboxylate (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).

Step 4: synthesis of Compounds 1-4

The compound tert-butyl 6-nitro-1-oxoisoindole-2-carboxylate (9.0 g,32.37mmol,1.0 eq.) was added to a methanol solution (200 mL), followed by Pd/C (1.10 g) and reacted for 16 hours at room temperature under hydrogen protection. After the reaction was completed, celite was rapidly filtered, and the filter cake was washed with methanol, and the filtrate was concentrated to give the crude product, tert-butyl 6-amino-1-oxoisoindole-2-carboxylate (7.80 g,97.0% yield), which was used directly in the next reaction. LCMS (TOF MS ES) ⁺ )m/z[M+H] ⁺ :249.12。

Step 5: synthesis of Compounds 1-5

Tert-butyl 6-amino-1-oxoisoindole-2-carboxylate (7.8 g,31.40mmol,1.0 eq.) and 4, 6-dichloro-5-nitropyrimidine (9.15 g,47.17mmol,1.50 eq.) were added to a solution of tetrahydrofuran (120 mL) and reacted at room temperature for 16 hours. After the completion of the reaction, the reaction mixture was added to water, extracted three times with methylene chloride (100 ml), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, the normal phase column was separated and purified, methylene chloride and ethyl acetate were separated and purified, and when the polarity was 10%, the product was eluted, and concentrated to give 6- [ (6-chloro-5-nitropyrimidin-4-yl) amino as a yellow solid]-1-oxo-isoindole-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(400MHz,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).

Step 6: synthesis of Compounds 1-6

By reacting 6- [ (6-chloro-5-nitropyrimidin-4-yl) amino group]-1-oxoisoindolesTert-butyl indole-2-carboxylate (7.8 g,19.26mmol,1.0 eq.) was added to a methanol solution (200 mL) followed by Pd/C (1.10 g) and reacted at room temperature under hydrogen protection for 40 hours. After the reaction is completed, the diatomite is quickly filtered, a filter cake is washed by methylene dichloride/methanol (10:1), the filtrate is concentrated, a normal phase column is used for separation and purification, methylene dichloride and methylene dichloride/methanol (10:1) systems, when the polarity is 80%, a product flows out, and the product is concentrated to obtain brown solid 6- [ (5-aminopyrimidin-4-yl) amino ]-tert-butyl 1-oxoisoindole-2-carboxylate (1.2 g,18.27% yield). LCMS (TOF MS ES) ⁺ )m/z[M+H] ⁺ :342.15。 ¹ H NMR(400MHz,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).

Step 7: synthesis of Compounds 1-7

Tert-butyl 6- [ (5-aminopyrimidin-4-yl) amino ] -1-oxoisoindole-2-carboxylate (200 mg,0.58mmol,1.0 eq.) was added to a solution of dichloromethane (5 mL), and a solution of triethylamine (120.0 mg,1.17mmol,2.0 eq.) and BTC (260 mg,0.87mmol,1.50 eq.) in dichloromethane were added sequentially at 0deg.C and reacted under nitrogen for 1 hour at 0deg.C. After completion of the reaction, the mixture was purified by column chromatography to give tert-butyl 1-oxo-6- (8-oxo-7, 8-dihydro-9H-purin-9-yl) isoindole-2-carboxylate (280 mg, crude) as a white solid. Directly used in the next reaction. LCMS (TOF MS ES+) M/z [ M+H ] +:368.13/312.13.

Step 8: synthesis of Compound 1

Tert-butyl 1-oxo-6- (8-oxo-7, 8-dihydro-9H-purin-9-yl) isoindole-2-carboxylate (280 mg,0.76mmol,1.0 eq.) 4-methoxyphenylboronic acid (695.4 mg,4.58mmol,6.0 eq.) anhydrous copper acetate (206.4 mg,1.14mmol,1.5 eq.) and anhydrous pyridine (180.4 mg,2.28mmol,3.0 eq.) were added to a solution of anhydrous N, N-dimethylformamide (8 mL) and reacted at room temperature under oxygen protection for 16 hours. After the completion of the reaction, the reaction mixture was filtered, the filter cake was washed with methanol, and the filtrate was concentrated and then added to ethyl acetate (50 mL), and then washed with water and saturated brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated after filtration. Prep-HPLC preparation of white solid 7- (4-methoxyphenyl) -9- (3-oxoisoindol-5-yl) -7, 9-dihydro-8H-purine-8 Ketone (13.4 mg, 10.17%). LCMS (TOF MS ES) ⁺ )m/z[M+H] ⁺ :374.13. ¹ H NMR(400MHz,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).

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

Step 1: synthesis of Compound 2-1

2, 6-dichloropurine (21.77 g,115.0mmol,1.0 eq), cyclohexene-1-boronic acid (17.28 g,138mmol,1.2 eq), anhydrous copper acetate (25.0 g,138.0mmol,1.2 eq), triethylamine (58.3 g,576.0mmol,5.0 eq) were dissolved in 500mL dichloromethane and stirred at room temperature for 2 hours and minutes under air protection. After completion of the reaction, celite was filtered, and the filtrate was concentrated. Separating with normal phase column, petroleum ether and ethyl acetate system with polarity of 40% to obtain 2, 6-dichloro-9-cyclohexyl-9H-purine (3.3 g,10.71% yield), ¹ H NMR(400MHz,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).

step 2: synthesis of Compound 2-2

2, 6-dichloro-9-cyclohexyl-9H-purine (2.7 g,10.0mmol,1.0 eq) was dissolved in tetrahydrofuran (10 mL), followed by addition of aqueous sodium hydroxide (1.0g,25.0mmol,l2.5 eq,30wt%) and reacted at room temperature for 16 hours. After the reaction is completed, 20mL of ethyl acetate is added, the organic layer is separated, filtered, washed by ethyl acetate and the solid is collected to obtain 2-Chloro-9-cyclohexyl-1, -dihydropyran-6-one (1.46 g,58.20% yield). ¹ H NMR(400MHz,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).

Step 3: synthesis of Compound 2-3

2-chloro-9-cyclohexyl-1, -dihydropyran-6-one (1.46 g,5.82mmol,1.0 eq), 2-aminopyridine (1.10 g,11.68mmol,2.0 eq), xant-Phos (1.01 g,1.75mmol,0.3 eq), palladium acetate (260.7 mg,1.16mmol,0.2 eq), cesium carbonate (3.77 g,11.60mmol,2.0 eq) were dissolved in a 1, 4-dioxane solution (50 mL), and reacted under nitrogen at 120℃for 16 hours. After completion of the reaction, celite was filtered, and the filtrate was concentrated, and the system was separated by a normal phase column (dichloromethane/(dichloromethane: methanol (10:1)) to give 9-cyclohexyl-1-en-1-yl-2-pyridin-2-amino-1, 9-dihydro-6H-purin-6-one (840 mg,47.42% yield) at a polarity of 60%, LCMS (ESI) [ M+H]+:309.14。 ¹ H NMR(400MHz,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)。

Step 4: synthesis of Compounds 2-4

9-cyclohexyl-1-en-1-yl-2-pyridin-2-amino-1, 9-dihydro-6H-purin-6-one (840 mg,2.76mmol,1.0 eq), p-methoxyphenylboronic acid (4.2 g,27.6mmol,10.0 eq), anhydrous copper acetate (749.3 mg,4.14mmol,1.5 eq) were dissolved in anhydrous DMF (25 ml), and then anhydrous pyridine (872.0 mg,11.04mmol,4.0 eq) was added and reacted at room temperature under oxygen protection for 16 hours. After the reaction is completed, filtering, washing filter cake with methanol, collecting solid to obtain9-cyclohexyl-1-en-1-yl-1- (4-methoxyphenyl) -2-pyridin-2-ylamino) -1, 9-dihydro-6H-purin-6-one (660 mg,57.97% yield). ¹ H NMR(400MHz,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)。

Step 5: synthesis of Compounds 2-5

N-bromosuccinimide (859.9 mg,4.83mmol,4.0 eq) was added to a 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,1.21mmol,1.0 eq) in dichloromethane (5 mL) and reacted at room temperature for 16 hours. After the reaction was completed, the reaction mixture was concentrated, and the mixture was separated by a normal phase column to give 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) at a polarity of 35%. LCMS (ESI) [ M+H ]]+:573.0. ¹ H NMR(400MHz,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).

Step 6: synthesis of Compound 2

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,0.43mmol,1.0 eq), phenylboronic acid (93.8 mg,0.77mmol,2.0 eq), pd (dppf) Cl ₂ (124.1 mg,0.15mmol,0.4 eq) and potassium phosphate (161.0 mg,0.77mmol,2.0 eq) were added to a mixed solvent of 1, 4-dioxane (3.0 mL) and water (0.3 mL), 100After the reaction is completed, diatomite is filtered, filtrate is concentrated, ethyl acetate is added for dilution, water washing, saturated saline water washing and anhydrous sodium sulfate drying are respectively carried out, and finally the filtrate is concentrated. The crude product (80 mg) was obtained by normal phase column separation, petroleum ether and ethyl acetate system, polarity 60%, and the white solid (5 mg) was obtained by preparative separation. LCMS (ESI) [ M+H ] ]+:489.20. ¹ H NMR(400MHz,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)。

The compounds of table 2 are obtained by reference to the procedure of example 1:

table 2: structure and characterization of Compounds 3 to 7

The compounds of table 3 are obtained by reference to the procedure of example 2:

table 3: structure and characterization of Compounds 8-29, 32-35

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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)

Step 1: synthesis of Compound 30-1

To 15mL of dimethylformamide solvent were added 1-bromo-2-fluoro-3-nitrobenzene (5 g,23.5mmol,1.0 eq), sodium carbonate (4.982 g,47mmol,2.0 eq) and methylamine hydrochloride (1.284 g,28.2mmol,1.7 eq) and reacted at room temperature for 16h. After the completion of the reaction, the reaction mixture was concentrated, and subjected to normal phase column chromatography (ethyl acetate 5%) to give 2-bromo-N-methyl-6-nitroaniline (5.09 g, yield 97.4%) as a red liquid. 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).

Step 2: synthesis of Compound 30-2

The compound 2-bromo-N-methyl-6-nitroaniline (2.0 g,8.70mmol,1.0 eq) was added to a solution of glacial acetic acid (20 mL), the temperature was raised to 50℃and reduced iron powder (1.46 g,26.1mmol,3 eq) was added in portions and reacted at 50℃for 1h. After completion of the reaction, the solid residue was filtered off, and reversed-phase column chromatography (25% acetonitrile) was performed to give 6-bromo-N-methylbenzene-1, 2-diamine (510.0 mg, yield 14.1%) as a yellow oil. LCMS (ESI) [ M+H ] ] ⁺ :201

Step 3: synthesis of Compound 30-3

6-bromo-N-methylbenzene-1, 2-diamine (510 mg,2.55mmol,1.0 eq) and p-toluenesulfonic acid monohydrate (51.0 mg, 0.292 mmol,0.12 eq) were dissolved in triethyl orthoformate (1.5 mL) and reacted at 85℃for 1h. After the reaction was completed, the reaction mixture was concentrated and subjected to reverse phase column chromatography (acetonitrile 15%) to obtain 7-bromo-1-methyl-1H-benzo [ d ] as a white solid]Imidazole (480.0 mg, 89.6% yield). 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).

Step 4: synthesis of Compound 30-4

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

Step 5: synthesis of Compound 30-5

The compound (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-benzo [ d ]]Imidazole (375 mg,1.45mmol,1.0 eq), 8-bromo-2-chloro-9- (cyclohex-1-en-1-yl) -9H-purin-6-ol (477.1 mg,1.45mmol,1.0 eq), pd (dppf) Cl ₂ (53.0 mg,0.0725mmol,0.05 eq.) and Cs ₂ CO ₃ (1.43 g,4.35mmol,3 eq) was added to a mixed solution of 1,4 dioxane (4 mL) and water (1 mL) and reacted at 80℃under nitrogen with stirring for 2h. After completion of the reaction, reverse phase column chromatography (50% acetonitrile) gives 2-chloro-9- (cyclohex-1-en-1-yl) -8- (1-methyl-1H-benzo [ d ] as a brown solid]Imidazol-7-yl) -1, 9-dihydro-6H-purin-6-one (213 mg, yield 38.7%). LCMS (ESI) [ M+H ]] ⁺ :381。

Step 6: synthesis of Compound 30-6

The 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,0.56mmol,1.0 eq), 2-aminopyridine (79.0 mg,0.84mmol,1.5 eq), pd ₂ (dba) ₃ (25.6 mg,0.028mmol,0.05 eq.) and Cs ₂ CO ₃ (551.0 mg,1.68mmol,3 eq) was added to a solution of 1,4 dioxane (5 mL) and reacted at 80℃under nitrogen with stirring for 4h. After completion of the reaction, reverse phase column chromatography (60% acetonitrile) gives 9- (cyclohex-1-en-1-yl) -8- (1-methyl-1H-benzo [ d) as a yellow solid]Imidazol-7-yl) -2- (pyridin-2-ylamino) -1, 9-dihydro-6H-purin-6-one (122 mg, yield 49.7%)

Step 7: synthesis of Compound 30

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

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)

Step 1: synthesis of Compound 31-1

To 500mL of the super-dry dichloromethane solvent was added 2, 6-dichloro-9H-urea (60 g,317.46mmol,1.0 eq), p-toluenesulfonic acid monohydrate (6.039 g,476.19mmol,1.5 eq), followed by adding 3, 4-dihydro-2H-pyran (40.05 g,476.19mmol,1.5 eq) dissolved in 100mL of the super-dry dichloromethane solvent slowly with stirring, and reacting at room temperature for 4H. After the reaction was completed, the reaction solution was filtered, the filter cake was washed with methylene chloride, 300ml of 10% sodium carbonate solution was added for extraction, the organic phase was collected, water was further added for extraction, the extracted organic layer was dried over anhydrous sodium sulfate, and the organic phase was concentrated to a solid after drying. Ethyl acetate: petroleum ether = 1:12 to give 2, 6-dichloro-9- (tetrahydro-2H-pyran-2-en) -9H-purine (57.4 g, 86.8% yield) as a white solid. 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).

Step 2: synthesis of Compound 31-2

2, 6-dichloro-9- (tetrahydro-2H-pyran-2-ene) -9H-purine (5 g,18.38mmol,1.0 eq) was dissolved in 50ml of ultra-dry tetrahydrofuran solvent under nitrogen protection, cooled to-78℃under dry ice ethanol conditions, then 2mol/L lithium diisopropylamide (13.78 ml,27.57mmol,1.5 eq) was slowly added dropwise and stirred for 30min after addition. 1, 2-dibromo-1, 2-tetrachloroethane (8.98 g,27.57mmol,1.5 eq) in 15ml of ultra-dry tetrahydrofuran was then slowly added dropwise, and the reaction was continued at-78℃for 1.5h after the addition. After the reaction was completed, 10ml of saturated ammonium chloride solution was slowly added to quench the reaction solution at a temperature not exceeding-30 ℃. The reaction mixture was concentrated, and subjected to normal phase column chromatography (ethyl acetate 10%) to give 8-bromo-2, 6-dichloro-9- (tetrahydro-2H-pyran-2-en) -9H-purine (2.2554 g yield 35.1%) as a yellow solid LCMS (ESI) [ M+H ]+:351 ¹ H 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).

Step 3: synthesis of Compound 31-3

8-bromo-2, 6-dichloro-9- (tetrahydro-2H-pyran-2-ene) -9H-purine (1 g,2.9mmol,1.0 eq) was dissolved in dichloromethane (10 mL) and then 2.5mL trifluoroacetic acid was added under ice-bath conditions and reacted at room temperature for 30min. After the reaction was completed, the reaction solution was concentrated, dissolved in methanol, and triethylamine was added to clarify the solution, followed by reverse phase column chromatography to give 8-bromo-2, 6-dichloro-9H-urea as a pale yellow solid (616 mg, yield 81.1%). LCMS (ESI) [ M+H ]] ⁺ :267

Step 4: synthesis of Compound 31-4

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

Step 5: synthesis of 2-nitro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridine

5-bromo-2-nitropyridine (1 g,4.95mmol,1.0 eq.) 4,4', 5',2 '-octamethyl-2' -bis (1, 3, 2-dioxaborane) (1.56 g,6.14mmol,1.24 eq.) Pd (dppf) Cl ₂ (182 mg,0.25mmol,0.05 eq.) and potassium acetate (1.44 g,24.8mmol,5.0 eq.) in dioxane (20 mL), N ₂ The reaction was allowed to proceed overnight at 90 ℃. After the reaction was completed, the reaction solution was filtered, washed with ethyl acetate, and the mother liquor was retained. After spin drying, purifying by reverse phase column, passing water (1%formic acid solution) and acetonitrile system through column; 50%; the desired product was eluted for 15min to give 2-nitro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) pyridine (707 mg, yield 56%) as a yellow solid. 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)

Step 6: synthesis of Compound 31-5

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

Step 7: synthesis of Compound 31-6

The compound 2-chloro-9- (cyclohex-1-en-1-yl) -8- (6-nitropyridin-3-yl) -9H-purin-6-ol (190 mg,0.51mmol,1.0 eq), 2-aminopyridine (71.9 mg,0.77mmol,1.5 eq), pd ₂ (dba) ₃ (23.3 mg,0.0225mmol,0.05 eq.) and Cs ₂ CO ₃ (501.8 mg,1.53mmol,3 eq) was added to a solution of 1,4 dioxane (5 mL) and reacted at 80℃under nitrogen with stirring for 4h. After completion of the reaction, reverse phase column chromatography (60% acetonitrile) gave 9- (cyclohex-1-en-1-yl) as a yellow solid-8- (6-nitropyridin-3-yl) -2- (pyridin-2-ylamino) -1, 9-dihydro-6H-purin-6-one (155 mg, yield 70.5%). LCMS (ESI) [ M+H ]]+:431

Step 8: synthesis of Compound 31-7

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

Step 9: synthesis of Compound 31

The compound 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,0.168mmol,1.0 eq) was added to a solution of glacial acetic acid (5 mL), heated to 50℃and reduced iron powder (47.1 mg,0.84mmol,5 eq) was added in portions and reacted at 50℃for 1H. After completion of the reaction, the solid residue was filtered off, and reversed-phase column chromatography (60% acetonitrile) to give 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%) as a white solid. LCMS (ESI) [ M+H ] +:507.2.

The compounds of table 4 are obtained by the method of reference example 31:

table 4: structure and characterization of Compounds 36 and 37

The procedure for the preparation of compound 36 was the same as in example 31, except that there was one more halogenation step.

Experimental example 1: binding Capacity test of Compounds of the invention to mat2a protein

The purpose of the experiment is as follows: detection of binding Capacity of Compounds to MAT2A protein by CETSA Experimental method

Background principle: CETSA assay is a molecular assay that measures the affinity of a drug for a target protein. The principle is that after the medicine is combined with the target protein, the structure is more stable. Using a cell or tissue sample at the candidate drug, if the candidate drug is an inhibitor of MAT2A, the candidate drug can be combined with MAT2A, so that MAT2A protein becomes more stable, after the sample is subjected to heat treatment, MAT2A protein in the sample is easier to detect by a specific antibody, and MAT2A protein is easier to detect by a Western blot experiment; conversely, the stability of MAT2A protein after heating will be worse and the amount of protein detected will be lower. Thereby evaluating the binding capacity of the drug to the target protein for screening of MAT2A protein inhibitors.

The specific experimental process comprises the following steps:

taking HCT116 cells in logarithmic growth phase (cell survival rate > 90%), washing the cells 3 times by PBS, and centrifuging for 2min at 2000 g; lysing with a cell lysate containing a protease inhibitor PMSF on ice for 30min; protein samples were assayed for concentration using BCA kit. Incubating the samples for 30min by using the candidate drugs, the control drugs and the control reagents respectively, and heating the set 10 temperature points of each group of samples; recovering room temperature, centrifuging the sample with 20000g, and collecting the supernatant; protein samples were denatured using sample buffer at 100℃for 10 min. After the sample is restored to room temperature, performing western blot detection on the sample; the protein loading was controlled at 20ug. After the mutation temperature is determined, the concentration gradient of the compound is set to be 9 points generally, the sample is incubated, and western blot detection is carried out by the same operation. Protein electrophoresis: the concentrated gel voltage is set to 60v, and the separation gel voltage is set to 120v; after the electrophoresis, the electrophoresis was started to be performed. The condition of the electric transfer is set to 250mA for 2 hours; blocking with 5% BSA for 1h; adding a specific 1 antibody, and incubating on a shaking table at 4 ℃ overnight; TBST is washed for 4 times, each time for 2.5min; incubating the mixture for 2 hours on a shaking table at room temperature; TBST is washed for 4 times, each time for 2.5min; development was performed using ECL to detect the amount of TYK2 protein expressed in different groups and at various temperature points. EC50 was calculated by converting the western blot bands by image J and GraphPad software.

EC50 is the half maximal effector concentration (concentration for 50%of maximal effect,EC50), which refers to the concentration of drug that is effective in 50% of individuals. AG270 (compound 153 reported in CN 201780066270.4) of An Jiao s pharmaceutical limited was used as a positive reference compound.

EC50 values for each compound were classified according to the following description:

"+" indicates an EC50 value greater than 100 μm;

"++" indicates that the EC50 value is less than 100. Mu.M and greater than 10. Mu.M;

"+". ++'s representation of EC50 the value is less than 10. Mu.M.

TABLE 5 EC50 results

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EC50 experimental data show that the compound of the invention has better binding ability with MAT2A protein, and compared with a positive control AG270 (prepared by referring to the preparation method of the compound 153 reported in CN 201780066270.4), the binding ability of the compound of the invention with MAT2A protein is equivalent, even stronger.

Experimental example 2: the compound of the invention is used for preparing HCT116 MTAP ^-/- Determination of cell proliferation

The purpose of the experiment is as follows: the purpose of this test case was to test the compound pair HCT116 MTAP ^-/- Cell proliferation.

Background principle: methionine adenosyltransferase 2A (MAT 2A) is considered a synthetic lethal target for cancers in which the methylthioadenosine phosphorylase (MTAP) gene is deleted, adjacent to the CDKN2A tumor suppressor, and co-deleted with CDKN2A in about 15% of cancers. Thus, by detecting the compound pair HCT116 MTAP ^-/- The inhibition rate of cell proliferation is used for screening MAT2A protein inhibitor.

The specific experimental process comprises the following steps:

HCT116 MTAP knockout cells were constructed and monoclonal was selected. HCT116 MTAP to be in logarithmic growth phase ^-/- And WT cells, seeded in 96-well plates at 90uL per well, 1000 per well, and incubated overnight at 37 ℃. The next day, 10. Mu.l of the compound (final DMSO concentration 1%) was added and incubated in an incubator at 37℃for 10 days. On day 10, the old medium was aspirated, 110ul medium (medium to CCK8 ratio 100:10) was added and incubated at 37℃for 1-4h. Absorbance was measured at 450nM, IC50 was calculated by GraphPad software treatment and compounds were screened by comparison with positive drug.

IC50 (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It indicates that a certain drug or substance (inhibitor) is inhibiting half of a certain biological process (or a certain substance contained in the process, such as an enzyme, a cellular receptor or a microorganism). AG270 of An Jiao s pharmaceutical limited was used as a positive reference compound.

The test results are shown in Table 6 below, wherein the IC50 values of the compounds are classified according to the following description:

+++ is less than 100nM of the total of all the above-mentioned materials, ++ between 100nM and 10. Mu.M, + is greater than 10 μm.

The test results are shown in Table 6 below:

TABLE 6 IC50 experimental data

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The results show that the compound can inhibit HCT116 MTAP-null cells, and the IC50 value reaches the nM level, which is equivalent to or smaller than that of a positive control medicine AG270. This strong inhibition is of great therapeutic interest for the treatment of conditions or diseases associated with MAT2A inhibition.

Experimental example 3: determination of the functional Effect of the Compounds of the invention on MAT2A protease

The purpose of the experiment is as follows: the purpose of this test case was to test the ability of the compounds to inhibit MAT2A protease function.

Background principle: met A2A, a metabolic enzyme, plays an important role in metabolism and epigenetic science because it is the primary producer of the universal methyl donor S-adenosylmethionine (SAM). ATP and L-Met under the action of MAT2A produce SAM and phosphate groups. Thus, after incubation of the compound, the ability of the compound to inhibit MAT2A enzyme function was evaluated by detecting the amount of SAM produced for use in screening MAT2A protein inhibitors.

The specific experimental process comprises the following steps:

MAT2A protein expression: full length MAT2A was cloned into pET24N vector with an N-terminal (His) 6x tag and a Tobacco Etch Virus (TEV) protease cleavage site. The constructed vector was transformed into E.coli BL21 (DE 3), shaken to OD 0.6, and incubated with 1mM IPTG at 18℃for 16h. Collecting thallus, ultrasonic crushing, centrifuging to obtain supernatant, purifying protein with Ni-NTA, and dialyzing to determine protein concentration and purity. SAM assay: the reaction system: 91-x uL of KCl 50mM Tris HCl pH 7.5,1.5uL (10/3M), 1.5uL (1M) MgCl2,1uL (100 mM) ATP,1uL (80 mM) L-Met,1uL (30 mM PH7.67) EDTA,1uL5% BSA,2uL of the DMSO-solubilized drug, and x uL of MAT2A protein were added sequentially. The experimental groups were prepared with different drug concentrations, 2uL (100 x) was taken out, added to the reaction system, and reacted at 37 ℃ for 18h, and solvent control group and blank control group were set, respectively. The reaction was terminated, 40uL of the system was removed, and the reaction was quenched by adding 4uL of 10% SDS. IC50 was calculated by GraphPad software treatment and compounds were screened by comparison with positive drug.

The test results are shown in Table 7 below, wherein the IC50 values of the compounds are classified according to the following description:

small+++. At a concentration of 10nM, ++ between 10nM and 1. Mu.M, + is greater than 1 μm.

The test results are shown in Table 7 below:

TABLE 7 IC50 experimental data

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The results show that the compound has very strong inhibition effect on MAT2A protease function, and the IC50 values reach nM level and are smaller than that of a positive control medicine AG270. This strong inhibition is of great therapeutic interest for the treatment of conditions or diseases associated with MAT2A inhibition.

The above embodiments are merely representative. From the above examples, it can be seen that the compounds of the present invention are ideal, highly potent MAT2A inhibitors, and are expected to be useful in the treatment or prevention of disorders or diseases associated with MAT2A inhibition.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical scheme and the inventive concept thereof within the scope of the present invention.

Claims (5)

1. A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,

wherein:

R ₃ selected from methoxyphenyl groups;

R ₅ selected from the group consisting of The groups are optionally substituted with one or more amino, methyl, methoxy, hydroxy, halogen, cyano groups.

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

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

4. Use of a compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 3, in the manufacture of a medicament for treating a disease or disorder in a subject suffering from said disease or disorder, wherein said disease or disorder is mediated through overexpression of MAT2A, said disease or disorder being cancer.

5. The pharmaceutical use according to claim 4, wherein the cancer is characterized by a decrease or a deletion of the expression of the methylthioadenosine phosphorylase (MTAP) gene, a deletion of the MTAP gene, or a decrease in MTAP protein function.