Heterocyclic compounds as MAT2A inhibitors

Abstract

The present invention relates to a novel compound as shown in formula I: a heterocyclic compound as a MAT2A inhibitor and its isomer, or a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition, and its use in preparing a drug for treating cancer in a subject suffering from cancer. The MAT2A inhibitor compound and its isomer, or a pharmaceutically acceptable salt thereof of the present invention can reduce the enzyme activity of MAT2A, and treat cancers with reduced or absent MTAP expression, absent MTAP gene, or reduced MTAP protein function.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to a heterocyclic compound and its isomers, or pharmaceutically acceptable salts thereof, as a MAT2A inhibitor, a preparation method thereof, a pharmaceutical composition thereof, and use thereof in preparing a compound for treating cancer in a subject suffering from 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 the rate-limiting step of the methionine cycle. SAM is an propylamino group donor in polyamine biosynthesis and the major methyl group donor for DNA methylation. It is involved in gene transcription and cell proliferation, as well as the production of secondary metabolites.

[0003] Human MAT consists of three isoforms: MAT1 and MAT3, which are expressed in liver tissue, and MAT2A, which is ubiquitously expressed in human cell types and the predominant form in human tumors. Crystal structure and mechanistic studies have revealed that despite their 85% amino acid sequence identity, their mechanisms of action differ significantly. In its purified active form, MAT2A forms a functional homodimer and binds to the regulatory protein MAT2B. MAT2B modulates MAT2A activity by increasing its sensitivity to inhibition by the ADOMet product, but without providing a significant rate enhancement. Cellular localization studies have demonstrated that MAT2A is present in both the cytoplasm and the nucleus. Nuclear condensation of MAT2A reportedly occurs during replication and the subsequent G2 phase, fulfilling the requirement for hypermethylation of DNA and histone methylation processes in the nucleus during S phase. ADOMet's activity in transmethylation reactions has been identified as a rate-limiting factor in lung cancer stem cell development, making MAT2A and related enzymes of the methionine cycle attractive targets for anticancer drugs. The methionine required for ADOMet production by MAT2A comes from dietary sources or the polyamine cycle, where 5-methylthio-D-ribose 1-phosphate, generated by S-methyl-5'-thioadenosine phosphorylase, is recycled to methionine. Increased expression of the MAT2A protein has been reported in cancers including colon, liver, stomach, blood, and liver cancers. Approximately 15% of human cancers display deletions in the MTAP (S-methyl-5'-thioadenosine phosphorylase) gene, thereby lacking the methionine recycling pathway for polyamine synthesis. Loss of MTAP at chr9p21 often includes deletion of the CDKN2a tumor suppressor gene locus. Comprehensive genetic lethality studies of MATP- / - cancer cells have demonstrated 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, results in lower SAM content, which provides a growth advantage for liver cancer cells. Because MAT2A plays a crucial 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 by, for example, antimetabolites such as L-alanosine.

[0006] MAT2A is dysregulated in other cancers lacking MTAP deletion, including hepatocellular carcinoma and leukemia. 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 produces antiproliferative effects in various cancer models. H. Chen et al., Gastroenterology 133 (2007) 207-218; Q. Liu et al., Hepatol Res. 37 (2007) 376-388.

[0007] Many human and mouse malignant cells lack MTAP activity. MTAP deficiency is not only found 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 lymphoma, 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, inhibitors of the cyclin D-dependent kinases cdk4 and cdk6, respectively.

[0008] Alternatively, the p16INK4A transcript can be an alternative reading frame (ARF) spliced ​​into a transcript encoding p14ARF. p14ARF binds to MDM2 and prevents the 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). Deletion usually inactivates 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 170 kb region containing MTAP, p14ARF and P16INK4A. Carson et al. (WO99 / 67634) reported a correlation between the stage of tumor progression and the loss of homozygosity for the gene encoding MTAP and the gene encoding p16. For example, it was reported that the loss of the MTAP gene, but not p16INK4A, predicted cancer in the early stages of development, while the loss of the genes encoding p16 and MTAP predicted cancer in the later stages of cancer development. In some osteosarcoma patients, the MTAP gene was present at diagnosis but was lost at a later time point (Garcia-Castellano et al., Clin. Cancer Res. 8 (3) 2002 782-787).

[0009] These findings have led to the search for effective drugs targeting these enzymes. Two MAT2A inhibitors have entered Phase 1 clinical trials. AG-270, developed by Servier Pharmaceuticals in France, is still in the very early stages of clinical development, while another inhibitor, IDE-397, is in the earlier stages of recruitment. Its clinical efficacy needs to be confirmed by further patient efficacy data.

[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 MAT2A overexpression; 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.

[0011] There is a need in this field to develop novel drugs that have potent inhibitory effects on MAT2A, fewer side effects, and better efficacy. Summary of the Invention SUMMARY OF THE INVENTION

[0013] For the reasons stated above, the present invention addresses a significant need for safe and effective compounds and methods for treating, preventing, and managing cancer while reducing or avoiding the toxicities and / or side effects associated with conventional therapies.

[0014] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0015] In one aspect, the present invention provides a compound represented by Formula I and its isomers, or a pharmaceutically acceptable salt thereof,

[0016]

[0017] wherein R1 is selected from a C5-C6-aromatic ring, a 5- to 6-membered heteroaromatic ring, each of which is optionally substituted with one or more substituents selected from the following: hydroxy, halogen, -NH2, carboxyl, -CN, C1-C6-alkyl, C1-C6-alkoxy;

[0018] R2 is selected from 5- to 12-membered heterocyclic ring, -C(O)NHR 2a 、-C(O)OR 2a ;

[0019] R 2a is selected from hydrogen, C5-C6-aromatic ring, 5- to 6-membered heteroaromatic ring;

[0020] R3 is selected from 5- to 9-membered aryl, 5- to 9-membered heteroaryl, -N(CH3)C(O)-5- to 6-membered heteroaryl ring, each of which is optionally substituted with one or more substituents selected from the following: hydroxy, halogen, -NH2, carboxyl, -CN, C1-C6-alkyl, C1-C6-alkoxy.

[0021] Further, R1 is selected from the following structures:

[0022]

[0023] Further, R2 is selected from the following structures:

[0024]

[0025] Further, R3 is selected from the following structures:

[0026]

[0027] Furthermore, the halogen is F, Cl, Br, I, preferably F; the C1-C6 alkyl is a straight-chain alkyl, a branched-chain alkyl or a cycloalkyl; the C1-C6 alkoxy is a straight-chain alkyloxy, a branched-chain alkyloxy or a cycloalkyloxy.

[0028] Furthermore, the C1-C6 alkoxy group is a methoxy group.

[0029] Furthermore, the general formula is selected from the following compounds:

[0030]

[0031] In a second aspect, the present invention provides a method for preparing the compound of formula I and its isomers, or pharmaceutically acceptable salts thereof, comprising the following steps:

[0032]

[0033] The starting material ii is subjected to an addition ring-closure reaction to obtain ii-1, electrophilic substitution to obtain ii-2, and deprotection to obtain ii-3; amine ester exchange to obtain ii-4, which is then subjected to halogenation reaction, halogen hydrolysis, Buchwald-Hartig coupling, nucleophilic substitution, and ether hydrolysis to obtain the compound shown in formula I.

[0034] In a third aspect, the present invention also provides a method for preparing the compound of formula I and its isomers, or a pharmaceutically acceptable salt thereof. The method comprises the following steps:

[0035]

[0036] The starting material iii is subjected to nucleophilic substitution to obtain iii-1, and amine ester exchange is performed to obtain ii-4, which is then subjected to halogenation reaction, halogen hydrolysis, Buchwald-Hartwig coupling, and ether hydrolysis to obtain the compound represented by formula I; wherein R1-R3 are as defined above.

[0037] In a fourth aspect, the present invention further provides a method for preparing the compound of formula I and its isomers, or pharmaceutically acceptable salts thereof, comprising the following steps:

[0038]

[0039] The starting material i is successively subjected to electrophilic substitution, palladium-carbon reduction, Buchwald-Hartig coupling, acid deprotection & ether hydrolysis to prepare the compound represented by formula I; wherein R1-R3 are as defined above.

[0040] The present invention also provides a pharmaceutical composition comprising the above-mentioned floral compound and its isomers, or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

[0041] The present invention also provides the use of the above-mentioned compound and its isomers, or pharmaceutically acceptable salts thereof, in the preparation of a method for treating cancer in a subject suffering from cancer, wherein the cancer is characterized by reduced or absent MTAP expression, absent MTAP gene, or reduced MTAP protein function.

[0042] The present inventors have discovered that these compounds are highly effective MAT2A inhibitors with extremely strong MAT2A inhibitory activity. They can be used to prepare drugs for the prevention and / or treatment of conditions associated with MAT2A inhibition, including cancers caused by reduced or absent MTAP expression, MTAP gene deletion, or decreased MTAP protein function. The present invention is based on these discoveries. Detailed Description of the Invention

[0044] Various aspects and features of the present invention are further described below.

[0045] All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail. The following are definitions of various terms used in the present invention, which apply to the terms used throughout the specification of this application, unless otherwise specified in the specific case.

[0046] The compounds according to the present invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

[0047] The compounds of the present invention possess asymmetric centers. Compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art will appreciate how to prepare optically active forms, such as by racemate resolution or synthesis from optically active starting materials. Unless otherwise indicated with respect to specific stereochemistry or isomeric forms, the present invention encompasses all chiral, diastereoisomer, and racemic forms. Methods for preparing the compounds of the present invention and intermediates thereto are also intended to be included in the present invention. All tautomers of the compounds of the present invention are also intended to be included in the present invention.

[0048] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent, as long as the valence of the particular atom is normal and the resulting compound after the substitution is stable.

[0049] 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 amino group, respectively, wherein the alkyl group is as described herein.

[0050] 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.

[0051] As used herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include its subgroups. For example, when referring to "C1-C6 alkyl", it may also include sub-ranges of groups represented by C1-C4 alkyl, C1-C3 alkyl, C2-C6 alkyl, C2-C4 alkyl, etc., as well as specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

[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. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom 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," "heterocyclo," "heterocyclic," and "heterocyclyl" include "heteroaryl" groups, as defined below.

[0053] 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.

[0054] 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, the heteroatom-containing ring preferably having 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 fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated, or unsaturated. The nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be quaternized. A bicyclic or tricyclic heteroaryl must include at least one fully aromatic ring, but the other one or more fused rings may be aromatic or non-aromatic. The heteroaryl group may be attached to any available nitrogen or carbon atom of any ring. If the other ring is cycloalkyl or heterocycle, it is further optionally substituted with =0 (oxo), where valence permits.

[0055] 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.

[0056] As used herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to a subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate during chiral resolution. Although the salt of this intermediate cannot be directly administered to a subject, the salt can play a role in obtaining the final product of the present invention.

[0057] As described herein, the term "disease" refers to a physical condition of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegenerative diseases described in the present invention.

[0058] Cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy, and hormone therapy.

[0059] "Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other sites in the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cell" refers to a cell that is undergoing an early, intermediate, or advanced stage of multistep neoplastic progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis and hereditary non-polyposis colorectal cancer, esophageal cancer, lip cancer, 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 lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid 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, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myeloma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma, lymphoma, pancreatic ductal adenocarcinoma.

[0060] The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0061] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0062] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known method of administration.

[0063] 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.

[0064] The solvents used in this application can be obtained commercially. The abbreviations used in this application are as follows:

[0065] Table 1: Meaning of abbreviations

[0066]

[0067] The compounds of the present invention were named manually or using Chemdraw 14.0 software, and commercially available compounds were named according to the supplier's catalog.

[0068] Beneficial technical effects

[0069] The inventors have discovered that the compounds of the present invention exhibit excellent MAT2A inhibitory activity, with IC50 values ​​lower than those of the positive control drug AG270. This invention provides a class of structurally novel and highly active MAT2A inhibitor compounds, which have promising applications in preventing and / or treating indications associated with MAT2A inhibition, such as decreased or absent MTAP expression, MTAP gene deletion, and decreased MTAP protein function. DETAILED DESCRIPTION

[0070] The following embodiments are intended to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the present invention in any way.

[0071] For all of the following examples, standard procedures and methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and / or mass spectroscopy (MS).

[0072] The structures of the compounds of the present invention were confirmed by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are expressed in parts per million (ppm). NMR measurements were performed using a Bruker Avance-400 NMR spectrometer. The solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), and deuterated chloroform (CDCl3), with tetramethylsilane (TMS) as the internal standard.

[0073] Liquid phase mass spectrometry LC-MS measurement The liquid phase part used ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part used Xevo G2-S Qtof mass spectrometer.

[0074] The starting materials used 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: 1-{3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-7-oxo-2-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)-3-(pyridin-2-yl}urea (Compound 1)

[0076] Synthesis route:

[0077]

[0078] Step 1: Synthesis of 1-(4-methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (Intermediate Compound 1-1)

[0079] Benzoylacetonitrile (20 g, 137.78 mmol, 1 eq), (4-methoxyphenyl)hydrazine hydrochloride (23.06 g, 151.56 mmol, 1.1 eq), AcOH (50 mL), and ethanol (150 mL) were added to a 500 mL reaction flask and refluxed at 85°C overnight. The pH was adjusted to neutral with 4 M NaOH solution, and the excess ethanol was removed by concentration. The mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and spin-dried. Ethyl acetate (10 mL) and petroleum ether (50 mL) were added to the slurry, and the mixture was filtered. The filter cake was dried to yield 1-(4-methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (33.2 g, 86.26%) as a yellow solid. LCMS (ESI) [M+H] + :280.14. 1 HNMR(400MHz,Chloroform-d)δ7.81(d,J=7.3Hz,2H),7.41(t,J=7.6Hz,2H),7.33(d,J=7.4H z, 1H), 7.21 (d, J = 8.6Hz, 2H), 6.90 (d, J = 8.6Hz, 2H), 5.93 (s, 1H), 5.25 (s, 2H), 3.82 (s, 3H).

[0080] Step 2: 4-(cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (intermediate Synthesis of Compound 1-2)

[0081] 1-(4-Methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (33 g, 118.94 mmol, 1 eq) and cyclohexanone (23.32 g, 237.88 mmol, 2 eq) were added to an acetic acid solution (100 mL) and reacted at room temperature for 48 hours. The pH was adjusted to neutral with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate. The mixture was dried over anhydrous sodium sulfate and concentrated. Separation by normal phase column chromatography with a mixture of ethyl acetate and petroleum ether (3:7) yielded the product, which was then dried to give 4-(cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (42 g, 72.44%). LCMS (ESI) [M+H] + :360.20. 1 H NMR (400MHz, DMSO-d6) δ7.55(d,J=7.1Hz,2H),7.32(t,J=7.5Hz,2H),7.25-7.15(m,3H),6.88(d,J=8.7Hz,2H ),5.58(s,1H),5.10(s,2H),4.90(s,2H),3.71(s,3H),2.14(p,J=3.0Hz,2H),1.95-1.85(m,2H),1.60(s,4H).

[0082] Step 3: Synthesis of 4-(cyclohex-1-en-1-yl)-3-phenyl-1H-pyrazol-5-amine (Intermediate Compound 1-3)

[0083] 4-(Cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-3-phenyl-1H-pyrazol-5-amine (30 g, 83.58 mmol, 1 eq) was dissolved in trifluoroacetic acid (18 mL). Trifluoromethanesulfonic anhydride (6 mL) was then added dropwise and allowed to react at room temperature for 3 hours. The pH was adjusted to 8 with saturated aqueous NaCO₃ at 0°C. The mixture was extracted with ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, and concentrated. The product was separated by a normal phase column using a mixture of ethyl acetate and petroleum ether (3:7) and dried to give 4-(cyclohex-1-en-1-yl)-3-phenyl-1H-pyrazol-5-amine (10 g, 50.06%). LCMS (ESI) [M+H] + :240.15. 1 H NMR(400MHz,Chloroform-d)δ7.55-7.47(m,2H),7.43-7.29(m,3H),5.81(tt,J=3.7,1 .7Hz, 1H), 2.25-2.14 (m, 2H), 1.99 (ddt, J = 8.4, 6.1, 2.7Hz, 2H), 1.64 (p, J = 3.0Hz, 4H).

[0084] Step 4: 3-(cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine Synthesis of pyridine-7(4H)-one (intermediate compound 1-4)

[0085] 4-(Cyclohex-1-en-1-yl)-3-phenyl-1H-pyrazol-5-amine (10 g, 41.81 mmol, 1 eq), dimethyl 2-(4-methoxyphenyl)malonate (19.91 g, 83.62 mmol, 2 eq), triethylamine (12.67 g, 125.43 mmol, 3 eq), and xylene (50 mL) were added to a 250 mL single-necked flask and refluxed for 3 hours. A yellow solid precipitated and was filtered. The filter cake was washed with tetrahydrofuran (10 mL) and 2M HCl (10 mL), respectively, and dried to give 3-(cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (2.5 g, 14.46%). LCMS (ESI) [M+H] + :414.18. 1 H NMR (400MHz, DMSO-d6) δ7.85-7.68(m,2H),7.51-7.29(m,5H),7.02-6.86(m,2H),5. 85-5.77(m,1H),3.78(s,3H),2.16(d,J=7.1Hz,2H),2.02(s,2H),1.71-1.54(m,4H).

[0086] Step 5: 5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a] Synthesis of pyrimidine (intermediate compound 1-5)

[0087] 3-(Cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (2.5 g, 6.05 mmol, 1 eq) and phosphorus oxychloride (10 mL) were added to a 50 mL single-necked flask and refluxed at 100°C for 3 hours. The pH was adjusted to 8 with saturated aqueous NaHCO₃. The liquid phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The product was separated by normal phase column separation using a 3:7 ratio of ethyl acetate and petroleum ether. The product was then dried to give 5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine (1.83 g, 67.44%). LCMS (ESI) [M+H] + :450.11. 1 HNMR(400MHz,DMSO-d6)δ7.88-7.81(m,2H),7.59-7.38(m,5H),7.16-7.08(m,2H ),5.92-5.86(m,1H),3.86(s,3H),2.22(d,J=5.9Hz,4H),1.72(q,J=8.0Hz,4H).

[0088] Step 6: 5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(4-methoxyphenyl)-2-phenylpyrrolidone Synthesis of oxazolo[1,5-a]pyrimidine (intermediate compound 1-6)

[0089] Under an ice bath, sodium methoxide (1.09 g, 20.35 mmol, 5 eq) dissolved in methanol (4 mL) was added dropwise to 5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine (1.83 g, 4.07 mmol, 1 eq) dissolved in dichloromethane (6 mL). The reaction was allowed to continue at this temperature for 1 hour. The mixture was extracted with water and ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated using a normal phase column using a mixture of ethyl acetate and petroleum ether (1:5). The product was then dried to afford 5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine (909.8 mg, 50.16%) as a yellow solid. LCMS (ESI) [M+H] +: 446.16.

[0090] Step 7: 3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a] Synthesis of pyrimidine-5-amine (intermediate compound 1-7)

[0091] 5-Chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine (900 mg, 2.02 mmol, 1 eq), tBuBrettphos Pd G3 (540 mg, 0.4 mmol, 0.2 eq), tBuBrettphos (98 mg, 0.2 mmol, 0.1 eq), t-BuONa (580 mg, 6.06 mmol, 3 eq), NH3 / 1,4-dioxane (0.4 M, 15 mL, 6.06 mmol, 3 eq), and 1,4-dioxane (4 mL) were added to a 40 mL microwave reaction vial and reacted at 60°C for 1 hour. Separation was performed on a reverse phase column using a water (1‰ formic acid) and acetonitrile system with a polarity of 35%-55% for 20 minutes and monitored at 254 nm. The reaction mixture was concentrated to give a yellow solid 3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-amine (371 mg, 43.05%). LCMS (ESI) [M+H]+: 427.21.

[0092] Step 8: 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5- Synthesis of pyrimidin-5-yl}-3-(pyridin-2-yl)urea (Intermediate Compound 1-8)

[0093] At 0°C, triphosgene (267 mg, 0.9 mmol, 1.1 eq) dissolved in dichloromethane (1.5 mL) was added dropwise to a solution of 3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-amine (350 mg, 0.82 mmol, 1 eq) dissolved in dichloromethane (1.5 mL). Triethylamine (248 mg, 2.46 mmol, 3 eq) dissolved in dichloromethane was then added dropwise. The mixture was stirred at room temperature for 30 minutes. The product was evaporated under reduced pressure, and the concentrated solid was dissolved in dichloromethane (5 mL). 2-aminopyridine (0.98 mmol, 1.2 eq) was then added and refluxed for 30 minutes. The product was concentrated and separated by reverse phase column separation using a water (1‰ formic acid) and acetonitrile system with a polarity of 40%-60% for 25 minutes, monitored at 254 nm. The product was dried and 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl}-3-(pyridin-2-yl)urea (105 mg, 30.68%) was obtained. LCMS (ESI) [M+H] + :547.24.

[0094] Step 9: 1-{3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-7-oxo-2-phenyl-4,7-dihydropyridine Synthesis of oxazolo[1,5-a]pyrimidin-5-yl)-3-(pyridin-2-yl}urea (Compound 1)

[0095] 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-5-yl}-3-(pyridin-2-yl)urea (100 mg, 0.18 mmol, 1 eq) and HCl / 1,4-dioxane (4M, 5 mL) were stirred at room temperature overnight and concentrated to give 1-{3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-7-oxo-2-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl}-3-(pyridin-2-yl)urea (90 mg, 92.37%). LCMS (ESI) [M+H] + :533.22.

[0096] The compounds in Table 2 were obtained by referring to the method of Example 1:

[0097] Table 2: Structure and characterization of compounds 2, 3, and 4

[0098]

[0099] Example 5: 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl}urea (Compound 5)

[0100] Synthesis route:

[0101]

[0102] Step 1: Synthesis of dimethyl 2-(pyridin-3-yl)malonate (Intermediate Compound 5-1)

[0103] Methyl pyridine-3-acetate (5 g, 33.1 mmol, 1 eq) was dissolved in dimethyl carbonate (20 mL). NaH (1.98 g, 82.75 mmol, 5 eq) was added portionwise at 0°C. After 30 minutes, the mixture was allowed to react at room temperature overnight. Water and ethyl acetate were added for extraction, and the organic phase was dried over anhydrous sodium sulfate and spin-dried. Separation was performed on a normal phase column using a 1:1 system of ethyl acetate and petroleum ether. The product was then spin-dried to give dimethyl 2-(pyridin-3-yl)malonate (3.3 g, 47.68%) as a yellow oil. LCMS (ESI) [M+H] + :210.07. 1 H NMR (400MHz, Chloroform-d) δ 8.61-8.54 (m, 2H), 7.84 (dt, J=8.0, 2.0Hz, 1H), 7.32 (ddd, J=8.0, 4.8, 0.8Hz, 1H), 4.66 (s, 1H), 3.77 (s, 6H).

[0104] Step 2: 3-(cyclohex-1-en-1-yl)-5-hydroxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine Synthesis of pyridine-7(4H)-one (intermediate compound 5-2)

[0105] Dimethyl 2-(pyridin-3-yl)malonate (3 g, 14.35 mmol, 2 eq), 4-(cyclohex-1-en-1-yl)-3-phenyl-1H-pyrazol-5-amine (1.7 g, 7.13 mmol, 1 eq), triethylamine (2.16 g, 21.39 mmol, 3 eq), and xylene (30 mL) were added to a 100 mL single-necked flask and refluxed for 3 hours. A yellow solid precipitated and was filtered. The filter cake was washed with tetrahydrofuran (5 mL) and 2M hydrochloric acid (5 mL), respectively, and dried to give 3-(cyclohex-1-en-1-yl)-5-hydroxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (1.36 g, 49.80%). LCMS (ESI) [M+H] + :385.16. 1H NMR (400MHz, DMSO-d6) δ15.02(s,1H),10.31(s,1H),9.70(d,J=2.1Hz,1H),9.44(d,J=8.6Hz,1H),8.36(d,J=5.4Hz,1H),7.83(dd,J=8.6,5.5H z,1H),7.77-7.66(m,2H),7.41(dd,J=8.2,6.7Hz,2H),7.36-7.27(m,1H),5.72-5.62(m,1H),2.13(s,2H),2.00(s,2H),1.64(p,J=2.8Hz,4H).

[0106] Step 3: 5,7-dichloro-3-(cyclohex-1-en-1-yl)-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine (intermediate compound Synthesis of 5-3)

[0107] 3-(Cyclohex-1-en-1-yl)-5-hydroxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-7(4H)-one (1.3 g, 3.12 mmol, 1 eq) and phosphorus oxychloride (8 mL) were added to a 50 mL single-necked flask and refluxed at 100°C for 3 hours. The pH was adjusted to 8 with saturated aqueous NaHCO₃. The liquid phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The product was separated by normal phase column separation using a 3:7 system of ethyl acetate and petroleum ether, and dried to give 5,7-dichloro-3-(cyclohex-1-en-1-yl)-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine (390 mg, 27.43%). LCMS (ESI) [M+H] + :421.09. 1 H NMR(400MHz, DMSO-d6)δ8.76-8.68(m,2H),7.98(dt,J=7.9,1.9Hz,1H),7.87-7.81(m,2H),7.62(dd d,J=7.8,4.9,0.9Hz,1H),7.58-7.44(m,3H),5.93-5.82(m,1H),2.20(d,J=5.7Hz,4H),1.71(s,4H).

[0108] Step 4: 5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1, Synthesis of 5-a]pyrimidine (intermediate compound 5-4)

[0109] Under an ice bath, sodium methoxide (192 mg, 3.55 mmol, 5 eq) dissolved in methanol (1.5 mL) was added dropwise to 5,7-dichloro-3-(cyclohex-1-en-1-yl)-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine (300 mg, 0.714 mmol, 1 eq) dissolved in dichloromethane (6 mL). The reaction was allowed to continue at this temperature for 1 hour. The mixture was extracted with water and ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated by normal phase column chromatography using a mixture of ethyl acetate and petroleum ether (1:4). The product was obtained by spin drying to obtain 5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine (164 mg, 55.18%) as a yellow solid. LCMS (ESI) [M+H] + :417.14. 1 H NMR (400MHz, DMSO-d6) δ8.68(d,J=6.1Hz,2H),7.95(dt,J=7.9,2.0Hz,1H),7.88-7.82(m,2H),7.61-7.56(m,1H),7.53(d,J =7.6Hz,2H),7.51-7.46(m,1H),5.90-5.81(m,1H),4.29(s,3H),2.22(d,J=6.1Hz,4H),1.72(td,J=14.3,13.6,6.1Hz,4H).

[0110] Step 5: 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5- Synthesis of pyrimidin-5-yl}urea (intermediate compound 5-5)

[0111] 5-Chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidine (100 mg, 0.24 mmol, 1 eq), urea (28 mg, 0.48 mmol, 2 eq), Xant-Phos (41 mg, 0.07 mmol, 0.3 eq), palladium acetate (11 mg, 0.05 mmol, 0.2 eq), cesium carbonate (156 mg, 0.48 mmol, 3 eq) and 1,4-dioxane solution (1.5 mL) were added to a microwave reaction tube and microwaved at 100°C for 45 minutes. Separation was performed on a reverse phase column using a water (1‰ formic acid) and acetonitrile system with a polarity of 75%-90% for 20 minutes and monitored at 254 nm. Concentration gave 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl}urea (35 mg, 33.08%). LCMS (ESI) [M+H] + :441.20.

[0112] Step 6: 1-{3-(cyclohex-1-en-1-yl)-7-oxo-2-phenyl-6-(pyridin-3-yl)-4,7-dihydropyrazole Synthesis of 5-[1,5-a]pyrimidin-5-yl}urea (Compound 5)

[0113] 1-{3-(cyclohex-1-en-1-yl)-7-methoxy-2-phenyl-6-(pyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl}urea (35 mg, 0.08 mmol, 1 eq), HCl (4 M in dioxane, 0.5 mL), and 1,4-dioxane (1.5 mL) were added to an 8 mL reaction vial and reacted overnight at room temperature. The pH was adjusted to 9 with saturated potassium carbonate. The mixture was extracted with ethyl acetate and the organic phase was dried by rotary evaporation. Separation was performed on a reversed-phase column using water (1‰ formic acid) and acetonitrile with a polarity of 75%-95% for 25 minutes, monitored at 254 nm. Yellow solid 1-{3-(cyclohex-1-en-1-yl)-7-oxo-2-phenyl-6-(pyridin-3-yl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl}urea (96 mg, 29.49%). LCMS (ESI) [M+H] + :427.18.

[0114] The following compounds in Table 3 were obtained by referring to the method of Example 5:

[0115] Table 3: Structure and characterization of compounds 6, 7, 8, and 9

[0116]

[0117]

[0118] Example 11 5-[(1H-1,2,4-triazol-3-yl)amino]-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (Compound 11)

[0119] Synthesis route:

[0120]

[0121] Step 1: 3-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazole (intermediate Synthesis of compound 11-1)

[0122] To 3-nitro-4H-1,2,4-triazole (1 g, 8.77 mmol, 1 eq) dissolved in tetrahydrofuran (5 mL) was added sodium hydride (526 mg, 21.92 mmol, 2.5 eq) in portions under an ice bath. The mixture was allowed to react at constant temperature for 10 minutes. 2-(Trimethylsilyl)ethoxymethyl chloride (1.75 g, 10.52 mmol, 1.2 eq) was then added and the mixture was allowed to react at room temperature for 1.5 hours. Water and ethyl acetate were added, and the mixture was extracted. The organic phase was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The product was separated by normal phase column chromatography using a 1:3 system of ethyl acetate and petroleum ether. The product was then dried to give 3-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazole (350 mg, 20.65%) as a yellow oil. LCMS (ESI) [M+H] + :245.11. 1 H NMR (400MHz, Chloroform-d) δ7.87(s,1H),5.28(s,2H),3.69-3.55(m,2H),0.99-0.86(m,2H),0.00(s,9H).

[0123] Step 2: 1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazol-3-amine (intermediate Synthesis of Compound 11-2)

[0124] 3-Nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazole (300 mg, 1.09 mmol, 1 eq) and Pd / C (30 mg) were added to methanol (10 mL) and reacted at room temperature under a hydrogen atmosphere for 3 hours. Filter through Celite to remove the palladium on carbon. The product was dried to give 1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazol-3-amine (235 mg, 89.52%). LCMS (ESI) [M+H] + :215.13. 1 H NMR (400MHz, DMSO-d6) δ8.16(s,1H),5.20(s,2H),3.57-3.48(m,2H),0.88-0.78(m,2H),-0.03(s,9H).

[0125] Step 3: 3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-[(1-{[2-(trimethylsilyl) [alkyl)ethoxy]methyl}-1H-1,2,4-triazol-3-yl)amino]pyrazolo[1,5-a]pyrimidin-7(4H)-one (intermediate Synthesis of compound 11-3)

[0126] 5-Chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidine (200 mg, 0.45 mmol, 1 eq), 1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazol-3-amine (192 mg, 0.9 mmol, 2 eq), Xant-Phos (78 mg, 0.13 mmol, 0.3 eq), palladium acetate (19 mg, 0.08 mmol, 0.2 eq), cesium carbonate (439 mg, 1.35 mmol, 3 eq) and 1,4-dioxane solution (4 mL) were added to a microwave reaction tube and microwaved at 120 ° C for 1 hour. Reverse phase column separation was performed using a water (1‰ formic acid) and acetonitrile system with a polarity of 60%-85% for 25 minutes and monitored at 254 nm. The product was spin-dried to give 3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-[(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazol-3-yl)amino]pyrazolo[1,5-a]pyrimidin-7(4H)-one (57.6 mg, 21.05%). LCMS (ESI) [M+H] + :610.30. 1 H NMR (400MHz, DMSO-d6) δ8.65(s,1H),8.27(s,1H),7.71(s,2H),7.50-7.36(m,3H),7.29(d,J=8.3Hz,2H),7.01(d,J=8.3Hz,2H), 5.87(s,1H),5.40(s,2H),3.82(s,3H),3.60-3.58(m,2H),2.24-2.20(m,4H),1.71-1.67(m,4H),0.88-0.86(m,2H),0.01(s,9H).

[0127] Step 4: 5-[(1H-1,2,4-triazol-3-yl)amino]-3-(cyclohex-1-en-1-yl)-6-(4-methoxybenzene Synthesis of 2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (Compound 11)

[0128] 3-(Cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenyl-5-[(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-1,2,4-triazol-3-yl)amino]pyrazolo[1,5-a]pyrimidin-7(4H)-one (50 mg, 0.08 mmol, 1 eq) was dissolved in trifluoroacetic acid (0.5 mL) and dichloromethane (1.5 mL) and allowed to react at room temperature for 2 hours. Aqueous ammonia was added dropwise to adjust the pH to neutral. The solution was concentrated to yield 5-[(1H-1,2,4-triazol-3-yl)amino]-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-2-phenylpyrazolo[1,5-a]pyrimidin-7(4H)-one (36.9 mg, 94.04%). LCMS (ESI) [M+H] + :480.21.

[0129] The following compounds were obtained by the method of Example 11:

[0130] Table 4: Structure and characterization of compound 13

[0131]

[0132] Example 12 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one (Compound 12)

[0133] Synthesis route:

[0134]

[0135] Step 1: Benzyl{5-[5-amino-1-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl}aminomethoxy Synthesis of acid ester (intermediate compound 12-1)

[0136] Benzyl [5-(2-cyanoacetyl)pyridin-2-yl] carbamate (20 g, 67.77 mmol, 1 eq), (4-methoxyphenyl)hydrazine hydrochloride (14.06 g, 74.55 mmol, 1.1 eq), AcOH (50 mL), and ethanol (150 mL) were added to a 500 mL reaction flask and refluxed at 85°C overnight. The pH was adjusted to neutral with 4 M NaOH solution, and the excess ethanol was removed by concentration. The mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and spin-dried. Ethyl acetate (15 mL) and petroleum ether (50 mL) were added to the slurry, and the mixture was filtered. The filter cake was dried to obtain benzyl {5-[5-amino-1-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl} carbamate (25.2 g, 86.63%) as a yellow solid. LCMS (ESI) [M+H] + :430.18.

[0137] Step 2: Benzyl {5-[5-amino-4-(cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-1H-pyrazol-3-yl] Synthesis of pyridin-2-yl}carbamate (intermediate compound 12-2)

[0138] Benzyl {5-[5-amino-1-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl}carbamate (40 g, 93.2 mmol, 1 eq) and cyclohexanone (18.29 g, 186.4 mmol, 2 eq) were added to an acetic acid solution (120 mL) and reacted at room temperature for 48 hours. The pH was adjusted to neutral with saturated sodium carbonate solution, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The product was separated by a normal phase column using a 1:2 system of ethyl acetate and petroleum ether. The product was then dried to obtain benzyl {5-[5-amino-4-(cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-1H-pyrazol-3-yl]pyridin-2-yl}carbamate (21.3 g, 44.87%).

[0139] LCMS (ESI) [M+H] + :510.24.

[0140] Step 3: Benzyl{5-[5-amino-4-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl}aminomethoxy Synthesis of acid ester (intermediate compound 12-3)

[0141] Benzyl {5-[5-amino-4-(cyclohex-1-en-1-yl)-1-(4-methoxybenzyl)-1H-pyrazol-3-yl]pyridin-2-yl}carbamate (15 g, 34.95 mmol, 1 eq) was dissolved in trifluoroacetic acid (21 mL). Trifluoromethanesulfonic anhydride (7 mL) was then added dropwise and allowed to react at room temperature for 3 hours. The pH was adjusted to 8 with saturated aqueous NaCO₃ at 0°C. The mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The product was separated by a normal phase column using a mixture of ethyl acetate and petroleum ether (1:2). The product was then dried to give benzyl {5-[5-amino-4-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl}carbamate (7.9 g, 68.73%).

[0142] LCMS (ESI) [M+H] + :390.19.

[0143] Step 4: Benzyl (5-{3-(cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-7-oxo-4,7-diol Synthesis of 1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (Intermediate Compound 12-4)

[0144] Dimethyl 2-(4-methoxyphenyl)malonate (8.56 g, 35.97 mmol, 2 eq) and benzyl {5-[5-amino-4-(cyclohex-1-en-1-yl)-1H-pyrazol-3-yl]pyridin-2-yl}carbamate (7 g, 17.98 mmol, 1 eq), triethylamine (5.45 g, 53.94 mmol, 3 eq) and xylene (30 mL) were added to a 100 mL single-necked bottle and refluxed for 3 hours. A yellow solid precipitated and was filtered. The filter cake was washed with tetrahydrofuran (8 mL) and 2M HCl (8 mL), respectively, and dried to give benzyl (5-{3-(cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (3.3 g, 32.57%).

[0145] LCMS (ESI) [M+H] + :564.22.

[0146] Step 5: Benzyl (5-{5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)pyrazolo[1,5-a] Synthesis of pyrimidin-2-yl}pyridin-2-yl)carbamate (intermediate compound 12-5)

[0147] Benzyl (5-{3-(cyclohex-1-en-1-yl)-5-hydroxy-6-(4-methoxyphenyl)-7-oxo-4,7-dihydropyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl) carbamate (2.5 g, 4.43 mmol, 1 eq) and phosphorus oxychloride (10 mL) were added to a 50 mL single-necked flask and refluxed at 100°C for 3 hours. The pH was adjusted to 8 with saturated aqueous NaHCO₃. The liquid phase was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The product was separated by normal phase column separation using a 1:2 system of ethyl acetate and petroleum ether, and the product was dried to give benzyl (5-{5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl) carbamate (1.8 g, 67.68%). LCMS (ESI) [M+H] + :600.16.

[0148] Step 6: Benzyl (5-{5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)pyrazolo[3-(4-yl) ... Synthesis of [1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (intermediate compound 12-6)

[0149] Under an ice bath, sodium methoxide (675 mg, 12.5 mmol, 5 eq) dissolved in methanol (7 mL) was added dropwise to benzyl (5-{5,7-dichloro-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (1.5 g, 2.5 mmol, 1 eq) dissolved in dichloromethane (6 mL). The reaction was allowed to continue at this temperature for 1 hour. The mixture was extracted with water and ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The product was separated using a normal phase column with a mixture of ethyl acetate and petroleum ether (1:7). The solution was dried to give a yellow solid benzyl (5-{5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (960 mg, 64.42%). LCMS (ESI) [M+H] + :596.21.

[0150] Step 7: Benzyl (5-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-5-(pyridin-2-yl) Synthesis of 2-amino)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (Intermediate Compound 12-7)

[0151] Benzyl (5-{5-chloro-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (800 mg, 1.34 mmol, 1 eq), Xant-Phos (232 mg, 0.4 mmol, 0.3 eq), palladium acetate (60 mg, 0.26 mmol, 0.2 eq) and cesium carbonate (1.09 g, 3.35 mmol, 2.5 eq) were added to a 1,4-dioxane solution (10 mL) and microwaved at 100°C for 1 hour. Reverse phase column separation was performed using a water (1‰ formic acid) and acetonitrile system with a polarity of 45%-55% for 20 minutes and monitored at 254 nm. The reaction mixture was concentrated to give benzyl (5-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (242 mg, 27.56%) as a yellow solid.

[0152] LCMS (ESI) [M+H] + :654.28.

[0153] Step 8: 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)- Synthesis of N-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5-amine (Intermediate Compound 12-8)

[0154] Benzyl (5-{3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-2-yl}pyridin-2-yl)carbamate (200 mg, 0.30 mmol, 1 eq) and palladium on carbon (20 mg) were added to methanol (5 mL) and reacted at room temperature under a hydrogen atmosphere for 3 hours. The palladium on carbon was removed by filtration through celite. 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-N-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5-amine (140 mg, 88.07%) was obtained as a yellow solid by spin drying. LCMS (ESI) [M+H] + :520.25.

[0155] Step 9: 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-5-(pyridine- Synthesis of 2-amino)pyrazolo[1,5-a]pyrimidin-7(4H)-one (Compound 12)

[0156] 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-7-methoxy-6-(4-methoxyphenyl)-N-(pyridin-2-yl)pyrazolo[1,5-a]pyrimidin-5-amine (120 mg, 0.23 mmol, 1 eq) and hydrochloric acid / 1,4-dioxane (4M, 5 mL) were stirred at room temperature overnight and concentrated to give 2-(6-aminopyridin-3-yl)-3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-5-(pyridin-2-ylamino)pyrazolo[1,5-a]pyrimidin-7(4H)-one (75 mg, 64.23%). LCMS (ESI) [M+H] + :506.23.

[0157] The following Table 5 compounds were obtained by referring to the method of Example 12:

[0158] Table 5: Structure and characterization of compounds 10, 14, and 15

[0159]

[0160]

[0161] Experimental Example 1: Binding ability test of the compound of the present invention to mat2a protein

[0162] The CETSA assay is a molecular test that measures the affinity of a drug for its target protein. The principle is that drug binding to the target protein stabilizes its structure. Cell or tissue samples are treated with a drug candidate. If the drug candidate is a mat2a inhibitor, it will 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 by Western blot. Conversely, heating will destabilize the mat2a protein, and the amount of protein detected will be lower. This method assesses the binding ability of the drug to the target protein and is used to screen for mat2a protein inhibitors.

[0163] The advantages of this method are:

[0164] (1) The types of test samples are more diverse: not only can the affinity of drugs for mat2a proteins in cell samples be tested, but also the effects of mat2a proteins in tissues and organs can be tested, which is convenient for testing drug distribution and toxic side effects in animals or humans, and can provide reference data for in vitro and in vivo activity testing, and provide reference guidance for preclinical and clinical research.

[0165] (2) The detection method is more convenient: the expression level of mat2a protein can be detected by western blot, a conventional experimental method, which is convenient for carrying out a large number of drug screening tests.

[0166] (3) More instructive significance: This experimental method can reflect the direct binding of drugs to mat2a protein, and can better reflect the interaction between drugs and targets, thus avoiding the occurrence of drug off-target phenomena.

[0167] Specific experimental process:

[0168] Take HCT116 cells in the logarithmic growth phase, wash the cells three times with PBS, and centrifuge at 2000g for 2 minutes. Use 0.4% trypan blue exclusion method to detect cell viability (viability>90%), use a cell counter to count the cells and adjust the cell density to 2×106cells / ml. Use cell lysis buffer containing protease inhibitor PMSF to lyse on ice for 30 minutes. Use BCA kit to measure the concentration of protein samples. Incubate the samples with candidate drugs, control drugs and control reagents for 30 minutes, respectively, and heat each group of samples at about 10 set temperature points. Return to room temperature, centrifuge the samples at 20000g, and collect the supernatant. Use NuPAGE TMThe protein samples were denatured by heating at 70°C for 10 minutes using LDS sample buffer (4×). After the samples returned to room temperature, they were analyzed by western blot. The protein loading amount was controlled at 20 μg. Protein electrophoresis: the voltage of the stacking gel was set to 60V, and the voltage of the separation gel was set to 120V. After the electrophoresis, electrotransfer was started. The electrotransfer conditions were set to 250 mA for 2 hours. Blocking was performed with 5% BSA for 1 hour. Specific primary antibody was added and incubated on a shaker at 4°C overnight. Wash with TBST 4 times, 2.5 minutes each time. Incubate with secondary antibody on a shaker at room temperature for 1 hour, wash with TBST 4 times, 2.5 minutes each time, and develop using ECL. The expression level of mat2a protein was detected in different groups and at different temperature points. Western blot bands were converted and processed using Image J and GraphPad software, and the Tm difference and EC50 were calculated. The dissociation constant Kd was obtained by fitting the change of Tm with compound concentration. EC50 stands for concentration for 50% of maximal effect (EC50), which refers to the drug concentration that elicits an effective response in 50% of individuals. Compound 153 (AG270) reported in CN201780066270.4 was used as a positive reference compound.

[0169] The test results are shown in Table 6 below, where the T m Values ​​are categorized as follows:

[0170] “+” indicates that the Kd value is greater than 10 μM;

[0171] “++” indicates that the Kd value is less than 10 μM and greater than 1 μM;

[0172] “+++” indicates that the Kd value is less than 1 μM;

[0173] The lower the Tm, the stronger the binding affinity of the compound to the mat2a protein.

[0174] The EC50 values ​​of each compound are classified according to the following description:

[0175] “+” indicates EC50 value greater than 1 μM;

[0176] “++” indicates that the EC50 value is less than 1 μM and greater than 100 nM;

[0177] “+++” indicates that the EC50 value is less than 100 nM and greater than 10 nM;

[0178] “++++” indicates that the EC50 value is less than 10 nM.

[0179] Table 6: K d and EC50 results

[0180] Compound number <![CDATA[K d ]]> EC50 1 ++ ++++ 2 +++ +++ 3 ++ +++ 4 ++ +++ 5 +++ ++++ 6 ++ ++++ 7 ++ ++++ 8 +++ +++ 9 ++ ++++ 10 +++ ++++ 11 ++ ++++ 12 +++ ++++ 13 ++ ++++ 14 +++ +++ 15 ++ ++++ Positive drug AG270 ++ ++

[0181] The Kd experimental results show that the compounds of the present invention have good ability to bind to the MAT2A protein, and their in vitro affinity is similar to that of the reference compound. Therefore, the compounds of the present invention can be used as an effective MAT2A protein regulator; EC50 experimental data show that the compounds of the present invention have good activity in stimulating MAT2A cytokines and have good MAT2A protein agonist function, especially compounds 1, 5, 6, 7, 9, 10, 11, 12, 13, 15, etc. Compared with the reference compound, the compounds of the present invention have comparable or even higher activity in stimulating immune cells to produce cytokines.

[0182] Experimental Example 2: Cell proliferation inhibition assay

[0183] Methionine adenosyltransferase (MAT2A), an enzyme that catalyzes the ubiquitous methyl donor S-adenosyl-L-methionine (SAM), plays a crucial role in metabolism and epigenetic inheritance. Studies have shown that methylthioadenosine phosphorylase (MTAP) and the adjacent tumor suppressor gene CDKN2A are co-deficient in approximately 15% of cancer patients. Tumor cells lacking MTAP are more sensitive to MAT2A inhibitors. Consequently, MAT2A has been identified as a synthetic lethal target in MTAP-deficient cancers. Based on this, the creation of an MTAP-knockout HCT116 stable cell line using CRISPR / CAS9 technology to screen for MAT2A inhibitors is considered a reliable and effective approach.

[0184] The advantages of this method are:

[0185] (1) Based on the research of the signaling pathway of mat2a, it was found that by constructing MTAP - / - The use of HCT116 cells as a screening tool for mat2a inhibition is widely practiced.

[0186] (2) When used in conjunction with an enzyme-labeled instrument, it can be used for large-scale screening of mat2a inhibitors and even high-throughput screening.

[0187] MTAP- / -HCT116 cell construction process:

[0188] (1) sgRNA design: Based on the literature data, the gRNA sequence was designed online using the GenCRISPR gRNA Design Tool. After BLAST verification on NCBI, one control gRNA and three target gRNAs were synthesized.

[0189] (2) Construction of lentiviral expression vector: Select Bsmbi restriction endonuclease and digest the vector: 37℃, 15min. Heat inactivate: 80℃, 20min. Perform nucleic acid electrophoresis on the linearized vector and recover the target band by gel cutting. Use T4 ligase to connect the linearized vector and the designed sgRNA sequence: 22℃, 1h. Heat inactivate: 70℃, 5min. Transform competent cells and culture overnight. Pick a single clone, shake the bacteria overnight, extract the plasmid, and after sequencing to verify the inserted gRNA is correct, amplify and extract the plasmid.

[0190] (3) Lentivirus packaging: Inoculate 293T cells into four 6-cm culture dishes to a cell density of 50%-80%. Package lentivirus: Co-transfect 293T cells with pMD2.G, pSPAX.2, and the vector plasmid at a mass ratio of 1.5:3:5. Collect lentivirus fluid: Collect the cell supernatant 48 and 72 hours after co-transfection. Centrifuge at 4000 rpm for 10 min at 4°C. Collect the supernatant and store at 4°C.

[0191] (4)MTAP - / - HCT116 cell line screening: HCT116 cells were seeded into 6-well plates. When the cells grew to 50%-70%, they were infected with lentiviral solution. After 48 hours, 1-2 μg / ml of puromycin was added for screening. After about 1-2 weeks of screening, a portion of the cells were taken for Western blotting. Complete knockout of MTAP was considered the standard for completion of screening.

[0192] (5) MTT experiment to detect the inhibitory effect of drugs: MTAP- / -HCT116 cells were seeded into 96-well plates with a cell density of 2000 cells / well. Different concentrations of drugs were added after 24 hours, and the absorbance of the drugs was detected at 24 hours, 48 ​​hours, and 72 hours. The inhibitory rate IC50 of the drugs on MAT2A at different concentrations and time points was calculated and scored. IC50 (halfmaximal inhibitory concentration) refers to the half-inhibitory concentration of the measured antagonist. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). Compound 153, i.e., AG270, reported in CN201780066270.4, was used as a positive reference compound.

[0193] The test results are shown in Table 7 below, where the IC50 values ​​of the compounds are classified according to the following description:

[0194] +++ less than 100 nM, ++ between 100 nM and 1 μM, + greater than 1 μM.

[0195] The test results are shown in Table 7 below:

[0196] Table 7: IC50 experimental data

[0197] Compound number 24h IC50 IC50 at 48h IC50 at 72h 1 +++ +++ +++ 2 ++ ++ +++ 3 ++ ++ +++ 4 ++ ++ +++ 5 +++ ++ +++ 6 ++ ++ +++ 7 + ++ +++ 8 ++ ++ +++ 9 ++ ++ +++ 10 + ++ +++ 11 ++ ++ +++ 12 + ++ +++ 13 ++ ++ +++ 14 ++ ++ +++ 15 ++ ++ +++ AG270 + ++ ++

[0198] The results showed that the compounds of the present invention have very strong inhibitory effects on MAT2A, with IC50 values ​​reaching the nM level, which is lower than that of the positive control drug AG270. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to MAT2A inhibition.

[0199] The above examples are merely 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 conditions or diseases associated with MAT2A inhibition.

[0200] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, and these changes should be covered by the scope of protection of the present invention.

Claims

1. A compound as described below or a pharmaceutically acceptable salt thereof:

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

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

4. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 or the composition according to claim 2 or 3 in the preparation of a medicament for treating cancer in a subject suffering from cancer, wherein the cancer is characterized by a reduction or absence of MTAP expression, a loss of the MTAP gene, or a reduction in MTAP protein function.

Images