Methionine adenosyltransferase inhibitor, preparation method and application thereof

Abstract

This application provides a methionine adenosyltransferase inhibitor, a pharmaceutical composition containing the same, and its use. The compounds of this application have excellent MAT2a enzyme inhibitory activity and also have excellent inhibitory effects on the growth of cancer cells. Therefore, the compounds of this application may have excellent therapeutic effects in MAT2a-related cancers or tumor diseases.

Description

Technical Field

[0001] The present application relates to the field of medicinal chemistry, and specifically to a methionine adenosyltransferase inhibitor, a preparation method thereof, and its application in the pharmaceutical field. Background Art

[0002] Loss-of-function mutations in tumor suppressor genes are extremely common, yet few therapies have been developed to selectively target these mutations. This is understandable because the missing protein is difficult to directly inhibit for therapeutic benefit. Targeted therapy for tumor suppressor genes inactivated by homozygous deletion is particularly challenging because the lack of residual protein renders therapeutic strategies that directly activate, stabilize, or repair the tumor suppressor gene ineffective.

[0003] 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 and is considered the rate-limiting step in the methionine cycle. SAM is the propylamino donor in polyamine biosynthesis and the primary methyl donor for DNA methylation. It is involved in gene transcription, cell proliferation, and the production of secondary metabolites. The MAT gene can be divided into the MAT1A gene and the MAT2A gene, encoding MAT, the only enzyme that can catalyze the synthesis of SAM. MAT has three isoenzymes: MAT I, MAT III, and MAT II. The first two are products encoded by the MAT1A gene, and the latter is encoded by the MAT2A gene. The MAT1A gene is primarily expressed in the adult liver, while the MAT2A gene is widely expressed in human tissues other than the liver. More and more studies have found that MAT2a protein is also highly expressed in other cancer tissues or cells, such as breast cancer, colorectal cancer, leukemia and lymphoma, and silencing of the MAT2A gene leads to the death of corresponding cancer cells, indicating that MAT2a protein has the potential to be a therapeutic target.

[0004] Methylthioadenosine phosphorylase (MTAP) is an enzyme expressed in all normal tissues that catalyzes the conversion of methylthioadenosine (MTA) to adenine and 5-methylthioglycoside-1-phosphate. Many malignant cell lines lack MTAP activity, and loss of MTAP activity has been detected in numerous primary lesions, including gliomas, melanomas, pancreatic cancer, non-small cell lung cancer, bladder cancer, astrocytomas, osteosarcomas, head and neck cancers, myxoid chondrosarcomas, ovarian cancer, endometrial cancer, breast cancer, soft tissue sarcomas, and non-Hodgkin's lymphoma. When MTAP is deficient, MTA accumulates to approximately 100 μM in cells and is excreted. Abnormal MTA accumulation leads to vulnerability of protein arginine methyltransferase 5 (PRMT5). Because PRMT5 utilizes SAM as a methyl donor substrate, inhibiting MAT2a activity reduces the intracellular concentration of SAM, thereby selectively reducing PRMT5 methylation activity in MTAP-deficient cells to below the threshold level required for growth. Therefore, inhibiting MAT2a activity could produce a synergistic lethal effect in MTAP-deficient cells by inhibiting PRMT5 activity, potentially providing therapeutic benefits for a variety of cancers. Summary of the Invention

[0005] One of the objectives of the present application is to provide a compound having MAT2a inhibitory activity.

[0006] Specifically, the present application provides a compound represented by the following formula I, and its pharmaceutically acceptable salts, hydrates, isomers, prodrugs and mixtures:

[0007]

[0008] Wherein, R1 and R2 are independently selected from H, -OH, -OR3, -CN, -NR4R5, 3 to 6 membered aliphatic heterocyclic group,

[0009] R3 is selected from -CH3, -CH2CH2OH, -CH2CH2NH2;

[0010] R4 and R5 are each independently selected from H, C1-C3 alkyl, cycloalkyl, -CH2CH2OH, -CH2CH2NH2.

[0011] In certain specific embodiments, the compound represented by the above formula I structure of the present application is not

[0012] In certain specific embodiments, R1 and R2 described herein are independently selected from H, -OH, -OR3, -CN, -NR4R5, 3 to 6 membered alicyclic group, And R1 and R2 are not H at the same time.

[0013] In certain specific embodiments, R1 described in the present application is selected from H, -OH, -OR3, -CN, -NR4R5, 3 to 6 membered alicyclic group, And the R2 is selected from -OH, -OR3, -CN, -NR4R5, 3 to 6 membered aliphatic heterocyclic group, Or R1 in this application is selected from -OH, -OR3, -CN, -NR4R5, 3 to 6 membered aliphatic heterocyclic group, And the R2 is selected from H, -OH, -OR3, -CN, -NR4R5, 3 to 6 membered aliphatic heterocyclic group,

[0014] In certain specific embodiments, R1 and R2 described herein are each independently selected from H, -OH, -OCH3, -OCH2CH2OH, -OCH2CH2NH2, -CN, -NH2, -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -N(CH3)2, -N(CH2CH3)2, -N(CH2CH2CH3)2, -NH-(C3-C6 cycloalkyl), -NHCH2CH2OH, -NHCH2CH2NH2,

[0015] In certain specific embodiments, R1 described herein is selected from H, -OH, -OR3 or -CN, and R2 is selected from -OH, -OR3 or -CN; or R1 described herein is selected from -OH, -OR3 or -CN, and R2 is selected from H, -OH, -OR3 or -CN.

[0016] In certain specific embodiments, R1 described herein is selected from H, -OH, -OCH3, -OCH2CH2OH, -OCH2CH2NH2 or -CN, and R2 is selected from -OH, -OCH3, -OCH2CH2OH, -OCH2CH2NH2 or -CN; or R1 described herein is selected from -OH, -OCH3, -OCH2CH2OH, -OCH2CH2NH2 or -CN, and R2 is selected from H, -OH, -OCH3, -OCH2CH2OH, -OCH2CH2NH2 or -CN.

[0017] In certain specific embodiments, R1 described herein is selected from H, -OH, -OCH2CH2OH or -CN, and R2 is selected from -OH, -OCH2CH2OH or -CN; or R1 described herein is selected from -OH, -OCH2CH2OH or -CN, and R2 is selected from H, -OH, -OCH2CH2OH or -CN.

[0018] In certain specific embodiments, the present application provides compounds represented by the following structures of Formula II, Formula III, Formula IIa, Formula IIIa, Formula IV, Formula V, and Formula VI, and pharmaceutically acceptable salts, hydrates, isomers, prodrugs, and mixtures thereof:

[0019]

[0020] Another object of the present application is to provide the use of the aforementioned compounds and pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof for the preparation of a medicament for treating MAT2a-related diseases. Alternatively, the present application provides the aforementioned compounds and pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof for treating MAT2a-related diseases. Alternatively, the present application provides a method for treating MAT2a-related diseases, comprising administering the aforementioned compounds and pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures thereof to a subject in need thereof.

[0021] Another object of the present application is to provide a pharmaceutical composition comprising a therapeutically effective dose of any one or more of the aforementioned compounds of the present application, or a pharmaceutically acceptable salt, hydrate, isomer, prodrug, or mixture thereof, and a pharmaceutically acceptable carrier. Alternatively, the aforementioned compounds of the present application and their pharmaceutically acceptable salts, hydrates, isomers, prodrugs, or mixtures are present in the form of a pharmaceutical composition.

[0022] The present application further provides the use of the above-mentioned pharmaceutical composition for preparing a medicament for treating a MAT2a-related disease. Alternatively, the present application provides the above-mentioned pharmaceutical composition for treating a MAT2a-related disease. Alternatively, the present application provides a method for treating a MAT2a-related disease, comprising administering the above-mentioned pharmaceutical composition to a subject in need thereof.

[0023] The MAT2a-related diseases described in the present application are cancers or tumors. Further, the cancers or tumors include neuroblastoma, intestinal cancer (such as rectal cancer, colon cancer, familial adenomatous polyposis cancer and hereditary non-polyposis colorectal cancer), esophageal cancer, lip cancer, laryngeal cancer, nasopharyngeal 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, testicular cancer, breast cancer, urinary system cancer, melanoma, brain tumors (such as glioblastoma, astrocytoma, meningioma, medulloblastoma) and ulcerative colitis. ectodermal tumors), Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia, hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmacytoma. In one embodiment, the cancer is lung cancer, non-small cell lung cancer (NSLC), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, Prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, chronic or acute leukemia, diffuse large B-cell lymphoma, central nervous system (CNS) tumors, spinal tumors, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwannomas, ependymomas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenomas, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

[0024] Another object of the present application is to provide a method for treating cancer or tumor diseases, comprising administering to a patient in need thereof one or more of the aforementioned pharmaceutical compositions or compounds of formula I or their pharmaceutically acceptable salts, hydrates, isomers, prodrugs or mixtures.

[0025] Definition of terms

[0026] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0027] As used herein, the term "pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0028] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present application, prepared by reacting the compounds of the present application with relatively nontoxic acids or bases, having specific substituents. When the compounds of the present application contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in a neat solution or a suitable inert solvent. When the compounds of the present application contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent.

[0029] The compounds of the present application may exist as specific tautomers. Unless otherwise indicated, the term "tautomer" refers to that at room temperature, different functional group isomers are in dynamic equilibrium and can quickly convert to each other. If tautomers are possible (such as in solution), the chemical equilibrium of the tautomers can be reached. For example, proton tautomers (proton tautomers) (also known as prototropic tautomers) include interconversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valence tautomers) include interconversions carried out by the reorganization of some bonding electrons. Representative tautomer examples in the present application are as follows: the compounds shown in the structures of Formula II and Formula IIa are tautomers to each other, and the compounds shown in the structures of Formula III and Formula IIIa are tautomers to each other.

[0030] The term "isomer" herein may include tautomers, geometric isomers, stereoisomers and atropisomers.

[0031] The compounds of the present invention may exist in specific geometric or stereoisomer or atropisomer forms. The term "stereoisomer" as used herein refers to isomers resulting from differences in the spatial arrangement of atoms in a molecule. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, all of which are within the scope of the present invention.

[0032] "Alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group, for example: C1-C3 alkyl refers to a saturated aliphatic hydrocarbon group containing 1 to 3 (including 1, 2 or 3) carbon atoms, including but not limited to methyl, ethyl, propyl, isopropyl, etc. and their various isomers.

[0033] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent. For example, "C3-C6 cycloalkyl" refers to a cycloalkyl group containing 3 to 6 (e.g., 3, 4, 5, or 6) carbon atoms. Typical C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl.

[0034] "Alicyclic group" may refer to a saturated monocyclic hydrocarbon substituent in which one or more ring atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon. Alternatively, the term may include unsaturated alicyclic groups. For example, a "3- to 6-membered alicyclic ring" may refer to a saturated cyclic hydrocarbon substituent containing 3-6 ring atoms, in which one or more ring atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, with the remaining ring atoms being carbon. Specific examples include, but are not limited to, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, morpholinyl, and the like.

[0035] The abbreviations used in this application are known to those skilled in the art and, unless otherwise specified, represent commonly understood meanings in the art. For example, DMF refers to N,N-dimethylformamide; THF refers to tetrahydrofuran; and Me refers to methyl.

[0036] Unless otherwise indicated, the terms "comprise, comprise, and comprising" or their equivalents (contain, contains, containing, include, includes, including) used herein are open-ended expressions and mean that in addition to the listed elements, components, and steps, other unspecified elements, components, and steps may also be included.

[0037] Unless otherwise indicated, all numbers used herein expressing amounts of ingredients, measurements, or reaction conditions are to be understood as modified in all instances by the term "about." When used in conjunction with a percentage, the term "about" can mean, for example, ±1%, preferably ±0.5%, and more preferably ±0.1%.

[0038] Unless the context clearly indicates otherwise, singular terms herein include plural referents and vice versa. Similarly, the word "or" herein is intended to include "and" unless the context clearly indicates otherwise.

[0039] Experiments have demonstrated that the compounds of the present application possess excellent MAT2a enzyme inhibitory activity and can inhibit the growth of cancer cells. Comparison of experimental results in MTAP-deficient (i.e., MTAP- / -) and wild-type cells revealed that the compounds of the present application (particularly the compound of Formula IV) exhibit highly selective activity against MTAP-deficient cancer cells. Furthermore, the compounds of the present application exhibit excellent metabolic stability in the liver, high bioavailability, good safety, and excellent solubility, resulting in excellent drugability. Therefore, the compounds of the present application have excellent application prospects in MAT2a-related cancers or tumor diseases. DETAILED DESCRIPTION

[0040] The following examples illustrate the synthesis methods of the compounds and intermediates of this application. The following examples are merely examples of this application and should not be construed as limiting the scope of this application. Unless otherwise specified, the raw materials and reagents involved in this application can be obtained through commercial channels, and the specific source of the channel does not affect the implementation of the technical solution of this application.

[0041] Preparation Example 1: Preparation of 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile

[0042]

[0043] 2-Chloro-6-(trifluoromethyl)nicotinonitrile (1.0 g) was dissolved in 1,4-dioxane (10 mL), and 4-chloroaniline (1.0 g) was added. The reaction was initiated by microwave at 120°C for 12 hours. LCMS showed that most of the starting materials had reacted completely. The reaction system was added to ethyl acetate and adjusted to a weak base with saturated sodium bicarbonate. The layers were separated, and the aqueous phase was extracted twice with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain 1.0 g of the title compound.

[0044] MS (ESI) m / z (M+H) + =298.1.

[0045] Example 1: Preparation of 4-amino-1-(4-chlorophenyl)-3-(3-hydroxyquinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0046]

[0047] Step 1: Preparation of 4-amino-3-nitrophenylacetic acid

[0048]

[0049] 4-Fluoro-3-nitrophenylacetic acid (1 g) was dissolved in 25% aqueous ammonia (20 mL), heated to reflux and stirred overnight. After the disappearance of the starting material as monitored by LCMS, the reaction system was concentrated under reduced pressure and the residue was purified by reverse phase column chromatography to obtain 800 mg of the target compound.

[0050] MS (ESI) m / z (M+H) + =197.1.

[0051] Step 2: Preparation of 2-ethoxy-2-oxoethyl 2-(4-((2-ethoxy-2-oxoethyl)amino)-3-nitrophenyl)acetate

[0052]

[0053] 4-Amino-3-nitrophenylacetic acid (800 mg) was added to ethyl bromoacetate (8 mL), followed by potassium carbonate (0.8 g). The temperature was raised to 140°C and the mixture was stirred overnight. After the starting material had mostly disappeared as monitored by LCMS, the reaction was quenched with water and extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to yield 950 mg of the title compound.

[0054] MS (ESI) m / z (M+H) + =369.1.

[0055] Step 3: Preparation of 2-ethoxy-2-oxoethyl 2-(3-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)acetate

[0056]

[0057] 2-Ethoxy-2-oxoethyl 2-(4-((2-ethoxy-2-oxoethyl)amino)-3-nitrophenyl)acetate (950 mg) was dissolved in tetrahydrofuran (30 mL), and 10% palladium on carbon (0.2 g) was added. The mixture was replaced with hydrogen three times and stirred at room temperature for 4 hours. The mixture was filtered and the filtrate was heated to 60°C and stirred for 1 hour. The disappearance of the starting material was monitored by LCMS. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give 680 mg of the target compound.

[0058] MS (ESI) m / z (M+H) + =293.1.

[0059] Step 4: Preparation of 2-ethoxy-2-oxoethyl 2-(3-hydroxyquinoxalin-6-yl)acetate

[0060]

[0061] 2-Ethoxy-2-oxoethyl 2-(3-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)acetate (680 mg) was dissolved in dichloromethane (20 mL), and 2,3-dichloro-5,6-dicyanobenzoquinone (750 mg) was added. The reaction was stirred at room temperature for 1 hour. After the disappearance of the starting material monitored by LCMS, the reaction was stopped and the system was quenched by adding saturated sodium bisulfite solution. The mixture was extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 550 mg of the target compound.

[0062] MS (ESI) m / z (M+H) + =291.1.

[0063] Step 5: Preparation of 2-(3-hydroxyquinoxalin-6-yl)acetic acid

[0064]

[0065] 2-Ethoxy-2-oxoethyl 2-(3-hydroxyquinoxalin-6-yl) acetate (550 mg) was dissolved in a mixed solution of tetrahydrofuran and water (THF:H2O=1:1 (v / v), 20 mL), and lithium hydroxide monohydrate (400 mg) was added. The reaction was stirred at room temperature for 1 hour. The starting material disappeared after monitoring by LCMS, and the reaction was stopped. The system was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography to obtain 350 mg of the target compound.

[0066] MS (ESI) m / z (M+H) + =205.1.

[0067] Step 6: Preparation of 4-amino-1-(4-chlorophenyl)-3-(3-hydroxyquinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0068]

[0069] 2-(3-Hydroxyquinoxalin-6-yl)acetic acid (110 mg) was dissolved in tetrahydrofuran (10 mL), followed by the addition of 1-hydroxybenzotriazole (110 mg) and N,N'-diisopropylcarbodiimide (100 mg). The mixture was stirred at room temperature for 6 hours. Separately, 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile (Preparation Example 1) (80 mg) was dissolved in tetrahydrofuran (10 mL). Sodium hydride (110 mg) was added under ice-cooling and stirred for 30 minutes. The 2-(3-hydroxyquinoxalin-6-yl)acetic acid system was then added dropwise to the 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile system. After the addition was complete, the reaction was stirred for another 30 minutes. LCMS monitoring revealed the disappearance of the starting material, and the reaction was stopped. 4M aqueous hydrochloric acid was added to adjust the pH to a weakly acidic state. The mixture was concentrated under reduced pressure, and the residue was purified by Prep-HPLC to yield 30 mg of the title compound.

[0070] MS (ESI) m / z (M+H) + =484.1.

[0071] 1 H NMR (400MHz, DMSO-d6) δ12.50(s,1H),8.87(d,J=8.2Hz,1H),8.19(s,1H),7.84(d,J=8.2Hz,1H ),7.78(d,J=8.2Hz,1H),7.57-7.53(m,2H),7.36-7.32(m,2H),7.31-7.26(m,2H),6.74(s,2H).

[0072] Example 2: Preparation of 4-amino-1-(4-chlorophenyl)-3-(2-hydroxyquinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0073]

[0074] Step 1: Preparation of ethyl 2-(4-amino-3-nitrophenyl)acetate

[0075]

[0076] 2-(4-Amino-3-nitrophenyl)acetic acid (500 mg) was dissolved in ethanol (10 mL), concentrated sulfuric acid (1 mL) was added, and the temperature was raised to 60°C with stirring for 4 hours. After the disappearance of the starting material as monitored by LCMS, the ethanol was removed by concentration under reduced pressure. 30 mL of water was added to the residue, and the mixture was adjusted to a weak base with saturated sodium carbonate. The mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 450 mg of the title compound.

[0077] MS (ESI) m / z (M+H) + =225.1.

[0078] Step 2: Preparation of 2-chloro-2-oxoethane-1,1-diacetic acid diester

[0079]

[0080] Glyoxylic acid aqueous solution (5.2 g, 50% w / w) was added to glacial acetic acid (8 mL), and then acetic anhydride (35 mL) was added. The temperature was raised to reflux and stirred for 2 hours. The solvent was removed by concentration under reduced pressure. Toluene (100 mL) was added to the residue, and the low-volatile solvent was removed by concentration under reduced pressure. Dichloromethane (50 mL) and thionyl chloride (9 mL) were added to the residue, and the temperature was raised to reflux and stirred for 1 hour. The solvent was removed by concentration under reduced pressure. Dichloromethane (100 mL) was added to the residue, and the low-volatile solvent was removed by concentration under reduced pressure to obtain the target compound (5.5 g). The crude product was used directly in the next step without purification.

[0081] Step 3: Preparation of 2-((4-(2-ethoxy-2-oxoethyl)-2-nitrophenyl)amino)-2-oxoethane-1,1-diacetic acid diester

[0082]

[0083] Ethyl 2-(4-amino-3-nitrophenyl)acetate (450 mg) was dissolved in dichloromethane (10 mL). Pyridine (480 mg) and 2-chloro-2-oxoethane-1,1-diacetic acid diester (500 mg) were added under ice-cooling and stirred for 0.5 hours. After the disappearance of the starting material as monitored by LCMS, water (30 mL) was added to the reaction system. The mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 400 mg of the title compound.

[0084] Step 4: Preparation of ethyl 2-(2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)acetate

[0085]

[0086] 2-((4-(2-Ethoxy-2-oxoethyl)-2-nitrophenyl)amino)-2-oxoethane-1,1-diacetic acid diester (400 mg) was dissolved in a mixture of ethanol and water (EtOH:H2O = 2:1 (v / v), 10 mL). Ammonium chloride (100 mg) and zinc powder (300 mg) were added, and the mixture was heated to reflux with stirring for 2 hours. After the disappearance of the starting material as monitored by LCMS, the mixture was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 180 mg of the title compound.

[0087] MS (ESI) m / z (M+H) + =235.1.

[0088] Step 5: Preparation of ethyl 2-(2-hydroxyquinoxalin-6-yl)acetate

[0089]

[0090] Ethyl 2-(2-oxo-1,2,3,4-tetrahydroquinoxalin-6-yl)acetate (180 mg) was dissolved in dichloromethane (10 mL), and 2,3-dichloro-5,6-dicyanobenzoquinone (200 mg) was added. The reaction was stirred at room temperature for 1 hour. After the disappearance of the starting material monitored by LCMS, the reaction was stopped and the system was quenched by adding saturated sodium bisulfite solution. The mixture was extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 150 mg of the target compound.

[0091] MS (ESI) m / z (M+H) + =233.1.

[0092] Step 6: Preparation of 2-(2-hydroxyquinoxalin-6-yl)acetic acid

[0093]

[0094] Ethyl 2-(2-hydroxyquinoxalin-6-yl)acetate (150 mg) was dissolved in a mixed solution of tetrahydrofuran and water (THF:H2O=1:1 (v / v), 10 mL), and lithium hydroxide monohydrate (130 mg) was added. The reaction was stirred at room temperature for 1 hour. The starting material disappeared after monitoring by LCMS, and the reaction was stopped. The system was concentrated under reduced pressure, and the residue was purified by reverse phase column chromatography to obtain 100 mg of the title compound.

[0095] MS (ESI) m / z (M+H) + =205.1.

[0096] Step 7: Preparation of 4-amino-1-(4-chlorophenyl)-3-(2-hydroxyquinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0097]

[0098] 2-(2-Hydroxyquinoxalin-6-yl)acetic acid (100 mg) was dissolved in tetrahydrofuran (10 mL), followed by the addition of 1-hydroxybenzotriazole (100 mg) and N,N'-diisopropylcarbodiimide (100 mg). The mixture was stirred at room temperature for 6 hours. Separately, 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile (Preparation Example 1) (80 mg) was dissolved in tetrahydrofuran (10 mL). Sodium hydride (110 mg) was added under ice-cooling and stirred for 30 minutes. The 2-(2-hydroxyquinoxalin-6-yl)acetic acid system was then added dropwise to the 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile system. After the addition was complete, the reaction was stirred for another 30 minutes. LCMS monitoring revealed the disappearance of the starting material, and the reaction was stopped. 4M aqueous hydrochloric acid was added to adjust the pH to a weakly acidic state. The mixture was concentrated under reduced pressure, and the residue was purified by Prep-HPLC to yield 35 mg of the title compound.

[0099] MS (ESI) m / z (M+H) + =484.1.

[0100] 1 H NMR (400MHz, DMSO-d6) δ11.98(brs,1H),8.86(d,J=8.2Hz,1H),8.21(s,1H),7.77(d,J=8.2Hz,1H) ,7.73(d,J=1.8Hz,1H),7.59-7.49(m,3H),7.38(d,J=8.4Hz,1H),7.36-7.30(m,2H),6.67(s,2H).

[0101] Example 3: Preparation of 7-(4-amino-1-(4-chlorophenyl)-2-oxo-7-(trifluoromethyl)-1,2-dihydro-1,8-naphthyridin-3-yl)quinoxaline-2-carbonitrile

[0102]

[0103] Step 1: Preparation of 2-ethoxy-2-oxoethyl 2-(3-(((trifluoromethyl)sulfonyl)oxy)quinoxalin-6-yl)acetate

[0104]

[0105] 2-Ethoxy-2-oxoethyl 2-(3-hydroxyquinoxalin-6-yl) acetate (synthesis see Step 4 in Example 1) (0.4 g) was added to N,N-dimethylformamide (12 mL), followed by N-phenylbis(trifluoromethanesulfonyl)imide (0.74 g) and triethylamine (0.21 g). The mixture was stirred at room temperature for 1 hour. After LCMS monitoring, most of the starting material disappeared, and the reaction was quenched with water. The mixture was extracted with ethyl acetate three times, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 0.32 g of the crude target compound.

[0106] MS (ESI) m / z (M+H) + =423.1.

[0107] Step 2: Preparation of 2-ethoxy-2-oxoethyl-2-cyanoquinoxaline-6-acetate

[0108]

[0109] 2-Ethoxy-2-oxoethyl 2-(3-(((trifluoromethyl)sulfonyl)oxy)quinoxalin-6-yl)acetate (0.32 g) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of zinc cyanide (0.17 g) and tetrakis(triphenylphosphine)palladium (0.04 g). The mixture was replaced with argon and stirred at 100°C for 4 hours. LCMS monitoring revealed the disappearance of most of the starting material, and the reaction was terminated. The system was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to yield 0.15 g of the title compound.

[0110] MS (ESI) m / z (M+H) + =300.1.

[0111] Step 3: Preparation of 2-(3-cyanoquinolin-6-yl)acetic acid

[0112]

[0113] 2-Ethoxy-2-oxoethyl-2-cyanoquinoxaline-6-acetate (150 mg) was dissolved in acetic acid (10 mL), and 4 M aqueous hydrochloric acid solution (2 mL) was added. The mixture was reacted in an oil bath at 80°C for 2 hours. After monitoring by LCMS, the starting material disappeared and the reaction was stopped. After cooling the system, sodium bicarbonate was added to adjust the pH to weak acidity. The system was slightly concentrated, and the residue was purified by reverse phase column chromatography to obtain 85 mg of the target compound.

[0114] MS (ESI) m / z (M+H) + =214.1.

[0115] Step 4: Preparation of 7-(4-amino-1-(4-chlorophenyl)-2-oxo-7-(trifluoromethyl)-1,2-dihydro-1,8-naphthyridin-3-yl)quinoxaline-2-carbonitrile

[0116]

[0117] 2-(3-Cyanoquinolin-6-yl)acetic acid (50 mg) was dissolved in tetrahydrofuran (6 mL), and 1-hydroxybenzotriazole (63 mg) and N,N'-diisopropylcarbodiimide (59 mg) were added. After stirring at room temperature for 8 hours, the system turned yellow. 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile (Preparation Example 1) (104 mg) was dissolved in tetrahydrofuran (10 mL), and sodium hydroxide (47 mg) was added under ice bath. ), stirred for 30 minutes, and then the 2-(3-cyanoquinolin-6-yl)acetic acid system was added dropwise to the 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile system. After the addition was complete, the reaction was stirred for 30 minutes. LCMS monitoring showed that the starting material disappeared and the reaction was stopped. A 4M aqueous hydrochloric acid solution was added to the system to adjust the pH to weak acidity, and the mixture was extracted with ethyl acetate three times. The organic phases were combined and concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain 15 mg of the target compound.

[0118] MS (ESI) m / z (M+H) + =493.1.

[0119] 1 H NMR (400MHz, DMSO-d6) δ9.39(s,1H),8.91(d,J=8.2Hz,1H),8.26(d,J=8.7Hz,1H),8.18(d,J=1.8Hz,1H) ,8.04(dd,J=8.8,1.9Hz,1H),7.82(d,J=8.1Hz,1H),7.59–7.53(m,2H),7.40–7.33(m,2H),6.95(s,2H).

[0120] Example 4: Preparation of 6-(4-amino-1-(4-chlorophenyl)-2-oxo-7-(trifluoromethyl)-1,2-dihydro-1,8-naphthyridin-3-yl)quinoxaline-2-carbonitrile

[0121]

[0122] Step 1: Preparation of ethyl 2-(2-(((trifluoromethyl)sulfonyl)oxy)quinoxalin-6-yl)acetate

[0123]

[0124] Ethyl 2-(2-hydroxyquinoxaline-6-yl)acetate (synthesis see Step 5 in Example 2) (300 mg), N-phenylbis(trifluoromethanesulfonyl)imide (690 mg), and N,N-diisopropylethylamine (250 mg) were dissolved in N,N-dimethylformamide (5 mL). The system was stirred at room temperature overnight. The disappearance of the starting material was monitored by LCMS. The reaction solution was diluted with ethyl acetate, the organic phase was washed with water, dried, concentrated, and purified by silica gel column chromatography to obtain 250 mg of a light yellow solid.

[0125] MS (ESI) m / z (M+H) + =365.0

[0126] Step 2: Preparation of ethyl 2-(2-cyanoquinoxalin-6-yl)acetate

[0127]

[0128] Ethyl 2-(2-(((trifluoromethyl)sulfonyl)oxy)quinoxalin-6-yl)acetate (300 mg), zinc cyanide (690 mg), and tetrakis(triphenylphosphine)palladium (250 mg) were dissolved in N,N-dimethylformamide (5 mL). The system was stirred at 100°C under an anhydrous, oxygen-free nitrogen atmosphere for 4 hours. LCMS monitoring revealed the disappearance of the starting material. The reaction mixture was diluted with ethyl acetate, and the organic phase was washed with water, dried, concentrated, and purified by silica gel column chromatography to yield 160 mg of a pale yellow solid.

[0129] MS (ESI) m / z (M+H) + =242.0.

[0130] Step 3: Preparation of 2-(2-cyanoquinoxalin-6-yl)acetic acid

[0131]

[0132] Ethyl 2-(2-cyanoquinoxalin-6-yl)acetate (160 mg) was dissolved in a mixed solvent of acetic acid (5 mL) and hydrochloric acid (1 mL, 4 M). The system was stirred at 80°C for 2 hours. The disappearance of the starting material was monitored by LCMS. The reaction solution was evaporated to dryness and purified by Prep-HPLC to give 65 mg of a white solid.

[0133] MS (ESI) m / z (M+H) + =214.0.

[0134] Step 4: Preparation of 6-(4-amino-1-(4-chlorophenyl)-2-oxo-7-(trifluoromethyl)-1,2-dihydro-1,8-naphthyridin-3-yl)quinoxaline-2-carbonitrile

[0135]

[0136] 2-(2-Cyanoquinolin-6-yl)acetic acid (65 mg) was dissolved in tetrahydrofuran (10 mL), and 1-hydroxybenzotriazole (81 mg) and N,N'-diisopropylcarbodiimide (75 mg) were added. After stirring at room temperature for 8 hours, the system was observed to turn yellow. 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile (Preparation Example 1) (270 mg) was dissolved in tetrahydrofuran (15 mL), and sodium hydroxide (36 mg) was added under ice-water bath. After stirring for 30 minutes, the 2-(2-cyanoquinolin-6-yl)acetic acid system was added dropwise to the 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile system. After the addition was completed, the reaction was continued with stirring for 30 minutes. LCMS monitoring showed that the starting material disappeared and the reaction was stopped. A 4M aqueous hydrochloric acid solution was added to the system to adjust the pH to weak acidity. The product was extracted with ethyl acetate three times, the organic phases were combined, and the product was concentrated under reduced pressure. The residue was purified by Prep-HPLC to obtain the target compound (5 mg, 3%).

[0137] MS (ESI) m / z (M+H) + =493.1.

[0138] 1 H NMR (400MHz, DMSO-d6) δ9.40(s,1H),8.91(d,J=8.2Hz,1H),8.26(d,J=8.7Hz,1H),8.20(d,J=1.8Hz,1H) ,8.01(dd,J=8.7,1.9Hz,1H),7.82(d,J=8.2Hz,1H),7.61–7.51(m,2H),7.43–7.30(m,2H),6.99(s,2H).

[0139] Example 5: Preparation of 4-amino-1-(4-chlorophenyl)-3-(3-(2-hydroxyethoxy)quinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0140]

[0141] Step 1: Preparation of 2-ethoxy-2-oxoethyl 2-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)acetate

[0142]

[0143] Take triphenylphosphine (499 mg) and dissolve it in tetrahydrofuran (15 mL). Add diisopropyl azodicarboxylate (385 mg) dropwise. Stir at room temperature for 15 minutes after addition. Then add 2-((tetrahydro-2H-pyran-2-yl)oxy)ethane-1-ol (278 mg). After 15 minutes, add the reaction solution dropwise to a tetrahydrofuran solution (10 mL) of 2-ethoxy-2-oxoethyl 2-(3-hydroxyquinoxalin-6-yl) acetate (synthesis see Step 4 in Example 1) (370 mg) and stir for 4 hours. LCMS shows no starting material remaining. Add water to quench the reaction and extract with ethyl acetate three times. After the combined filtrate is dried and concentrated, the crude product is purified by column chromatography to obtain the target compound (360 mg).

[0144] MS (ESI) m / z (M-84) + =335.1

[0145] Step 2: Preparation of 2-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)acetic acid

[0146]

[0147] 2-Ethoxy-2-oxoethyl 2-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)acetate (360 mg) was dissolved in a mixed solution of tetrahydrofuran and water (20 mL, 3:1), and lithium hydroxide monohydrate (361 mg) was added. The reaction was stirred at room temperature for 1 hour. The starting material disappeared after LCMS monitoring, and the reaction was stopped. The system was concentrated under reduced pressure and the pH was adjusted to 4 with 2M hydrochloric acid. The solid was collected and washed to give the target product (320 mg). The crude product was directly used in the next reaction.

[0148] MS (ESI) m / z (M-84) + =249.1.

[0149] Step 3: Preparation of 4-amino-1-(4-chlorophenyl)-3-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0150]

[0151] 2-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)acetic acid (120 mg) was dissolved in tetrahydrofuran (10 mL), and N,N'-diisopropylcarbodiimide (59 mg) and 1-hydroxybenzotriazole (63 mg) were added. The mixture was stirred at room temperature for 2 hours. Separately, 2-((4-chlorophenyl)amino)-6-(trifluoromethyl)nicotinonitrile (Preparation Example 1) (107 mg) was dissolved in tetrahydrofuran (10 mL), and sodium hydride (72 mg, 60%) was added under ice bath. After stirring for 30 minutes, the above reaction solution was added dropwise. The reaction was continued to stir for 30 minutes. The starting material disappeared after LCMS monitoring. The reaction was stopped and 2M hydrochloric acid aqueous solution was added to the system to adjust the pH to weak acidity. The mixture was extracted with ethyl acetate three times, washed with saturated brine, and the organic phases were combined and dried and concentrated to obtain the target product (150 mg). The crude product was used directly in the next reaction.

[0152] MS (ESI) m / z (M-84) + =528.1

[0153] Step 4: Preparation of 4-amino-1-(4-chlorophenyl)-3-(3-(2-hydroxyethoxy)quinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one

[0154]

[0155] 4-Amino-1-(4-chlorophenyl)-3-(3-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)quinoxalin-6-yl)-7-(trifluoromethyl)-1,8-naphthyridin-2(1H)-one (150 mg) was treated with 4M hydrogen chloride in 1,4-dioxane (10 mL) for 1 hour. The reaction mixture was concentrated and purified by prep-HPLC to afford the desired product (2 mg).

[0156] MS (ESI) m / z (M+1) + =528.1

[0157] 1 H NMR (400MHz, DMSO-d6) δ8.89(d,J=8.3Hz,1H),8.61(s,1H),8.05(d,J=8.5Hz,1H),7.83–7.74(m,2H),7.59(dd,J=8.5,1 .9Hz,1H),7.58–7.52(m,2H),7.37–7.32(m,2H),6.74(s,2H),4.95(s,1H),4.49(t,J=5.0Hz,2H),3.81(t,J=5.1Hz,2H).

[0158] Biological tests

[0159] Related biological test studies have shown that the compounds of the present application may have excellent MAT2a inhibitory activity, which is significantly better than existing compounds with the same target. The inventors of the present application found in previous studies that existing small molecule compounds with the same target performed poorly in cell inhibitory activity tests, and their selective activity in MTAP-deficient and MTAP wild-type cells was not ideal. Although a few compounds have made certain breakthroughs in the above-mentioned activities, the compounds themselves have poor water solubility, poor metabolic stability, strong inhibition of liver microsomal enzymes, which is not conducive to drug combination, poor membrane permeability, which is not conducive to oral administration, or potential hepatotoxicity and cardiotoxicity and many other problems, which cannot meet the requirements of drugability. After repeated experiments and investigations, the inventors unexpectedly found that the MAT2a small molecule inhibitors in the specific embodiments of the present application can comprehensively solve the above-mentioned defects, have advantages such as good pharmaceutical activity, good safety, and good drugability, and have great clinical application prospects.

[0160] Experimental Example 1: Enzyme activity test

[0161] Colorimetric assay was used to detect the IC of the test compounds against MAT2a. 50 value.

[0162] The specific steps are as follows: Compounds are tested at a starting concentration of 1 μM or 10 μM, and a three-fold serial dilution is performed to yield 10 concentration points (including a 0 concentration point as a positive control). 250 nL of each of the 10 different test compound solutions is added to a 384-well plate. A 20 μg / mL solution of MAT2a enzyme (BPS Bioscience Inc., catalog #71401-1) is prepared in assay buffer (50 mM Tris, 50 mM KCl, 10 mM MgCl2, 0.05% polyoxyethylene lauryl ether, pH 8.0). 15 μL of this 20 μg / mL MAT2a enzyme solution is added to the wells containing the test compound at different concentrations. 15 μL of assay buffer is added to the negative control wells. The plates are shaken to mix thoroughly and incubated for 15 minutes. Prepare substrate mixed solution (containing 400 μM ATP and 600 μM L-Methionine) with Assay buffer, add 10 μL of substrate mixed solution to the positive control well, test compound well, and negative control well respectively, and start the reaction for 150 minutes. Then add 50 μL of stop reaction solution (BIOMOL Green TM The reaction was terminated with 4% paraformaldehyde (5% paraformaldehyde) and centrifuged at 1000 rpm for 60 seconds, followed by incubation for 15 minutes. OD620 was measured and the data processed.

[0163] Calculation formula:

[0164] Inhibition%=(OD620 阳性对照孔 -OD620 待测化合物孔 ) / (OD620 阳性对照孔 -OD620 阴性对照孔 )×100

[0165] The log value of the concentration was used as the X-axis and the percentage inhibition rate (Inhibition%) was used as the Y-axis. The log (inhibitor) vs. response-Variable slope analysis software GraphPad Prism 8 was used to fit the dose-effect curve to obtain the IC value of each compound on the enzyme activity. 50 The experimental results are shown in Table 1 below:

[0166] Table 1 IC values ​​of the compounds of the present application for MAT2a 50 value

[0167]

[0168] Conclusion: The above experiments show that the compound of the present application has excellent MAT2a enzyme inhibitory activity.

[0169] Experimental Example 2: Activity test of HCT116 MTAP gene homozygous deletion cells (Source: Horizon)

[0170] On day 1, cell plating: After trypsinization, cells were resuspended in complete medium (RPMI-1640 containing 10% FBS. FBS brand is EXCELL, cat. no. FND500; RPMI-1640 brand is ATCC, cat. no. 30-2001) to the desired density. Mix well and add 100 μL / well to a 96-well plate at a cell density of 1000-3000 cells per well. Return the plate to the incubator to allow cells to adhere and grow. Meanwhile, wells containing only complete medium without cells were set up as controls. On day 2, test compounds were added: Prior to compound addition, cells were starved with serum-free medium for 4 hours. Then, complete medium containing the corresponding concentration of compound was added and incubated at 37°C, 5% CO2 for 120 hours. Meanwhile, wells containing cells but with the same volume of DSMO were set up as controls. On day 7, the compound-treated cells were removed and equilibrated to room temperature. 50 μL of CellTiter-Glo (Promega, Cat. No. G7571) reagent was added to each well. The cells were shaken at room temperature for 2 minutes to fully lyse the cells. The cells were then incubated for another 60 minutes and the fluorescence intensity was measured. The percentage inhibition rate was calculated using the formula:

[0171] Inhibition% = 100% - (Signal of the test compound well - Signal of the well containing no cells and only culture medium) / (Signal of the well containing cells but no compound - Signal of the well containing no cells)

[0172] cells (signal in wells containing culture medium only) × 100%.

[0173] The IC50 values ​​of each compound's cell activity were obtained by fitting dose-effect curves using the analysis software GraphPad Prism 5. The experimental results show that the compounds of the present application have outstanding cancer cell inhibition activity. The compounds of the present application generally have IC50 values ​​below 200 nM, preferably below 100 nM. In particular, the compound of Example 3 has excellent activity. The results are shown in Table 2 below:

[0174] Table 2: Inhibitory activity of HCT116 MTAP gene homozygous deletion cells

[0175]

[0176] NA means not determined.

[0177] Experimental Example 3: HCT116WT Cell Activity Test (Source: Nanjing Kebai)

[0178] On Day 1, cell plating: After trypsinization, cells were resuspended in complete medium (McCoy 5A containing 10% FBS. FBS brand is EXCELL, Cat. No. FND500; McCoy 5A brand is BOSTER, Cat. No. PYG0025) to the desired density. Mix thoroughly and add 100 μL / well to a 96-well plate at a cell density of 500-1000 cells per well. Return the plate to the incubator to allow cells to attach and grow. Meanwhile, wells containing only complete medium without cells were set up as controls. On Day 2, test compounds were added: Prior to compound addition, cells were starved with serum-free medium for 4 hours. Then, complete medium containing the corresponding concentration of compound was added and incubated at 37°C, 5% CO2 for 120 hours. Meanwhile, wells containing cells but with the same volume of DSMO were set up as controls. On day 7, the compound-treated cells were removed and equilibrated to room temperature. 50 μL of CellTiter-Glo (Promega, Cat. No. G7571) reagent was added to each well. The cells were shaken at room temperature for 2 minutes to fully lyse the cells. The cells were then incubated for another 60 minutes and the fluorescence intensity was measured. The percentage inhibition rate was calculated using the formula:

[0179] Inhibition%=100%-(signal of the well with test compound-signal of the well without cells and containing only culture medium) / (signal of the well with cells but no compound added-signal of the well without cells and containing only culture medium)×100%.

[0180] The IC50 values ​​of each compound on cell activity were obtained by fitting the dose-effect curve using the analysis software GraphPad Prism 5. The compounds in the examples of the present application all had IC50 values ​​greater than 10 μM on HCT116WT cells.

[0181] Experimental Example 4: Liver microsome metabolic stability test

[0182] The source information of liver microsomes is as follows Table 3:

[0183]

[0184] The test compound was diluted to 10 mM in DMSO, and 2 μL was added to 198 μL of a 50% acetonitrile / 50% aqueous solution to obtain a 100 μM solution. Liver microsomes were prepared and diluted to 0.5 mg / mL in 1× PBS. NADPH cofactor (final concentration: 1 mM) was added, and the mixture was preheated at 37°C for 10 minutes. 2.5 μL of the prepared test compound was added to 222.5 μL of the preheated mixture, and the reaction was initiated in a 37°C water bath. At 0.5, 15, 30, and 45 minutes, the incubated centrifuge tubes were removed and 25 μL of the incubated liver microsomal suspension (containing the compound) was aspirated. The reaction was terminated by adding 5 volumes of stop solution, and the samples were centrifuged at 3220 g for 40 minutes. The supernatant was collected and the residual compound content in the samples at each time point was determined by LC-MS / MS. The half-life (t½) of the compound in liver microsomes was calculated by nonlinear linear regression of the % residual drug against time.

[0185] Table 4 Metabolic stability of the test compounds in human, dog, rat and mouse liver microsomes

[0186]

[0187] The metabolic half-life t1 / 2=∞ in the above table means that the compound is not metabolized by liver microsomes under the experimental conditions.

[0188] Experimental Example 5: Hepatocyte metabolic stability test

[0189] Hepatocyte source information:

[0190]

[0191]

[0192] The test compound was diluted to 10 mM in DMSO, and 2 μL was added to 198 μL of a 50% acetonitrile / 50% aqueous solution to obtain a 100 μM solution. After cryopreserved hepatocytes were resuspended in culture medium (William's E Medium, Gibco, Cat. No. 22551032) to a density of 500,000 cells / mL. 198 μL of the hepatocyte suspension was transferred to a 96-well plate, 2 μL of the prepared 100 μM test compound solution was added, and the plate was incubated in an incubator. At 0.5, 15, 30, 60, 90, and 120 minutes, the incubated 96-well plate was removed and 25 μL of the cell suspension (containing the compound) was aspirated. The reaction was terminated by adding 6 volumes of stop solution and centrifuged at 3220 g for 45 minutes. The supernatant was collected and the remaining compound content in the sample at each time point was analyzed by LC-MS / MS. The half-life (t1 / 2) of the compound in hepatocytes was calculated by performing nonlinear linear regression on the ratio of % remaining drug to time.

[0193] Table 5 Metabolic stability of the test compounds in human, dog, rat and mouse hepatocytes

[0194]

[0195] The metabolic half-life t1 / 2=∞ in the above table means that the compound is not metabolized by hepatocytes under the experimental conditions.

[0196] Experimental Example 6: Pharmacokinetic Study in SD Rats

[0197] 1. Experimental Animals

[0198] Species: SD rats. Source: Weitong Lihua Laboratory Animal Technology Co., Ltd. Quantity: 3 rats for each dosage form.

[0199] 2. Preparation of test samples:

[0200] Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% normal saline (all percentages by volume) in sequence, and vortex or sonicate to mix thoroughly to obtain a dosing solution with a test sample concentration of 0.2 mg / mL for intravenous (IV) administration.

[0201] Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% normal saline (all percentages by volume) in sequence, and vortex or sonicate to mix thoroughly to obtain a dosing solution with a test sample concentration of 0.5 mg / mL for oral gavage (PO) administration.

[0202] 3. Experimental Design

[0203]

[0204] 4. Dosage

[0205] Weigh the patient before administration and calculate the dosage based on body weight. Administer the drug intravenously or orally via gavage.

[0206] 5. Blood collection time

[0207] Before administration and 0.083h, 0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 8h, and 24h after administration.

[0208] 6. Sample Collection and Disposal

[0209] On the day of the experiment, 150 μL of blood was collected via the jugular sinus at each designated time point. The whole blood samples were placed in tubes containing EDTA-K2 anticoagulant. The whole blood samples were centrifuged at 1500 g for 10 minutes to separate the plasma, and the supernatant plasma was collected into sample tubes. The biological samples were stored at -40 to -20°C until analysis.

[0210] 7. Biological analysis and data processing

[0211] According to the requirements of SOP-BA-002 (liquid chromatography-mass spectrometry analysis of biological samples) of Suzhou Shengsu New Drug Development Co., Ltd., an LC-MS / MS analytical method for determining the concentration of compounds in rat plasma was established and used to determine the concentration of compounds in the biological samples obtained in this experiment.

[0212] The pharmacokinetic parameters were calculated using the non-compartmental model in Pharsight Phoenix 8.0.

[0213] Table 6 In vivo pharmacokinetic study data of the test compounds administered intravenously and orally to SD rats

[0214]

[0215] Experimental Example 7: Pharmacokinetic Study in ICR Mice

[0216] 1. Experimental Animals

[0217] Species: ICR mice, SPF grade. Source: Shanghai Xipul-Bikai Laboratory Animal Co., Ltd. Quantity: 3 mice per dosage form.

[0218] 2. Preparation of test samples

[0219] Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% normal saline (all percentages by volume) in sequence, and vortex or sonicate to mix thoroughly to obtain a dosing solution with a test sample concentration of 0.2 mg / mL for intravenous (IV) administration.

[0220] Accurately weigh an appropriate amount of the test sample, add 5% DMSO, 10% polyethylene glycol-15 hydroxystearate, and 85% normal saline (all percentages by volume) in sequence, and vortex or sonicate to mix thoroughly to obtain a dosing solution with a test sample concentration of 1 mg / mL for oral gavage (PO) administration.

[0221] 3. Experimental Design

[0222]

[0223] 4. Dosage

[0224] Weigh the patient before administration and calculate the dosage based on body weight. Administer the drug intravenously or orally via gavage.

[0225] 5. Blood collection time

[0226] Before administration and 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, and 24h after administration.

[0227] 6. Sample Collection and Disposal

[0228] Blood samples (approximately 0.04 mL per sample) were collected via the submandibular vein or other appropriate means. Whole blood samples were placed in anticoagulant tubes containing EDTA-K2. Whole blood samples were centrifuged at 1500 g for 10 minutes to separate plasma. The supernatant plasma was collected and transferred to a sample tube. Biological samples were stored at -40 to -20°C until analysis.

[0229] 7. Biological analysis and data processing

[0230] When measuring test substance plasma concentrations and plotting plasma drug concentration-time curves, the BLQ (lower limit of detection) is recorded as 0. When calculating pharmacokinetic parameters, the concentration before administration is calculated as 0; the BLQ before Cmax (including the "No peak") is calculated as 0; and the BLQ after Cmax (including the "No peak") is not included in the calculation. Pharmacokinetic parameters such as AUC(0-t), T1 / 2, and Cmax are calculated using WinNonlin using plasma drug concentration data at different time points.

[0231] Table 7 In vivo pharmacokinetic study data of the test compounds administered intravenously and orally to ICR mice

[0232]

[0233] Experimental Example 8: In vitro permeability test of Caco-2 cells

[0234] 1. Cell Culture and Plating

[0235] In HTS TranswellTM Caco-2 cells were seeded in a cell culture plate (Brand: Corning; Catalog No.: 3391). The volume of culture medium in the plate wells was 50 μL, and the culture medium was replaced every 24 h.

[0236] 2. Detection resistor

[0237] After 14-16 days of culture, Caco-2 cells formed a confluent monolayer and the resistance was measured. The transmembrane resistance of the monolayer reached 230Ω·cm. 2 , which can be used for the next penetration test.

[0238] 3. Penetration Testing

[0239] First, prepare the compound working solution. Use DMSO to prepare the compound to be tested to 2mM, and then further dilute it to 10μM with HBSS buffer (10mM HEPES buffer containing 4% BSA, pH 7.4) to obtain the test working solution. TM The cell culture plate was washed three times with 37°C HBSS buffer, followed by addition of 37°C HBSS buffer and incubation in a 37°C incubator for 30 minutes. For A→B permeability testing, the buffer solution was aspirated and a working solution containing the test drug was added to the upper chamber (apical, A side) as the supply solution. A blank HBSS buffer was added to the lower chamber (basolateral, B side) as the receiving solution. For B→A permeability (efflux) testing, the buffer solution was aspirated and a blank HBSS buffer was added to the A side as the receiving solution. A working solution containing the compound was added to the B side as the dosing solution.

[0240] 4. Sampling and testing

[0241] At Transwell TM An appropriate amount of the dosing solution was taken from the plate as the T0 sample and then incubated in a 37°C incubator for 2 hours. After incubation, a sample was taken at the receiving end. The sample was analyzed using LC-MS / MS.

[0242] 5. Data Analysis

[0243] Apparent permeability coefficient (P app , unit: ×10 -6 cm / s):

[0244] V R is the volume of the receiving end solution (i.e., receiving solution) (A→B: the receiving end is the base end; B→A: the receiving end is the top end), Area is the area of ​​the Transwell-96 well plate membrane (0.0804 cm 2), Time is the incubation time (unit: s), C R is the drug concentration at the sample receiving end, and C0 is the drug concentration at the initial T0 point of the sample.

[0245] Efflux rate (ER):

[0246] P app (B→A) is the apparent permeability coefficient from the base to the top, P app (A→B) is the apparent permeability coefficient from the top to the base. Table 8 Caco-2 cell in vitro permeability test data

[0247]

[0248] Note: The test compound cannot be detected from the A→B receiving end, that is, it is below the detection limit. The cutoff value is calculated based on the detection limit of the test compound.

[0249] Experimental Example 9: Compound Solubility Test

[0250] 1. Compound Preparation

[0251] The test compound was prepared into a 10 mM solution using DMSO.

[0252] 2. Solubility test

[0253] Place 15 μL of the test compound at a 10 mM concentration in a 96-well plate and add 485 μL of 1× PBS (pH 7.4). Seal the plate wells and incubate on a shaker at 25°C, 1100 rpm for 2 hours. After 2 hours, transfer 500 μL of the incubated sample to a filter plate and filter using a vacuum manifold. Take 5 μL of the filtrate, mix with 5 μL of DMSO, and then add 490 μL of ultrapure water. The compound concentration is then measured by LC-MS.

[0254] 3. Standard products

[0255] Take 6 μL of the test compound with a concentration of 10 mM in a 96-well plate, add 194 μL of DMSO solution, mix well, take 5 μL of the resulting solution and mix with 5 μL of PBS buffer, then add 490 μL of ultrapure water and mix well. Use LC-MS to test the compound concentration simultaneously with the sample in step 2.

[0256] 4. Data Analysis

[0257] The injection concentration of the standard is known to be 10 μM. The solubility is calculated by comparing the peak areas of the test compound and the standard in LC-MS. The specific formula is as follows:

[0258]

[0259] Table 9 Solubility test data

[0260]

[0261] Therefore, the compounds of the present application can exhibit desirable solubility.

[0262] For the purposes of description and disclosure, all patents, patent applications, and other publications are expressly incorporated herein by reference. These publications are provided solely because their disclosure predates the filing date of the present application. All statements regarding the dates of these documents or the representations of their contents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates of these documents or the contents of these documents. Furthermore, any citation of these publications herein does not constitute an admission that such publications constitute part of the common general knowledge in the art in any country.

[0263] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific implementation plans and examples described above, but that various modifications, replacements, or recombinations can be made without departing from the spirit of the present application, and these adjusted solutions fall within the scope of protection of the present application.

Claims

1. A compound represented by formula I, a pharmaceutically acceptable salt thereof, or a mixture thereof: in, R1 is selected from -OH, -OR3, -CN or -NR4R5, R2 is H, and R3 is selected from -CH3, -CH2CH2OH or -CH2CH2NH2; or R1 is H, R2 is selected from -OH, -OR3, -CN or -NR4R5, and R3 is -CH3; R4 and R5 are each independently H.

2. The compound according to claim 1, its pharmaceutically acceptable salt, or a mixture thereof, wherein The compound is a compound represented by the structure of Formula II, Formula III, Formula IIa, Formula IIIa, Formula IV, Formula V or Formula VI:

3. Use of the compound according to claim 1 or 2, a pharmaceutically acceptable salt thereof, or a mixture thereof for preparing a medicament for treating MAT2a-related diseases.

4. A pharmaceutical composition comprising a therapeutically effective dose of the compound according to claim 1 or 2, a pharmaceutically acceptable salt thereof, or a mixture thereof, and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition according to claim 4 for preparing a medicament for treating MAT2a-related diseases.

6. The use according to claim 3 or 5, wherein The MAT2a-related disease is cancer or tumor.

7. The use according to claim 6, wherein The cancer or tumor includes intestinal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, thyroid cancer, kidney cancer, ovarian cancer, cervical cancer, choriocarcinoma, pancreatic cancer, prostate cancer, testicular cancer, breast cancer, melanoma, brain tumor, Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic or acute leukemia, hepatocellular carcinoma, gallbladder cancer, bronchial cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, craniopharyngioma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, Fibrosarcoma, Ewing sarcoma, plasmacytoma, lung cancer, bone cancer, skin cancer, head and neck cancer, anal cancer, uterine cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethra cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvis cancer, mesothelioma, biliary tract cancer, central nervous system tumors, neurilemmoma, ependymoma, medulloblastoma, peripheral neuroectodermal tumor, pituitary adenoma, refractory forms of any of the foregoing cancers or tumors, or a combination of one or more of the foregoing cancers or tumors.

8. The use according to claim 7, wherein The intestinal cancer includes rectal cancer, colon cancer, familial adenomatous polyposis cancer and hereditary non-polyposis colorectal cancer, small intestine cancer, the thyroid cancer includes medullary thyroid cancer and papillary thyroid cancer, the melanoma includes skin or intraocular melanoma, the brain tumor is meningioma, the chronic or acute leukemia includes acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia, the sarcoma is rhabdomyosarcoma, the lung cancer includes small cell lung cancer and non-small cell lung cancer, the central nervous system tumor includes neuroblastoma, spinal tumor, brain stem glioma, multiforme keratinoma, astrocytoma.

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