Novel substituted thienopyrimidine compounds, processes for their preparation and their use

Abstract

The present application relates to novel substituted thienopyrimidine compounds and preparation method and application thereof, the substituted thienopyrimidine compound has the structural formula shown in general formula I, as a kind of antitumor drug, with significant EZH2 histone methyltransferase inhibitory activity.The present application also provides the preparation method of the compound, and the pharmaceutical composition and use containing them.The novel substituted thienopyrimidine compound obtained by the present application has more excellent antitumor activity and safety, can be used in EZH2, PRC2, EZH2 / PRC2 mediated diseases, especially as antitumor agent has great value.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and particularly relates to novel substituted thiophene-pyrimidine compounds that inhibit tumor cell growth and exert anti-tumor effects, as well as their pharmaceutically acceptable salts, preparation methods, and pharmaceutical uses. It is a compound with therapeutic effects on EZH2, PRC2, and EZH2 / PRC2-mediated diseases. Background Technology

[0002] Malignant tumors are a major threat to human health and a leading cause of death. Epigenetics maintains normal bodily functions by regulating the chemical modifications of histones and DNA. In the occurrence and development of cancer, transcriptional dysregulation caused by abnormal histone modifications has become a major mode of epigenetic regulatory disorder. Among forty-nine human histone methyltransferases, the H3K27 methyltransferase EZH2 leads to transcriptional repression and target gene silencing by catalyzing the trimethylation of histone H3 lysine 27 (H3K27). Trimethylation of H3K27 has been shown to be frequently mutated in cancers of the hematologic and digestive systems, and its overexpression in various human solid tumors is closely associated with disease progression. For example, in breast cancer, the expression level of EZH2 is highly correlated with its invasiveness and proliferation rate; this overexpression is closely related to the pRB-E2F, PI3K / Akt, and estrogen receptor pathways in cancer cells. In other tumors, EZH2 also leads to malignant proliferation of tumor cells by silencing related tumor signaling pathways, such as VEGF-A / AKT, mTOR, and TGF-β-Smad-ASCL1. Therefore, EZH2 shows great promise as a molecular therapeutic target.

[0003] Tazemetostat (EPZ-6438), as the only marketed EZH2 inhibitor, has demonstrated its immense potential in the treatment of epithelioid sarcoma and follicular lymphoma. Although several competitive EZH2 inhibitors remain in preclinical or clinical trials, their poor clinical efficacy and resistance mutations highlight the significant importance of developing more potent and bioavailable compounds. These compounds play a crucial role in further exploring the pathobiological functions of EZH2 and as potential second-generation EZH2 inhibitors for clinical application. Summary of the Invention

[0004] The purpose of this invention is to provide a novel class of compounds containing substituted thienopyrimidine structures and their use in EZH2, PRC2, and EZH2 / PRC2-mediated disease drugs. After in vitro activity screening, this compound exhibits significant antitumor activity.

[0005] This invention relates to novel substituted thiophene-pyrimidine compounds of general formula I and their pharmaceutically acceptable salts, hydrates, solvates or prodrugs.

[0006]

[0007] in,

[0008] A is selected from primary amine compounds;

[0009] L is selected from C5-C6 aryl or heteroaryl; wherein the heteroaryl contains 1-2 heteroatoms selected from N, O or S, and L is substituted by 1-2 R2 atoms; R2 is selected from secondary amines or structures containing secondary amine substitutions;

[0010] R1 is selected from hydrogen, alkoxy, halogen, or structures containing halogen elements.

[0011] Furthermore, the aforementioned novel substituted thiophene-pyrimidine compounds and their pharmaceutically acceptable salts,

[0012] A is selected from 3-aminomethyl-4,6-dimethylpyridin-2(1H)one, 4-aminomethyl-1-methyl-5,6,7,8-tetrahydroisoquinoline-3(2H)one, 3-aminomethyl-6-methyl-4-propylpyridin-2(1H)one, 3-aminomethyl-6-methyl-4-methylthiopyridin-2(1H)one, 3-aminomethyl-4-methoxy-6-methylpyridin-2(1H)one, 3-aminopiperidin-2,6-dione, 2-(1H-imidazol-4-yl)ethane-1-amine, or 2-(1H-indol-3-yl)ethane-1-amine;

[0013] R2 is selected from dimethylamino, diethylamino, piperidinyl, piperazinyl, morpholinyl, tetrahydropyrrolyl, N-methylpiperazinyl, N-ethylpiperazinyl, 4-methylpiperridinyl, N-phenylpiperazinyl, 4-dimethylaminopiperridinyl, 1-(2-methoxyethyl)piperazinyl, or 4-(2-methoxyethyl)morpholinyl;

[0014] R1 is selected from hydrogen, methoxy, ethoxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, monofluoromethoxy, difluoromethoxy, or trifluoromethoxy.

[0015] This invention preferably relates to novel substituted thiophene-pyrimidine compounds of general formula (I) and their pharmaceutically acceptable salts, hydrates, solvates, or prodrugs, specifically with the following structures, but these compounds are not intended to limit the invention in any way:

[0016]

[0017]

[0018]

[0019] A pharmaceutical composition comprising the above-mentioned substituted thiophene pyrimidine compounds and their pharmaceutically acceptable salts as active ingredients, combined with a pharmaceutically acceptable carrier.

[0020] Preferably, in the above-described pharmaceutical composition, the pharmaceutically acceptable carrier is selected from one or more fillers, disintegrants, binders, and lubricants.

[0021] Preferably, the above-mentioned pharmaceutical composition is formulated into dosage forms such as tablets, capsules, granules, sprays, or injections.

[0022] According to some common methods in the field to which this invention pertains, the novel substituted thiophene-pyrimidine compounds represented by Formula I in this invention can react with acids to form pharmaceutically acceptable salts. Pharmaceutically acceptable addition salts include addition salts of inorganic and organic acids, with salts reacting with the following acids being particularly preferred: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, etc.

[0023] Furthermore, the present invention also includes prodrugs derived from the present invention. These prodrugs are derivatives of general formula I, which may themselves have weak or no activity, but are converted into their corresponding biologically active forms under physiological conditions (e.g., through metabolism, solvation, or other means) after administration.

[0024] The present invention relates to the use of novel substituted thiophene-pyrimidine compounds and their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of medicaments for treating EZH2, PRC2 or EZH2 / PRC2-mediated diseases.

[0025] Preferably, the novel substituted thiophene-pyrimidine compounds and their pharmaceutically acceptable salts or pharmaceutical compositions provided by the present invention are used in the preparation of EZH2 as a molecular therapeutic target drug.

[0026] The above-mentioned compounds and their pharmaceutically acceptable salts are used as active ingredients in the preparation of antitumor drugs. Tumor types mainly include liver cancer, nasopharyngeal carcinoma, colorectal cancer, melanoma, bladder cancer, esophageal cancer with leukemia, breast cancer, gastric cancer, prostate cancer, pancreatic cancer, lung cancer, ovarian cancer, epithelioid sarcoma, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma, etc.

[0027] The beneficial effects of this invention are: the novel thienopyrimidine compounds and their pharmaceutically acceptable salts obtained by this invention possess excellent antitumor activity and safety. They can be used to treat neoplastic diseases such as epithelioid sarcoma, lymphoma, prostate cancer, and breast cancer, as well as other autoimmune diseases. They have a high anticancer spectrum and a broad therapeutic window, making them of great application value in the pharmaceutical field. Detailed Implementation

[0028] The examples and preparation methods provided below further illustrate and demonstrate the compounds of the present invention and their preparation methods. It should be understood that the scope of the following examples and preparation methods does not limit the scope of the present invention in any way.

[0029] The following synthetic route describes a method for preparing novel substituted thiophene-pyrimidine compounds of general formula I of this invention.

[0030] All raw materials were prepared by methods well known to those skilled in the art of organic chemistry, or were commercially available, as described in the synthetic routes below. All final compounds of this invention were prepared by methods described in the synthetic routes below or by similar methods well known to those skilled in the art of organic chemistry. All variable factors used in the synthetic routes below are as defined below or as defined in the claims.

[0031] The examples are intended to illustrate, and not limit, the scope of the invention. The proton NMR spectra of the compounds were determined using a Bruker ARX-400 or ARX-600, and the mass spectrometry was performed using an Agilent 1100 LC / MSD; all reagents used were analytical grade or chemically pure.

[0032] The synthetic route for compound Z1-25 of general formula (I) according to the present invention is as follows, wherein the functional groups of A, L, and R1 are as described in the claims:

[0033]

[0034] Example 1: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 1)

[0035]

[0036] Step 1: Synthesis of thieno[3,2-d]pyrimidine-2,4-diol (intermediate 2)

[0037] 100 g (1 eq) of methyl 3-amino-2-thiophenecarboxylate and 191 g (5 eq) of urea were added to a 2 L three-necked flask and heated and stirred in an oil bath at 190 °C. After 1 hour, the reaction system reached a molten state, and after 4 hours, the reaction was complete, precipitating a large amount of viscous, deep yellow solid. After cooling to room temperature, the solid was transferred to 1 L of 20% sodium hydroxide solution and stirred. After stirring for half an hour, diatomaceous earth was used to filter out solid impurities. The pH of the filtrate was adjusted to 2 with 2N hydrochloric acid, precipitating a large amount of yellow solid. 68.5 g of a light yellow solid product was obtained by filtration, with a yield of 64.21%, which was intermediate 2.

[0038] Step 2: Synthesis of 2,4-dichlorothiopheno[3,2-d]pyrimidine (intermediate 3)

[0039] Intermediate 2 (68 g, 1 eq) was placed in a 1 L three-necked flask, and 400 mL of phosphorus oxychloride was added. The mixture was heated to reflux at 180 °C for 8 h. After the reaction was complete, most of the phosphorus oxychloride was removed by rotary evaporation. The residue was slowly poured into ice water and stirred vigorously, resulting in the precipitation of a large amount of yellow solid. The solid was filtered, and the filter cake was washed with water until neutral. After drying, 62.3 g of a pale yellow solid was obtained, with a yield of 75.45%, which was intermediate 3.

[0040] Step 3: Synthesis of 2-chloro-4-(1-ethoxyvinyl)thiopheno[3,2-d]pyrimidine (intermediate 4)

[0041] Intermediate 3 (45 g, 1 eq) was added to a 2 L three-necked flask and dissolved in 800 mL of dioxane. Then, 300 mL of potassium carbonate solution (60.88 g, 2 eq) was added. Nitrogen gas was bubbled into the reaction system for 30 min, followed by the addition of ethyl tributyltin carboxylate (79.63 g, 1 eq) and Pd(Pph3)2Cl2 (7.74 g, 0.05 eq). The mixture was then evaporated under nitrogen protection at 100 °C. After approximately 3 h, the reaction was complete. The reaction solution was collected, and dioxane was removed by evaporation. 300 mL of dichloromethane and 100 mL of water were added for extraction. The organic phases were combined, washed twice with water, twice with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. Column chromatography purification yielded 35.2 g of a white solid, 66.49% yield, which was intermediate 4.

[0042] MS (ESI) m / z (%): 241.1[M+H]+, 279.2[M+K]+.

[0043] Step 4: Synthesis of ethyl 2-chlorothiopheno[3,2-d]pyrimidine-4-carboxylate (intermediate 5)

[0044] Sodium periodate (64.8 g, 2 eq) was dissolved in 600 mL of purified water and stirred at room temperature until clear (pH approximately 4). This solution was added to a solution of dioxane containing intermediate 4 (36 g, 1 eq), followed by the addition of potassium permanganate (7.11 g, 0.3 eq) in portions. The reaction was allowed to proceed at room temperature. After 3 hours, the reaction was complete. The reaction mixture was collected and filtered through diatomaceous earth. Dichloromethane was added to the filtrate, and the mixture was extracted with water. The organic phases were combined, washed twice with water, twice with saturated brine, and dried over anhydrous sodium sulfate. The resulting dark yellow solid was evaporated to dryness. The solid was stirred in an anhydrous ethanol solution containing a small amount of ethyl acetate for 2 hours and then filtered to obtain 19.4 g of a pale yellow solid (53.45% yield), which was intermediate 5.

[0045] MS(ESI) m / z (%): 243.0 [M+H] + 265.0 [M+Na] + .

[0046] Step 5: Synthesis of ethyl 2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxylate (intermediate 6)

[0047] Intermediate 5 (3.5 g, 1 eq), furan-2-boric acid (1.94 g, 1.2 eq), and Cs₂CO₃ (14.14 g, 3 eq) were added to a 100 mL three-necked flask and dissolved in 30 mL of dioxane. Nitrogen gas was applied for 30 min, then Pd(dppf)₂Cl₂ (0.529 g, 0.05 eq) was added. The mixture was then evacuated and reacted in an oil bath at 100 °C under nitrogen protection. The reaction was completed after approximately 3 h. The reaction solution was collected and filtered through diatomaceous earth. Dichloromethane was added to the filtrate, and the mixture was extracted with water. The organic phases were combined, washed twice with water, twice with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography yielded 1.23 g of a pale yellow solid (30.9% yield), which was intermediate 6.

[0048] Step 6: Synthesis of 2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxylic acid (intermediate 7)

[0049] Intermediate 6 (1.23 g, 1 eq) was added to a 50 mL round-bottom flask and dissolved in 20 mL of methanol. Then, 5 mL of NaOH solution (0.269 g, 1.5 eq) was added, and the reaction was carried out at 50 °C. The reaction was complete after 2 hours. Methanol was removed by rotary evaporation, and 20 mL of water was added. The pH was adjusted to 5 with 1 N HCl, and the mixture was stirred at room temperature for half an hour. Filtering yielded 1.02 g of a brown solid (92.7% yield), which was intermediate 7.

[0050] MS (ESI) m / z (%): 247.10 [M+H]+, 269.00 [M+Na]+.

[0051] Step 7: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 1)

[0052] Intermediate 7 (1.02 g, 1 eq) and 3-aminomethyl-4,6-dimethylpyridin-2(1H)one HATU (3.15 g, 2 eq) were added to a 50 mL round-bottom flask and dissolved in DMF. Triethylamine (544 mg, 1.3 eq) was then added. The reaction was carried out at 25 °C for 24 h until complete. The reaction solution was added to 50 mL of ice water, and a grayish-white solid precipitated. Filtering yielded 1.15 g of solid, with a yield of 73.2%, which was compound 1.

[0053] MS (ESI) m / z (%): 381.10 [M+H]+. 1H NMR(600MHz,DMSO)δ11.73(s,1H),9.44(s,1H),8.63(s,1H),7.98(s,1H),7.69(s,1H), 7.53(s,1H),6.77(s,1H),5.93(s,1H),4.48(d,J=5.8Hz,2H),2.26(s,3H),2.13(s,3H).

[0054] Example 2: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-(morpholinomethyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carbamoylhydrazide (Compound 2)

[0055]

[0056] Compound 1 (150 mg, 1 eq) was added to a 25 mL round-bottom flask and dissolved in glacial acetic acid. Formaldehyde solution (237 mg, 20 eq) and morpholine (172 mg, 5 eq) were then added. The mixture was heated to 120 °C and refluxed for approximately 8 hours. After the reaction was complete, the glacial acetic acid was removed by rotary evaporation, and the pH was adjusted to 8-9 with saturated sodium carbonate solution. The organic phase was collected, and the aqueous phase was extracted by reverse extraction. The organic phases were combined. The mixture was washed twice with water and twice with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain an oily substance. Column chromatography yielded 37 mg of a grayish-white solid, with a yield of 18.31%, which was compound 2.

[0057] MS (ESI) m / z (%): 480.1691[M+H]+, 502.1511[M+Na]+. 1H NMR (600MHz, DMSO) δ11.75(s,1H),9.51(s,1H),8.66(s,1H),7.73(s,1H),7.53(s,1H),6. 64(s,1H),5.96(s,1H),4.51(s,2H),3.67(s,7H),2.55(s,4H),2.29(s,3H),2.17(s,3H).

[0058] By replacing appropriate raw materials and reagents, and following the preparation scheme of Example 2, Examples 3-7 (compounds 3-7) were finally obtained.

[0059] Example 3: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-(pyrrolidone-1-ylmethyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 3)

[0060]

[0061] MS (ESI) m / z (%): 464.20 [M+H]+. 1H NMR (600MHz, DMSO) δ11.75(s,1H),9.52(t,J=5.6Hz,1H),8.68(d,J=5.5Hz,1H),7.75(d,J=5.5Hz,1H),7.55(d,J=2.9Hz,1H),6 .68(s,1H),5.98(s,1H),4.53(d,J=5.6Hz,2H),3.91(s,2H),2.71(s,3H),2.31(s,3H),2.19(s,3H),1.80(s,3H),1.28(s,2H).

[0062] Example 4: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-((4-methylpiperazin-1-yl)methyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 4)

[0063]

[0064] MS (ESI) m / z (%): 493.20 [M+H]+. 1H NMR(600MHz,DMSO)δ11.71(s,1H),9.47(s,1H),8.61(s,1H),7.70(s,1H),7.49(s,1H),6.58(s,1H),5.92(s ,1H),4.47(s,2H),3.62(s,2H),2.46(s,4H),2.22(s,7H),2.12(s,4H),1.22(s,1H),0.92(t,J=48.1Hz,1H).

[0065] Example 5: N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-((4-ethylpiperazin-1-yl)methyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 5)

[0066]

[0067] MS (ESI) m / z (%): 507.200[M+H]+, 529.200[M+Na]+. 1H NMR(600MHz,DMSO)δ11.71(s,1H),9.45(s,1H),8.65(s,1H),7.70(s,1H),7.48(s,1H),6.55(s, 1H), 5.91 (s, 1H), 4.46 (s, 2H), 3.62 (s, 2H), 2.23 (t, J = 66.6Hz, 15H), 1.22 (s, 1H), 0.97 (s, 3H).

[0068] Example 6: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-((4-(dimethylamino)piperidin-1-yl)methyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 6)

[0069]

[0070] MS (ESI) m / z (%): 521.2333[M+H]+, 543.2156[M+Na]+. 1H NMR (600MHz, DMSO) δ10.64(d,J=5.2Hz,1H),9.52(s,1H),8.52(d,J=5.4Hz,1H),7.66(d,J=5.3Hz,1H), 7.29(d,J=3.0Hz,1H),6.54(d,J=2.5Hz,1H),3.64(s,2H),3.09-2.96(m,4H),2.66(s,7H),2.07(t,J=1 1.2 Hz, 3H), 1.98 (d, J = 10.9 Hz, 3H), 1.66 (d, J = 10.3 Hz, 3H), 1.22 (s, 3H), 0.93–0.81 (m, 1H). Example 7, N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(5-((4-(2-methoxyethyl)piperazin-1-ylmethyl)furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 7)

[0071]

[0072] MS (ESI) m / z (%): 537.2288[M+H]+, 559.2097[M+Na]+. 1H NMR (600MHz, DMSO) δ11.80(s,1H),9.55(d,J=11.3Hz,1H),8.68(s,1H),7.77(s,1H),7.55(s,1H),6.64(s,1H),5.98(s,1H),4 .99(s,1H),4.53(s,2H),3.67(s,2H),3.45(s,3H),3.43(s,2H),2.57(s,6H),2.34(d,J=7.9Hz,3H),2.31(s,3H),2.19(s,3H).

[0073] Example 8: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-morpholinomethylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 8)

[0074]

[0075] Step 1: Ethyl 2-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyrimidine-4-carboxylate (intermediate 8a1)

[0076] In a 50 mL three-necked flask, intermediate 5 (1.5 g, 1 eq), 4-benzylmorpholinophenylboronic acid pinacol ester (2.25 g, 1.2 eq), and 30 mL of dioxane (containing a small amount of water) were added and dissolved. Cs₂CO₃ (4.02 g, 3 eq) was added. Nitrogen gas was applied for 30 minutes, and Pd(dppf)₂Cl₂ (0.529 g, 0.05 eq) was added. The mixture was then evacuated under nitrogen protection at 100 °C. The reaction was complete after approximately 3 hours. The reaction solution was collected and filtered through diatomaceous earth. Dichloromethane was added to the filtrate, and the mixture was extracted with water. The organic phases were combined, washed twice with water, twice with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated. Column chromatography yielded 576 mg of a yellow solid, 30.67%, which was intermediate 8a1.

[0077] MS (ESI) m / z (%): 384.1371[M+H]+, 406.1199[M+Na]+.

[0078] Step 2, 2-(4-(morpholinomethyl)phenyl)thieno[3,2-d]pyrimidine-4-carboxylic acid (intermediate 9a1)

[0079] Intermediate 8a1 (570 mg, 1 eq) and sodium hydroxide (128 mg, 2 eq) solution were added to a 50 mL flask and reacted at 50 °C. The reaction was completed after about 2 hours. After removing methanol by evaporation, 15 mL of water was added, and the pH was adjusted to about 6 with 1 N HCl. A solid precipitated out, which was filtered to give 196 mg of solid product, with a yield of 35.77%, which was intermediate 9a1.

[0080] MS (ESI) m / z (%): 342.0913[M+H]+, 364.0727[M+Na]+. 1 H NMR (600MHz, DMSO) δ8.43(d,J=7.3Hz,3H),7.58(d,J=5.4Hz,1H),7.03(d,J=8.5Hz,2H),3.75(s,4H),3.21(s,4H),2.51(s,2H).

[0081] Step 3, N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-morpholinomethylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 8)

[0082] In a 25 mL round-bottom flask, intermediate 9a1 (90 mg, 1 eq), 3-aminomethyl-4,6-dimethylpyridin-2(1H)one (46 mg, 1.2 eq), and HATU (193 mg, 2 eq) were added and dissolved in DMF. Triethylamine (33 mg, 1.3 eq) was then added. The reaction was carried out at 25 °C for 20 h until complete. The reaction solution was collected, and 50 mL of water was added. Extraction was performed with 50 mL of dichloromethane. The combined organic phases were washed twice with water and twice with saturated brine. The mixture was dried over anhydrous sodium sulfate and evaporated to dryness to obtain an oily liquid. Column chromatography yielded 37 mg of a white solid, 32.17% in yield, which was compound 8.

[0083] MS(ESI)m / z(%):490.2[M+H]+, 512.2[M+Na]+.1H NMR(600MHz,DMSO)δ11.72(s,1H),9.65(s,1H),8.63(s,1H),8.54(s,2H),7.70(s,1H),7.48(s,2H),5 .92(s,1H),4.49(s,2H),3.60(s,6H),2.81(d,J=95.8Hz,1H),2.40(s,3H),2.26(s,3H),2.12(s,3H).

[0084] Example 9: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-morpholinylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 9)

[0085]

[0086] Step 1: Ethyl 2-(4-morpholinylphenyl)thieno[3,2-D]pyrimidine-4-carboxylate (intermediate 8a2)

[0087] In a 50 mL three-necked flask, intermediate 5 (417 mg, 1 eq), 4-phenylmorpholine benzyl borate pinacol ester (286 mg, 1.2 eq), and 10 mL of dioxane (containing a small amount of water) were added and dissolved. Cs₂CO₃ (809 mg, 3 eq) was added. Nitrogen gas was applied for 30 minutes, followed by the addition of Pd(Pph₃)₂Cl₂ (29 mg, 0.05 eq). The mixture was then evacuated and reacted at 100 °C under nitrogen protection. The reaction was completed after approximately 3 hours. The reaction solution was collected and filtered through diatomaceous earth. The addition of 30 mL of saturated saline solution to the filtrate resulted in the precipitation of a yellow solid. Filtration yielded 380 mg of the yellow solid, with a yield of 59.84%, which was intermediate 8a₂.

[0088] MS (ESI) m / z (%): 370.10 [M+H]+.

[0089] Step 2, 2-(4-morpholinylphenyl)thieno[3,2-D]pyrimidine-4-carboxylic acid (intermediate 9a2)

[0090] Using intermediate 8a2 (180 mg, 1 eq) and sodium hydroxide (29 mg, 1.5 eq) as raw materials, and following the preparation method of intermediate 9a1, 105 mg of a pale yellow solid product was obtained, with a yield of 63.25%, which was intermediate 9a2. MS (ESI) m / z (%): 342.0913 [M+H]+. 1 H NMR (600MHz, DMSO) δ8.43(d,J=7.3Hz,3H),7.58(d,J=5.4Hz,1H),7.03(d,J=8.5Hz,2H),3.75(s,4H),3.21(s,4H),2.51(s,2H).

[0091] Step 3, N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-morpholinylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (compound 9)

[0092] Using intermediate 9a2 (120 mg, 1 eq), 3-aminomethyl-4,6-dimethylpyridin-2(1H)one (64 mg, 1.2 eq), HATU (268 mg, 2 eq), and triethylamine (53 mg, 1.5 eq) as raw materials, and following the preparation method of compound 8, 81 mg of a white solid product was obtained, with a yield of 34.32%, which was compound 9.

[0093] MS (ESI) m / z (%): 476.2000[M+H]+, 498.1000[M+Na]+. 1 H NMR(600MHz,DMSO)δ11.72(s,1H),9.58(s,1H),8.58(s,1H),8.45(s,2H),7.65(s,1H),7. 07(s,2H),5.93(s,1H),4.49(s,2H),3.78(s,4H),3.28(s,4H),2.27(s,3H),2.14(s,3H).

[0094] Example 10: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(2-morpholinylethoxy)phenyl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 10)

[0095]

[0096] Step 1: Ethyl 2-(4-(2-morpholinylethoxy)phenyl)thieno[3,2-D]pyrimidine-4-carboxylate (intermediate 8a3)

[0097] Using intermediates 5 (2 g, 1 eq), 4-(phenoxy)ethyl)morpholinoboronic acid pinacol ester (2.75 g, 1.3 eq), Cs2CO3 (7.83 g, 3 eq), and Pd(Pph3)2Cl2 (30 mg, 0.05 eq) as raw materials, 987 mg of white solid was obtained with a yield of 39.79% by referring to the preparation method of intermediate 8a1, which is intermediate 8a3.

[0098] MS (ESI) m / z (%): 414.10[M+H]+, 436.10[M+Na]+.

[0099] Step 2, 2-(4-(2-morpholinylethoxy)phenyl)thiopheno[3,2-d]pyrimidine-4-carboxylic acid (intermediate 9a3).

[0100] Using intermediate 8a3 (500 mg, 1 eq) and sodium hydroxide (44 mg, 1.5 eq) as raw materials, and following the preparation method of intermediate 9a1, 350 mg of a white solid product was obtained, with a yield of 75.10%, which was intermediate 9a3.

[0101] Step 3: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(2-morpholinylethoxy)phenyl)thieno[3,2-d]pyrimidine-4-carboxamide (compound 10)

[0102] Using intermediate 9a3 (130 mg, 1 eq), 3-aminomethyl-4,6-dimethylpyridin-2(1H)one (61 mg, 1.2 eq), HATU (268 mg, 2 eq), and triethylamine (51 mg, 1.5 eq) as raw materials, and following the preparation method of compound 8, 125 mg of a white solid product was obtained, with a yield of 71.42%, which was compound 10.

[0103] MS (ESI) m / z (%): 520.20[M+H]+, 542.20[M+Na]+. 1H NMR (600MHz, DMSO) δ11.66(s,1H),9.87(s,1H),9.56(t,J=5.8Hz,1H),8.62(d,J=5.5Hz,1H),8.59(d,J=8.8Hz,2H),7.69(d,J=5.5Hz,1H),7. 18(d,J=8.8Hz,2H),5.93(s,1H),4.50(d,J=5.8Hz,2H),4.48(s,1H),4 .02(s,2H),3.79–3.55(m,6H),3.25(s,2H),2.28(s,3H),2.14(s,3H).

[0104] Example 11: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(6-methoxypyridin-3-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 11)

[0105]

[0106] Step 1: Ethyl 2-(6-methoxypyridin-3-yl)thieno[3,2-D]pyrimidine-4-carboxylate (intermediate 8b)

[0107] Using intermediate 5 (2.5 g, 1 eq), (6-methoxypyridin-3-yl)boronic acid (1.88 g, 1.2 eq), Cs2CO3 (10.10 g, 3 eq), and Pd(dppf)2Cl2 (378 mg, 0.05 eq) as raw materials, 1.17 g of white solid was obtained with a yield of 36.00% by following the preparation method of intermediate 8a1, which is intermediate 8b.

[0108] MS (ESI) m / z (%): 316.10[M+H]+, 385.10[M+Na]+.

[0109] Step 2, 2-(6-methoxypyridin-3-yl)thieno[3,2-D]pyrimidine-4-carboxylic acid (intermediate 9b)

[0110] Using intermediate 8b (1 g, 1 eq) and sodium hydroxide (190 mg, 1.5 eq) as raw materials, and following the preparation method of intermediate 9a1, 786 mg of a white solid product was obtained, with a yield of 91.11%, which was intermediate 9b.

[0111] Step 3: N-(4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(6-methoxypyridin-3-yl)thieno[3,2-d]pyrimidine-4-carboxamide (compound 11)

[0112] Using intermediate 9b (150 mg, 1 eq), 3-aminomethyl-4,6-dimethylpyridin-2(1H)one (95 mg, 1.2 eq), HATU (397 mg, 2 eq), and triethylamine (79 mg, 1.5 eq) as starting materials, and following the preparation method of compound 8, a white solid product of 95 mg was obtained with a yield of 43.18%, which was compound 11.

[0113] MS(ESI) m / z (%): 422.10 [M+H] + . 1 H NMR (600MHz, DMSO) δ11.74 (s, 1H), 9.76 (t, J=

[0114] 5.6Hz,1H),9.40(d,J=2.1Hz,1H),8.78(dd,J=8.7,2.3Hz,1H),8.64(d,J=5.5Hz,1H),7.71(d,J=5.5Hz ,1H),6.99(d,J=8.7Hz,1H),5.93(s,1H),4.50(d,J=5.7Hz,2H),3.96(s,3H),2.25(s,3H),2.13(s,3H).

[0115] Example 12: N-(2,6-dioxopiperidin-3-yl)-2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 12)

[0116]

[0117] Using intermediate 7 (200 mg, 1 eq), 3-aminopiperidine-2,6-dione hydrochloride (160 mg, 1.2 eq), HATU (618 mg, 2 eq), and triethylamine (246 mg, 3 eq) as raw materials, and following the preparation method of compound 1, 90 mg of a white solid product was obtained, with a yield of 41.47%, which was compound 12.

[0118] MS (ESI) m / z (%): 357.00 [M+H] + 379.10 [M+Na] + . 1H NMR (600MHz, DMSO) δ11.02(s,1H),9.56(d,J=8.7Hz,1H),8.65(d,J=5.5Hz,1H),7.99(s,1H),7.71(t,J=4.8Hz,2H),6.8 0(dd,J=3.3,1.7Hz,1H),4.95–4.89(m,1H),2.89–2.83(m,1H),2.62–2.56(m,1H),2.40–2.34(m,1H),2.09–2.04(m,1H).

[0119] Example 13: N-(2,6-dioxopiperidin-3-yl)-2-(4-(morpholinomethyl)phenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 13)

[0120]

[0121] Using intermediate 9a1 (150 mg, 1 eq) and 3-aminopiperidine-2,6-dione (65 mg, 1.2 eq) as raw materials, and following the preparation method of compound 8, 84 mg of a white solid product was obtained, with a yield of 42.86%, which was compound 13.

[0122] MS(ESI) m / z (%): 466.1 [M+H] + . 1H NMR(600MHz,DMSO)δ11.03(s,1H),9.69(s,1H),8.66(s,3H),7.74(s,1H),7.50(s,2H),4.95(s ,1H),3.59(s,4H),3.57(s,2H),2.89(s,1H),2.59(d,J=17.1Hz,1H),2.39(s,5H),2.06(s,1H).

[0123] Example 14: N-(2,6-dioxopiperidin-3-yl)-2-(4-morpholinylphenyl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 14)

[0124]

[0125] Using intermediate 9a2 (100 mg, 1 eq), 3-aminopiperidine-2,6-dione hydrochloride (82 mg, 1.2 eq), HATU (315 mg, 2 eq), and triethylamine (167 mg, 4 eq) as starting materials, and following the preparation method of compound 9, 99 mg of a white solid product was obtained, with a yield of 75.00%, which was compound 14.

[0126] MS (ESI) m / z (%): 452.10 [M+H]+ 464.20 [M+Na] + . 1 H NMR (600MHz, DMSO) δ11.02(s,1H),9.66(d,J=8.3Hz,1H),8.59(d,J=5.8Hz,2H),7.68(d,J=4.9Hz,1H),7.09(d,J=8.1Hz,2H),4.94(s, 1H),3.78(s,4H),3.35(s,4H),2.88(t,J=12.9Hz,1H),2.60(d,J=17.2Hz,1H),2.51(s,3H),2.44–2.36(m,1H),2.09(d,J=4.4Hz,1H).

[0127] Example 15: N-(2,6-dioxopiperidin-3-yl)-2-(4-(2-morpholinylethoxy)phenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 15)

[0128]

[0129] Using intermediate 9a3 (120 mg, 1 eq), 3-aminopiperidine-2,6-dione hydrochloride (61 mg, 1.2 eq), HATU (237 mg, 2 eq), and triethylamine (94 mg, 3 eq) as starting materials, and following the preparation method of compound 10, 36 mg of a white solid product was obtained, with a yield of 23.37%, which was compound 15.

[0130] MS (ESI) m / z (%): 496.20 [M+H] + . 1 H NMR (600MHz, DMSO) δ11.04 (s, 1H), 9.69 (d, J=

[0131] 8.6Hz,1H),8.73(d,J=8.6Hz,2H),8.64(d,J=5.5Hz,1H),7.72(d,J=5.5Hz,1H),7.21(d,J=8.6Hz,2H),4.99–4.91(m,1H),4.49(s, 2H),4.01(s,2H),3.78–3.54(m,6H),3.25(s,2H),2.92–2.86(m,1H),2.61(d,J=17.0Hz,1H),2.44–2.36(m,1H),2.12–2.06(m,1H).

[0132] Example 16: N-(2,6-dioxopiperidin-3-yl)-2-(6-methoxypyridin-3-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 16)

[0133]

[0134] Using intermediate 9b (150 mg, 1 eq), 3-aminopiperidine-2,6-dione hydrochloride (129 mg, 1.5 eq), HATU (397 mg, 2 eq), and triethylamine (158 mg, 3 eq) as starting materials, and following the preparation method of compound 11, 160 mg of a white solid product was obtained, with a yield of 77.29%, which was compound 16.

[0135] MS (ESI) m / z (%): 398.10 [M+H] + , 420.10[M+Na]+. 1 H NMR (600MHz, DMSO) δ11.03(s,1H),9.76(d,J=8.8Hz,1H),9.57(d,J=2.2Hz,1H ),8.89(dd,J=8.7,2.4Hz,1H),8.66(d,J=5.5Hz,1H),7.74(d,J=5.5Hz,1H),7 .02(d,J=8.7Hz,1H),4.96(ddd,J=13.9,8.8,5.5Hz,1H),3.98(s,3H),2.90(s ,1H),2.61(d,J=17.2Hz,1H),2.39(qd,J=13.1,4.4Hz,1H),2.10–2.05(m,1H).

[0136] Example 17: N-(2-(1H-imidazol-4-yl)ethyl)-2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 17)

[0137]

[0138] Using intermediate 7 (150 mg, 1 eq) as the starting material, histamine hydrochloride (179 mg, 1.2 eq) was added as the starting material, and the preparation method of compound 1 was followed to obtain 39 mg of white solid product with a yield of 19.12%, thus obtaining compound 17.

[0139] MS (ESI) m / z (%): 340.10 [M+H] + . 1 H NMR (600MHz, DMSO) δ9.59 (s, 1H), 8.68 (d, J = 5.3

[0140] Hz,1H),8.03(s,1H),7.84–7.59(m,3H),6.91(d,J=81.6Hz,2H),3.70(d,J=6.3Hz,2H),2.92(t,J=6.9Hz,2H).

[0141] Example 18: N-(2-(1H-imidazol-4-yl)ethyl)-2-(4-(morpholinomethyl)phenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 18)

[0142]

[0143] Using intermediate 9a1 (150 mg, 1 eq) and histamine hydrochloride (56 mg, 1.2 eq) as raw materials, and following the preparation method of compound 8, 50 mg of a white solid product was obtained, with a yield of 27.32%, yielding compound 18.

[0144] MS(ESI) m / z (%): 449.2 [M+H] + . 1 H NMR (600MHz, DMSO) δ9.77 (s, 1H), 8.68 (d, J = 7.5

[0145] Hz,2H),8.63(d,J=5.0Hz,1H),7.77–7.70(m,2H),7.53(d,J=7.4Hz,2H),6.98(s,1H), 3.66(d,J=6.0Hz,1H),3.59(d,J=13.0Hz,5H),2.88(s,2H),2.41(s,4H),1.22(s,2H).

[0146] Example 19: N-(2-(1H-imidazol-4-yl)ethyl)-2-(4-morpholinylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 19)

[0147]

[0148] Using intermediate 9a2 (100 mg, 1 eq), histamine hydrochloride (65 mg, 1.2 eq), HATU (222 mg, 2 eq), and triethylamine (118 mg, 4 eq) as raw materials, and following the preparation method of compound 9, 18 mg of a white solid product was obtained, with a yield of 14.17%, thus yielding compound 19.

[0149] MS (ESI) m / z (%): 435.20 [M+H] + . 1 H NMR (600MHz, DMSO) δ9.72 (t, J = 5.6Hz, 1H),

[0150] 8.58(dd,J=12.3,7.2Hz,3H),7.89(s,1H),7.65(d,J=5.5Hz,1H),7.11(d,J=9.0Hz,2H),7.04(s,1H),3 .80–3.77(m,4H),3.67(dd,J=13.1,6.8Hz,2H),3.30–3.29(m,4H),2.89(t,J=7.0Hz,2H),1.23(s,2H).

[0151] Example 20: N-(2-(1H-imidazol-4-yl)ethyl)-2-(4-(2-morpholinylethoxy)phenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 20)

[0152]

[0153] Using intermediate 9a3 (130 mg, 1 eq), histamine hydrochloride (75 mg, 1.2 eq), HATU (257 mg, 2 eq), and triethylamine (136 mg, 4 eq) as raw materials, and following the preparation method of compound 10, 20 mg of a white solid product was obtained, with a yield of 12.42%, thus yielding compound 20.

[0154] MS (ESI) m / z (%): 479.20 [M+H] + 501.20[M+Na] + . 1 HNMR(600MHz,DMSO)δ9.80(t,J=5.2Hz,1H),8.74(d,J=8.7Hz,2H),8.67(d,J=5.5Hz,1H),8.29(s,1H),7.75(d,J=5.5Hz,1H), 7.26-7.19(m,3H),4.33(t,J=5.1Hz,2H),3.75(d,J=6.3Hz,2H),3.71(s,4H),3.00(t,J=6.7Hz,2H),2.93(s,2H),2.69(s,4H).

[0155] Example 21: N-(2-(1H-imidazol-4-yl)ethyl)-2-(6-methoxypyridin-3-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 21)

[0156]

[0157] Using intermediate 9b (150 mg, 1 eq), histamine hydrochloride (115 mg, 1.2 eq), HATU (397 mg, 2 eq), and triethylamine (211 mg, 4 eq) as raw materials, and following the preparation method of compound 11, 23 mg of a white solid product was obtained, with a yield of 11.62%, thus yielding compound 21.

[0158] MS(ESI) m / z (%): 381.10 [M+H] + 403.10 [M+Na] + . 1 HNMR (600MHz, DMSO) δ9.82(t,J=5.6Hz,1H),9.56(d,J=2.0Hz,1H),8.92(dd,J=8.7,2.4Hz,1H),8.64(d,J=5.5Hz,1H),7. 72(d,J=5.6Hz,2H),7.04(d,J=8.7Hz,1H),6.97(s,1H),3.99(s,3H),3.66(dd,J=13.4,7.0Hz,2H),2.89(t,J=7.2Hz,2H).

[0159] Example 22: N-(2-(1H-indol-3-yl)ethyl)-2-(furan-2-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 22)

[0160]

[0161] Using intermediate 7 (150 mg, 1 eq) as the starting material, and adding tryptophan (117 mg, 1.2 eq) as the starting material, and following the preparation method of compound 1, 37 mg of a white solid product was obtained, with a yield of 15.67%, thus yielding compound 22.

[0162] MS (ESI) m / z (%): 389.10 [M+H] + . 1 HNMR(600MHz,DMSO)δ10.89(s,1H),9.39(t,J=

[0163] 6.0Hz,1H),8.63(d,J=5.5Hz,1H),7.98(d,J=0.7Hz,1H),7.71–7.66(m,3H),7.36(d,J=8.1Hz,1H),7.27(d,J=2.1Hz,1H), 7.08(t,J=7.1Hz,1H), 7.00(t,J=7.4Hz,1H), 6.79(dd,J=3.3,1.7Hz,1H), 3.71(dd,J=14.9,6.6Hz,2H), 3.09–3.06(m,2H).

[0164] Example 23: N-(2-(1H-indol-3-yl)ethyl)-2-(4-morpholinylphenyl)thiopheno[3,2-d]pyrimidine-4-carboxamide (Compound 23)

[0165]

[0166] Using intermediate 9a2 (150 mg, 1 eq), tryptophan (84 mg, 1.2 eq), HATU (334 mg, 2 eq), and triethylamine (67 mg, 1.5 eq) as raw materials, and following the preparation method of compound 9, 138 mg of a white solid product was obtained, with a yield of 65.09%, yielding compound 23.

[0167] MS (ESI) m / z (%): 484.20 [M+H] + . 1 H NMR (600MHz, DMSO) δ10.90 (s, 1H), 9.45 (t, J=

[0168] 6.0Hz,1H),8.57(d,J=5.5Hz,1H),8.54(d,J=8.9Hz,2H),7.69(d,J=7.9Hz,1H),7.65(d,J=5.5Hz,1H),7.37(d,J=8.1Hz,1H),7.28(d,J=2.0Hz,1 H),7.09(dd,J=7.9,5.5Hz,3H),7.00(t,J=7.4Hz,1H),3.80–3.78(m,4H),3.73(dd,J=14.7,6.7Hz,2H),3.30–3.28(m,4H),3.08(t,J=7.6Hz,2H).

[0169] Example 24: N-(2-(1H-indol-3-yl)ethyl)-2-(4-(2-morpholinylethoxy)phenyl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 24)

[0170]

[0171] Using intermediate 9a3 (120 mg, 1 eq), tryptophan (60 mg, 1.2 eq), HATU (237 mg, 2 eq), and triethylamine (47 mg, 1.5 eq) as raw materials, and following the preparation method of compound 10, 30 mg of a white solid product was obtained, with a yield of 18.29%, thus yielding compound 24.

[0172] MS (ESI) m / z (%): 528.20 [M+H] + 550.20 [M+Na]+ . 1 H NMR (600MHz, DMSO) δ10.97(s,1H),9.57(t,J=6.0Hz,1H),8.68(dd,J=13.5,7.2Hz,3H),7. 75(dd,J=6.8,3.1Hz,2H),7.44(d,J=8.1Hz,1H),7.35(d,J=2.0Hz,1H),7.20(d,J=8.9Hz, 2H),7.16(t,J=7.1Hz,1H),7.07(t,J=7.1Hz,1H),4.29(t,J=5.6Hz,2H),3.80(dd,J=14.7 ,6.7Hz,2H),3.69–3.66(m,4H),3.15(t,J=7.6Hz,2H),2.82(s,2H),2.58(d,J=1.7Hz,4H).

[0173] Example 25: N-(2-(1H-indol-3-yl)ethyl)-2-(6-methoxypyridin-3-yl)thieno[3,2-d]pyrimidine-4-carboxamide (Compound 25)

[0174]

[0175] Using intermediate 9b (150 mg, 1 eq), tryptophan (100 mg, 1.2 eq), HATU (397 mg, 2 eq), and triethylamine (79 mg, 1.5 eq) as raw materials, and following the preparation method of compound 11, 39 mg of a white solid product was obtained, with a yield of 17.03%, thus yielding compound 25.

[0176] MS (ESI) m / z (%): 430.20 [M+H] + 452.20 [M+Na] + . 1 H NMR (600MHz, DMSO) δ10.92(s,1H),9.62(d,J=51.5Hz,2H),8.90(d,J=7.8Hz,1H),8.67(d,J=5.1Hz,1H),7.73(dd,J=12.2,6.6Hz,2H),7. 41(d,J=7.8Hz,1H),7.31(s,1H),7.12(t,J=7.1Hz,1H),7.05(d,J=7.8Hz,2H),4.02(s,3H),3.76(d,J=6.2Hz,2H),3.13(d,J=6.9Hz,2H).

[0177] Example 26: In vitro antitumor cell activity

[0178] Compounds containing substituted thiophene-pyrimidine structures were screened for their in vitro inhibitory activity against human B lymphoma cells (SU-DHL-6), human chronic myeloid leukemia cells (K562), and human embryonic kidney cells (HEK 2993T).

[0179] (1)Planning:

[0180] Take test cells in the logarithmic growth phase, centrifuge, discard the supernatant, and add 1 mL of culture medium. Dilute 100 μL of cells to 900 μL of culture medium (10-fold dilution), count cells using a counting chamber, and seed 100 μL of cells (1 × 10⁻⁶) into 96-well plates. 4 Place the well (in a single well) in an incubator and incubate for 24 hours.

[0181] (2) Sample preparation:

[0182] Accurately weigh the compound to be tested. First, add 100 μL of DMSO to dissolve it completely, then add 50 μL of Tween 80, and finally add RPMI-1640 basal medium to bring the volume to 2 mL. After shaking well, the solution should be clear and transparent, and the stock solution concentration is 1000 μmol / L.

[0183] (3) Adding medication:

[0184] The drug was serially diluted 10-fold to five concentrations: 100 μmol / L, 10 μmol / L, 1 μmol / L, 0.1 μmol / L, and 0.01 μmol / L. 100 μL of the drug solution was added to each well and diluted twice to a final concentration of 50 μmol / L, 5 μmol / L, 0.5 μmol / L, 0.05 μmol / L, and 0.005 μmol / L. The samples were then incubated for 48 hours before detection.

[0185] (4) Detection:

[0186] 20 μL of 0.5% MTT solution was pipetted into a 96-well plate in the dark and incubated at 37°C for 4 h. The plate was then centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 150 μL of DMSO was added to the 96-well plate and the plate was shaken for 30 s using a microplate reader to completely dissolve the blue-purple crystals. The optical density (OD) was measured at 490 nm. The cell inhibition rate and half-maximal inhibitory concentration (IC50) were calculated for each treatment group based on the data. 50 value.

[0187] Table 1

[0188]

[0189]

Claims

1. A substituted thiophene-pyrimidine compound and its pharmaceutically acceptable salt, characterized in that, It has a general structural formula as shown in (Ⅰ): A is selected from ; L is selected from C5-C6 aryl or C5-C6 heteroaryl; wherein the C5-C6 heteroaryl contains 1-2 heteroatoms selected from N, O or S, and L can be substituted by 1-2 R2 atoms; R2 is selected from dimethylamino, diethylamino, piperidinyl, piperazinyl, morpholinyl, tetrahydropyrrolyl, N-methylpiperazinyl, N-ethylpiperazinyl, 4-methylpiperridinyl, N-phenylpiperazinyl, 4-dimethylaminopiperridinyl, 1-(2-methoxyethyl)piperazinyl, or 4-(2-methoxyethyl)morpholinyl; R1 is selected from hydrogen, methoxy, ethoxy, trifluoromethyl, monofluoromethoxy, difluoromethoxy, or trifluoromethoxy.

2. Substituted thienopyrimidine compounds and pharmaceutically acceptable salts thereof, characterized in that, It has the following structural formula: 。 3. A pharmaceutical composition comprising a compound of claim 1 or 2 and a pharmaceutically acceptable carrier. It is prepared by combining the substituted thiophene-pyrimidine compound as described in claim 1 or 2 and its pharmaceutically acceptable salt as the active ingredient with a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein The pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, binders, and lubricants.

5. The pharmaceutical composition according to claim 3, characterized in that, Formulating a pharmaceutical composition into a dosage form such as tablets, capsules, granules, sprays, or injections.

6. The use of the substituted thiophene-pyrimidine compound of claim 1 or 2 and its pharmaceutically acceptable salt, or the pharmaceutical composition of any one of claims 3-5, in the preparation of an antitumor drug, wherein the tumor is leukemia, renal cell carcinoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, and follicular lymphoma.

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