4-amino-2-methylbenzamide HBV capsid protein inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a 4-amino-2-methylbenzamide HBV capsid protein inhibitor as well as a preparation method and application thereof. The compound has a structure as shown in a general formula I. The invention further discloses a preparation method and application of the 4-amino-2-methylbenzamide HBV capsid protein inhibitor. The invention also relates to a preparation method of the compound with the structure as shown in the general formula I, a pharmaceutical composition and application of the compound in preparation of anti-HBV drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry, specifically relating to a 4-amino-2-methylbenzamide-based inhibitor of hepatitis B virus (HBV) capsid protein, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus (HBV), commonly known as hepatitis B, is an infectious disease primarily affecting the liver, caused by persistent HBV infection. Further progression can lead to complications such as liver metabolic disorders, liver failure, cirrhosis, and liver cancer. Currently, the anti-HBV drugs approved by the US FDA for clinical use mainly include pegylated interferon-alpha (PEG-IFNα) and nucleoside analogs. PEG-IFNα primarily exerts its antiviral effect through immunomodulation; however, its effectiveness in only some patients and varying degrees of side effects limit its widespread clinical application. Nucleoside analogs competitively inhibit the activity of viral polymerase, thereby suppressing HBV genome replication and ultimately achieving an anti-HBV effect. However, they cannot completely eliminate the HBV from the body, requiring long-term medication and are prone to drug resistance, with a high relapse rate after discontinuation. Clinically, the combined use of nucleoside analogs and interferon can significantly inhibit viral replication, improving patients' quality of life and lifespan, but it cannot completely cure HBV, requiring long-term medication. Therefore, there is an urgent need to explore new treatment strategies and develop new antiviral drugs.

[0003] Currently, with the continuous development of structural biology research on HBV capsid proteins, HBV capsid proteins have become a promising new target in the field of anti-hepatitis B virus drug research. Currently, capsid protein inhibitors under development can be divided into two categories: Type I capsid protein inhibitors interfere with the normal assembly of the viral nucleocapsid, causing it to form various non-capsid structures, such as heteroaryl dihydropyrimidines (Bay 41-4109 and GLS4, etc.); Type II capsid protein inhibitors promote the formation of empty capsids with "normal" structures but without pregenomic RNA (pgRNA), such as phenylacrylamides (AT-130, etc.), benzenesulfonamides (JNJ-6379 and NVR3-778, etc.), and oxaloylpyrrolamides (ALG-001084). Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a 4-amino-2-methylbenzamide.

[0005] This invention provides HBV capsid protein inhibitors and their preparation methods. It also provides activity screening results and applications of the above compounds as non-nucleoside HBV inhibitors.

[0006] The technical solution of the present invention is as follows:

[0007] I. 4-Amino-2-methylbenzamide-type HBV capsid protein inhibitors

[0008] A 4-amino-2-methylbenzamide-based HBV capsid protein inhibitor has the structure shown in general formula I:

[0009]

[0010] Wherein, L represents an amide group, a sulfonamide group, a urea group, or a thiourea group;

[0011] M is a substituted phenyl, cycloalkyl, thienyl, substituted -3-pyridyl, chain aliphatic, or 4-morpholinyl group; the substituent is selected from halogens, aliphatic hydrocarbons, nitro groups, methoxy groups, cyano groups, or trifluoromethyl groups.

[0012] R is a hydrogen atom or an aliphatic acyl group.

[0013] According to a preferred embodiment of the present invention, in the general formula, L is... M is R is -H,

[0014] According to a further preferred embodiment of the present invention, the 4-amino-2-methylbenzamide-based HBV capsid protein inhibitor is characterized in that it is a compound having one of the following structures:

[0015]

[0016]

[0017] II. Preparation method of 4-amino-2-methylbenzamide HBV capsid protein inhibitors

[0018] A method for preparing 4-amino-2-methylbenzamide-based HBV capsid protein inhibitors, using 2-methyl-4-fluorobenzoic acid as a raw material, and preparing the compounds disclosed in this invention via one of the following synthetic routes;

[0019] Synthetic route I is as follows:

[0020]

[0021] Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0℃, 12h, rt; (ii) HATU, DIPEA, CH2Cl2, 20h, 0℃-rt; (iii) 4-methoxybenzylamine, DIPEA, DMF, 95℃, reflux, 12h; (iv) Fe, NH4Cl, MeOH, H2O, 12h, 90℃, reflux; (v, viii) different types of acyl chlorides or sulfonyl chlorides, pyridine, CH2Cl2, 4h, rt; (vi, ix, xi) CF3COOH and CH2Cl2 with v1:v2 = 1:4, 0.5h; (vii, xii) acetyl chloride, pyridine / triethylamine, CH3CN, 12h, 60℃; (x) different types of isocyanates, CH2Cl2, 10h, rt.

[0022] Among them, M1, M2 and M3 are each independently selected from 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-butylphenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-fluorophenylethyl, cyclopropyl, cyclopentyl, cyclohexyl, 2-thienyl, 6-chloro-3-pyridyl, 4-methoxycarbonylbutyl, 4-morpholinyl, 4-methoxyphenyl, 4-nitrophenyl;

[0023] The different types of acyl chlorides mentioned are selected from 4-fluorobenzoyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, 4-methylbenzoyl chloride, 4-butylbenzoyl chloride, 4-trifluoromethylbenzoyl chloride, 4-cyanobenzoyl chloride, 4-fluorophenylacetyl chloride, cyclopropylformyl chloride, cyclopentylformyl chloride, cyclohexylformyl chloride, 2-thiopheneformyl chloride, 6-chloro-3-pyridineformyl chloride, methyl chloroformylbutyrate, and chloroformyl-4-morpholine;

[0024] The different types of sulfonyl chlorides mentioned are selected from 4-methoxybenzenesulfonyl chloride, 4-nitrobenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride, and 4-bromobenzenesulfonyl chloride;

[0025] The different types of isocyanates mentioned are selected from 4-fluorophenyl isocyanate and 4-bromophenyl isocyanate;

[0026] Synthetic route II is as follows:

[0027]

[0028] Reagents and conditions: (i) NH4OH, 130℃, 1h; (ii) di-tert-butyl dicarbonate, NaH, DMF, 0℃-rt; (iii) Fe, NH4Cl, MeOH, H2O, 12h, 90℃, reflux; (iv) different types of thioisocyanates, THF, 60℃; (v) potassium carbonate, THF, cooled, 2h; (vi) DIPEA, potassium carbonate, THF, 60℃; (vii) hydrochloric acid-1,4-dioxane solution, rt.

[0029] M4 is isopropyl, allyl, or 4-bromophenyl;

[0030] The different types of thioisocyanates mentioned are isopropyl thioisocyanate or 4-bromophenyl isocyanate.

[0031] III. Application of 4-amino-2-methylbenzamide-based HBV capsid protein inhibitors

[0032] This invention discloses the screening results of the anti-HBV activity of 4-amino-2-methylbenzamide-based HBV capsid protein inhibitors and their application as anti-HBV inhibitors. Experiments demonstrate that the 4-amino-2-methylbenzamide compounds of this invention can be used as classic non-nucleoside inhibitors of HBV.

[0033] As shown in Table 1, lead compound 17i was selected as a positive control. The in vitro anti-HBV activity of the synthesized target compounds 7a-7n, 8a, 10a-10d, 12a-12b and 13a was evaluated. The in vitro cytotoxicity of the drugs was determined by the CCK-8 assay. At the same time, the inhibitory activity of the drugs on HBV DNA replication was determined by quantitative PCR.

[0034] The 4-amino-2-methylbenzamide-based HBV capsid protein inhibitor of the present invention is a novel type of non-nucleoside HBV inhibitor and can be used as a lead compound for anti-HBV treatment.

[0035] The 4-amino-2-methylbenzamide-based HBV capsid protein inhibitor of the present invention can be used as a non-nucleoside HBV inhibitor. Specifically, it can be used as an HBV inhibitor to prepare anti-hepatitis B drugs.

[0036] An anti-HBV pharmaceutical composition comprising the 4-amino-2-methylbenzamide HBV capsid protein inhibitor of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0037] This invention discloses 4-amino-2-methylbenzamide-type HBV capsid protein inhibitors, their preparation methods, anti-HBV activity screening results, and their first application as anti-HBV inhibitors. Experiments have shown that 4-amino-2-methylbenzamide-type HBV capsid protein inhibitors can be used as HBV inhibitors in the preparation of anti-hepatitis B drugs. Detailed Implementation

[0038] The following examples are helpful for understanding the present invention, but they are not intended to limit the scope of the invention. In the following examples, all target compounds are numbered the same as above.

[0039] Synthesis Scheme 1

[0040]

[0041] Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0℃, 12h, rt; (ii) HATU, DIPEA, CH2Cl2, 20h, 0℃-rt; (iii) 4-methoxybenzylamine, DIPEA, DMF, 95℃, reflux, 12h; (iv) Fe, NH4Cl, MeOH, H2O, 12h, 90℃, reflux; (v, viii) different types of acyl chlorides or sulfonyl chlorides, pyridine, CH2Cl2, 4h, rt; (vi, ix, xi) CF3COOH and CH2Cl2 with v1:v2 = 1:4, 0.5h; (vii, xii) acetyl chloride, pyridine / triethylamine, CH3CN, 12h, 60℃; (x) different types of isocyanates, CH2Cl2, 10h, rt.

[0042] Example 1: Preparation of Intermediate 2. Under ice bath conditions, 22.5 mL of concentrated sulfuric acid was added to a 100 mL round-bottom flask. 4-Fluoro-2-methylbenzoic acid (5 g, 25.11 mmol) was slowly added to the concentrated sulfuric acid. After stirring until homogeneous, 2.25 mL of concentrated nitric acid was added dropwise. The reaction was then transferred to room temperature and allowed to proceed for 12 h. After the reaction was completed as detected by TLC, the reaction solution was added dropwise to a beaker containing an ice-water mixture (200 mL). The precipitated solid was filtered, and the filter cake was washed with water (20 mL × 3). The filter cake was then removed and dried in an oven at 50 °C to obtain Intermediate 2, a white solid of 6.26 g. No purification was required, and the mixture was proceeded to the next step.

[0043] Example 2: Preparation of Intermediate 3. Under ice bath conditions, Intermediate 2 (6 g, 30 mmol) was added to a 250 mL round-bottom flask and dissolved in 80 mL of CH2Cl2. Then, HATU (17.18 g, 45 mmol) and DIPEA (7.87 mL, 45 mmol) were added, and the mixture was activated under ice bath conditions for 30 min. Next, 3-methyl-4-fluoroaniline (3.77 g, 30 mmol) was added, and the reaction was allowed to proceed to room temperature overnight. After TLC analysis, the reaction mixture was poured into a 1000 mL separatory funnel, and 600 mL of water and 100 mL of ethyl acetate were added. The mixture was extracted three times to remove a large amount of HATU. The EA layer was collected, and the solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization from dichloromethane / petroleum ether yielded Intermediate 3, a white solid of 5.22 g, with a yield of 56%.

[0044] Example 3: Preparation of Intermediate 4. In a 100 mL round-bottom flask, intermediate 3 (1.6 g, 5.22 mmol) was dissolved in 10 mL of N,N-dimethylformamide. 4-Methoxybenzylamine (717 mg, 5.22 mmol) was added. The mixture was heated in an oil bath to 95 °C and refluxed for 12 h. After the reaction was complete as detected by TLC, the reaction solution was cooled to room temperature and then poured into a 500 mL separatory funnel. 300 mL of water was added, and the mixture was extracted three times with 50 mL of ethyl acetate. The organic layer was collected and washed once with 150 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization from ethyl acetate / petroleum ether yielded intermediate 4, a yellow solid of 1.34 g, with a yield of 60%.

[0045] Example 4: Preparation of Intermediate 5. In a 100 mL round-bottom flask, intermediate 4 (1 g, 2.36 mmol) was dissolved in a mixture of methanol and water (20 mL: 2 mL). Iron powder (462 mg, 8.27 mmol) and ammonium chloride (189 mg, 3.54 mmol) were added, and the mixture was refluxed in an oil bath at 90 °C for 12 h. After the reaction was complete as detected by TLC, the reaction mixture was filtered while hot through a vacuum funnel lined with diatomaceous earth and washed three times with hot methanol (20 mL). The solvent in the collected filtrate was removed by rotary evaporation to obtain intermediate 5, 0.8 g of a pink solid, with a yield of 86%. No purification was required; the mixture was directly added to the next step.

[0046] Example 5: Synthesis of important intermediates 6a-6n. In a 50 mL round-bottom flask, intermediate 5 (150 mg, 0.38 mmol) was dissolved in 10 mL of dichloromethane. Different types of acyl chlorides (0.38 mmol) and pyridine (0.38 mmol) were added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete as detected by TLC, the reaction mixture was transferred to a 125 mL separatory funnel, and 30 mL of water and 1 mL of 3N HCl were added. The mixture was extracted three times with 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization was then performed using dichloromethane / n-hexane to obtain the important intermediates 6a-6n.

[0047] Example 6: Synthesis of target compound 7a. In a 50 mL round-bottom flask, the important intermediate 6a was added to a mixed solution of trifluoroacetic acid and dichloromethane (1 mL:4 mL). The mixture was stirred at room temperature for 30 min. After the reaction was complete as detected by TLC, the mixture was directly subjected to rotary evaporation under reduced pressure. Then, 15 mL of dichloromethane was added and the mixture was rotary evaporated again, repeated three times. The mixture was then further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Finally, recrystallization was performed using dichloromethane / n-hexane to obtain target compound 7a. The product was a white solid with a yield of 99% and a purity of 99.6%. 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.78(s,1H),7.95(d,J=7.8Hz,2H),7.74(d,J=7.7Hz,2H),7.66(s,1 H),7.51(s,1H),7.40(s,1H),7.07(s,1H),6.65(s,1H),5.34(s,2H),2.34(d,J=1.9Hz,3H),2.22(s,3H). 13 C NMR(101MHz,DMSO-d6)δ167.71,165.15,158.15,155.78,145.56,136.24( d,J=2.6Hz),135.91,134.16,131.75,130.43,127.60,125.64,124.44,12 4.23 (d, J = 6.5Hz), 123.05 (d, J = 4.5Hz), 120.02, 119.25 (d, J = 7.5Hz), 117.92, 115.15 (d, J = 22.8Hz), 20.34, 14.89 (d, J = 3.1Hz). ESI-MS: calculated for C 22 H 19 BrFN3O2[MH] - 455.06447.found 454.0574.

[0048] Example 7: Preparation of compound 7b, the procedure was the same as in Example 6, except that 4-bromobenzoyl chloride was replaced with 4-fluorobenzoyl chloride. White solid, yield: 62%, purity: 99.0%. 1H NMR (400MHz, DMSO-d6) δ9.92(s,1H),9.72(s,1H),8.08(dd,J=8.2,5.1Hz,2H),7.65(d,J=6.7Hz,1H),7.5 4–7.47(m,1H),7.41–7.31(m,3H),7.11–7.02(m,1H),6.64(s,1H),5.32(s,2H),2.33(s,3H),2.21(s,3H). 13 C NMR(101MHz,DMSO-d6)δ167.73,165.72,164.99,163.25,158.15,155.78,145.56,1 36.25(d,J=2.8Hz),135.82,131.50(d,J=3.1Hz),130.97(d,J=9.0Hz),127.61,124 .44,124.25(d,J=2.4Hz),123.05(d,J=4.3Hz),120.19,119.25(d,J=7.6Hz),117.9 4,115.76,115.55,115.27,115.04,20.33,14.88(d,J=3.1Hz).ESI-MS:calculated for C 22 H 19 F2N3O2[MH] - 395.14453.found394.136.

[0049] Example 8: Preparation of compound 7c, the procedure was the same as in Example 7, except that 4-fluorobenzoyl chloride was replaced with 4-methylbenzoyl chloride. White solid, yield: 52%, purity: 99.4%. 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.64(s,1H),7.90(d,J=7.8Hz,2H),7.65(d,J=7.2Hz,1H),7.54–7.47(m,1H),7.40 (s,1H),7.32(d,J=7.8Hz,2H),7.06(t,J=9.2Hz,1H),6.64(s,1H),5.28(s,2H),2.38(s,3H),2.33(s,3H),2.21(s,3H). 13C NMR(101MHz,DMSO-d6)δ167.77,165.89,158.14,155.77,145.39,141.84, 136.26(d,J=2.5Hz),135.60,132.18,129.28,128.29,127.42,124.43,124 .29(d,J=7.0Hz),123.04(d,J=4.5Hz),120.54,119.24(d,J=7.5Hz),118.07,115.27,115.04,21.48,20.30,14.88(d,J=3.0Hz).ESI-MS:calculated for C 23 H 22 FN3O2[MH] - 391.16961.found 390.161.

[0050] Example 9: Preparation of compound 7d, the procedure was the same as in Example 8, except that 4-methylbenzoyl chloride was replaced with 4-butylbenzoyl chloride. White solid, yield: 95%, purity: 98.4%. 1 H NMR (400MHz, DMSO-d6) δ9.94 (s, 1H), 9.65 (s, 1H), 7.91 (d, J = 7.7Hz, 2H), 7.6 5(d,J=7.1Hz,1H),7.53–7.48(m,1H),7.40(s,1H),7.33(d,J=7.8Hz,2H),7. 06(t,J=9.2Hz,1H),6.65(s,1H),5.88–4.68(m,2H),2.63(t,J=7.7Hz,2H),2 .33(s,3H),2.21(s,3H),1.62(h,J=7.4Hz,2H),0.90(td,J=7.3,1.8Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ167.78,165.98,158.15,155.78,146.42,136.25,132.48,128.71,128.30,127.39,124.44,124.27, 123.06,120.61,119.24(d,J=7.7Hz),118.12,115.27,115.04,37.51,24.37,14.88(d,J=3.0Hz),14.05.ESI-MS:calculated forC 25 H 26 FN3O2[MH] - 419.20091.found 418.193.

[0051] Example 10: Preparation of compound 7e, the procedure was the same as in Example 9, except that 4-butylbenzoyl chloride was replaced with 4-trifluoromethylbenzoyl chloride. White solid, yield: 77%, purity: 98.4%. 1 H NMR (400MHz, DMSO-d6)δ

[0052] 9.93(s,1H),9.91(s,1H),8.20(d,J=8.0Hz,2H),7.90(d,J=8.0Hz,2H),7.65(d,J=7.1Hz,1H),7.56–7.48(m, 1H),7.41(d,J=2.0Hz,1H),7.06(td,J=9.4,2.0Hz,1H),6.65(s,1H),5.38(s,2H),2.33(s,3H),2.21(s,3H). 13 C NMR(101MHz,DMSO-d6)δ167.70,164.98,158.16,155.79,138.91,136.23(d ,J=2.9Hz),136.05,131.87,131.55,129.21,127.65,125.91–125.57(m),12 4.44, 124.24 (d, J = 5.0Hz), 123.06 (d, J = 4.9Hz), 119.79, 119.26 (d, J = 7.7Hz), 117.88, 115.27, 115.04, 20.34, 14.87 (d, J = 3.1Hz). ESI-MS: calculated for C 23 H 19 F4N3O2[MH] - 445.14134.found 444.133.

[0053] Example 11: Preparation of compound 7f, the procedure was the same as in Example 10, except that 4-trifluoromethylbenzoyl chloride was replaced with 4-chlorobenzoyl chloride. White solid, yield: 51%, purity: 97.8%. 1 H NMR (400MHz, DMSO-d6) δ9.92(s,1H),9.77(s,1H),8.09–7.98(m,2H),7.65(d,J=7.2Hz,1H),7.63–7.57(m,2H) ,7.54–7.48(m,1H),7.39(s,1H),7.12–7.02(m,1H),6.64(s,1H),5.47–5.21(m,2H),2.33(s,3H),2.21(s,3H). 13C NMR (101MHz, DMSO-d6) δ167.72,165.03,158.15,155.78,145.56,136.71,136.24(d,J=2.6Hz),135.90,133.80,128.80,127.61,124.44,124 .24(d,J=3.9Hz),123.05(d,J=4.4Hz),120.05,119.25(d,J=7.5Hz),117.93,115.27,115.04,20.34,14.88(d,J=3.2Hz).ESI-MS:calculated for C 22 H 19 ClFN3O2[MH] - 411.11498.found 410.107.

[0054] Example 12: Preparation of 7g of compound, following the same procedure as in Example 11, except that 4-chlorobenzoyl chloride was replaced with 4-fluorophenylacetyl chloride. White solid, yield: 52%, purity: 99.1%. 1 H NMR (400MHz, DMSO-d6) δ9.91(s,1H),9.19(s,1H),7.63(d,J=7.1Hz,1H),7.54–7.46(m,1H),7.37–7.27(m,3H),7.06(t,J =9.2Hz,1H),6.90(d,J=8.0Hz,2H),6.43(s,1H),4.32(d,J=5.7Hz,2H),3.73(d,J=1.9Hz,2H),2.26(s,3H),2.21(s,3H). 13 C NMR(101MHz,DMSO-d6)δ175.39,158.64,158.14,155.77,143.92,136.23,135.03,131.79,128.69,126.22,124.45,124.27,124.07, 122.97(d,J=4.3Hz),121.29,119.18(d,J=7.5Hz),115.27,115.04,114.23,55.50,46.06,45.11,30.66,26.19.ESI-MS:calculated for C 23 H 21 F2N3O2[MH] - 409.16018.found408.152.

[0055] Example 13: Preparation of compound 7h, the procedure was the same as in Example 12, except that 4-fluorophenylacetyl chloride was replaced with cyclopropylformyl chloride. White solid, yield: 48%, purity: 97.5%. 1 H NMR (400MHz, DMSO-d6) δ9.90(s,1H),9.46(s,1H),7.64(d,J=6.7Hz,1H),7.49(tt,J=4.5,2.0Hz,1H),7.44(d,J=1.7H z,1H),7.11–7.02(m,1H),6.59(s,1H),5.22(s,2H),2.28(s,3H),2.21(s,3H),1.87–1.80(m,1H),0.81–0.74(m,4H). 13 C NMR (101MHz, DMSO-d6) δ172.20,167.88,158.12,155.75,143.75,136.26(d,J=2.7Hz),134.54,125.51,124.42(d,J=3.6Hz),124. 26,122.95(d,J=4.4Hz),120.95,119.16(d,J=7.6Hz),117.92,115.28,115.05,20.13,14.99–14.53(m),7.50.ESI-MS:calculated for C 19 H 20 FN3O2[M+H] + 341.15396.found 342.161.

[0056] Example 14: Preparation of compound 7i, the procedure was the same as in Example 13, except that cyclopropylformyl chloride was replaced with cyclopentylformyl chloride. White solid, yield: 60%, purity: 98.5%. 1 H NMR(400MHz,DMSO-d6)δ9.91(s,1H),9.13(s,1H),7.64(d,J=7.2Hz,1H),7.52– 7.47(m,1H),7.40(d,J=1.9Hz,1H),7.06(td,J=9.2,1.9Hz,1H),6.59(s,1H),5. 18(s,2H),2.82(p,J=7.9,7.5Hz,1H),2.28(d,J=1.8Hz,3H),2.21(s,4H),1.86 (q,J=8.4,6.5Hz,3H),1.71(dp,J=19.0,7.0Hz,6H),1.56(q,J=8.6,7.2Hz,3H). 13C NMR (101MHz, DMSO-d6) δ175.00,167.91,158.14,155.76,143.90,134.58,125.78,124.49(d,J=11.0Hz),124.26,122.99(d,J=4. 4Hz), 120.93, 119.20 (d, J = 7.6Hz), 117.91, 115.27, 115.04, 45.12, 30.70, 26.20, 20.11, 14.88 (d, J = 3.0Hz). ESI-MS: calculated for C 21 H 24 FN3O2[MH] - 369.18526.found 368.177.

[0057] Example 15: Preparation of compound 7j, the procedure was the same as in Example 14, except that cyclopentylformyl chloride was replaced with cyclohexylformyl chloride. White solid, yield: 96%, purity: 97.1%. 1 H NMR (400MHz, DMSO-d6) δ9.91(s,1H),9.05(s,1H),7.63(d,J=7.2Hz,1H),7.53–7.46(m,1H),7.39(d,J=1.8Hz,1H),7.06(t,J=8.8Hz,1H),6.58(s,1 H),5.17(s,2H),2.42–2.32(m,1H),2.28(s,3H),2.21(s,3H),1.86–1.71( m,5H),1.65(d,J=11.7Hz,1H),1.41(q,J=12.2Hz,2H),1.32–1.11(m,3H). 13 C NMR (101MHz, DMSO-d6) δ174.89,167.94,158.14,155.76,143.89,136.25(d,J=2.6Hz),134.53,125.76,124.59,124.44,124.26,122.99(d,J=4. 4Hz), 120.86, 119.20 (d, J = 7.7Hz), 117.87, 115.27, 115.04, 44.72, 29.81, 25.84 (d, J = 17.0Hz), 20.09, 14.88 (d, J = 3.1Hz). ESI-MS: calculated for C 22 H 26 FN3O2[MH] - 383.20091.found 382.193.

[0058] Example 16: Preparation of compound 7k, the procedure was the same as in Example 15, except that cyclohexylformyl chloride was replaced with 2-thiopheneformyl chloride. White solid, yield: 86%, purity: 96.5%. 1 H NMR (400MHz, DMSO-d6) δ9.93(s,1H),9.76(s,1H),8.00(t,J=2.6Hz,1H),7.83(d,J=5.0Hz,1H),7.65(d,J=6.9Hz,1H),7.50(tt,J=4.9,2. 1Hz,1H),7.37(d,J=1.9Hz,1H),7.22(d,J=4.9Hz,1H),7.11–7.02(m,1H),6.65(s,1H),4.29(d,J=55.5Hz,2H),2.33(s,3H),2.21(s,3H). 13 C NMR(101MHz,DMSO-d6)δ167.64,160.71,158.17,155.80,136.40–135.72(m),131.88,129.62,128.49,127.67,124.48(d,J=6.8Hz), 124.27, 123.08 (d, J = 4.4Hz), 119.95, 119.28 (d, J = 7.6Hz), 118.16, 115.27, 115.04, 20.33, 14.88 (d, J = 3.0Hz). ESI-MS: calculated for C 20 H 18 FN3O2S[MH] - 383.11038.found382.102.

[0059] Example 17: Preparation of compound 7l, the procedure was the same as in Example 16, except that 2-thiophenecarboxyl chloride was replaced with 6-chloro-3-pyridinyl chloride. White solid, yield: 73%, purity: 99.5%. 1 H NMR (400MHz, DMSO-d6) δ9.91(d,J=3.9Hz,1H),8.99(d,J=2.6Hz,1H),8.37(dd,J=8.2,2.9Hz,1H),7.70(d,J=8.3Hz,1H),7.64(d,J =7.1Hz,1H),7.50(dt,J=7.7,3.2Hz,1H),7.38(s,1H),7.06(t,J=9.2Hz,1H),6.63(s,1H),5.43(s,2H),2.33(s,3H),2.21(s,3H). 13C NMR (101MHz, DMSO-d6) δ167.67,163.64,158.15,155.78,149.98,145.79,139.65,136.22,130.15,127.83,124.45(d,J=2.9Hz),12 4.26,124.06,123.06(d,J=4.5Hz),119.27(d,J=10.8Hz),117.70,115.27,115.04,20.38,14.88(d,J=3.0Hz).ESI-MS:calculated for C 20 H 18 ClFN4O2[M+H] + 412.11023.found 413.117.

[0060] Example 18: Preparation of compound 7m, performed as in Example 17, except that 6-chloro-3-pyridinecarboxyl chloride was replaced with methyl chloroformylbutyrate. White solid, yield: 57%, purity: 95.6%. 1 H NMR(400MHz, DMSO-d6)δ9.91(s,1H),9.14(s,1H),7.64(d,J=7.2Hz,1H),7.53–7.47(m,1H),7.37(s,1H),7.04(s,1H),6 .58(s,1H),5.21(s,2H),3.60(d,J=1.9Hz,3H),2.37(q,J=7.1Hz,4H),2.28(s,3H),2.21(s,3H),1.84(p,J=7.4Hz,2H). 13 C NMR (101MHz, DMSO-d6) δ173.60,171.21,167.89,158.14,155.77,144.14,136.24(d,J=2.6Hz),134.78,126.03,124.44,124.27,122.99(d,J =4.3Hz),120.59,119.20(d,J=7.7Hz),117.75,115.27,115.04,51.75,35.06,33.18,21.00,20.13,14.88(d,J=3.0Hz).ESI-MS:calculated for C 21 H 24 FN3O4[M+H] + 401.17508.found 402.182.

[0061] Example 19: Preparation of compound 7n, performed as in Example 18, except that methyl chloroformylbutyrate was replaced with morpholine-4-acyl chloride. White solid, yield: 62%, purity: 97.5%. 1 H NMR (400MHz, DMSO-d6) δ9.88(s,1H),7.91(s,1H),7.65(d,J=7.2Hz,1H),7.50(dq,J=7.5,2.5Hz,1H),7.22(d,J=2.0Hz,1H),7.06(td,J =9.3, 2.0Hz, 1H), 6.56 (d, J = 2.0Hz, 1H), 5.15 (s, 2H), 3.60 (t, J = 5.0Hz, 4H), 3.42 (d, J = 5.0Hz, 4H), 2.29 (d, J = 2.0Hz, 3H), 2.21 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ167.88,158.10,156.37,155.73,145.58,136.30(d,J=2.5Hz),134.66,124.40,124.20(d,J=4.2Hz),122.9 9(d,J=4.3Hz),121.83,119.20(d,J=7.5Hz),117.80,115.24,115.02,66.46,44.63,20.25,14.89(d,J=3.1Hz).ESI-MS:calculated for C 20 H 23 FN4O3[MH] - 386.17542.found 385.1678.

[0062] Example 20: Preparation of compound 8a. Compound 7a (0.22 mmol) was dissolved in 10 mL acetonitrile in a 50 mL round-bottom flask. Triethylamine (0.44 mmol) was added, and the mixture was stirred in an ice bath for 15 min. Acetyl chloride (0.44 mmol) was then added dropwise, and the mixture was heated to reflux at 60 °C for 12 h. After the reaction was complete as detected by TLC, the solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization was then performed using dichloromethane / n-hexane to obtain the target product 8a. The product was a white solid with a yield of 41% and a purity of 95.0%. 1H NMR (400MHz, DMSO-d6) δ10.27(s,1H),9.97(s,1H),9.72(s,1H),7.92(d,J=8.1Hz,2H),7.77(d,J=8.2Hz,2H), 7.72(s,1H),7.67(d,J=7.2Hz,1H),7.53(s,2H),7.10(t,J=9.2Hz,1H),2.38(s,3H),2.23(s,3H),2.10(s,3H). 13 C NMR(101MHz,DMSO-d6)δ169.75,167.31,164.90,156.08,135.71,133.96,133.62,133.32,133.14,131.99,130.21,127.69,126.50, 126.02,125.48,124.61(d,J=18.1Hz),119.40(d,J=7.6Hz),115.46,115.23,24.16,19.64,14.87(d,J=3.0Hz).ESI-MS:calculated for C 24 H 21 BrFN3O2[MH] - 497.07503.found496.067.

[0063] Example 21: Preparation of important intermediates 9a-9d. In a 50 mL round-bottom flask, intermediate 5 (150 mg, 0.38 mmol) was dissolved in 10 mL of dichloromethane. Different types of sulfonyl chlorides (0.38 mmol) and pyridine (0.38 mmol) were added dropwise, and the mixture was stirred at room temperature for 4 h. After the reaction was complete as detected by TLC, the reaction mixture was transferred to a 125 mL separatory funnel, and 30 mL of water and 1 mL of 3N HCl were added. The mixture was extracted three times with 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization was then performed using dichloromethane / n-hexane to obtain important intermediates 9a-9d.

[0064] Example 22: Synthesis of target compound 10a. In a 50 mL round-bottom flask, the important intermediate 9a was added to a mixed solution of trifluoroacetic acid and dichloromethane (1 mL:4 mL). The mixture was stirred at room temperature for 30 min. After the reaction was complete as detected by TLC, the mixture was directly subjected to rotary evaporation under reduced pressure. Then, 15 mL of dichloromethane was added and the mixture was rotary evaporated again, repeated three times. The mixture was then further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Finally, recrystallization was performed using dichloromethane / n-hexane to obtain target compound 9a. The product was a white solid with a yield of 71% and a purity of 97.7%. 1 HNMR(400MHz,DMSO-d6)δ9.81(s,1H),9.16(s,1H),7.69–7.62(m,2H),7.60(d,J=7.2Hz,1H),7.46(dt,J=7 .1,2.7Hz,1H),7.12–6.99(m,4H),6.45(s,1H),5.20(s,2H),3.78(d,J=1.9Hz,3H),2.22(t,J=2.3Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ167.62,162.78,158.19,155.82,145.86,136.15–136.00(m),132.56,129.43,127.88,124.68,124.52,124.34,1 22.94(d,J=4.4Hz),119.15(d,J=7.6Hz),118.13,117.23,115.32,115.09,114.61,56.03,20.11,14.87(d,J=3.0Hz).ESI-MS:calculated for C 22 H 22 FN3O4S[MH] - 443.13151.found 442.123.

[0065] Example 23: Preparation of compound 10b, following the same procedure as in Example 22, except that 4-methoxybenzenesulfonyl chloride was replaced with 4-nitrobenzenesulfonyl chloride. Pale yellow solid, yield: 73%, purity: 96.1%. 1H NMR (400MHz, DMSO-d6) δ9.83(d,J=2.0Hz,1H),9.68(s,1H),8.37(dd,J=8.8,2.2Hz,2H),7.96(dd,J=8.8,2.2Hz,2H),7.57(d, J=6.7Hz,1H),7.45(q,J=4.6,4.2Hz,1H),7.05(dd,J=8.6,2.1Hz,2H),6.46(d,J=2.0Hz,1H),5.19(s,2H),2.26–2.19(m,6H). 13 C NMR (101MHz, DMSO-d6) δ167.41,158.21,155.84,150.17,146.52,146.09,136.89,136.03(d,J=2.8Hz),128.92,128.43,124.83(d,J=7.0Hz) ,124.54,124.37,122.97(d,J=4.6Hz),119.17(d,J=7.6Hz),117.47,116.99,115.31,115.09,20.15,14.85(d,J=3.1Hz).ESI-MS:calculated for C 21 H 19 FN4O5S[MH] - 458.10602.found457.098.

[0066] Example 24: Preparation of compound 10c, following the same procedure as in Example 23, except that 4-nitrobenzenesulfonyl chloride was replaced with 4-fluorobenzenesulfonyl chloride. White solid, yield: 85%, purity: 97.1%. 1 H NMR (400MHz, DMSO-d6) δ9.81(s,1H),9.33(s,1H),7.74(ddt,J=6.9,5.1,1.9Hz,2H),7.58(d,J=7.2Hz,1H),7.45(dt ,J=7.4,3.0Hz,1H),7.36(td,J=8.9,1.9Hz,2H),7.10–6.98(m,2H),6.44(s,1H),5.18(s,2H),2.21(d,J=5.8Hz,6H). 13C NMR(101MHz,DMSO-d6)δ167.53,166.02,163.52,158.21,155.83,145.99,137.22 (d,J=2.9Hz),136.39,136.06(d,J=2.8Hz),130.28(d,J=9.6Hz),128.21,124.76 ,124.53,124.35,122.97(d,J=4.4Hz),119.18(d,J=7.8Hz),117.62,117.32,116 .78,116.55,115.33,115.10,20.14,14.87(d,J=3.0Hz).ESI-MS:calculatedfor C 21 H 19 F2N3O3S[MH] - 431.11152.found 430.103.

[0067] Example 25: Preparation of compound 10d, following the same procedure as in Example 24, except that 4-fluorobenzenesulfonyl chloride was replaced with 4-bromobenzenesulfonyl chloride. White solid, yield: 75%, purity: 97.0%. 1 H NMR(400MHz,DMSO-d6)δ9.83(s,1H),9.42(s,1H),7.76(dd,J=8.5,1.9Hz,2H),7.66–7.57(m,3H) ,7.47(dt,J=7.3,3.1Hz,1H),7.12–6.99(m,2H),6.46(s,1H),5.20(s,2H),2.23(d,J=4.9Hz,6H). 13 C NMR (101MHz, DMSO-d6) δ158.21,155.83,145.95,140.19,136.51,136.04(d,J=2.7Hz),132.59,129.28,128.22,127.04,124.71,124.53,12 4.36,122.99(d,J=4.4Hz),119.20(d,J=7.6Hz),117.39(d,J=10.4Hz),115.33,115.10,20.15,14.89(d,J=3.0Hz).ESI-MS:calculatedfor C 21 H 19 BrFN3O3S[MH] - 491.03145.found 490.024.

[0068] Example 26: Preparation of important intermediates 11a-11b. In a 50 mL round-bottom flask, intermediate 5 (0.38 mmol) was dissolved in 10 mL of dichloromethane. Different types of isocyanates (0.38 mmol) were added dropwise, and the mixture was stirred at room temperature for 10 h. After the reaction was complete as detected by TLC, the solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization was then performed using dichloromethane / n-hexane to obtain important intermediates 11a-11b.

[0069] Example 27: Preparation of compound 12a. In a 50 mL round-bottom flask, the important intermediate 11a was added to a mixed solution of trifluoroacetic acid and dichloromethane (1 mL:4 mL). The mixture was stirred at room temperature for 30 min. After the reaction was complete as detected by TLC, the mixture was directly subjected to rotary evaporation under reduced pressure. 15 mL of dichloromethane was added, and the mixture was evaporated again. This process was repeated three times. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization was then performed using dichloromethane / n-hexane to obtain the target compound 12a. The compound was a white solid with a yield of 68% and a purity of 97.8%. 1 H NMR(400MHz,DMSO-d6)δ9.93(s,1H),8.77(s,1H),7.75(s,1H),7.65(d,J=7.2Hz,1H),7.55–7 .41(m,4H),7.09(dt,J=14.4,8.9Hz,3H),6.62(s,1H),5.21(s,2H),2.30(s,3H),2.22(s,3H). 13 C NMR(101MHz,DMSO-d6)δ168.02,158.84,158.14,156.47,155.77,153.87,14 3.73,136.84(d,J=2.4Hz),136.25(d,J=2.8Hz),133.78,125.04,124.85,124 .46,124.28,122.99(d,J=4.2Hz),121.49,120.17(d,J=7.6Hz),117.80,115 .81,115.59,115.28,115.05,20.08,14.88(d,J=3.1Hz).ESI-MS:calculated for C 22 H 20 F2N4O2[MH] - 410.15543.found 409.147.

[0070] Example 28: Preparation of compound 12d, following the same procedure as in Example 27, except that 4-fluorophenyl isocyanate was replaced with 4-bromophenyl isocyanate. White solid, yield: 78%, purity: 97.0%. 1 H NMR(400MHz, DMSO-d6)δ9.92(d,J=2.0Hz,1H),8.89(d,J=2.0Hz,1H),7.78(s,1H),7.68–7.62(m,1H),7.55–7.46(m,2H),7 .43(d,J=1.9Hz,4H),7.07(td,J=9.3,2.0Hz,1H),6.60(d,J=1.9Hz,1H),5.21(s,2H),2.29(d,J=1.9Hz,3H),2.22(s,3H). 13 C NMR (101MHz, DMSO-d6) δ167.97,158.13,155.76,153.63,143.88,139.96,136.26(d,J=2.7Hz),133.92,131.95,125.13,124.74,124.45,124.2 7,122.97(d,J=4.4Hz),121.23,120.35,119.18(d,J=7.7Hz),117.75,115.28,115.05,113.34,20.11,14.89(d,J=3.1Hz).ESI-MS:calculated for C 22 H 20 BrFN4O2[MH] - 470.07537.found 469.0677.

[0071] Example 29: Preparation of compound 13a. In a 50 mL round-bottom flask, under ice bath conditions, compound 12a (120 mg, 0.25 mmol) was dissolved in 10 mL acetonitrile, followed by the addition of pyridine (40 mg, 0.51 mmol), and then dropwise addition of acetyl chloride (40 mg, 0.51 mmol). The mixture was heated to reflux at 60 °C for 12 h. After the reaction was complete as detected by TLC, the reaction mixture was transferred to a 125 mL separatory funnel, and 30 mL of water and 1 mL of 3N HCl were added. The mixture was extracted with dichloromethane (20 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the mixture was further purified by silica gel column chromatography. A specific ratio of petroleum ether / ethyl acetate was selected as the eluent, and recrystallization was performed using dichloromethane / n-hexane to obtain the target product 13a. The product was a white solid with a yield of 50% and a purity of 97.4%. 1H NMR (400MHz, DMSO-d6) δ10.27(s,1H),9.73(s,1H),9.38(s,1H),7.91(s,2H),7.68(d,J=7.1Hz,1H),7.56– 7.49(m,1H),7.44(d,J=1.9Hz,4H),7.19(s,1H),7.15–7.06(m,1H),2.31(s,3H),2.23(s,3H),2.13(s,2H). 13 C NMR(101MHz,DMSO-d6)δ169.80,167.69,158.42,156.04,152.99,139.66 ,135.76,134.81,132.02,131.19,130.49,130.04,128.17,124.61(d,J=1 7.9Hz), 123.07 (d, J = 4.4Hz), 122.10, 120.52, 119.31 (d, J = 7.9Hz), 115.46, 115.23, 113.70, 23.80, 19.18, 14.88 (d, J = 3.1Hz). ESI-MS: calculated forC 24 H 22 BrFN4O3[MH] - 512.08593.found 511.0782.

[0072] Synthesis Scheme 2:

[0073]

[0074] Reagents and conditions: (i) NH4OH, 130℃, 1h; (ii) di-tert-butyl dicarbonate, NaH, DMF, 0℃-rt; (iii) Fe, NH4Cl, MeOH, H2O, 12h, 90℃, reflux; (iv) different types of thioisocyanates, THF, 60℃; (v) potassium carbonate, THF, cooled, 2h; (vi) DIPEA, potassium carbonate, THF, 60℃; (vii) hydrochloric acid-1,4-dioxane solution, rt.

[0075] Example 30: Preparation of Intermediate 14. A 250 mL pressure-resistant tube was used. Intermediate 3 (5 g, 16.33 mmol) was dissolved in 100 mL ammonium hydroxide. The reaction was carried out in an oil bath at 130 °C for 1 h. After the reaction was completed by TLC, the reaction solution was cooled to room temperature and then filtered. The filter cake was dried in an oven to obtain Intermediate 14, a yellow solid of 4.63 g, with a yield of 93%. No further processing was required; it was directly used in the next step.

[0076] Example 31: Preparation of Intermediate 15. In a 50 mL round-bottom flask, intermediate 14 (2.0 g, 6.59 mmol) was dissolved in 10 mL of N,N-dimethylformamide. Under ice bath conditions, 60% sodium hydride (791 mg, 19.77 mmol) was slowly added. After stirring and activation for 1 h, di-tert-butyl dicarbonate (1.73 g, 7.91 mmol) was added dropwise. The reaction mixture was then transferred to room temperature and allowed to stand overnight. After TLC detection, the reaction mixture was transferred to a 250 mL separatory funnel, 150 mL of water was added, and the mixture was extracted three times with 25 mL of ethyl acetate. The combined organic phases were added back to the separatory funnel and washed with 100 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. The solution was then recrystallized from dichloromethane / petroleum ether to give intermediate 15, a white solid weighing 1.95 g, with a yield of 77%.

[0077] Example 32: Preparation of Intermediate 16. In a 100 mL round-bottom flask, intermediate 15 (1.55 g, 3.84 mmol) was dissolved in a mixture of methanol and water (30 mL: 3 mL). Iron powder (751 mg, 13.45 mmol) and ammonium chloride (617 mg, 11.52 mmol) were added, and the mixture was refluxed in an oil bath at 90 °C for 12 h. After the reaction was complete as detected by TLC, the reaction mixture was filtered while hot through a vacuum funnel lined with diatomaceous earth and washed three times with hot methanol (20 mL). The solvent in the collected filtrate was removed by rotary evaporation to obtain intermediate 16, 1.24 g of white solid, with a yield of 86%. No purification was required; the mixture was directly added to the next step.

[0078] Example 33: Preparation of Intermediate 18. In a 50 mL round-bottom flask, under ice bath conditions, phenyl thiochloroformate (17,250 mg, 1.45 mmol) was dissolved in 10 mL of tetrahydrofuran. Potassium carbonate (300 mg, 2.17 mmol) and allylamine hydrochloride (207 mg, 2.17 mmol) were added, and the mixture was transferred to room temperature and reacted for 2 h. After TLC analysis, the reaction mixture was transferred to a 125 mL separatory funnel, and 30 mL of water and 2 mL of 3N HCl were added. The mixture was extracted three times with 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, intermediate 18, a transparent oily product, was obtained in 100% yield.

[0079] Example 34: Preparation of important intermediate 19a. In a 50 mL round-bottom flask, intermediate 18 (279 mg, 1.44 mmol) was dissolved in 15 mL of tetrahydrofuran. Intermediate 16 (323 mg, 0.87 mmol), DIPEA (280 mg, 2.17 mmol), and potassium carbonate (299 mg, 2.17 mmol) were added. The mixture was heated to reflux at 60 °C for 12 h. After the reaction was complete as detected by TLC, the reaction mixture was transferred to a 125 mL separatory funnel. 30 mL of water and 5 mL of 3N HCl were added. The mixture was extracted three times with 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. The mixture was then recrystallized from dichloromethane / n-hexane to obtain important intermediate 19a.

[0080] Example 35: Preparation of important intermediates 19b-19c. In a 50 mL round-bottom flask, intermediate 16 (150 mg, 0.40 mmol) was dissolved in 10 mL of tetrahydrofuran. Different types of thioisocyanates (0.48 mmol) were added. The reaction was carried out in an oil bath and refluxed at 60 °C for 10 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation. The mixture was further purified by silica gel column chromatography, using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization from dichloromethane / n-hexane yielded important intermediates 19b-19c.

[0081] Example 36: Preparation of compound 20a. In a 50 mL round-bottom flask, intermediate 19a (100 mg) was dissolved in 5 mL of 1,4-dioxane hydrochloric acid solution. The mixture was stirred at room temperature for 2 h. After the reaction was complete as detected by TLC, the reaction mixture was transferred to a 125 mL separatory funnel. 30 mL of water and 5 mL of 4N NaOH were added. The mixture was extracted three times with 20 mL of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the mixture was further purified by silica gel column chromatography using a specific ratio of petroleum ether / ethyl acetate as the eluent. Recrystallization from dichloromethane / n-hexane yielded the important intermediate 20a. The product was a white solid with a yield of 74% and a purity of 95.7%. 1H NMR(400MHz, DMSO-d6)δ9.86(s,1H),8.90(s,1H),7.64(d,J=7.2Hz,1H),7.58–7.40(m,2H),7.23(s,1H),7.07(t,J=9.3Hz,1H),6.62(s,1H ),5.87(ddt,J=16.3,10.6,5.4Hz,1H),5.25(s,2H),5.16(d,J=17.3Hz,1H),5.06(d,J=10.4Hz,1H),4.14(s,2H),2.32(s,3H),2.22(s,3H). 13 C NMR (101MHz, DMSO-d6) δ 181.75, 167.48, 158.15, 155.78, 146.38, 136.81, 136.20 (d, J = 2.7Hz), 135.59, 128.80, 124.49, 124.31, 124. 20,122.98(d,J=4.4Hz),119.19(d,J=7.5Hz),117.87,115.81,115.19(d,J=22.9Hz),46.91,20.42,14.90,14.87.ESI-MS:calculated for C 19 H 21 FN4O3[MH] - 372.14201.found371.13445.

[0082] Example 37: Preparation of compound 20b, performed in the same manner as in Example 36, except that isopropyl thioisocyanate was replaced with thioallylcarbamate O-phenyl ester. White solid, yield: 66%, purity: 95.3%. 1 H NMR(400MHz, DMSO-d6)δ9.86(s,1H),8.68(s,1H),7.65(d,J=7.1Hz,1H),7.51–7.46(m,1H),7.24(s,2H),7.07( t,J=9.2Hz,1H),6.60(s,1H),5.34–5.04(m,2H),4.41(s,1H),2.31(s,3H),2.22(s,3H),1.15(d,J=6.6Hz,6H). 13C NMR (101MHz, DMSO-d6) δ180.37, 167.57, 155.77, 146.07, 136.22 (d, J = 3.3Hz), 124.38 (d, J = 19.0Hz), 122.98 (d, J = 4.3 Hz), 119.18 (d, J = 7.6Hz), 117.91, 115.18 (d, J = 22.9Hz), 46.34, 22.53, 20.38, 14.88 (d, J = 3.1Hz). ESI-MS: calculated for C 19 H 23 FN4OS[MH] - 374.15776.found 373.1489.

[0083] Example 38: Preparation of compound 20c, performed in the same manner as in Example 37, except that isopropyl thioisocyanate was replaced with 4-bromophenyl thioisocyanate. White solid, yield: 83%, purity: 96.8%. 1 H NMR (400MHz, DMSO-d6) δ9.87(s,1H),9.67(s,1H),9.15(s,1H),7.65(d,J=7.4Hz,1H),7.51(s ,6H),7.32(s,1H),7.07(t,J=9.1Hz,1H),6.61(s,1H),5.34(s,2H),2.33(s,3H),2.21(s,3H). 13 C NMR(101MHz,DMSO-d6)δ180.82,167.53,158.16,155.78,146.36,139.59,136.71,136.22,136.19,131.56,129.04,126.16,124.45,124.27,124 .11,123.08(d,J=4.4Hz),120.87,119.28(d,J=7.6Hz),117.86,116.76,116.30,115.27,115.04,20.50,14.88(d,J=3.1Hz).ESI-MS:calculated for C 22 H 20 BrFN4OS[MH] - 486.05252.found 485.04496.

[0084] Example 39: In vitro anti-HBV cell activity assay of the target compound

[0085] HBV cell line and culture conditions

[0086] HepAD38 cells were resuscitated until they were in good condition and reached confluence. After digestion and cell counting, Tetracycline (final concentration 300 ng / mL) and G418 (final concentration 400 μg / mL) were added to the culture medium. Virus expression was inhibited in the presence of Tetracycline. The culture medium was diluted to a concentration of 2 × 10⁻⁶ cells using DMEM / F-12K medium containing 10% FBS (containing Tetracycline at a final concentration of 300 μg / mL, G418 at a final concentration of 400 μg / mL, and 1% penicillin antibody). 5 Cell suspension was seeded at a rate of 100 μL per well in a 96-well plate (covering the entire plate), and incubated at 37°C in a 5% CO2 incubator for 24 h. After 24 h, the old culture medium was discarded, and 200 μL of fresh DMEM / F-12K medium containing 2% FBS and 1% antibiotics was added.

[0087] (1) Cytotoxicity test

[0088] Compound preparation and cell treatment in in vitro cytotoxicity assays: Dissolve the compound to 20 mM using DMSO. Add 1 μL of serially diluted compound to each well of the cell plate as described above, with the highest final concentration being 100 μM (200-fold dilution). Staurosporine (Selleck, CAS No. 62996-74-1) was used as a positive control compound, with a maximum concentration of 1 μM. 1 μL of DMSO was added to the negative control wells, resulting in a final concentration of 0.5%.

[0089] After 72 hours, the old culture medium was discarded, and culture medium containing 10% CCK8 solution was added. After incubation for 20-40 minutes, the OD value was obtained by microplate reader. The data were exported to calculate the inhibition rate. The experimental data were analyzed and processed using Graphpad Prism 8 software. Quantitative data that followed a normal distribution were statistically described using mean ± standard error (Means ± SEM).

[0090] (2) HBV DNA activity inhibition assay (quantitative PCR method)

[0091] Compound preparation and cell treatment in the antiviral experiment: The compound was dissolved in DMSO to 20 mM, and then diluted 4-fold in 8 separate dilutions, with the highest concentration being 20 μM, in duplicate. QPCR was performed using the Sansure Biotech 48-person (PCR-fluorescent probe method) one-step hepatitis B virus nucleic acid quantitative detection kit. 2.5 μL of supernatant was aspirated for Q-PCR. Before use, the kit reagents were thawed and vortexed to mix. After centrifugation, the enzyme mixture was placed on ice, and subsequent steps were ensured to be performed on ice. 2.5 μL of sample release agent and 2.5 μL of test sample supernatant were added to each well of the Q-PCR plate (experimental group, control group, standard curve group). The viral DNA copy number was obtained after the Q-PCR reaction. Experimental data were analyzed using Graphpad Prism 8 software. Quantitative data following a normal distribution were statistically described using mean ± standard error (Means ± SEM).

[0092] Table 1. Anti-hepatitis B virus activity of the directed-synthesized compound and lead compound 17i

[0093]

[0094] The activity results showed that the 4-amino-2-methylbenzamide HBV capsid protein inhibitors of the present invention exhibited significant anti-HBV activity. Among them, compound 20a (EC... 50 =0.059μM, CC 50 (>100μM) has the potential for further research.

Claims

1. A 4-amino-2-methylbenzamide class HBV capsid protein inhibitor, characterized in that, has the structure shown in the general formula L is an amide group, a sulfonamide group, a urea group, a thiourea group; M includes a substituted phenyl group, a cycloalkyl group, a thiophene group, a substituted-3-pyridyl group, a chain aliphatic group, a 4-morpholinyl group, and the substituent is selected from a halogen, an aliphatic hydrocarbon, a nitro group, a methoxy group, a cyano group, a trifluoromethyl group, and the like; R includes a hydrogen atom and an aliphatic acyl group.

2. The 4-amino-2-methylbenzamide HBV capsid protein inhibitor of claim 1, wherein In the general formula, L is M is R is -H, 3. The 4-amino-2-methylbenzamide HBV capsid protein inhibitor of claim 2, wherein is one of the compounds having the following structure 4. The process for the preparation of 4-amino-2-methylbenzamides HBV capsid protein inhibitors according to claim 3, characterized in that 2-methyl-4-fluorobenzoic acid as a raw material, prepared by one of the following synthetic routes: Synthetic route I is as follows: Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0 °C, 12 h, r.t; (ii) HATU, DIPEA, CH2Cl2, 20 h, 0 °C - rt; (iii) 4-methoxybenzylamine, DIPEA, DMF, 95 °C, reflux, 12 h; (iv) Fe, NH4Cl, MeOH, H2O, 12 h, 90 °C, reflux; (v, viii) different types of acyl chloride or sulfonyl chloride, pyridine, CH2Cl2, 4 h, rt; (vi, ix, xi) v1: v2 = 1:4 CF3COOH and CH2Cl2, 0.5 h; (vii, xii) acetyl chloride, pyridine / triethylamine, CH3CN, 12 h, 60 °C; (x) different types of isocyanate, CH2Cl2, 10 h, rt. M1, M2and M3are each independently selected from 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-methylphenyl, 4-butylphenyl, 4-trifluoromethylphenyl, 4-cyanophenyl, 4-fluorophenethyl, cyclopropyl, cyclopentyl, cyclohexyl, 2-thiophenyl, 6-chloro-3-pyridyl, 4-methoxycarbonylbutyl, 4-morpholinyl, 4-methoxyphenyl, 4-nitrophenyl; the different types of acyl chloride are selected from 4-fluorobenzoyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, 4-methylbenzoyl chloride, 4-butylbenzoyl chloride, 4-trifluoromethylbenzoyl chloride, 4-cyanobenzoyl chloride, 4-fluorophenylacetyl chloride, cyclopropylcarbonyl chloride, cyclopentylcarbonyl chloride, cyclohexylcarbonyl chloride, 2-thiophenecarbonyl chloride, 6-chloro-3-pyridinecarbonyl chloride, chloroformyl butyric acid methyl ester, chloroformyl-4-morpholine; the different types of sulfonyl chloride are selected from 4-methoxybenzenesulfonyl chloride, 4-nitrobenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride; the different types of isocyanate are selected from 4-fluorophenyl isocyanate, 4-bromophenyl isocyanate; Synthetic route II is as follows: Reagents and conditions: (i) NH4OH, 130 °C, 1 h; (ii) di-tert-butyl dicarbonate, NaH, DMF, 0 °C - rt; (iii) Fe, NH4Cl, MeOH, H2O, 12 h, 90 °C, reflux; (iv) different types of thiocyanate, THF, 60 °C; (v) potassium carbonate, THF, cooled, 2 h; (vi) DIPEA, potassium carbonate, THF, 60 °C; (vii) hydrochloric acid-1,4-dioxane solution, rt. M4is an isopropyl group, an allyl group, or a 4-bromophenyl group; The different types of thioisocyanate are isopropyl thioisocyanate or 4-bromophenyl isocyanate.

5. Use of the 4-amino-2-methylbenzamide HBV capsid protein inhibitor according to any one of claims 1-3 in the manufacture of a medicament for the treatment of hepatitis B.

6. A pharmaceutical composition for the treatment of HBV, comprising the 4-amino-2-methylbenzamide HBV capsid protein inhibitor according to any one of claims 1-3 and one or more pharmaceutically acceptable carriers.

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