Cyclopropyl-substituted benzamide HBV capsid protein inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a cyclopropyl-substituted benzamide 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 cyclopropyl-substituted benzamide 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. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a cyclopropyl-substituted benzamide hepatitis B virus (HBV) capsid protein inhibitor, a preparation method thereof, and an application thereof. Background Art

[0002] Hepatitis B virus (HBV), abbreviated as hepatitis B, is an infectious disease mainly characterized by liver damage caused by continuous HBV infection, and further development will cause complications such as abnormal liver metabolism, liver failure, liver cirrhosis, and liver cancer. At present, the anti-HBV drugs approved by the US FDA for clinical use mainly include two categories: polyethylene glycol interferon α (PEG-IFNα) and nucleotide analogs. PEG-IFNα mainly exerts antiviral effects through immunomodulation. Since it is only effective for some patients and has varying degrees of side effects, its extensive clinical application is limited. Nucleoside analogs inhibit the replication of the HBV genome by competitively inhibiting the activity of viral polymerase, and ultimately achieve the effect of anti-hepatitis B virus. However, they cannot completely clear the hepatitis B virus in the body, require long-term medication, are prone to drug resistance, and have a high recurrence rate after drug withdrawal. Clinically, the combined use of nucleoside analogs and interferon can significantly inhibit virus replication, improve the quality of life and lifespan of patients, but cannot completely cure HBV and requires 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 the structural biology research of HBV capsid protein, the HBV capsid protein has become a new target attracting much attention in the field of anti-hepatitis B virus drug research. Currently, the in-research capsid protein inhibitors can be divided into two categories: type I capsid protein inhibitors interfere with the normal assembly of viral nucleocapsids, causing them to form non-capsid structures of various morphologies, such as heteroaryl dihydropyrimidines (Bay 41-4109 and GLS4, etc.); type II capsid protein inhibitors promote the formation of "normal" but empty capsids without pre-genomic RNA (pgRNA), such as phenylacrylamides (AT-130, etc.) and benzenesulfonamides (JNJ-6379 and NVR3-778, etc.). Summary of the Invention

[0004] In order to overcome the defects of the above-mentioned existing technologies, the present invention provides a cyclopropyl-substituted benzamide HBV capsid protein inhibitor and a preparation method thereof. The present invention also provides the activity screening results and applications of the above compounds as non-nucleoside HBV inhibitors.

[0005] The technical solution of the present invention is as follows:

[0006] I. Cyclopropyl-substituted benzamide HBV capsid protein inhibitor

[0007] A cyclopropyl-substituted benzamide HBV capsid protein inhibitor has the structure shown in the following general formula I:

[0008]

[0009] Wherein,

[0010] X is a carbonyl group or a thiocarbonyl group;

[0011] Y is a phenyl group substituted with a cyano group, a 5-membered heteroaryl group, a 3- to 5-membered cycloalkyl group, a C 2-4 aliphatic amino group, an alkenyl-substituted amino group, or a 3- to 5-membered cycloalkyl-substituted aliphatic amino group;

[0012] Preferably according to the present invention, in the general formula, X is Y is

[0013] More preferably according to the present invention, the cyclopropyl-substituted benzamide HBV capsid protein inhibitor is characterized in that it is one of the compounds having the following structures:

[0014]

[0015] II. Preparation method of cyclopropyl-substituted benzamide HBV capsid protein inhibitor

[0016] The preparation method of the cyclopropyl-substituted benzamide HBV capsid protein inhibitor uses 2-bromo-4-fluorobenzoic acid as a raw material and prepares the compounds disclosed in the present invention through one of the following synthetic routes;

[0017] Synthetic route 1 is as follows:

[0018]

[0019] Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0 °C, 12 h, r.t., 73%; (ii) thionyl chloride, DMF, 6 h, 80 °C; (iii) acetonitrile, 3,4,5-trifluoroaniline, 8 h, 60 °C, 73%; (iv) toluene, H2O, cyclopropylboronic acid, Pd(OAc)2, tricyclohexylphosphine, K3PO4, 10 h, 90 °C, 62%; (v) MeOH, H2O, Fe, NH4Cl, 10 h, 90 °C, 74%; (vi) dichloromethane, pyridine, different types of acyl chlorides, 4 h, r.t., 42 - 50%.

[0020] Wherein, Y is selected from

[0021] The different types of acyl chlorides described above are selected from: 4-cyanobenzoyl chloride, 2-thiophenecarbonyl chloride, cyclopropylcarbonyl chloride, cyclopentylcarbonyl chloride.

[0022] Synthetic route 2 is as follows:

[0023]

[0024] Reagents and their conditions: (i) Dichloromethane, pyridine, phenyl chlorothionoformate, 5 h, r.t., 42%; (ii) Dichloromethane, different types of amines, DIPEA, 4 h, r.t., 36 - 47%.

[0025] Among them, Y is selected from

[0026] The different types of amines described above are selected from: ethylamine, allylamine, isopropylamine, 1,1,1-trifluoropropan-2-amine, N-methylisopropylamine, cyclopentylamine, cyclopropylamine.

[0027] III. Application of cyclopropyl-substituted benzamide HBV capsid protein inhibitors

[0028] The present invention discloses the anti-HBV activity screening results of cyclopropyl-substituted benzamide HBV capsid protein inhibitors and their application as anti-HBV inhibitors. It is proved by experiments that the cyclopropyl-substituted benzamide compounds of the present invention can be used as classical HBV non-nucleoside inhibitors.

[0029] As shown in Table 1, taking lead compound 17i as a positive control, the in vitro anti-HBV activities of the synthesized target compounds 7a - 7d, 9a - 9g were evaluated, and the in vitro cytotoxicity of the drugs was determined by the CCK-8 method; meanwhile, the drug's inhibitory activity on HBV DNA replication was determined by quantitative PCR.

[0030] The cyclopropyl-substituted benzamide HBV capsid protein inhibitors of the present invention are a novel class of non-nucleoside HBV inhibitors and can be used as anti-HBV lead compounds.

[0031] The cyclopropyl-substituted benzamide HBV capsid protein inhibitors of the present invention can be used as non-nucleoside HBV inhibitors. Specifically, they are used as HBV inhibitors to prepare anti-hepatitis B drugs.

[0032] An anti-HBV drug composition comprising the cyclopropyl-substituted benzamide HBV capsid protein inhibitor of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0033] The present invention discloses such cyclopropyl-substituted benzamide HBV capsid protein inhibitors, their preparation methods, anti-HBV activity screening results and their first application as anti-HBV inhibitors. Experiments prove that cyclopropyl-substituted benzamide HBV capsid protein inhibitors can be used as HBV inhibitors for the preparation of anti-hepatitis B drugs. Detailed implementation manners

[0034] The following examples are helpful for understanding the present invention, but cannot limit the content of the present invention. In the following examples, the numbers of all target compounds are the same as above.

[0035] Synthesis Scheme 1

[0036]

[0037] Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0 °C, 12 h, r.t., 73%; (ii) thionyl chloride, DMF, 6 h, 80 °C; (iii) acetonitrile, 3,4,5-trifluoroaniline, 8 h, 60 °C, 73%; (iv) toluene, H2O, cyclopropylboronic acid, Pd(OAc)2, tricyclohexylphosphine, K3PO4, 10 h, 90 °C, 62%; (v) MeOH, H2O, Fe, NH4Cl, 10 h, 90 °C, 74%; (vi) dichloromethane, pyridine, different types of acyl chlorides, 4 h, r.t., 42% - 50%.

[0038] Example 1: Preparation of Intermediate 2. Under ice bath conditions, 2-bromo-4-fluorobenzoic acid (1.00 g, 4.57 mmol) was dissolved in concentrated sulfuric acid (8 mL), and concentrated nitric acid (224 μL, 5.02 mmol) was added dropwise. The reaction was carried out at room temperature for 12 h; detected by TLC. After the reaction was completed, the reaction solution was added dropwise to an ice-water mixture (100 mL). The mixture was filtered by suction, and the filter residue was washed with water (20 mL × 2) and dried in an oven to obtain Intermediate 2, a white powder, 879 mg, with a yield of 73%.

[0039] Example 2: Preparation of Intermediate 3. Intermediate 2 (700 mg, 2.65 mmol) was dissolved in SOCl2 (8 mL, 72.91 mmol), and the reaction was heated at 80 °C for 5 h; detected by TLC. After the reaction was completed, the reaction solution was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure to obtain Intermediate 3.

[0040] Example 3: Preparation of Intermediate 4. Intermediate 3 was dissolved in acetonitrile (8 mL), and 3,4,5-trifluoroaniline (331 mg, 2.65 mmol) was slowly added. The reaction was heated at 60 °C for 8 h. After TLC detection and completion of the reaction, the reaction solution was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. Using petroleum ether / ethyl acetate as the eluent, it was further purified by silica gel column chromatography and then recrystallized from dichloromethane / n-hexane to obtain Intermediate 4 as a white solid, 649 mg, with a yield of 73%.

[0041] Example 4: Preparation of Intermediate 5. Intermediate 4 (800 mg, 2.03 mmol) was dissolved in toluene (7 mL), cyclopropylboronic acid (174 mg, 2.44 mmol), potassium phosphate (864 mg, 4.07 mmol) were added, and the mixture was purged with nitrogen three times. Palladium acetate (45.69 mg, 0.20 mmol) and tricyclohexylphosphine (114 mg, 0.41 mmol) were quickly added, and the mixture was purged with nitrogen three times. The reaction was heated at 90 °C for 10 h. After TLC detection and completion of the reaction, the reaction solution was cooled to room temperature, the catalyst was removed by filtration, and the solvent was removed by rotary evaporation under reduced pressure. Using petroleum ether / ethyl acetate as the eluent, it was further purified by silica gel column chromatography and then recrystallized from dichloromethane / n-hexane to obtain Intermediate 5 as a pale yellow solid, 450 mg, with a yield of 62%.

[0042] Example 5: Preparation of Intermediate 6. Intermediate 5 (450 mg, 1.27 mmol) was dissolved in a mixed solution of methanol / water (1:1, 8 mL), iron powder (248 mg, 4.45 mmol) and ammonium chloride (102 mg, 1.91 mmol) were added, and the reaction was heated at 90 °C for 10 h. After TLC detection and completion of the reaction, the reaction solution was filtered through diatomaceous earth while hot, and the filtrate was evaporated to dryness under reduced pressure to obtain Intermediate 6 as a pale yellow solid, 305 mg, with a yield of 74%.

[0043] Example 6: Preparation of Compound 7a. Intermediate 6 (0.56 mmol) was dissolved in dichloromethane (5 mL), 4-cyanobenzoyl chloride (0.62 mmol) was slowly added, and pyridine (0.84 mmol) was added as an acid-binding agent. The reaction was carried out at room temperature for 4 h. After TLC detection and completion of the reaction, the reaction solution was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. 2N dilute hydrochloric acid solution (20 mL) was added, and it was extracted with dichloromethane (15 mL × 3). The organic phases were collected and combined, washed with saturated brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Using petroleum ether / ethyl acetate as the eluent, it was further purified by silica gel column chromatography and then recrystallized from dichloromethane / n-hexane to obtain the target compound 7a. The product was a white solid powder, with a yield of 42%, melting point: 226.5 - 228.9 °C, purity: 97.31%. 11H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.47 (s, 1H), 8.17–8.08 (m, 2H), 8.07–8.00 (m, 2H), 7.67 (td, J = 10.4, 9.2, 4.1 Hz, 3H), 6.99 (d, J = 11.9 Hz, 1H), 2.22–2.11 (m, 1H), 1.02–0.94 (m, 2H), 0.77 (d, J = 5.3 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 167.56, 164.74, 157.11 (d, J = 250.5 Hz), 151.88–151.53 (m), 149.43–149.04 (m), 142.10 (d, J = 7.9 Hz), 138.22, 135.83 (d, J = 3.4 Hz), 133.69 (d, J = 3.2 Hz), 133.05, 129.10, 125.91, 122.86 (d, J = 13.3 Hz), 118.72, 114.68, 112.59 (d, J = 21.1 Hz), 104.40 (d, J = 24.6 Hz), 12.90, 9.93. ESI-MS: calculated for C 24 H 15 F4N3O2 [M-H] - 453.11004, found 452.103.

[0044] Example 7: Preparation of compound 7b. The procedure was the same as in Example 6, except that 4-cyanobenzoyl chloride was replaced with 2-thiophenecarbonyl chloride. The product was a white solid powder with a yield of 49%, melting point: 221.7 - 223.1 °C, purity: 98.84%. 1 1H NMR (400 MHz, DMSO-d6) δ 10.80 (s, 1H), 10.22 (s, 1H), 8.04–7.99 (m, 1H), 7.88 (d, J = 5.0 Hz, 1H), 7.65 (td, J = 9.2, 8.2, 4.0 Hz, 3H), 7.23 (s, 1H), 6.97 (d, J = 11.9 Hz, 1H), 2.16 (t, J = 7.1 Hz, 1H), 0.96 (d, J = 8.1 Hz, 2H), 0.77 (d, J = 5.3 Hz, 2H). 1313C NMR (100 MHz, DMSO-d6) δ 167.57, 160.53, 157.17 (d, J = 249.8 Hz), 151.75 (dd, J = 9.6, 4.8 Hz), 149.32 (dd, J = 9.6, 5.0 Hz), 141.86 (d, J = 7.7 Hz), 139.31, 135.91 (dd, J = 12.2, 3.4 Hz), 133.64 (d, J = 2.9 Hz), 132.65, 130.20, 128.67, 126.12 (d, J = 2.5 Hz), 122.80 (d, J = 13.4 Hz), 112.57 (d, J = 21.2 Hz), 104.41 (d, J = 24.1 Hz), 12.88, 9.89. ESI-MS: calculated for C 21 H 14 F4N2O2S [M-H] - 434.07121, found 433.064.

[0045] Example 8: Preparation of Compound 7c. The procedure was the same as in Example 6, except that 4-cyanobenzoyl chloride was replaced with cyclopropylcarbonyl chloride. The product was a white solid powder with a yield of 44%, melting point: 213.2 - 214.8 °C, purity: 99.03%. 1 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 10.07 (s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.71–7.58 (m, 2H), 6.91 (dd, J = 12.5, 1.9 Hz, 1H), 2.11 (d, J = 6.8 Hz, 1H), 2.04–1.95 (m, 1H), 0.92 (d, J = 8.1 Hz, 2H), 0.83–0.78 (m, 4H), 0.71 (d, J = 5.3 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 172.74, 167.83, 153.45, 151.74 (dd, J = 10.6, 5.6 Hz), 149.31 (dd, J = 9.8, 5.5 Hz), 139.42 (d, J = 7.1 Hz), 135.86 (d, J = 3.6 Hz), 133.50 (d, J = 3.1 Hz), 124.03 (d, J = 12.5 Hz), 122.89, 112.17 (d, J = 20.8 Hz), 104.59–104.00 (m), 14.43, 12.71, 9.63, 7.96. ESI-MS: calculated for C 20 H 16 F4N2O2 [M-H]- 392.11479, found 391.108.

[0046] Example 9: Preparation of Compound 7d. The procedure was the same as in Example 6, except that 4-cyanobenzoyl chloride was replaced with cyclopropylcarbonyl chloride. The product was a white solid powder with a yield of 50%, melting point: 218.2 - 220.3 °C, and purity: 98.91%. 1 H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 9.71 (s, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.70–7.59 (m, 2H), 6.90 (dd, J = 12.4, 1.9 Hz, 1H), 2.97–2.87 (m, 1H), 2.12 (t, J = 6.9 Hz, 1H), 1.84 (t, J = 7.5 Hz, 2H), 1.68 (h, J = 7.6, 7.1 Hz, 4H), 1.54 (d, J = 7.5 Hz, 2H), 0.92 (d, J = 8.0 Hz, 2H), 0.71 (d, J = 5.4 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 175.46, 167.83, 155.10 (d, J = 248.1 Hz), 151.78 (dd, J = 6.1, 3.6 Hz), 149.32 (dd, J = 9.9, 5.5 Hz), 139.68 (d, J = 7.5 Hz), 135.94 (dd, J = 11.7, 3.5 Hz), 133.48 (d, J = 2.9 Hz), 124.02 (d, J = 12.6 Hz), 123.31 (d, J = 2.6 Hz), 112.18 (d, J = 21.0 Hz), 104.55–104.19 (m), 44.94, 30.58, 26.19, 12.72, 9.64. ESI-MS: calculated for C 22 H 20 F4N2O2 [M-H] - 420.14609, found 419.139.

[0047] Synthesis Scheme 2

[0048]

[0049] Reagents and conditions: (i) dichloromethane, pyridine, phenyl chloroformate, 5 h, r.t., 42%; (ii) dichloromethane, different types of amines, DIPEA, 4 h, r.t., 36% - 47%.

[0050] Example 10: Preparation of Intermediate 8. Dissolve Intermediate 6 (500 mg, 1.54 mmol) in dichloromethane solution (7 mL), slowly add phenyl chlorothionoformate (319 mg, 1.85 mmol), and add pyridine (183 mg, 2.31 mmol) as an acid-binding agent, and react at room temperature for 5 h. Detected by TLC. After the reaction is completed, concentrate the reaction solution under reduced pressure to remove the solvent. Add 2N dilute hydrochloric acid solution (20 mL), extract with dichloromethane (15 mL×3), collect and combine the organic phases, wash with saturated brine (20 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Using petroleum ether / ethyl acetate as the eluent, further purify by silica gel column chromatography, and then recrystallize with dichloromethane / n-hexane to obtain Intermediate 8, a pale yellow solid, 298 mg, with a yield of 42%.

[0051] Example 11: Preparation of Compound 9a. Dissolve Intermediate 8 (0.43 mmol) in dichloromethane (5 mL), add ethylamine (0.51 mmol) and DIPEA (0.86 mmol), and react at room temperature for 4 h. Detected by TLC. After the reaction is completed, cool the reaction solution to room temperature and concentrate under reduced pressure to remove the solvent. Add 1N dilute hydrochloric acid solution (20 mL), extract with ethyl acetate (15 mL×3), collect and combine the organic phases, wash with saturated brine (20 mL), dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Using petroleum ether / ethyl acetate as the eluent, further purify by silica gel column chromatography, and then recrystallize with dichloromethane / n-hexane to obtain the target compound 9a. A white solid powder, with a yield of 47%, melting point: 231.8 - 234.3 °C, purity: 90.30%. 1 H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 9.21 (s, 1H), 7.91 (s, 1H), 7.75 (s, 1H), 7.68–7.55 (m, 2H), 6.89 (d, J = 12.0 Hz, 1H), 3.57–3.35 (m, 2H), 2.16 (t, J = 6.5 Hz, 1H), 1.11 (t, J = 7.1 Hz, 3H), 0.94 (d, J = 8.1 Hz, 2H), 0.73 (d, J = 5.3 Hz, 2H). 1313C NMR (100 MHz, DMSO-d6) δ 181.42, 167.61, 161.49, 158.34 (d, J = 3.5 Hz), 156.26–155.90 (m), 151.92–151.57 (m), 149.34, 141.36 (d, J = 5.5 Hz), 135.93 (dd, J = 11.6, 3.8 Hz), 133.21 (d, J = 3.2 Hz), 126.74–126.55 (m), 112.41 (d, J = 21.4 Hz), 104.39 (d, J = 24.3 Hz), 40.40, 14.58, 12.84, 9.83. ESI-MS: calculated for C 19 H 17 F4N3OS [M-H] - 411.10285, found 410.095.

[0052] Example 12: Preparation of Compound 9b. Similar to Example 11, using allylamine instead of ethylamine, a white solid powder was obtained with a yield of 43%, melting point: 237.5 - 239.1 °C, purity: 93.32%. 1 1H NMR (400 MHz, DMSO-d6) δ 10.73 (s, 1H), 9.33 (s, 1H), 8.05 (s, 1H), 7.83–7.61 (m, 3H), 6.90 (d, J = 12.0 Hz, 1H), 5.95–5.83 (m, 1H), 5.15 (dd, J = 32.3, 13.8 Hz, 2H), 4.13 (s, 2H), 2.16 (t, J = 4.8 Hz, 1H), 0.95 (d, J = 8.0 Hz, 2H), 0.73 (d, J = 5.3 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 181.99, 171.80, 167.58, 150.50 (d, J = 243.8 Hz), 141.63, 136.60, 135.86 (dd, J = 12.1, 8.8 Hz), 135.08, 133.25, 126.86, 124.43 (dd, J = 9.6, 3.3 Hz), 116.26, 112.41 (d, J = 21.4 Hz), 104.39 (d, J = 24.3 Hz), 46.83, 12.85, 9.87. ESI-MS: calculated for C 20 H 17 F4N3OS [M-H] - 423.10285, found 422.096.

[0053] Example 13: Preparation of compound 9c. Similar to Example 11, replace ethylamine with isopropylamine to obtain a white solid powder with a yield of 44%, melting point: 230.7 - 231.4 °C, and purity: 96.58%. 1 H NMR(400MHz,DMSO-d6)δ10.71(s,1H),9.06(s,1H),7.87(d,J=7.9Hz,2H),7.66(dd,J=10.4,6.5Hz,2H),6.88(d,J=12.1Hz,1H),4.46–4.25(m,1H),2.16(d,J=7.6Hz,1H),1.16(dd,J=6.6,1.9Hz,6H),0.94(d,J=8.1Hz,2H),0.73(d,J=5.3Hz,2H). 13 C NMR(100MHz,DMSO-d6)δ180.50,167.68,151.88–151.53(m),149.32(dd,J=10.0,4.8Hz),136.73,136.59,134.16,133.11,126.57–125.70(m),124.80(d,J=11.1Hz),112.28(d,J=21.4Hz),104.38(d,J=24.5Hz),46.11,22.39,12.83,9.75.ESI-MS:calculated for C 20 H 19 F4N3OS[M-H] - 425.11850,found424.111.

[0054] Example 14: Preparation of compound 9d. Similar to Example 11, replace ethylamine with 1,1,1-trifluoropropan-2-amine to obtain a white solid powder with a yield of 36%, melting point: 245.2 - 247.8 °C, and purity: 95.44%. 1 H NMR(400MHz,DMSO-d6)δ10.74(s,1H),9.43(s,1H),8.49(d,J=9.1Hz,1H),7.78(d,J=7.8Hz,1H),7.66(dd,J=10.4,6.6Hz,2H),6.92(d,J=12.0Hz,1H),5.34(s,1H),2.16(t,J=6.7Hz,1H),1.32(d,J=7.0Hz,3H),0.95(d,J=8.1Hz,2H),0.75(d,J=5.3Hz,2H). 1313C NMR (100 MHz, DMSO-d6) δ 183.01, 167.51, 157.35 (d, J = 251.3 Hz), 151.91–151.52 (m), 149.39–149.12 (m), 142.01 (d, J = 7.6 Hz), 135.85, 133.23, 127.65, 126.81–126.66 (m), 124.33 (d, J = 12.3 Hz), 112.39 (d, J = 20.8 Hz), 104.43 (d, J = 24.5 Hz), 55.38, 14.05, 12.89, 9.93. ESI-MS: calculated for C 20 H 16 F7N3OS [M-H] - 479.09023, found 478.083.

[0055] Example 15: Preparation of Compound 9e. Similar to Example 11, replace ethylamine with N-methylisopropylamine to obtain a white solid powder with a yield of 47%, melting point: 247.7 - 249.6 °C, purity: 97.23%. 1 1H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 8.81 (s, 1H), 7.72–7.64 (m, 2H), 7.30 (dd, J = 7.8, 1.9 Hz, 1H), 6.85 (dd, J = 11.6, 1.9 Hz, 1H), 5.34 (s, 1H), 3.03 (d, J = 1.9 Hz, 3H), 2.16 (t, J = 6.9 Hz, 1H), 1.14 (dd, J = 6.7, 1.9 Hz, 6H), 0.95 (d, J = 8.1 Hz, 2H), 0.74 (d, J = 5.3 Hz, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 181.72, 167.65, 158.87 (d, J = 249.8 Hz), 151.71 (dd, J = 10.3, 4.9 Hz), 149.29 (dd, J = 10.2, 5.3 Hz), 142.13 (d, J = 8.0 Hz), 136.01–135.82 (m), 133.20 (d, J = 3.2 Hz), 129.10, 126.62 (d, J = 12.6 Hz), 112.27 (d, J = 22.0 Hz), 104.46 (d, J = 24.3 Hz), 51.30, 31.32, 19.68, 12.97, 9.88. ESI-MS: calculated for C 21 H 21 F4N3OS [M+H] +439.13415, found 440.1466.

[0056] Example 16: Preparation of Compound 9f. Similar to Example 11, replace ethylamine with cyclopentylamine to obtain a white solid powder with a yield of 42%, melting point: 253.6 - 255.1 °C, purity: 98.48%. 1 H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.04 (s, 1H), 8.00 (d, J = 39.0 Hz, 2H), 7.70–7.60 (m, 2H), 6.88 (d, J = 12.1 Hz, 1H), 4.49 (s, 1H), 2.14 (t, J = 6.8 Hz, 1H), 1.93 (dd, J = 12.5, 6.2 Hz, 2H), 1.65 (t, J = 5.8 Hz, 2H), 1.51 (dt, J = 34.7, 6.7 Hz, 4H), 0.93 (d, J = 8.1 Hz, 2H), 0.72 (d, J = 5.3 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6) δ 181.02, 167.72, 151.97–151.75 (m), 149.62–149.17 (m), 135.84 (d, J = 12.3 Hz), 134.15, 133.45–132.81 (m), 126.94 (d, J = 7.4 Hz), 125.95 (d, J = 6.8 Hz), 125.08 (t, J = 6.4 Hz), 112.22 (d, J = 21.1 Hz), 104.38 (d, J = 24.0 Hz), 55.38, 32.47, 23.83, 12.83, 9.73. ESI-MS: calculated for C 22 H 21 F4N3OS [M+Na] + 451.13415, found 474.1312.

[0057] Example 17: Preparation of Compound 9g. Similar to Example 11, replace ethylamine with cyclopropylamine to obtain a white solid powder with a yield of 40%, melting point: 247.2 - 249.9 °C, purity: 96.59%. 11H NMR (400 MHz, DMSO-d6) δ 10.75 (s, 1H), 9.11 (s, 1H), 8.58–8.01 (m, 1H), 7.67 (dd, J = 10.5, 6.6 Hz, 3H), 6.89 (dd, J = 11.9, 1.8 Hz, 1H), 2.80 (d, J = 101.0 Hz, 1H), 2.15 (s, 1H), 0.95 (d, J = 8.0 Hz, 2H), 0.74 (t, J = 5.7 Hz, 4H), 0.58 (s, 2H). 13 13C NMR (100 MHz, DMSO-d6) δ 182.95, 167.64, 151.73 (dd, J = 9.4, 5.5 Hz), 149.30 (dd, J = 9.5, 5.7 Hz), 136.59, 135.87 (d, J = 3.7 Hz), 134.15, 133.50–132.80 (m), 126.20, 115.74, 112.28 (d, J = 21.6 Hz), 104.42 (d, J = 24.0 Hz), 35.47, 12.91, 9.86, 7.32. ESI-MS: calculated for C 20 H 17 F4N3OS [M-H] - 423.10285, found 422.096.

[0058] Example 18: In vitro anti-HBV cell activity experiment of the target compound

[0059] HBV cell line and culture conditions

[0060] Resuscitate HepAD38. When the cell state is good and after it has grown to confluence, digest it, count the cells, add Tetracycline (final concentration 300 ng / mL) and G418 (final concentration 400 μg / mL) to the culture medium. The virus does not express in the presence of Tetracycline. Dilute it with DMEM / F-12K medium containing 10% FBS (containing Tetracycline at a final concentration of 300 μg / mL and G418 at a final concentration of 400 μg / ml, 1% double antibody) to a cell suspension with a concentration of 2×10 5 / mL, and inoculate 100 μL per well into a 96-well plate (the whole plate is filled), and incubate it in a constant temperature incubator at 37 °C and 5% CO2 for 24 h. After 24 h, discard the old culture medium and add 200 μL of fresh DMEM / F-12K medium containing 2% FBS and 1% double antibody.

[0061] (1) Cytotoxicity experiment

[0062] Compound preparation and cell treatment in in vitro cytotoxicity assay: Dissolve the compound in DMSO to 20 mM. Add 1 μL of serially diluted compound to each well of the above cell plate, and the highest final concentration in the experiment is 100 μM (200-fold dilution). Staurosporine (Selleck, CAS No. 62996-74-1) is used as a positive control compound, and the highest concentration is 1 μM. Add 1 μL of DMSO to the negative control wells, and the final concentration is 0.5%.

[0063] After 72 h, discard the old medium, add the medium containing 10% CCK8 solution, incubate for 20 - 40 min, detect with an enzyme-linked immunosorbent assay (ELISA) reader to obtain the OD value, export the data to calculate the inhibition rate, and analyze and process the experimental data with Graphpad Prism 8 software. Quantitative data that follows a normal distribution is statistically described using the mean ± standard error (Means ± SEM).

[0064] (2) Inhibitory activity assay of HBV DNA (quantitative PCR method)

[0065] Compound preparation and cell treatment in antiviral assay: Dissolve the compound in DMSO to 20 mM, and then perform 4-fold dilution for 8 dilutions, with the highest concentration being 20 μM and 2 replicates. Use the hepatitis B virus nucleic acid quantitative detection kit (48 tests per kit, PCR-fluorescence probe method) from Shengxiang Biotech to perform QPCR. Pipette 2.5 μL of the supernatant for Q-PCR. After the reagents in the kit are melted, vortex and mix well before use, centrifuge, place the enzyme mixture on ice for later use, and ensure that the subsequent steps are completed on ice. Add 2.5 μL of sample release agent and 2.5 μL of the test sample supernatant (experimental group, control group, standard curve group) to each well of the Q-PCR plate. After Q-PCR reaction, the virus DNA copy number in each well is obtained. Analyze and process the experimental data with Graphpad Prism 8 software. Quantitative data that follows a normal distribution is statistically described using the mean ± standard error (Means ± SEM).

[0066] Table 1 Anti-hepatitis B virus activities of the directionally synthesized compounds and the lead compound 17i

[0067]

[0068] The activity results show that the newly synthesized cyclopropyl-substituted benzamide HBV capsid protein inhibitors exhibit significant anti-HBV activity. Among them, compound 9e (EC 50 = 0.033 μM, CC 50 > 100 μM) is worthy of further study.

Claims

1. A cyclopropyl-substituted benzamide HBV capsid protein inhibitor, characterized in that: It has the structure shown in the following general formula I: in, X is a carbonyl group or a thiocarbonyl group; Y is a phenyl group substituted with a cyano group, a 5-membered heteroaryl group, a 3-5-membered cycloalkyl group, a C 2-4 Fatty amine, alkenyl-substituted amine, 3-5-membered cycloalkyl-substituted fatty amine.

2. The cyclopropyl-substituted benzamide HBV capsid protein inhibitor according to claim 1, characterized in that: In the general formula, X is Y is 3. The cyclopropyl-substituted benzamide HBV capsid protein inhibitor according to claim 1 or 2, characterized in that: It is one of the compounds having the following structures:

4. The method for preparing the cyclopropyl-substituted benzamide HBV capsid protein inhibitor according to claim 3, characterized in that Using 2-bromo-4-fluorobenzoic acid as raw material, it is prepared by one of the following synthetic routes: Synthesis route 1 is as follows: Reagents and conditions: (i) concentrated HNO3, concentrated H2SO4, 0℃, 12h, rt, 73%; (ii) thionyl chloride, DMF, 6h, 80℃; (iii) acetonitrile, 3,4,5-trifluoroaniline, 8h, 60℃, 73%; (iv) methylbenzene, H2O, cyclopropylboronic acid, Pd(OAc)2, tricyclohexylphosphine, K3PO4, 10h, 90℃, 62%; (v) MeOH, H2O, Fe, NH4Cl, 10h, 90℃, 74%; (vi) dichloromethane, pyridine, different types of acyl chlorides, 4h, rt, 42-50%; Among them, Y is selected from The different types of acyl chlorides are selected from: 4-cyanobenzoyl chloride, 2-thiophenecarbonyl chloride, cyclopropylcarbonyl chloride, cyclopentylcarbonyl chloride; Synthesis route 2 is as follows: Reagents and conditions: (i) dichloromethane, pyridine, phenyl chlorothioformate, 5h, rt, 42%; (ii) dichloromethane, different types of amines, DIPEA, 4h, rt, 36-47%; Among them, Y is selected from The different types of amines are selected from: ethylamine, allylamine, isopropylamine, 1,1,1-trifluoropropane-2-amine, N-methylisopropylamine, cyclopentylamine, and cyclopropylamine.

5. Use of the cyclopropyl-substituted benzamide HBV capsid protein inhibitor according to any one of claims 1 to 3 in the preparation of anti-hepatitis B drugs.

6. An anti-HBV pharmaceutical composition comprising the cyclopropyl-substituted benzamide HBV capsid protein inhibitor according to any one of claims 1 to 3 and one or more pharmaceutically acceptable carriers.

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