2-deoxy-2-fluoro-4-azido-N-hydroxycytidine as well as preparation method and application thereof

Abstract

The invention discloses 2-deoxy-2-fluoro-4-azido-N-hydroxycytidine shown as a formula (F4) as well as a preparation method and application of the 2-deoxy-2-fluoro-4-azido-N-hydroxycytidine. Compared with the prior art, the 2-deoxy-2-fluoro-4-azido-N-hydroxycytidine (F4) and the phosphate prodrugs (F6 and F12) thereof provided by the invention show relatively strong cell viability to HepG2 / 2.2. 15 cells capable of continuously and stably expressing HBV (Hepatitis B Virus), the cell viability can reach a nanomole level, and the 2-deoxy-2-fluoro-4-azido-N-hydroxycytidine (F4) and the phosphate prodrugs (F6 and F12) thereof can be used for remarkably inhibiting HBV DNA (Deoxyribonucleic Acid) replication to serum and liver of a hepatitis B virus model mouse; the compound has a liver targeting property and has a good application prospect in the aspect of treating HBV (Hepatitis B Virus). The development of the medicine lays a foundation for the research of fluorine-containing nucleoside analogues. And (F4).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of anti-HBV drugs, and particularly relates to 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine as well as a preparation method and application thereof. BACKGROUND

[0002] Chronic hepatitis B is a serious global public health problem caused by hepatitis B virus (HBV) infection. According to the World Health Organization, there are about 296 million chronic HBV infections worldwide, and about 820,000 people die every year, mainly due to end-stage liver disease such as cirrhosis, liver failure and hepatocellular carcinoma. Although an effective preventive vaccine has been widely used, the treatment of infected people is still a great challenge. At present, the drugs used for the treatment of chronic hepatitis B in the clinic mainly include two categories: interferon-alpha and nucleos(t)ide analogues. Interferon-alpha inhibits HBV replication through immune regulation and antiviral effect, however, it needs subcutaneous injection, which is inconvenient to administer; the incidence of adverse reactions is high, and it is contraindicated for some patients (such as decompensated cirrhosis), and the overall response rate is limited. Nucleos(t)ide analogues (NAs) are the first-line drugs for antiviral therapy of chronic hepatitis B, including entecavir (ETV), tenofovir disoproxil (TDF) and tenofovir alafenamide (TAF), etc. This class of drugs, as a chain terminator of HBV reverse transcriptase, can effectively inhibit the replication of viral DNA, and has the advantages of strong antiviral activity, convenient oral administration and good tolerance. However, there are still limitations such as difficulty in clearing covalently closed circular DNA, high recurrence rate after drug withdrawal, low functional cure rate, and risk of drug resistance.

[0003] Phosphate prodrug technology is a classic and key strategy to improve the properties of nucleoside drugs. Many nucleoside drugs (such as tenofovir, adefovir) have poor oral absorption, and by designing them as phosphate prodrugs (such as TDF, TAF), the oral bioavailability and liver-targeted delivery can be significantly improved. Although some small molecule compounds for treating HBV have been reported, there is still room for improvement in the pharmaceutical properties of these compounds. For example, the oral bioavailability is not ideal, the in vivo metabolism is fast or the distribution is not ideal, and the activity needs to be further improved.

[0004] In summary, there is an urgent need in the art to develop a new small molecule compound with a completely new or more optimal chemical structure, stronger anti-HBV activity, better pharmacokinetic properties (especially oral bioavailability and liver targeting), and which can overcome the defects of existing therapeutic drugs. SUMMARY

[0005] Invention purposes: In order to solve the problems existing in the prior art, the present application provides a 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine and a preparation method and application thereof. The compound of the present application can stably and continuously express HBV virus HepG2 / 2.2.15 cells, show strong cell activity, significantly inhibit the replication of HBV DNA in the serum and liver of a hepatitis B virus model mouse, have liver targeting, and can be further used in potential antiviral drugs.

[0006] Technical solutions: In order to achieve the above-mentioned purposes of the present application, the technical solutions adopted by the present application are as follows: In a first aspect, the present application provides a 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof represented by formula (F4): ; (F4).

[0007] In a second aspect, the present application provides a preparation method of the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine, comprising the following steps: ; (1) Compound 1 is reacted with 1,2,4-triazole to synthesize compound 2; (2) Compound 2 is reacted with hydroxylamine hydrochloride to synthesize compound 3; (3) Compound 3 is synthesized under the condition of NH3 / MeOH to synthesize compound F4.

[0008] Preferably, the preparation is carried out by using the following reaction formula: .

[0009] In a third aspect, the present application provides a phosphate prodrug of the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof, and the phosphate prodrug is selected from a compound represented by formula (F6): ; (F6).

[0010] In a fourth aspect, the present application provides a preparation method of the phosphate prodrug, comprising the following steps: ; Compound F4 is reacted with compound 5 under the condition of a Grignard reagent to synthesize compound F6.

[0011] Preferably, the preparation is carried out by using the following reaction formula: .

[0012] In a fifth aspect, the present application provides another said phosphate prodrug of 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof, which is selected from a compound of the following formula (F12): ; (F12).

[0013] In a sixth aspect, the present application provides a preparation method of the phosphate prodrug, comprising the following steps: ; reacting compound F4 with compound 11 under the condition of formic reagent to synthesize compound F12.

[0014] As a specific embodiment, the preparation method of compound 11 comprises the following steps: ; (1) reacting compound 7 with phosphorus oxychloride to synthesize compound 8; (2) reacting compound 8 with a compound of formula I to synthesize compound 9; (3) reacting compound 9 with a compound of formula II to synthesize compound 10; (4) recrystallizing compound 10 to obtain compound 11.

[0015] Preferably, the above compound F12 is prepared by the following reaction: ; .

[0016] In a seventh aspect, the present application provides a composition comprising the said 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; or comprising one or two of the said phosphate prodrugs or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0017] In an eighth aspect, the present application provides the use of the said 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof, the said phosphate prodrug or a pharmaceutically acceptable salt thereof, and the said composition in the preparation of a medicine for inhibiting HBV.

[0018] In a ninth aspect, the present application provides the use of the said 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof, the said phosphate prodrug or a pharmaceutically acceptable salt thereof, and the said composition in the preparation of a medicine for treating chronic hepatitis B.

[0019] Beneficial effects: Compared with the prior art, the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxyl cytidine (F4) and phosphate prodrugs (F6 and F12) provided by the application show strong cell activity on HepG2 / 2.2.15 cells which can stably express HBV virus, and can reach the nanomolar level. The serum and liver of the HBV model mice can significantly inhibit the replication of HBV DNA, have liver targeting, and have good application prospects in the treatment of HBV. The development of such drugs lays a foundation for the research of fluorine-containing nucleoside analogs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Time flow chart for the experiment in Example 2.

[0021] Figure 2 Experimental steps for the biological activity test of the compound in Example 2.

[0022] Figure 3 HBV DNA relative expression amount of the screening compound in Example 2.

[0023] Figure 4 HBsAg expression amount of the screening compound in Example 2.

[0024] Figure 5 HBeAg expression amount of the screening compound in Example 2.

[0025] Figure 6 Cell activity and cytotoxicity chart of compound F4 and blank control in Example 2.

[0026] Figure 7 Cell activity and cytotoxicity chart of compound F6 and blank control in Example 2.

[0027] Figure 8 Cell activity and cytotoxicity chart of compound F12 and blank control in Example 2.

[0028] Figure 9 Mouse serum HBV DNA replication detection results in Example 2.

[0029] Figure 10 Mouse liver HBV DNA replication detection results in Example 2. DETAILED DESCRIPTION

[0030] In order to make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of the present application.

[0031] Embodiment 1

[0032] 1. Preparation of compound F4 To a solution of compound 1 (1100 mg, 2.08 mmol) in CH2Cl2(25 mL) was added 1,2,4-triazole (1580 mg, 22.88 mmol) and pyridine (1.7 mL, 20.80 mmol) successively at 0 °C, and phosphorus oxychloride (0.96 mL, 10.40 mmol) was added dropwise at 0 °C. After stirring for 10 min, the reaction was warmed to reflux for 5 h. After the reaction was cooled to room temperature, H2O (2 mL) was added to quench the reaction at 0 °C, and saturated sodium chloride (20 mL) was added. After stirring for 10 min, the organic phase was extracted with ethyl acetate (20 mL x 3), combined, and dried over anhydrous sodium sulfate. The organic phase was concentrated to give crude compound 2 (1.3 g).

[0033] To a solution of compound 2 (2.0 mmol) in CH2Cl2(25 mL) was added hydroxylamine hydrochloride (347 mg, 5 mmol) and N,N-diisopropylethylamine (1292 mg, 10 mmol) successively at room temperature. After reaction for 4 h, the reaction was quenched by adding 5% citric acid aqueous solution (20 mL), and extracted with CH2Cl2(20 mL x 3). The organic phase was combined, dried over anhydrous sodium sulfate, and concentrated. The residue was separated by silica gel column chromatography (dichloromethane / methanol 60:1) to give compound 3 (501 mg, 46%). 1 H NMR (400 MHz, DMSO) δ 10.20 (s, 1H),10.03 (d, J = 2.1 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.87 – 7.79 (m, 2H), 7.75 –7.68 (m, 2H), 7.57 (t, J = 7.8 Hz, 2H), 7.48 (t, J = 7.8 Hz, 1H), 7.09 (dd, J =8.2, 2.3 Hz, 1H), 6.54 (dd, J= 11.7, 6.1 Hz, 1H), 6.23 (dd, J = 21.8, 4.9 Hz,1H), 5.82 (dt, J = 52.9, 5.6 Hz, 1H), 5.61 (dd, J = 8.2, 2.1 Hz, 1H), 5.00 – 4.77(m, 2H); 13 C NMR (100 MHz, DMSO) δ 164.90, 164.15, 149.46, 143.14, 134.72,134.12, 134.03, 131.30, 130.98, 130.15, 129.41, 129.26, 128.45, 99.17, 94.03,93.41, 93.32, 92.08, 77.75, 77.48, 66.60; 19 F NMR (376 MHz, DMSO) δ -201.30;HRMS (ESI)(m / z):(M+H) + Theoretical value of C 23 H 19 ClFN6O7545.0982, the actual measured value 545.0982.

[0034] To compound 3 (470 mg, 0.86 mmol) was added saturated ammonium hydroxide solution (30 mL), and the reaction was carried out at room temperature for 18 h. The reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane / methanol 13:1) to obtain the target compound F4 (white solid, 237 mg, 91%). 1 H NMR (400 MHz, CD3OD) δ 6.88 (dd, J = 8.3, 1.8 Hz, 1H), 6.32 (dd, J = 11.1, 5.6Hz, 1H), 5.49 (d, J = 8.3 Hz, 1H), 5.05 (dt, J = 54.2, 5.4 Hz, 1H), 4.39 (dd, J =22.7, 5.2 Hz, 1H), 3.79 – 3.62 (m, 2H); 13C NMR (100 MHz, CD3OD) δ 149.74,144.46, 130.94, 129.45, 97.53, 96.32, 96.23, 95.93, 94.00, 81.46, 81.29,75.07, 74.83, 61.76, 31.68, 29.36, 29.08, 28.94, 26.71, 22.36, 13.07; 19 F NMR (376 MHz, CD3OD) δ -202.96; HRMS (ESI) (m / z): (M+H) + Theoretical calculation value C9H 12 FN6O5303.0848, actual measured value 303.0849.

[0035] 2. Preparation of compound F6 F4 (151 mg, 0.5 mmol) and prodrug intermediate 5 (272 mg, 0.6 mmol) were weighed into a 50 mL round-bottom flask and dried under vacuum at 50 °C for 30 min (to remove residual solvent from the raw materials). After cooling, the solution was dissolved in anhydrous tetrahydrofuran (10 mL) under vacuum and N2 protection. Tert-butylmagnesium chloride (1.05 mL, 1.05 mmol) was slowly added dropwise to the above solution at 0 °C. After stirring for 10 min, the mixture was moved to room temperature and reacted for 12 h. The reaction was quenched with saturated NH4Cl solution (10 mL), extracted with ethyl acetate (20 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and separated by silica gel column chromatography. The eluent was initially ethyl acetate / petroleum ether 1:2, then dichloromethane / methanol 40:1 to obtain the target compound F6 (white solid, 186 mg, 66%). 1 H NMR (400MHz, CDCl3) δ 7.36 – 7.30 (m, 2H), 7.19 (dd, J = 17.1, 8.0 Hz, 3H), 6.67 (d, J =6.4 Hz, 1H), 6.46 (dd, J = 13.1, 5.2 Hz, 1H), 5.70 (d, J = 8.3 Hz, 1H), 5.24 –5.07 (m, 1H), 5.03 – 4.96 (m, 1H), 4.73 – 4.60 (m, 1H), 4.50 (dd, J = 21.7, 4.4Hz, 1H), 4.35 (ddd, J= 38.7, 11.5, 7.9 Hz, 2H), 3.94 (td, J = 9.4, 6.8 Hz, 1H), 1.34 (d, J = 7.1 Hz, 3H), 1.21 (dd, J = 6.3, 4.6 Hz, 6H); 13 C NMR (100 MHz, CDCl3)δ 173.26, 173.20, 150.28, 150.21, 149.56, 144.63, 130.82, 129.93, 125.45,120.00, 119.95, 98.78, 95.17, 94.69, 94.61, 94.53, 93.25, 81.90, 77.26,75.79, 69.77, 65.75, 50.35, 31.52, 30.14, 29.72, 22.61, 21.69, 21.62, 21.58,20.77, 20.71; 31 P NMR (162 MHz, CDCl3) δ 3.58; HRMS (ESI) (m / z): (MH) - Theoretical calculated value C 21 H 26 FN7O9P 570.1519, actual measured value 570.1515.

[0036] 3. Preparation of compound F12 At -78°C, an anhydrous dichloromethane solution of 2-methylbenzyl alcohol (1 g, 0.008 mol) in 2 mL and a triethylamine solution (1.2 mL, 0.008 mol) in 15 mL of phosphorus oxychloride (1.26 g, 0.091 mol) were added dropwise to the reaction mixture. After stirring at a constant temperature for 3 hours, L-alanine isopropyl hydrochloride (1.37 g, 0.008 mol) was slowly added to the reaction mixture. After 15 minutes, an anhydrous dichloromethane solution of triethylamine (2.4 mL, 0.8 mol) in 1.5 mL was added dropwise. The mixture was stirred at -78°C for 1 hour, and then allowed to rise to room temperature for 1 hour. Pentafluorophenol (0.89 g, 0.005 mol) and triethylamine (1.37 mL, 0.011 mol) were dissolved in anhydrous dichloromethane (1.4 mL) beforehand and added to the above reaction mixture. The mixture was stirred overnight at room temperature. The filtrate was concentrated. Ethyl acetate (20 ml) and water (10 ml) were added for extraction. The organic phase was separated, and the aqueous phase was extracted again with ethyl acetate (2 × 20 ml). The organic phases were combined, washed once with brine (20 ml), dried over anhydrous sodium sulfate, and concentrated. Separation was performed by silica gel column chromatography (ethyl acetate / petroleum ether = 1:7~1:5) to give compound 10 (~1.3:1), an isomer of compound 11. This compound was further recrystallized from diisopropyl ether (3 ml) and petroleum ether (6 ml) under reflux to obtain pure prodrug intermediate compound 11 (2.4 g, Sp:Rp > 10:1), with an overall yield of 60%. 1 H NMR (400 MHz, CDCl3) δ 7.35 – 7.30(m, 1H), 7.28 – 7.17 (m, 3H), 5.23 (d, J = 7.2 Hz, 2H), 5.00 (hept, J = 6.2 Hz,1H), 4.06 – 3.94 (m, 1H), 3.80 – 3.68 (m, 1H), 2.37 (s, 3H), 1.43 (d, J = 7.0Hz, 3H), 1.21 (dd, J = 6.3, 3.4 Hz, 6H); 13 C NMR (100 MHz, CDCl3) δ 172.71,172.63, 137.03, 133.22, 133.15, 130.50, 129.13, 129.08, 126.15, 69.46, 68.02,67.96, 58.47, 50.36, 50.34, 21.59, 21.58, 20.97, 20.92, 18.67, 18.45;19 F NMR (376 MHz, CDCl3) δ -153.70 (dd, J = 21.7, 4.0 Hz), -159.98 (td, J = 22.2, 3.6Hz), -162.29 (td, J = 22.4, 4.4 Hz); 31 P NMR (162 MHz, CDCl3) δ 3.99; HRMS (ESI) (m / z): (M + H) + Theoretical calculated value C 20 H 20 F5NO5P 480.0994, actual measured value 480.0995.

[0037] F4 (300 mg, 0.99 mmol) and prodrug intermediate 11 (573.3 mg, 1.2 mmol) were weighed into a 50 mL round-bottom flask and dried under vacuum at 50 °C for 30 min (to remove residual solvent from the raw materials). After cooling, the solution was dissolved in anhydrous tetrahydrofuran (10 mL) under vacuum and N2 protection. Tert-butylmagnesium chloride (2 mL, 2 mmol) was slowly added dropwise to the above solution at 0 °C. After stirring for 10 min, the mixture was moved to room temperature and reacted for 12 h. The reaction was quenched with 10 mL of saturated NH4Cl solution, extracted with ethyl acetate (20 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was concentrated and separated by silica gel column chromatography. The eluent was initially ethyl acetate / petroleum ether 1:2, then changed to dichloromethane / methanol 40:1 to obtain the target compound F12 (white solid, 371 mg, 62%). 1 H NMR (400MHz, CD3OD): δ7.37 (d, J = 8.0 Hz, 1H), 7.26–7.17 (m, 3H), 6.86 (dd, J = 8,2 Hz, 1H), 6.44 (dd, J = 13.6, 5.2 Hz, 1H), 5.53 (d, J = 8.4 Hz, 1H), 5.23–4.95 (m, 3H), 4.98 (p, J = 6.3 Hz, 1H), 4.49 (dd, J = 20.8, 4.4 Hz, 1H), 4.27– 4.19 (m, 2H), 3.86 – 3.78 (m, 1H), 2.38 (s, 3H), 1.33 (d, J = 7.2 Hz, 3H), 1.24 – 1.21 (m, 6H); 13C NMR (100 MHz, CD3OD) δ 177.30, 177.25, 153.55,148.21, 140.68, 137.98, 137.90, 134.98, 133.95, 132.55, 132.45, 129.66,101.76, 99.71, 98.71, 97.79, 86.21, 86.05, 80.06, 79.81, 72.70, 70.80, 70.75,69.77, 54.03, 24.52, 24.45, 23.03, 22.96, 21.46; 19 F NMR (376MHz, CD3OD): δ -198.7; 31 P NMR (162MHz, CD3OD): δ -12.24; HRMS (ESI) (m / z): (M + Na) + Theoretical calculated value C 23 H 31 FN7NaO9P 622.1797, actual measured value 622.1796.

[0038] Example 2

[0039] This embodiment describes the detection process and results of the compound described in Example 1 inhibiting HBV virus replication within cells and inhibiting HBV DNA replication in the serum and liver of a hepatitis B virus model mouse. Unless otherwise specified, all materials and consumables listed in this embodiment are commercially available. Cells and viruses were obtained from cell and microbial resource banks of CTCC or other relevant institutions, and the hepatitis B virus model mice were purchased from Guangzhou Huateng Biomedical Technology Co., Ltd. The experimental methods in this embodiment are standard molecular biology, cell biology, or virology procedures, which researchers in the field can easily understand and operate.

[0040] The specific steps are as follows: 1. Cell HepG2 / 2.2.15 (inserted HBV ayw, D isotype, 3182bp, stably expressed), culture conditions: DMEM + 10% FBS + 1% Penicillin / Streptomycin + 380μg / mL G418 + 0.2% L-Glutamine + 0.2% non-essential amino acids.

[0041] 2. Main reagents DMEM (Gibco, cat: C11995500BT); Fetal bovine serum FBS (Gibco, cat: 10270-106); Penicillin / Streptomycin (10000U / mL) (M&C gene biotechnology, cat: G2723M3); The positive compound was tenofovir disoproxil fumarate (TDF).

[0042] Tguide Smart Universal Genomic DNA Extraction Kit (TIANGEN, mat: 4995051) Hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunosorbent assay) (KHB) Hepatitis B virus e antigen detection kit (enzyme-linked immunosorbent assay) (KHB) 3. Animals Hepatitis B virus model mice (C57BL / 6 mice, female, weighing 20-25g, plasmid AAV8-HBV1.3 (D subtype), titer 1×10⁻⁶ per mouse). 11 -1×10 12 The animal samples (vg / mL) were purchased from Guangzhou Huateng Biomedical Technology Co., Ltd. Prior to the experiment, the animals were housed in transparent plastic cages with free access to water and food in an environment with a temperature of 20-26℃ and a relative humidity of 40%-70%. The light-dark period was 12 hours, lasting for 7 days. All experimental procedures were approved by the Animal Ethics Committee of Zhengzhou University, with ethics approval number ZZUIRB-2025-030.

[0043] 4. Experimental Procedure 4.1 Preliminary in vitro screening of compounds to inhibit HBV activity Two control groups were set up in the experiment: the j-positive drug group (treated with 10 µM TDF) and the k-virus control group (treated with only DMSO without the compound); the experimental groups were F4, F6, F12, and compounds FNC, 1a, 1b, 1c, 1d, 1e, and 1f from previous studies of this research group. The specific structural formulas of the compounds are shown in the table below:

[0044] HepG2 / 2.2.15 cells, 3 × 10⁻⁶ 5 cells / well, 24-well plate, 375 μL / well; After 12-16 hours, once the cells have fully adhered to the culture medium, add 375 μL of culture medium containing the test compound, with a final compound concentration of 10 µM. On day 4 of incubation at 37 ℃, 125 μL of culture medium containing the test compound was added, bringing the final concentration of the compound to 10 µM. On day 6 of incubation at 37 ℃, 125 μL of culture medium containing the test compound was added, bringing the final concentration of the compound to 10 µM. On day 8 of incubation at 37 ℃, cells and culture supernatant were collected. DNA was extracted from the samples using an automated nucleic acid extractor, and the inhibition rate of the compound against HBV virus was determined by qPCR. The expression levels of HBsAg and HBeAg were detected by ELISA using an HBsAg and HBeAg detection kit.

[0045] qPCR detection Primer: F Primer: 5´-GGTGTCTTTCGGAGTGTGGA-3´ R Primer: 5´-GACCTGCCTCGTCGTCTAAC-3´ Amplified gene: C gene Amplification conditions: 95 ℃, 10 min; 95 ℃, 10s; 60 ℃, 30s; 72 ℃, 30s×40 4.2 Concentration-dependent anti-HBV activity of effective compounds 3×10 5 HepG2 / 2.2.15 cells were seeded in 24-well plates and compounds F4, F6 and F12, which showed good initial screening effects, were added to make the final concentrations of the compounds 0.00064, 0.0032, 0.016, 0.08, 0.4, 2 and 10 μM. Each concentration was set up in 3 replicates, and a control without the compounds was also set up. On day 4 of incubation at 37 ℃, 125 μL of culture medium containing the test compound was added, and the final concentrations of the compound were 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10 μM. On day 6 of incubation at 37 ℃, 125 μL of culture medium containing the test compound was added, and the final concentrations of the compound were 0.00064, 0.0032, 0.016, 0.08, 0.4, 2, and 10 μM. On day 8 of incubation at 37 ℃, cells and culture supernatant were collected. DNA was extracted from the samples using an automated nucleic acid extractor, and the concentration-dependent anti-HBV activity of the compound was determined by qPCR. The EC50 (50% effective concentration) for inhibiting DNA replication was also calculated.

[0046] 4.3 MTT colorimetric assay for detecting compound cytotoxicity 5×10 4HepG2 / 2.2.15 cells were seeded in 96-well cell culture plates. 100 μL of compound solution was added to 100 μL of culture medium to make the final concentrations of compounds F4, F6 and F12 500, 100, 20 and 4 μM, respectively, with 3 replicates for each concentration. A control without the compound was also set up. After 72 h of cell culture, discard 100 μL of supernatant, add 20 μL of MTT (5 mg / mL), and incubate at 37℃ for 4 h; centrifuge, discard 100 μL of supernatant, add 100 μL of DMSO, and incubate at room temperature in the dark, shaking for 15 minutes until the blue Formazen dissolves; use a microplate reader to detect OD595, with OD630 as the reference wavelength, and calculate CC50 (50% cytotoxic concentration).

[0047] SI: Calculate SI (Selective index) by dividing CC50 by EC50.

[0048] 4.4 In vivo pharmacodynamic experiments Mice were randomly divided into 6 groups (n=23): (1) Mock blank group; (2) Positive model group; (3) Prototype F4 group: 93.2558 μg / kg / d; (4) Prodrug F6 group: 187.3803 μg / kg / d; (5) Prodrug F12 group: 190.2704 μg / kg / d; (6) Positive drug ETV group: 100 μg / kg / d. Gavage was administered once daily from week 1 to week 3. Mice in the Mock blank group and the Positive model group were given an equal volume of PBS solution. The dosage administered via gavage to mice was based on the ETV mouse experimental protocol, at a molar concentration of 338.6501 nM / kg / d.

[0049] Time flow diagram as follows Figure 1 As shown.

[0050] Detection of serum HBV DNA replication: Blood was collected from the jaws of mice at weeks 1, 2, and 3 to detect the number of HBV DNA copies in the serum.

[0051] Detection of HBV DNA replication in the liver: Five mice were sacrificed at each of the weeks 1, 2 and 3, and liver tissue DNA was extracted to detect the expression level of HBV DNA in the liver tissue.

[0052] 4.5 Test Results The anti-HBV activity of the primary screening compounds at a single concentration (10 μM) Figures 3-5 ) (Compound concentration: 10 μM, control drug TDF concentration: 10 μM) Concentration-dependent anti-HBV activity of compounds F4, F6 and F12 Figures 6-8 ) Results of HBV DNA replication detection in mouse serum and liver ( Figures 9-10 ) 4.6 Conclusion In in vitro cell experiments, at a concentration of 10 µM, compounds F4, F6, and F12 reduced HBV DNA expression to 9%, 12%, and 11%, respectively, compared with compounds FNC and 1a-1f, demonstrating extremely significant inhibition of DNA replication. Compared with prodrugs F6 and F12, the original F4 showed better inhibitory effects on HBV DNA in in vitro cells. At a concentration of 10 µM, compounds FNC and 1a-1f did not inhibit the expression of HBsAg, but compounds F4, F6, and F12 significantly inhibited the expression of HBsAg. Prototype F4 had a stronger inhibitory effect on HBsAg in vitro than prodrugs F6 and F12.

[0053] At a concentration of 10 µM, none of the initial screening compounds inhibited the expression of HBeAg. Compound F4 did not show cytotoxicity at 500 μM, with a selectivity index (SI) > 742942.05; The selectivity index SI of compound F6 is 127800.94; The selectivity index SI of compound F12 is 5286.67; In in vivo animal experiments, regarding HBV DNA replication in mouse serum, after one week of administration, the prototype F4, prodrug F6, prodrug F12, and ETV significantly inhibited serum HBV DNA. After two weeks of administration, the prototype F4, prodrug F6, and prodrug F12 were all more effective than ETV, with the prodrug being more effective than the prototype.

[0054] Regarding HBV DNA replication in mouse liver, after 2 weeks of administration, the original F4, prodrug F6, and prodrug F12 showed significantly better inhibition of HBV DNA in the liver than ETV, with the prodrug showing better effect than the original; indicating that compounds F4, F6, and F12 have liver-targeting properties.

[0055] Note: Compounds F6 and F12 are insoluble in DMEM cell culture medium at 500 μM in cytotoxicity experiments and are in suspension.

[0056] The embodiments of the present invention have been described in detail above with reference to specific examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine of formula (F4) or a pharmaceutically acceptable salt thereof: ###0001### (F4) comprising the following steps: (1) reacting compound 1 with 1,2,4-triazole to synthesize compound 2; (2) reacting compound 2 with hydroxylamine hydrochloride to synthesize compound 3; (3) reacting compound 3 under NH3 / MeOH conditions to synthesize compound F4. ； （F4）。 2. The process for the preparation of 2'-deoxy-2'-fluoro-4'-azido-N- hydroxycytidine according to claim 1, characterized in that, The phosphate prodrug is selected from the following compound of formula (F6): ###0002### (F6) comprising the following steps: reacting compound F4 with compound 5 under the condition of formyl reagent to synthesize compound F6. ； The phosphate prodrug is selected from the following compound of formula (F12): ###0003### (F12) comprising the following steps: reacting compound F4 with compound 11 under the condition of formyl reagent to synthesize compound F12. The preparation method of compound 11 comprises the following steps: (1) reacting compound 7 with phosphorus oxychloride to synthesize compound 8; (2) reacting compound 8 with a compound of formula I to synthesize compound 9; (3) reacting compound 9 with a compound of formula II to synthesize compound 10; (4) recrystallizing compound 10 to obtain compound 11.

8. A composition comprising the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant; or comprising the phosphate prodrug of claim 3 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant; or comprising the phosphate prodrug of claim 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

3. The phosphate prodrug of 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, 9. Use of the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine of claim 1 or a pharmaceutically acceptable salt thereof, the phosphate prodrug of claim 3 or 5, or a pharmaceutically acceptable salt thereof, or the composition of claim 8 in the preparation of a medicament for inhibiting HBV. ； （F6）。 4. The method of claim 3, wherein the phosphonate prodrug is prepared by the process comprising:

10. Use of the 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine of claim 1 or a pharmaceutically acceptable salt thereof, the phosphate prodrug of claim 3 or 5, or a pharmaceutically acceptable salt thereof, or the composition of claim 8 in the preparation of a medicament for treating chronic hepatitis B. ； ​ 5. The phosphate prodrug of 2'-deoxy-2'-fluoro-4'-azido-N-hydroxycytidine or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, ​ ； （F12）。 6. The method of claim 5, wherein the phosphonate prodrug is prepared by the process comprising: ​ ； ​ 7. The method for preparing the phosphate prodrug according to claim 6, characterized in that, ​ ； ​ ​ ​ ​ ​ ​ ​

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