Application of small molecule compound in inhibition of HBV replication and treatment of hepatitis B

Abstract

The invention discloses an application of a small molecule compound in inhibiting HBV (Hepatitis B Virus) replication and treating hepatitis B. According to the application of the small molecule compound C18H12F3N3OS in resisting hepatitis B virus (HBV) provided by the invention, systematic in-vitro and in-vivo experiments prove that the compound can effectively inhibit the replication and expression of the HBV through a multi-target action mechanism. Experimental data show that the compound can significantly reduce the total virus RNAs level in HBV infected cells and the expression level of HBVpgRNA and HBc proteins under the micromolar concentration, and can reduce the expression of HBeAg in a dose-dependent manner. The small molecule compound has the advantages of good chemical structure solubility and low toxicity (CC50gt; and the compound is expected to be applied to clinical transformation of anti-HBV drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a small molecule compound in inhibiting HBV replication and treating hepatitis B. Background Technology

[0002] Hepatitis B virus (HBV) infection can lead to chronic disease, resulting in progressive liver fibrosis, cirrhosis, and hepatocellular carcinoma. HBV delivers a portion of its double-stranded viral genome (i.e., relaxed circular DNA, rcDNA) to hepatocytes. In the nucleus, cellular DNA repair factors convert rcDNA into covalently closed circular DNA (cccDNA). HBV replicates via an RNA intermediate—pregenomic RNA (pgRNA). The mature nucleocapsid, along with the HBV rcDNA, is packaged by envelope proteins and released from the cell, or recycled back into the cccDNA pool in the nucleus. Nucleoside (acid) analogues (NUCs) can block the reverse transcription of pgRNA but do not eliminate viral gene expression, at least in part due to the long half-life of cccDNA. During years of NUC treatment, the cccDNA pool decreases but, in most cases, does not disappear, leading to relapse of chronic infection after treatment cessation.

[0003] The current goal of HBV treatment is "functional cure," which refers to the persistent undetectable presence of serum hepatitis B surface antigen and HBV DNA after a limited course of treatment, seroconversion of hepatitis B e antigen, residual cccDNA, and a significant reduction in the incidence of end-stage liver disease. This is the ideal treatment goal recommended by current domestic and international guidelines. However, currently available treatments have relatively low functional cure rates: after one year of treatment, the HBV surface antigen loss rate with interferon is 3-7%, and with nucleoside analogs it is 0-3%. These treatments also have other drawbacks. While interferon can modulate the immune system and achieve relatively high seroconversion rates of e antigen and surface antigen, it has significant side effects and is not suitable for patients with decompensated cirrhosis or pregnant women. Furthermore, its treatment efficacy varies depending on the HBV genotype. Nucleoside (nucleotide) analogs can strongly inhibit HBV viral replication and rapidly achieve HBV DNA seroconversion, playing a crucial role in preventing the progression of cirrhosis and reducing the incidence of liver cancer. However, they have no direct effect on cccDNA, have low seroconversion rates of e antigen and surface antigen, and require long treatment cycles with strict discontinuation criteria. In particular, patients with chronic hepatitis B who are e antigen-negative may require lifelong antiviral treatment. Therefore, there is an urgent need to develop new drugs and therapies to cure chronic hepatitis B. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing an application of a small molecule compound in inhibiting HBV replication and treating hepatitis B.

[0005] To achieve its objective, the present invention employs the following technical solution:

[0006] The first aspect of the present invention provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of an HBV inhibitor or a medicament for treating hepatitis B.

[0007]

[0008] The compound represented by Formula I or its pharmaceutically acceptable salt inhibits HBV transcription.

[0009] The compound represented by Formula I or its pharmaceutically acceptable salt reduces the expression levels of HBV RNAs, HBV pgRNA and HBc protein.

[0010] The compound represented by Formula I, or its pharmaceutically acceptable salt, reduces the production level of HBeAg in hepatitis B.

[0011] A second aspect of the present invention provides a pharmaceutical composition for inhibiting HBV replication or treating hepatitis B, comprising a compound of formula I or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] In the pharmaceutical composition described above, the active ingredient exerts its effects by inhibiting the expression levels of HBV RNAs, HBV pgRNA and HBc protein, and by reducing the production level of HBeAg.

[0013] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] Preferably, the dosage form of the pharmaceutical composition is a capsule, tablet, pill, ointment, granule, oral solution, microcapsule, or injection.

[0015] A third aspect of the present invention provides the use of the above-described pharmaceutical composition in the preparation of HBV inhibitors or in the preparation of medicaments for treating hepatitis B.

[0016] In the application technology solution, the active ingredient in the pharmaceutical composition inhibits the expression levels of HBV RNAs, HBV pgRNA and HBc protein, and reduces the HBeAg production level.

[0017] The beneficial effects of this invention are:

[0018] This invention provides a small molecule compound C 18 H 12The application of F3N3OS against hepatitis B virus (HBV) has been validated through systematic in vitro and in vivo experiments. This compound effectively inhibits HBV replication and expression through a multi-target mechanism. Experimental data show that even at micromolar concentrations, this compound significantly reduces the levels of total viral RNAs, HBV pgRNA, and HBc protein expression in HBV-infected cells, while also dose-dependently downregulating HBeAg expression. This small molecule compound possesses advantages such as good chemical solubility and low toxicity (CC50 > 100 μM), making it a promising candidate for clinical translation into anti-HBV drugs. Attached Figure Description

[0019] Figure 1 The results show: A: CC50 and IC50 of the compound in different cell lines: CC50 = 152.7 μM in HepG2-NTCP cells; B: CC50 = 148.6 μM in HepAD38 cells; C: CC50 = 549.3 μM in HepG2 cells; D: CC50 = 62.6 μM in Huh7 cells; E: CC50 = 1356 μM in HepG2.2.15 cells; F: CC50 = 1536 μM in MHCC-97H cells; G: IC50 = 1 μM in HepG2-NTCP cells.

[0020] Figure 2 The study demonstrated the anti-HBV effect of small molecule compounds in an in vitro HBV infection model: A: ELISA analysis of the compound's effect on HBeAg secretion; B: RT-qPCR analysis of the compound's effect on HBV pgRNA expression levels in HepAD38 cells; C: RT-qPCR analysis of the compound's effect on total HBV RNA expression levels in HepAD38 cells; D: Northern blot analysis of the compound's effect on total HBV RNA expression levels in HepAD38 cells; E: Western blot analysis of the compound's effect on HBc protein expression levels in HepAD38 cells.

[0021] Figure 3 The study demonstrated the anti-HBV effect of small molecule compounds in an HBV replication model: A: ELISA analysis of the compound's effect on HBeAg secretion; B: RT-qPCR analysis of the compound's effect on HBV pgRNA expression levels in HepG2 cells; C: RT-qPCR analysis of the compound's effect on total HBV RNA expression levels in HepG2 cells; D: Northern blot analysis of the compound's effect on total HBV RNA expression levels in HepG2 cells; E: Western blot analysis of the compound's effect on HBc protein expression levels in HepG2 cells.

[0022] Figure 4 The study demonstrated the anti-HBV effects of small molecule compounds in an HBV replication model: A: ELISA analysis of the compound's effect on HBeAg secretion; B: RT-qPCR analysis of the compound's effect on HBV pgRNA expression levels in HepG2-NTCP; C: RT-qPCR analysis of the compound's effect on total HBV RNA expression levels in HepG-NTCP; D: Northern blot analysis of the compound's effect on total HBV RNA expression levels in HepG-NTCP; E: Western blot analysis of the compound's effect on HBc protein expression levels in HepG2-NTCP.

[0023] Figure 5 The study demonstrated the anti-HBV effect of small molecule compounds in the ALB-Cre mouse model: A: ELISA analysis of the effect of the compounds on HBsAg secretion; B: RT-qPCR analysis of the effect of the compounds on HBV pgRNA expression levels in ALB-Cre mice; C: RT-qPCR analysis of the effect of the compounds on total HBV RNA expression levels in ALB-Cre mice. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] The main reagents and materials used in the embodiments of this application are sourced from:

[0027] The plasmid template pCH9 / 3091 was constructed by Michael Nassal of the University of Freiburg, Germany, and is described in Chinese patent application CN202110351152.6 (publication number CN 113025651 B);

[0028] The plasmid template prcccDNA was constructed and kindly provided by the Shanghai Pasteur Institute of the Chinese Academy of Sciences, and is described in Chinese patent CN201310280198.9 (publication number CN104278055B).

[0029] 5×SDS-PAGE buffer: Yisheng, China;

[0030] HBeAg antigen kit, HBsAg antigen kit: Shanghai Kehua Bioengineering Co., Ltd., China;

[0031] Lipofectamine™ 3000 transfection reagent: L3000015, Thermo Fisher Scientific, USA;

[0032] Trizol reagent: Thermo Fisher Scientific, USA;

[0033] Northern blot assay kit, qPCR kit: Roche, Germany;

[0034] Roche (11417240001), a German manufacturer of nylon membrane.

[0035] DIG Northern Starter Kit Roche (12039672910) Germany;

[0036] Agarose gel, Invitrogen (75510-019), USA;

[0037] 37% Formaldehyde Sigma (F8775) USA;

[0038] Fuji X-ray film (4741019274) Japan;

[0039] Compound: Purchased from Shanghai Taoshu Biotechnology Co., Ltd. (China);

[0040] β-actin antibody: Abclonal (AC026), China;

[0041] HBc antibody: A generous gift from Professor Cai Xuefei of Chongqing Medical University;

[0042] prcccDNA plasmid: kindly provided by Professor Ren Fang of Chongqing Medical University;

[0043] Universal tissue fixative: purchased from Servicebio (G1101), China;

[0044] Alb-Cre transgenic mice (C57BL / 6-Tg[Alb-cre]21Mgn / J) were purchased from Shanghai Southern Model Organisms Center, China.

[0045] The PCR primer sequences used in this embodiment of the invention are as follows:

[0046]

[0047]

[0048] Unless otherwise specified, all other reagents are conventional reagents in this field and can be obtained commercially.

[0049] Example 1

[0050] Small molecule compound C was screened from the compound library. 18 H 12F3N3OS(0411)

[0051] 5-(4-methoxyphenyl)-2-(thiophen-2-yl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine, CAS No.: 883004-80-6, chemical formula: C 18 H 12 F3N3OS has the chemical structural formula shown in Formula I.

[0052]

[0053] First, 2.8 × 10 5 Human hepatocellular carcinoma HepG2-NTCP cells were seeded per well in 12-well plates. After 24 hours, the medium was replaced with PMM medium (hepatocyte maintenance medium containing: Transferrin, Hydrocortisone, Dexamethasone, Epidermal growth factor, Insulin-Transferrin-Selenium, PS stock solution, GlutaMAX, DMSO, Williams E medium), and different compounds were added to treat the cells at a concentration of 10 μM. After 24 hours of treatment, the cells were cultured in a mixture of infection medium (Williams E medium, 10% fetal bovine serum, 1% penicillin-streptomycin mixture, 1% L-glutamine and 2% DMSO), 40% PEG8000 and concentrated HBV (infection coefficient of 1000 genomic equivalent), and incubated at 37°C for 24 hours. The cells were washed three times with PBS buffer and then replaced with infection medium, and incubated at 37°C for the appropriate time, with the medium changed every other day. The secretion level of HBeAg in the cell culture supernatant after 7 days was detected according to the instructions of the enzyme-linked immunosorbent assay (ELISA) kit. Using a 50% fold change in HBeAg inhibition as the selection threshold, the aforementioned HepG2-NTCP cells were treated, and the expression levels of total HBV RNAs / HBV pgRNA in the cells were detected by RT-PCR. Using a fold change in HBV RNA inhibition >50% as the selection threshold, small molecule compound C was comprehensively selected. 18 H 12 F3N3OS.

[0054] We performed CC50 assays using HepG2-NTCP, HepG2, HepAD38, MHCC-97H, HepG2.2.215, and Huh7 cells. Before the experiment, each cell line in logarithmic growth phase was cultured at 1 × 10⁻⁶ cells per well. 4Cells were seeded at a density of 100 μL of complete culture medium in each well of a 96-well cell culture plate. After 24 hours, when the cell density in each well reached 80%-90%, the original culture medium in each well was removed, and fresh complete culture medium containing the target concentration of the compound was added to each well at 100 μL. The final test concentration gradient of the compound was set as follows: 800 μM, 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM, with at least 3 replicates for each concentration (n≥3). The cells were incubated for another 48 hours, and then 100 μL of 10% CCK-8 reagent was added to each well for detection. We performed IC50 assays using HepG2-NTCP cells. HepG2-NTCP cells were co-cultured with the test compounds at concentrations of 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, and 0 μM for 5 days. After culture, cells were collected and total RNA was extracted. The level of HBV pregenomic RNA (pgRNA) in cells was quantitatively detected using real-time quantitative reverse transcription PCR (RT-qPCR). Based on the RT-qPCR results, the inhibition rate (%) of HBV pgRNA expression at each compound concentration was calculated as follows: Inhibition rate (%) = [1 - (pgRNA level in the compound treatment group / pgRNA level in the solvent control group)] × 100%. Results are as follows: Figure 1 As shown, A: CC50 = 152.7 μM in HepG2-NTCP cells; B: CC50 = 148.6 μM in HepAD38 cells; C: CC50 = 549.3 μM in HepG2 cells; D: CC50 = 62.6 μM in Huh7 cells; E: CC50 = 1356 μM in HepG2.2.15 cells; F: CC50 = 1536 μM in MHCC-97H cells; G: IC50 = 1 μM in HepG2-NTCP cells.

[0055] We treated HepAD38 cells with a compound: first, we cultured them in Tet-off medium (tetracycline-free DMEM / F12 + 10% FBS + 1% Pen / Strep) for 6 consecutive days to ensure sustained activation of the HBV genome. In the absence of tetracycline, HepAD38 cells replicated HBV DNA at high levels and secreted viral particles. Cell status was observed daily, and the supernatant was collected on day 6 of culture. HBV DNA copy number (≥10) was detected by qPCR. 8 HepAD38 cells were then seeded into 12-well plates at a density of 2.5 × 10⁻⁶ copies / mL and ELISA was used to detect HBsAg / HBeAg secretion levels, confirming efficient viral replication.5 Cells were seeded in collagen-coated 12-well plates (seeding volume: 1 mL / well) and incubated at 37°C in a 5% CO2 incubator for 24 h to allow complete cell adhesion (adhesion rate > 95%). After 24 h, the original medium was aspirated and replaced with fresh Tet-off medium (1 mL / well) containing graded concentrations of compounds (0 μM, 10 μM), and a solvent control group (0.1% DMSO) was set up. Forty-eight h after the initial treatment, the medium in the wells was gently aspirated (avoiding disturbance of the cell layer), and pre-warmed (37°C) Tet-off medium (1 mL / well) was added, with corresponding concentrations of compounds (0 μM, 10 μM) added simultaneously to maintain consistent exposure dose, and culture continued.

[0056] On day 6, 200 μL of supernatant was collected from the 12-well plate, and the HBeAg secretion level in the cell culture supernatant was detected according to the instructions of the enzyme-linked immunosorbent assay kit (Kehua Biotechnology, China). Figure 2 The results show that small molecule compound C 18 H 12 F3N3OS significantly inhibited HBeAg secretion. RT-qPCR was performed to detect HBV pgRNA and HBV RNA levels using the Trizol method. The reaction system is as follows:

[0057]

[0058]

[0059] <![CDATA[HotStart TM 2XSYBRGreenqPCRMasterMix]]> 5μL cDNA 1μL HBVRNAS-F (7.2μM) 0.5μL HBVRNAS-R (7.2μM) 0.5μL <![CDATA[ddH2O]]> 3μL

[0060] The reaction conditions are as follows:

[0061]

[0062] from Figure 2 Results B and C show that small molecule compound C 18 H 12F3N3OS significantly reduced the levels of HBV pgRNA and HBVRNAS. Extracted RNA was separated using a denaturing agarose gel containing formaldehyde (gel composition: 6 ml 10×MOPS, 4 ml 37% formaldehyde, 50 ml DEPC water, 1 g agarose). Total RNA samples were mixed with an equal volume of formaldehyde loading buffer (containing 50% formamide, 1×MOPS, 0.5% SDS, 0.1% bromophenol blue), denatured at 65°C for 10 minutes, and then rapidly cooled on ice. Electrophoresis was performed at a constant voltage of 90V for 80 minutes in 10×MOPS electrophoresis buffer, and the electrophoresis progress was monitored by the bromophenol blue migration distance. After electrophoresis, the gel was briefly rinsed with DEPC water and placed on a 20×SSC saturated filter paper bridge (Whatman 3MM). This bridge was then covered sequentially with a positively charged nylon membrane (Roche, Cat#11417240001), three layers of moistened SSC filter paper, and a stack of absorbent paper, with weights ensuring close contact. 20×SSC buffer (3M NaCl, 0.3M sodium citrate, pH 7.0) was used as the transfer solution, and capillary transfer was performed overnight (approximately 16 hours). After transfer, the nylon film was irradiated with UV light (254nm, 120mJ / cm²). 2 The covalently fixed RNA membrane was placed in a prehybridization buffer containing 50% formamide, 5×SSC, 0.1% sodium dodecyl sarcosinate, 0.02% SDS, and 2% blocking reagent (Roche), and incubated at 68°C with shaking for 1 hour. In vitro transcription labeling was performed using a digoxigenin-labeled RNA probe (Roche, Cat#12039672910) according to the manufacturer's instructions. After prehybridization, the buffer was replaced with fresh hybridization buffer containing the probe (20-50 ng / mL), and hybridized overnight at 68°C (16-18 hours). The membrane was then thoroughly washed sequentially with 2×SSC / 0.1% SDS (room temperature, 5 min) and 0.5×SSC / 0.1% SDS (68°C, 15 min × 2 times). After washing, the membrane was incubated with a 1:10,000 diluted anti-digoxigenin-AP antibody (Roche) at 37°C for 30 minutes. The film was treated with CDP-Star or CSPD luminescent substrate (Roche), exposed to medium-pressure X-ray film in a darkroom, and the signal was analyzed after development and fixing. From Figure 2 Results D show that small molecule compound C 18 H 12 F3N3OS significantly reduced HBV RNA levels at 3.5 kb and 2.4 / 2.1 kb. Cells were lysed using 1×SDS-PAGE loading buffer and boiled in a 95°C metal bath for 10 min. Cell proteins were extracted for Western blot experiments, and HBc protein expression levels were subsequently detected using anti-HBc antibody. Figure 2 The results showed that the expression level of HBc protein decreased significantly after treatment with the compound, indicating a good antiviral effect.

[0063] Example 2

[0064] We treated HepG2 and HepG2-NTCP cells with compounds: HepG2 and HepG2-NTCP cells were cultured to the logarithmic growth phase (DMEM high glucose (containing 10% FBS and 1% penicillin-streptomycin), 12-well plates were pre-coated with 0.1 mg / mL collagen (Type I) (500 μL per well, incubated at room temperature for 1 h), washed twice with PBS, and air-dried for later use. Logarithmic growth phase HepG2 cells were digested, counted, and their density adjusted to 2.8 × 10⁻⁶. 5 cells / mL. Inoculate each well with 1 mL of cell suspension (i.e., 2.8 × 10⁻⁶ cells / mL). 5 Add cells / well, gently shake to mix, and incubate statically. Observe under a microscope after 24 hours; the adhesion rate should be >95% (non-adherent cells can be removed by changing the medium). Dissolve 1 μg of pCH9 / 3091 plasmid in 50 μL of medium. Serum-free culture medium, and another 2 μL of Lipofectamine 3000 + 50 μL After incubating at room temperature for 5 minutes, mix the plasmid with the transfection reagent (total volume 100 μL) and incubate at room temperature for 15 minutes to form a complex. Remove the original culture medium and add 900 μL of fresh DMEM (containing 10% FBS, antibiotic-free) to each well. Add 100 μL of the complex dropwise to each well and gently mix. 24 h post-transfection (ensuring sufficient HBV transcription and protein expression). Dilute with DMEM (containing 2% FBS) to 0, 5, and 10 μM (final DMSO concentration ≤0.1%), and dilute again with DMEM (containing 2% FBS) to 0, 5, and 10 μM (final DMSO concentration ≤0.1%). Add 1 mL of fresh culture medium containing the corresponding concentration of the compound to each well and continue culturing for 48 h (observe cell status during this period to avoid toxic effects).

[0065] On day 6, 200 μL of supernatant was collected from the 12-well plate, and the HBeAg secretion level in the cell culture supernatant was detected according to the instructions of the enzyme-linked immunosorbent assay kit (Kehua Biotechnology, China). Figure 3 , 4 The results from A show that small molecule compound C 18 H 12 F3N3OS significantly inhibited HBeAg secretion. RT-qPCR experiments using Trizol method to extract cellular RNA and detect HBV pgRNA and HBV RNAs levels were performed; the reaction system and conditions are shown in Example 2. Figure 3 , 4 The BC results show that small molecule compound C 18 H 12 F3N3OS significantly reduced the levels of HBV pgRNA and HBV RNAS. Figure 3 , 4Results D show that small molecule compound C 18 H 12 F3N3OS significantly reduced HBV RNA levels at 3.5 kb and 2.4 / 2.1 kb. Cellular proteins were extracted using a 1× SDS-PAGE loading buffer for Western blot analysis, followed by detection of HBc protein expression levels using an anti-HBc antibody. Figure 3 , 4 E). The results showed that the expression level of HBc protein decreased significantly after treatment with the compound.

[0066] Example 3

[0067] We used an HBV-infected mouse model. Alb-Cre transgenic mice (Shanghai Southern Model Organisms Center, China), 6-8 weeks old males, weighing 20-25g, were SPF-fed and specifically expressed Cre recombinase in their livers, suitable for HBV cccDNA research. Temperature: 22±2℃, humidity: 50±10%, 12h / 12h light / dark cycle, with free access to sterilized feed and drinking water. Plasmid concentration: 4μg (dissolved in 2mL physiological saline, filtered sterile), injected rapidly over 5-8 seconds using a 1mL syringe (injection volume was 8-10% of the mouse's body weight, approximately 1.6-2.0mL). The mice were observed after injection (successful recovery within 30 minutes). Seven days after injection, 100 μL of blood was collected from the orbital venous plexus, centrifuged (3000 rpm, 10 minutes) to separate the serum, and ELISA was used (OD value > 1.0 was considered successful infection). Control group (n=3): 0 mg / kg (corn oil solvent only), experimental group (n=3): 10 mg / kg compound (dissolved in corn oil, final concentration 1 mg / mL). Intraperitoneal injection (ip) was administered every 48 hours for a total of 7 times (14 days). Blood was collected from the orbital venous plexus every 7 days (days 0, 7, and 14 of administration) to detect HBsAg levels and assess the dynamic changes in viral load. Mice were sacrificed on day 14, and RNA was extracted from their livers and fixed with 4% paraformaldehyde.

[0068] from Figure 5 Results A show that, over time, the small molecule compound C... 18 H 12 F3N3OS gradually reduced HBsAg levels. RT-qPCR was performed to detect HBV pgRNA and HBV RNAs levels using the Trizol method. The reaction system and conditions are shown in Table 2. Figure 5 The BC results show that small molecule compound C 18 H 12 F3N3OS significantly reduced the levels of HBV pgRNA and HBV RNAs.

Claims

1. The use of the compound represented by Formula I or its pharmaceutically acceptable salt in the preparation of HBV inhibitors or medicaments for the treatment of hepatitis B.

2. The application according to claim 1, characterized in that: The compound represented by Formula I or its pharmaceutically acceptable salt inhibits HBV transcription.

3. The application according to claim 2, characterized in that: The compound represented by Formula I or its pharmaceutically acceptable salt reduces the expression levels of HBV RNAs, HBV pgRNA and HBc protein.

4. The application according to claim 2, characterized in that: The compound represented by Formula I, or its pharmaceutically acceptable salt, reduces the production level of HBeAg in hepatitis B.

5. A pharmaceutical composition for inhibiting HBV replication or treating hepatitis B, characterized in that: This includes the compound represented by Formula I in claim 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

6. The pharmaceutical composition according to claim 5, characterized in that: The active ingredient exerts its effects by inhibiting the expression levels of HBV RNAs, HBV pgRNA and HBc protein, as well as reducing the production level of HBeAg.

7. The pharmaceutical composition according to claim 5, characterized in that: It also includes pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to claim 7, characterized in that: The pharmaceutical composition is available in the following dosage forms: capsules, tablets, pills, ointments, granules, oral solutions, microcapsules, or injections.

9. The use of the pharmaceutical composition of claim 5 in the preparation of HBV inhibitors or in the preparation of medicaments for treating hepatitis B.

10. The application according to claim 9, characterized in that: The active ingredient in the pharmaceutical composition inhibits the expression levels of HBV RNAs, HBV pg RNA and HBc protein, and reduces the production level of HBeAg.

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