Application of N-(quinoline-8-yl) quinoline-8-sulfonamide compound in preparation of medicine for resisting hepatitis B virus

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an N-(quinoline-8-yl) quinoline-8-sulfonamide compound to preparation of a medicine for resisting hepatitis B virus. The structure of the compound comprises a salt as shown in (I) or pharmaceutically acceptable salt of the compound. The compound disclosed by the invention has a good inhibition effect on HBV virus replication on the cell level of HBV infection models such as HepG2.2. 15 and Huh7-NTCP, has anti-hepatitis virus activity, provides a good choice for treating viral hepatitis, and also has important significance for developing more ideal medicines for treating hepatitis B. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the use of N-(quinolin-8-yl) quinoline-8-sulfonamide compounds in the preparation of drugs against hepatitis B virus. Background Art

[0002] As a hepadnavirus, the HBV virus specifically binds to the Na+ / taurocholate cotransporting polypeptide (hNTCP) specifically expressed on the surface of hepatocytes in the body and enters the hepatocytes. Uncoating occurs in the cytoplasm, and relaxed circular DNA (rcDNA) is released into the nucleus. HBV rcDNA is repaired to form a stable fragment called covalently closed circular DNA (cccDNA). The HBV genome is a partially double-stranded DNA molecule, consisting of an enveloped circular DNA of approximately 3.2 kb in size, containing 4 overlapping open reading frames (ORFs): S, C, P, and X. According to structure and function, the S region can be divided into three regions: preS1 / preS2 / S, encoding three proteins: S, M (S+preS2), and L (S+preS2+preS1); ORF-C can also be divided into two regions, encoding two regions: preC and HBcAg. HBcAg assembles into the viral nucleocapsid, and preC forms the secreted protein HBeAg after translation and processing. ORF-P encodes a polymerase that can be divided into ribonuclease (RH), reverse transcriptase (RT), and terminal protein (TP) domains. The P region is the target of many nucleoside analogs for the treatment of HBV; the X region encodes the HBx protein. Currently, the drugs approved for the treatment of chronic hepatitis B are pegylated interferon (PEG-IFN) and nucleoside (nucleotide) analogs (NAs). NAs mainly play an antiviral role by inhibiting the reverse transcriptase activity of HBV DNA polymerase.

[0003] N-(quinolin-8-yl) quinoline-8-sulfonamide compounds are described in the patent with application number 2024102593999 filed by the applicant on March 7, 2024. This compound has stable properties, and its molecular formula is C18H12FN3O2S. Previously, N-(quinolin-8-yl) quinoline-8-sulfonamide compounds have been reported to achieve the treatment of breast cancer. Currently, there is no report on the use of N-(quinolin-8-yl) quinoline-8-sulfonamide compounds in anti-hepatitis B virus. Summary of the Invention

[0004] Objective of the Invention: The objective of the present invention is to provide the use of an N-(quinolin-8-yl) quinoline-8-sulfonamide compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for anti-hepatitis B virus.

[0005] To achieve the above technical objectives and reach the above technical effects, the present invention is realized through the following technical solutions:

[0006] The present invention provides the use of a compound shown in formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for anti-hepatitis B virus:

[0007]

[0008] Furthermore, the drug is a drug for inhibiting hepatitis B virus infection, replication, and / or proliferation.

[0009] Furthermore, the drug is a drug for reducing the expression level of at least one of hepatitis B surface antigen HbsAg and hepatitis B e antigen HBeAg.

[0010] Furthermore, the drug is a drug for treating and / or preventing hepatitis, liver cirrhosis, or liver cancer diseases caused by hepatitis B virus.

[0011] Furthermore, the drug uses the compound shown in formula (I) or a pharmaceutically acceptable salt thereof as a prodrug.

[0012] Furthermore, the pharmaceutically acceptable salt is an acid addition salt of the compound shown in formula (I), and the acids used for salting include inorganic acids and organic acids. The inorganic acids include hydrochloric acid, sulfuric acid, and phosphoric acid, and the organic acids include acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, di-gluconic acid, aspartic acid, and tartaric acid.

[0013] Furthermore, the drug also contains pharmaceutically acceptable additives or / and excipients or / and carriers.

[0014] Furthermore, the drug is a tablet, granule, or liquid preparation.

[0015] On the other hand, the present invention provides an antiviral drug, and the active ingredient of the drug includes the above compound or a pharmaceutically acceptable salt thereof.

[0016] On the other hand, the present invention provides a pharmaceutical composition, which contains the above compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0017] In the above technical solution, N-(quinolin-8-yl) quinoline-8-sulfonamide compounds promote the degradation of SND1 by inhibiting the MTDH-SND1 protein-protein interaction.

[0018] Specifically, N-(quinolin-8-yl) quinoline-8-sulfonamide compounds bind to the hydrophobic pocket of tryptophan at position 401 of MTDH in the MTDH-SND1 protein complex on the SN1 / 2 domain of SND1, thereby blocking the MTDH-SND1 protein-protein interaction.

[0019] More specifically, arginine at position 255, tryptophan at position 279, and asparagine at position 281 in the SN1 / 2 domain of SND1 are important amino acid residues for N-(quinolin-8-yl) quinoline-8-sulfonamide compounds to bind to the SND1 protein and produce the inhibitory effect on the MTDH-SND1 protein-protein interaction.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: N-(quinolin-8-yl) quinoline-8-sulfonamide compounds can produce an anti-hepatitis B virus effect by inhibiting the MTDH-SND1 protein-protein interaction. At the same time, these compounds have the characteristics of simple preparation process and easy availability of raw materials, providing a new option for the treatment of hepatitis B virus. Description of the Drawings

[0021] Figure 1 It is a schematic diagram for evaluating the inhibitory activity of the compound on the MTDH-SND1 protein-protein interaction.

[0022] Figure 2 It is the binding mode of C19 to the SN1 / SN2 domain of the SND1 protein predicted by molecular docking.

[0023] Figure 3 It is the detection of the degradation effect of C19 on the SND1 protein in the HBV infection model established based on the Huh7-NTCP cell line by Western blot of the protein immunoblotting test. Among them, Mock is the negative control; C19 (0.3 μM), C19 (1 μM), and C19 (3 μM) are the results of extracting protein samples after treating Huh7-NTCP-infected HBV cells with compound C19 at 0.3, 1, and 3 μM for 4 days, respectively.

[0024] Figure 4To evaluate the release levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) by enzyme-linked immunosorbent assay (ELISA) for compound C19, where a is the graph showing the effect of different concentrations of C19 on the release level of hepatitis B surface antigen (HBsAg) in the HBV model established with the HepG2.2.15 cell line; b is the graph showing the effect of different concentrations of C19 on the release level of hepatitis B e antigen (HBeAg) in the HBV model established with the HepG2.2.15 cell line; c is the graph showing the effect of different concentrations of C19 on the release level of hepatitis B surface antigen (HBsAg) in the HBV model established with the Huh7-NTCP cell line; d is the graph showing the effect of different concentrations of C19 on the release level of hepatitis B e antigen (HBeAg) in the HBV model established with the Huh7-NTCP cell line. Among them, Mock / NC is the negative control; the HepG2.2.15 HBV-infected cell model was treated with 0.3, 1, 3 μM of compound C19 for 4 days, and then the supernatant was collected to detect HBsAg and HBeAg; the Huh7-NTCP HBV-infected cell model was treated with 0.3, 1, 3 μM of compound C19 for 6, 8, 10 days respectively, and then the supernatant was collected to detect HBsAg and HBeAg; *** indicates that the P value of the t-test between the experimental group and the negative control is < 0.001; **** indicates that the P value of the t-test between the experimental group and the negative control is < 0.0001. Detailed implementation mode

[0025] The N-(quinolin-8-yl)quinoline-8-sulfonamide compound in the embodiment of the present invention is the compound shown in formula (I), that is, compound C19, which is described in the patent with the application number 2024102593999 applied by the applicant on March 7, 2024, and its structure is consistent with the structure of compound C-4 in this patent.

[0026]

[0027] The common synthesis method of N-(quinolin-8-yl)quinoline-8-sulfonamide compounds is the room-temperature reaction of quinoline sulfonyl chloride and 8-quinolinamine with pyridine as the solvent, as shown in formula (II)

[0028]

[0029] Example 1

[0030] The purpose of this example is to evaluate the inhibitory effect of the compound on the MTDH-SND1 protein-protein interaction.

[0031] This example verified that N-(quinolin-8-yl) quinoline-8-sulfonamide compounds promote the degradation of SND1 by inhibiting the MTDH-SND1 protein-protein interaction. In the HEK-293 cell line, the C-terminal fusion protein of MTDH-firefly luciferase (CLuc-MTDH) and the N-terminal fusion protein of SND1-firefly luciferase (SND-NLuc) were transfected respectively. Under normal physiological conditions, while the protein-protein interaction between MTDH and SND1 occurs, the two fragments of firefly luciferase fused with MTDH and SND1 respectively exhibit firefly luciferase activity due to the proximity effect, catalyzing the luminescent substrate to emit fluorescence that can be quantitatively measured. When a compound with inhibitory activity against the MTDH-SND1 protein-protein interaction blocks the binding of MTDH-SND1, the C-terminal and N-terminal firefly luciferases that fail to approach each other will not have the activity to catalyze the substrate to emit light, and the inhibitory activity of the compound against the MTDH-SND1 protein-protein interaction is quantitatively measured in the above manner( Figure 1 ).

[0032] When evaluating the inhibitory effect of compound C19 on the MTDH-SND1 protein-protein interaction, HEK-293T cells co-expressing SND-NLuc and CLuc-MTDH in the logarithmic growth phase were seeded at 3000-5000 cells / well in an opaque 96-well plate and cultured at 37 °C and 5% CO2 for 24 hours; 100 μL of the test compound solution with different concentrations diluted in gradient was added to the culture plate, and the culture plate was incubated in a 37 °C and 5% CO2 incubator for 48 hours; 100 μL of the medium was aspirated from each well and 100 μL of a mixture of Bright-Lite Luciferase Assay Buffer and the luminescent substrate Bright-Lite Luciferase Assay Substrate (Vazyme, DD1204) was added, incubated at room temperature for 3 minutes, read on a microplate reader, and the luminescence was recorded. The IC50 value of each compound against the enzyme activity was calculated using the analysis software GraphPad Prism. According to the above test method, the IC50 of C19 against the MTDH-SND1 protein-protein interaction was 487 ± 89 nM. This result indicates that compound C19 can effectively block the MTDH-SND1 protein-protein interaction.

[0033] Example 2

[0034] In this example, the applicant used the molecular simulation software Discovery Studio for molecular docking to predict the potential binding mode of compound C19 with the SND1 protein. The SND1 protein structure (PDB ID: 7KNX) from the Protein Data Bank (PDB) was isolated and set as the receptor; the structure of compound C19 was set as the docking ligand after energy and initial conformation optimization using the ligand preparation module; molecular docking was performed using the semi-flexible docking module CDOCK, and the docking results were optimized using the simulated annealing algorithm equipped with the module program. The results showed that N-(quinolin-8-yl)quinoline-8-sulfonamide compounds blocked the MTDH-SND1 protein-protein interaction by binding to the hydrophobic pocket of tryptophan at position 401 of MTDH in the MTDH-SND1 protein complex. The quinoline ring near the sulfonyl end of compound C19 tightly bound to the hydrophobic pocket of SND1 ( Figure 2 ). Currently, a large amount of evidence has proven that the hydrophobic pocket on the surface of SND1 at the SN1 / SN2 junction available for the binding of tryptophan at position 401 of MTDH is a significant druggable pocket.

[0035] The results of molecular docking showed that the hydrophobic pocket on the surface of SND1 bound to MTDH W401 was occupied by C19 and formed hydrogen bonds, π-π stacking, halogen bonds and other interactions with H279, N281, and R255 of SND1.

[0036] Specifically, arginine at position 255, tryptophan at position 279, and asparagine at position 281 in the SND1 SN1 / 2 domain are important amino acid residues for the binding of N-(quinolin-8-yl)quinoline-8-sulfonamide compounds to the SND1 protein and for producing the inhibitory effect on the MTDH-SND1 protein-protein interaction ( Figure 2 ). The quinoline ring at the sulfonyl end of compound C19 formed a π-π stacking interaction with tryptophan at position 279 of SND1 ( Figure 2 shown by the purple dashed line in ); the nitrogen atom on the quinoline ring at the sulfonyl end of C19, the nitrogen atom on the quinoline ring at the amino end, and the oxygen atom of the sulfonyl group formed hydrogen bond interactions with asparagine at position 281, arginine at position 255, and asparagine at position 281 of SND1 respectively ( Figure 2 shown by the green dashed line in ); the fluorine atom of C19 formed a halogen bond with asparagine at position 281 of SND1 ( Figure 2 shown by the blue dashed line in ).

[0037] Example 3

[0038] Given the important role of SND1 in HBV replication, the applicant used Western blot to verify the effects of the MTDH-SND1 protein-protein interaction inhibitor C19 on the expression of HBV Core protein and S protein in the HBV infection model of Huh7-NTCP cell line.

[0039] The implementation steps of Western blot are as follows: Extract protein samples from the HBV infection model of Huh7-NTCP cell line treated with different concentrations of compound C19; Prepare gels with corresponding concentrations according to the molecular weights of the target proteins; Measure the protein concentrations of the samples and perform electrophoresis for 60 minutes at a voltage of 120 V; Cut a polyvinylidene fluoride membrane (PVDF membrane) and soak it in methanol for 5 minutes for activation. Take out the gel from the electrophoresis tank and place it on the PVDF membrane. Add filter papers and sponges on both sides of the gel and the PVDF membrane, and start wet transfer after pressing to remove air bubbles; Transfer proteins with a current of 340 mA and add ice cubes outside the transfer tank for cooling; After the transfer is completed, take out the PVDF membrane and soak it in 5% skim milk for 2 hours for blocking; Take out the PVDF membrane and soak it in the primary antibody solution prepared with 1% bovine serum albumin, and incubate overnight at 4°C; Wash the PVDF membrane 5 times with TBST buffer solution, 6 minutes each time; Take out the PVDF membrane and soak it in the secondary antibody solution prepared with 1% fetal bovine serum albumin, and incubate at room temperature for 2 hours; Take out the PVDF membrane and wash it 5 times with TBST buffer solution; Prepare the exposure solution and use an exposure instrument to expose the PVDF membrane.

[0040] The Western blot results showed that high concentrations of C19 significantly inhibited the expression of HBV Core protein and S protein.

[0041] Example 4

[0042] The applicant used an enzyme-linked immunosorbent assay (ELISA) kit from Shanghai Kehua Bio-Engineering Co., Ltd. to detect the effects of compound C19 on the release levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the HBV infection models established with HepG2.2.15 and Huh7-NTCP cell lines ( Figure 4 ).

[0043] The method for detecting the inhibitory effect of compound C19 on the release of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) is as follows: The sample extracted from HBV-infected cells treated with compound C19 is diluted 5-fold with PBS, and the washing solution is diluted 25-fold with purified water; 75 μL of the sample to be tested is added to each well, and the plate is sealed with a cover slip and incubated at 37 °C for 60 minutes; 50 μL of the enzyme conjugate is added to each well, and after shaking the plate for 10 seconds, it is incubated at 37 °C for 30 minutes; the liquid in the well is removed and the plate is washed 5 times with the washing solution, 1 minute each time, and the reaction plate is patted dry on the absorbent paper; 50 μL of each of chromogenic reagents A and B is immediately added to each well, mixed and shaken for 10 seconds, and then incubated at 37 °C for 30 minutes; 50 μL of the stop solution is added to each well, and after shaking the plate for 5 seconds, the absorbance is measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0044] The method for detecting the inhibitory effect of compound C19 on the release of hepatitis B e antigen (HBeAg) is as follows: The sample extracted from HBV-infected cells treated with compound C19 is diluted 5-fold with PBS, and the washing solution is diluted 25-fold with purified water; 50 μL of the sample to be tested is added to each well, then 50 μL of the enzyme conjugate is added, the plate is sealed with a cover slip and incubated at 37 °C for 30 minutes; the liquid in the well is removed, the plate is washed 5 times with the washing solution, 1 minute each time, and finally the reaction plate is patted dry on the absorbent paper; 50 μL of each of chromogenic reagents A and B is immediately added to each well, mixed and shaken for 10 seconds, and then incubated at 37 °C for 15 minutes; 50 μL of the stop solution is added to each well, and after shaking the plate for 5 seconds, the absorbance is measured at a wavelength of 450 nm using an ELISA reader.

[0045] The results of the ELISA test showed that when the cells were treated with the compound for 4 days, 0.3 μM of C19 could significantly inhibit the release of hepatitis B surface antigen in the HepG2.2.15-infected HBP cell model; at concentrations of 1 μM and 3 μM, the release of hepatitis B surface antigen was inhibited to extremely low levels; 1 μM and 3 μM of C19 could significantly inhibit the release of hepatitis B e antigen in the HepG2.2.15-infected HBP cell model; when the cells were treated with the compound for 6, 8, and 10 days, C19 significantly inhibited the release of hepatitis B surface antigen and hepatitis B e antigen in the Huh7-NTCP-infected HBP cell model, and the dose-dependent effect became gradually more obvious with the increase in the compound treatment time. The above results indicate that the MTDH-SND1 protein-protein interaction inhibitor has good anti-hepatitis B virus infection activity.

[0046] The examples shown above in the description of the present invention are only used to help illustrate the present invention. The examples do not describe all the details in detail, nor do they limit the invention to the specific embodiments.

Claims

1. Use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-hepatitis B virus drug:

2. The use according to claim 1, characterized in that: The drug is a drug for inhibiting the infection, replication and / or proliferation of hepatitis B virus.

3. The use according to claim 1, characterized in that: The drug is a drug for reducing the expression amount of at least one of hepatitis B surface antigen HbsAg and hepatitis B e antigen HBeAg.

4. The use according to claim 1, characterized in that: The medicine is a medicine for treating and / or preventing hepatitis, liver cirrhosis or liver cancer caused by hepatitis B virus.

5. The use according to claim 1, characterized in that: The drug uses the compound represented by formula (I) or a pharmaceutically acceptable salt thereof as a prodrug.

6. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt is an acid addition salt of the compound represented by formula (I), wherein the acid used for salt formation includes inorganic acid and organic acid, wherein the inorganic acid includes hydrochloric acid, sulfuric acid, phosphoric acid, and the organic acid includes acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid and tartaric acid.

7. The use according to any one of claims 1 to 6, characterized in that: The medicine further contains pharmaceutically acceptable additives and / or excipients and / or carriers.

8. The use according to claim 7, characterized in that: The medicine is in the form of tablets, granules or liquid preparations.

9. An antiviral drug, characterized in that: The active ingredient of the drug comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof.

10. A pharmaceutical composition, characterized in that The invention comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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