Use of N-(quinolin-8-yl)quinoline-8-sulfonamide compounds in the preparation of a drug for resisting hepatitis B virus

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to application of N-(quinoline-8-yl) quinoline-8-sulfonamide compounds in preparation of anti-hepatitis B virus drugs. The structure comprises as shown in (I) or a pharmaceutically acceptable salt thereof. The compound disclosed in the application has good inhibitory effect on HBV virus replication at the level of HBV infection model cells 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 hepatitis B treatment drugs.

Description

TECHNICAL FIELD

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

[0002] HBV virus, as a hepadnavirus, specifically binds to the surface of hepatocytes to enter the liver cells, and occurs in the cytoplasm to release the relaxed circular DNA (rcDNA) to 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 composed of an envelope circular DNA of about 3.2 kb in size, containing four overlapping open reading frames (ORF): S, C, P and X. According to the structure and function, the S region can be divided into preS1 / preS2 / S three regions, encoding S, M (S+preS2), L (S+preS2+preS1) three proteins; ORF-C can also be divided into two regions, encoding preC and HBcAg two regions, and HBcAg is assembled into a viral nuclear envelope, while preC is processed into a secreted protein HBeAg. ORF-P encodes a polymerase that can be divided into ribonuclease (RH), reverse transcriptase (RT) and terminal protein (TP) domains, and the P region is the target of numerous nucleoside analogs for treating HBV; the X region encodes the HBx protein. The drugs currently approved for the treatment of chronic hepatitis B are pegylated-interferon (PEG-IFN) and nucleos(t)ide 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 the application number 2024102593999 applied by the applicant on March 7, 2024, and the compound has stable properties, with a molecular formula of C18H12FN3O2S. Previously, N-(quinolin-8-yl)quinoline-8-sulfonamide compounds were reported to achieve treatment of breast cancer. Currently, there is no report on N-(quinolin-8-yl)quinoline-8-sulfonamide compounds in anti-hepatitis B virus. SUMMARY

[0004] The application aims to provide an application of N-(quinoline-8-yl) quinoline-8-sulfonamide or a pharmaceutically acceptable salt thereof in preparing an anti-hepatitis B virus drug.

[0005] To achieve the above technical purposes and effects, the application is implemented by the following technical scheme.

[0006] The application provides an application of a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof in preparing an anti-hepatitis B virus drug.

[0007]

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

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

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

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

[0012] Further, the pharmaceutically acceptable salt is an acid addition salt of the compound as shown in formula (I), wherein the acid for salt formation includes inorganic acid and organic acid, the inorganic acid includes hydrochloric acid, sulfuric acid and 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.

[0013] Further, the drug further contains a pharmaceutically acceptable additive or / and excipient or / and carrier.

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

[0015] In another aspect, the application provides an anti-virus drug, wherein the effective component of the drug includes the above-mentioned compound or a pharmaceutically acceptable salt thereof.

[0016] In another aspect, the application provides a pharmaceutical composition containing the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

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

[0018] Specifically, the N-(quinolin-8-yl)quinoline-8-sulfonamide compound blocks the MTDH-SND1 protein-protein interaction by binding to the hydrophobic pocket of the MTDH 401 tryptophan on the SND1 SN1 / 2 domain in the MTDH-SND1 protein complex.

[0019] More specifically, the 255 arginine, 279 tryptophan, and 281 asparagine of the SND1 SN1 / 2 domain are important amino acid residues for the binding of the N-(quinolin-8-yl)quinoline-8-sulfonamide compound to the SND1 protein and the inhibition of the MTDH-SND1 protein-protein interaction.

[0020] Advantages: Compared with the prior art, the present application has the following significant advantages: the N-(quinolin-8-yl)quinoline-8-sulfonamide compound can produce an anti-HBV effect by inhibiting the MTDH-SND1 protein-protein interaction, and at the same time, the compound has the characteristics of simple preparation process and easy-to-obtain raw materials, providing a new choice for the treatment of anti-HBV. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Schematic diagram for evaluating the MTDH-SND1 protein-protein interaction inhibition activity of the compound.

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

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

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

[0025] The N-(quinolin-8-yl)quinoline-8-sulfonamide compound of the embodiment of the application is a compound shown in formula (I), i.e., compound C19, which is described in the patent with application number 2024102593999 filed by the applicant on March 7, 2024, and has the same structure as the C-4 compound in the patent.

[0026]

[0027] The common synthesis method of N-(quinolin-8-yl)quinoline-8-sulfonamide compound is to react quinoline sulfuryl chloride with 8-quinoline amine at room temperature with pyridine as the solvent, as shown in formula (II)

[0028]

[0029] Example 1

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

[0031] This example demonstrates that N-(quinolin-8-yl)quinoline-8-sulfonamides compounds promote SND1 degradation by inhibiting MTDH-SND1 protein-protein interaction. HEK-293 cell line was transfected with MTDH- firefly luciferase C-terminal fusion protein (CLuc-MTDH) and SND1-firefly luciferase N-terminal fusion protein (SND-NLuc) respectively. Under normal physiological conditions, MTDH interacts with SND1, and 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 on MTDH-SND1 protein-protein interaction blocks the combination of MTDH-SND1, the C-terminal and N-terminal firefly luciferase that cannot approach each other will not have catalytic substrate luminescence activity, and the inhibitory activity of the compound on MTDH-SND1 protein-protein interaction is quantitatively determined in the above manner. Figure 1

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

[0033] Example 2

[0034] ​In this embodiment, the applicant utilizes the molecular modeling software Discovery Studio to perform molecular docking to predict the potential binding mode of compound C19 to the SND1 protein. The SND1 protein structure (PDB ID: 7KNX) derived from the Protein Data Bank (PDB) is isolated and set as the receptor; the structure of compound C19 is set as the docking ligand after energy and initial conformation optimization with the Ligand Preparation module; the molecular docking is performed with the semi-flexible docking module CDOCK, and the docking results are optimized with the simulated annealing algorithm equipped in the module program. The results show that the N-(quinolin-8-yl)quinoline-8-sulfonamide compound blocks the MTDH-SND1 protein-protein interaction by binding to the hydrophobic pocket of the SND1 SN1 / 2 domain on the MTDH 401 tryptophan. Figure 2 There is currently a large amount of evidence that the hydrophobic pocket on the SND1 surface at the SN1 / SN2 junction where the MTDH 401 tryptophan binds is a significant druggable pocket.

[0035] The results of molecular docking show that the hydrophobic pocket on the SND1 surface where the MTDH W401 binds is occupied by C19 and forms hydrogen bonding, π-π stacking, halogen bonding, etc. interactions with the H279, N281, R255 of SND1.

[0036] Specifically, the 255 arginine, 279 tryptophan and 281 asparagine of 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 the inhibition effect of MTDH-SND1 protein-protein interaction. Figure 2 The quinoline ring at the sulfonamide end of compound C19 forms a π-π stacking interaction with the 279 tryptophan of SND1 (indicated by a purple dashed line in Figure 2 ); the nitrogen atom on the quinoline ring at the sulfonamide end, the nitrogen atom on the quinoline ring at the amino end and the oxygen atom of the sulfonamide form hydrogen bonding interactions with the 281 asparagine, 255 arginine and 281 asparagine of SND1, respectively (indicated by a green dashed line in Figure 2 ); the fluorine atom of C19 forms a halogen bond with the 281 asparagine of SND1 (indicated by a blue dashed line in Figure 2 ).

[0037] Example 3

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

[0039] The steps for performing Western blot are as follows: extract protein samples from the Huh7-NTCP cell line HBV infection model treated with different concentrations of compound C19; prepare gels of corresponding concentrations according to the molecular weight of the target protein; measure the protein concentration of the sample, and electrophorese for 60 minutes at a voltage of 120 volts; cut the polyvinylidene fluoride membrane (PVDF membrane) and soak it in methanol for 5 minutes, remove the gel from the electrophoresis tank and place it on the PVDF membrane, add filter paper and sponge on both sides of the gel and PVDF membrane, and start wet transfer after compaction to remove air bubbles; transfer the protein at a current of 340 milliamps, and add ice blocks to the outside of the transfer tank for cooling; after the transfer is complete, remove the PVDF membrane and soak it in 5% skimmed milk for 2 hours; remove the PVDF membrane and soak it in a primary antibody solution prepared with 1% bovine serum albumin, and incubate at 4°C overnight; wash the PVDF membrane with TBST buffer solution 5 times, each time for 6 minutes; remove the PVDF membrane and soak it in a secondary antibody solution prepared with 1% fetal bovine serum albumin, and incubate at room temperature for 2 hours; remove the PVDF membrane and wash it with TBST buffer solution 5 times; prepare the exposure solution, and use an exposure instrument to expose the PVDF membrane.

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

[0041] Example 4

[0042] The applicant used the enzyme-linked immunosorbent (Elisa) kit of Shanghai Kewei Bio to detect the effect of compound C19 on the release levels of hepatitis B surface antigen (HBsAg) and hepatitis B E antigen (HBeAg) in the HBV infection model established by HepG2.2.15, Huh7-NTCP cell lines Figure 4 ).

[0043] The detection method of 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 of the HBV infected cell treated by compound C19 is diluted 5 times with PBS, and the washing solution is diluted 25 times with purified water; 75 μL of the sample to be detected is added to each well, and the plate is sealed with a cover glass and incubated at 37℃ for 60 minutes; 50 μL of enzyme conjugate is added to each well, and after shaking for 10 seconds, the plate is incubated at 37℃ for 30 minutes; the liquid in the well is removed, and the plate is washed 5 times with the washing solution, each time for 1 minute, and finally the reaction plate is dried on the absorbent paper; 50 μL of color developing agent A and B are immediately added to each well, mixed and shaken for 10 seconds, and then incubated at 37℃ for 30 minutes; 50 μL of stop solution is added to each well, shaken for 5 seconds, and then the absorbance value is detected at 450 nm wavelength by using an enzyme-labeled instrument.

[0044] The detection method of the inhibitory effect of compound C19 on the release of hepatitis B E antigen (HBeAg) is as follows: the sample of the HBV infected cell treated by compound C19 is diluted 5 times with PBS, and the washing solution is diluted 25 times with purified water; 50 μL of the sample to be detected is added to each well, and then 50 μL of enzyme conjugate is added, the plate is sealed with a cover glass and incubated at 37℃ for 30 minutes; the liquid in the well is removed, and the plate is washed 5 times with the washing solution, each time for 1 minute, and finally the reaction plate is dried on the absorbent paper; 50 μL of color developing agent A and B are immediately added to each well, mixed and shaken for 10 seconds, and then incubated at 37℃ for 15 minutes; 50 μL of stop solution is added to each well, shaken for 5 seconds, and then the absorbance value is detected at 450 nm wavelength by using an enzyme-labeled instrument.

[0045] The results of the Elisa test show that when the compound is treated for 4 days, 0.3 μM of C19 can significantly inhibit the release of hepatitis B surface antigen in the HepG2.2.15 infected HBP cell model; at the concentrations of 1 μM and 3 μM, the release of hepatitis B surface antigen is inhibited to a very low level; 1 μM and 3 μM of C19 can significantly inhibit the release of hepatitis B E antigen in the HepG2.2.15 infected HBP cell model; when the compound is treated for 6, 8 and 10 days, C19 significantly inhibits 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 gradually becomes obvious as the treatment time of the compound increases. The above results show that the MTDH-SND1 protein-protein interaction inhibitor has good anti-hepatitis B virus infection activity.

[0046] The above embodiments of the present application are only used to help illustrate the present application. The embodiments do not describe all the details, nor limit the present application to only the specific embodiments.

Claims

1. Use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting hepatitis B virus infection, replication and / or proliferation. 。 2. Use according to claim 1, characterized in that, The medicament is a medicament for inhibiting hepatitis B virus infection, replication and / or proliferation.

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

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

5. The use according to claim 1, characterized in that, The pharmaceutically acceptable salt is an acid addition salt of the compound of formula (I), wherein the acid used for salt formation includes inorganic acids and organic acids, the inorganic acids include hydrochloric acid, sulfuric acid, 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, digluconic acid, aspartic acid and tartaric acid.

6. Use according to any one of claims 1 to 5, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

7. Use according to claim 6, characterized in that, The medicament is a tablet, granule or liquid preparation.

Images