KR101475056B1 - Pharmaceutical composition for preventing or treating hepatitis C comprising sulfonamide compound - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of hepatitis C comprising a sulfonamide compound as an active ingredient. The sulfonamide compound according to the present invention has a very low toxicity to hepatocytes and exhibits an excellent effect of selectively inhibiting the replication of the genome of HCV, so that it can be very useful for the prevention or treatment of hepatitis C virus.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for preventing or treating chronic hepatitis C comprising a sulfonamide compound as an active ingredient,

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of hepatitis C comprising a sulfonamide compound as an active ingredient.

Liver cancer and liver disease are one of the four major diseases with the highest number of deaths per 100,000 Koreans, along with cerebrovascular disease, heart disease and respiratory disease. Liver cancer and liver disease in Koreans are caused by hepatitis B and C virus Sexual hepatitis accounts for 83% of the total. Among them, hepatitis C virus is a very important medically important pathogen with about 170 million people worldwide. Hepatitis C was classified as non-A non-A post-transfusion associated hepatitis (NANB) after non-A type hepatitis B until the mid-1980s, The virus has been found to be a disease caused by infection by viruses, and then researches and developments have been actively conducted thereafter. Hepatocellular carcinoma of the hepatitis C virus is most likely chronic hepatitis and is fatal because it develops into chronic liver disease such as liver cirrhosis and liver cancer for a long period of 15 to 20 years. , Hepatitis B Virus), chronic hepatitis, liver cirrhosis and hepatocellular carcinoma are reported to be killed by 8,000 to 10,000 people in the United States every year. Most hepatitis C patients die from liver transplantation because they are not waiting for liver transplantation. Statistically, in Korea, about 1.5% of the total population is known to be infected with hepatitis C virus .

Interferon-alpha and ribavirin, which are commonly used as anti-hepatitis C drugs, are indirect-acting antivirals (IAAs) that indirectly attack viruses through internal immunity enhancement and inhibition of nucleic acid biosynthesis, respectively. , The combination of the two drugs together resulted in a sustained virologic response (SVR) of less than 50% due to the genotype of the virus after intravenous administration of 52 weeks of intravenous administration and oral administration In addition, many serious adverse reactions such as suicidal thoughts, depression, anemia, and toxicity have caused many patients with hepatitis C to give up treatment moderately. Therefore, there is a great demand for the development of a more effective and safe drug to replace antiviral drugs against the existing hepatitis C virus.

Korean Patent Application No. 10-2000-0027165 Korean Patent Application No. 10-2002-7013965

http://preview.britannica.co.kr/bol/topic.asp?article_id=b12s4159a, Encyclopedia of BRITANNICA Immunity in chronic hepatitis C infection, BioWave Vol. 8 No. 23 2006 Research Trends of Hepatitis C Virus, Biochemical Molecular Biology Society Webzine Feb 2009 Studies on the Regulation of Cell Cycle-Related Gene Expression and the Role of the Core Gene of Hepatitis C Virus in the Development of Hepatocellular Carcinoma, Dissertation (Ph.D.), Konkuk University, 2001

The present inventors have conducted studies to search for a substance having the preventive or therapeutic effect of hepatitis C, and found that the sulfoneamide-based compound has a very low toxicity to hepatocytes and has an excellent effect of selectively inhibiting the replication of the genome of HCV And completed the present invention.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis C comprising a sulfonamide compound as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating hepatitis C comprising a sulfonamide compound as an active ingredient.

The sulfonamide compound according to the present invention has a very low toxicity to hepatocytes and exhibits an excellent effect of selectively inhibiting the replication of the genome of HCV, so that it can be very useful for the prevention or treatment of hepatitis C virus.

Figure 1 shows the genome structure of the FL-J6 / JFH-5C19Rluc2AUbi HCV reporter virus used to measure genomic replication of hepatitis C virus.
FIG. 2 is a graph showing the effect of the sulfonamide compound of formula (1-2) of the present invention on RNA genome replication and cell viability of hepatitis C virus (red line: cell viability, blue line: genomic replication rate).
FIG. 3 is a graph showing the effect of the sulfonamide compound of Formula 1-3 of the present invention on RNA genome replication and cell viability of hepatitis C virus (red line: cell survival rate, blue line: genomic replication rate).

The present invention provides a pharmaceutical composition for the prevention or treatment of hepatitis C comprising, as an active ingredient, a sulfonamide compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

Wherein R ₁ is H or halogen, R ₂ is C ₁ -C ₁₀ alkyl or C ₁ -C ₁₀ alkoxy and R ₃ is -SO ₂ -NH- (C ₆ H ₃ ) R ₄ R ₅ or -CH ₂ -A- (C ₆ -C ₂₀ aryl), wherein R ₄ and R ₅ are each independently equal or different and are halogen or CF ₃ , and A is S, O, or NH.

Preferably, in Formula 1, R ₁ is H or Cl,

R ₂ is CH ₃ or OMe, R ₃ is SO ₂ -NH- (C ₆ H ₃ ) R ₄ R ₅ or -CH ₂ -A-phenyl,

Wherein R ₄ and R ₅ are independently the same or different and are Cl or CF ₃ and A is S.

Most preferably, the compound of Formula 1 may be a compound of Formula 1-1 or a compound of Formula 1-3.

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

The sulfonamide compound of the present invention can be used in the form of a pharmaceutically acceptable salt such as a salt with a metal salt, an organic base, a salt with an inorganic acid, a salt with an organic acid, and a salt with a basic or acidic amino acid. Suitable metal salts include alkali metal salts such as sodium salts, potassium salts and the like; Alkaline earth metal salts such as calcium salts, magnesium salts, barium salts and the like; Aluminum salts and the like, and suitable organic base salts include, for example, trimethylamine,

There are salts with triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N-dibenzylethylenediamine and the like. Examples of suitable salts with inorganic acids include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Examples of suitable salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, Maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and the like. Suitable examples of salts with basic amino acids include salts with, for example, arginine, lysine, ornithine and the like. Suitable examples of salts with acidic amino acids include salts with, for example, aspartic acid, glutamic acid and the like. Particularly preferred salts include inorganic salts such as alkali metal salts (for example, sodium salts and potassium salts) and alkaline earth metal salts (for example, calcium salts, magnesium salts and barium salts) when the compound has an acidic functional group therein, And salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like, organic acids such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, Succinic acid, methanesulfonic acid, p-toluenesulfonic acid and the like.

Hereinafter, the present invention will be described in detail.

The sulfonamide compound, which is an effective component of the composition of the present invention, has very low toxicity to hepatocytes and thus is highly safe and has an excellent effect of inhibiting replication of the genome of HCV.

The HCV is the only virus classified as hepacivirus in the Flaviviridae group and has a virus genome of RNA of a strand of about 9600 nucleotides. The RNA is translated into a polyprotein of about 3,000 amino acids in the hepatocyte, and the translated polyprotein is expressed in the signal peptidase and non-structural proteins of the virus, It is expressed in 10 different viral proteins by one NS3 protease. Structural proteins such as envelope glycoproteins E1, E2, and capsid proteins are used to make virus particles, while NS2, NS3 Non-structural proteins such as NS4A, NS4B, NS5A, and NS5B are used to create the viral genome replication complex necessary to replicate the genome of a virus. HCV replicates the RNA genome of a virus by creating a viral genomic clone from the membrane of the endoplasmic reticulum. The viral genomic clones are invaginated by clumping to form an independent structure separated from the external environment, expressing the non-structural proteins of the virus in the structure, and expressing the non-structural proteins as viral RNA polymerase Together with the NS5B protein, acts as a genomic cloning site for the virus. However, it is not known how this viral genomic clone is created and maintained. NS4B proteins, among the nonstructural proteins of viruses, have been shown to play a crucial role in making the membranous web, a multi-vesicular structure that is essential for the genome replication of viruses in hepatocytes. The structure of the membrane made by NS4B is believed to provide the physical structure necessary to make viral genomic clones. In addition, the NS5A protein is constructed by assembling the viral genome replicase using non-structural proteins of other viruses, transferring the viral genome to a structure called lipid droplet, where the core protein of the virus is combined with the viral genome It is known to play an important role in creating real virus particles. In particular, protein-protein interaction between non-structural proteins of viruses is known to play a crucial role in the replication of viral genomes, but it is not yet known exactly what role they play at the molecular level.

Examples of antiviral agents that can treat viral diseases include indirect-acting antivirals (IAAs) that indirectly attack viruses and direct-acting antiviral agents that directly attack viruses , DAAs). Interferon alpha, which is secreted in the body by pathogenic external microorganisms such as viruses, is a typical antiviral immune enhancer that indirectly attacks viruses. It is an immune response of host cells parasitized with virus rather than attacking virus itself. Since it inhibits the intracellular proliferation of viruses, it is effective against various kinds of viral diseases, and since it exhibits antiviral activity by controlling the function of the protein of the host cell in which the virus is parasitic, it has resistance and resistance There is a relatively low probability that a mutant virus is present. However, since the antiviral specificity, which can only exhibit antiviral activity for a specific virus, is significantly lowered, a much higher amount of interferon than the amount of interferon that is secreted naturally in vivo to overcome it Such as suicidal thoughts, depression, anemia, and other serious side effects and toxicity.

On the other hand, antiviral agents that directly attack viruses, unlike interferon, selectively inhibit the specific functions of viral proteins essential for the life cycle of viruses, thereby exhibiting high antiviral activity even at relatively low concentrations. Compared to antiviral agents that indirectly attack viruses, they are less toxic and have higher safety. Reverse transcriptase inhibitors and protease inhibitors of the human immunodeficiency virus (HIV) are typical antiviral agents that directly attack viruses. These viruses are known as reverse transcriptase Enzymes and proteolytic enzymes are inhibited to suppress intracellular proliferation of viruses. However, the possibility that the virus can exhibit resistance and resistance to existing antiviral agents by changing the amino acid sequence of the virus protein targeted by the antiviral agent through the mutation is relatively large, and in particular, the RNA polymerase of the hepatitis C virus polymerase) does not have 5'-3 'endonuclease activity, which is a function of correcting a wrongly inserted nucleic acid in the replication of the RNA genome. Therefore, mutant viruses are easily produced in the course of replication of RNA, the genome of hepatitis C virus And it is easy to have resistance and resistance against antiviral agents that directly attack viruses.

Therefore, the sulfonamide compound, which is an effective component of the composition of the present invention, has an excellent effect of selectively blocking genomic replication in the RNA genome replication step of hepatitis C virus, and therefore, the generation of mutant viruses While exhibiting less side effects and toxicity. Therefore, the sulfonamide-based compound of the present invention can be usefully used as a medicament for the prevention or treatment of hepatitis C, which is safe and highly specific for HCV.

The composition of the present invention may contain one or more known active ingredients having a prophylactic or therapeutic effect of hepatitis C together with a sulfonamide compound as an effective ingredient.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the intended method, and the dose may be appropriately determined depending on the patient's weight, age, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The daily dose of the sulfonamide compound is about 10 to 100 mg / kg, and it is preferable that the daily dose is administered once to several times a day.

The composition of the present invention can be used alone or in combination with methods for the prevention or treatment of hepatitis C, or using surgery, hormone therapy, drug therapy and biological response modifiers.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Analysis of hepatocyte toxicity of the sulfonamide compound of the present invention

In order to confirm the hepatocyte toxicity of the sulfonamide compound of the present invention, the following experiment was conducted using the EZ-Cytox cell viability assay (Daeil Lab Service). The sulfonamide-based compound used was a compound of a small molecule library owned by the College of Pharmacy, Dongguk University.

1-1. The hepatocellular toxicity of the sulfonamide compound of formula (1)

96 well-per well was added to 1.7X10 ⁴ 2.0X10 ⁴ Huh7.5 of human lung cancer cells to the plate and incubated at 37 ℃ incubator for 24 hours. The cells were treated with dimethyl sulfoxide (DMSO) as a control group and sulfonamide-based compounds of the formulas 1-1 to 1-3 at various concentrations (0.00001-100 μM) in an experimental group and cultured for 72 hours. The culture medium was discarded, washed with PBS, and 100 μl of a 1/10 (v / v) dilution prepared by adding a cell culture medium to an EZ-Cytox reagent containing a water-soluble tetrazolium salt was added, and the mixture was incubated for 3 hours Lt; / RTI > After the reaction was completed, the absorbance of the cells was measured at a wavelength of 450 nm using a spectrophotometer. The relative absorbance at the time of treating the compound of Formulas 1-1 to 1-3 was shown based on the absorbance at 100 DMSO treatment.

The results are shown in Table 1.

Cell survival rate by concentration of sulfonamide compound of formula (1) Concentration (μM) Relative Cell Survival (%) (1-1) 1-2 1-3 100 - 10.87 72.45 10 62.4 100.34 88.42 8 - 107.22 - 4 - 100.09 - 2 - 102.67 - One - 102.90 87.28 0.1 - 97.86 100.83 0.01 - 102.12 114.39 0.001 - 100.04 111.70 0.0001 - 99.57 108.17 0.00001 - 100.44 118.96 The control (DMSO) 100.00 100.00 100.00

As shown in Table 1, the sulfonamide compound of formula (1) exhibits excellent cell survival rate at a concentration of 0.00001 μM or more and less than 100 μM, indicating that the heparin is not toxic to hepatocytes, and thus the safety is high.

Example 2 Measurement of hepatitis C virus genome replication inhibitory activity of the sulfonamide compound of the present invention

In order to measure the hepatitis C virus genome replication inhibitory activity of the sulfonamide compound of the present invention, the following experiment was conducted.

2-1. Preparation of reporter viruses to measure genomic replication of hepatitis C virus

To measure genomic replication of hepatitis C virus, FL-J6 / JFH-5C19Rluc2AUbi HCV replicon belonging to genotype 2a was used as a reporter virus. The genome structure of the reporter virus is shown in Fig.

As shown in FIG. 1, the reporter virus includes the entire genome of the hepatitis C virus belonging to genotype 2 as a cDNA and contains the internal ribosome entry site (IRES) A Ubi sequence of a protein of the foot and mouth disease virus 2A is attached between the core protein of Renilla luciferase and the self cleaving of the FL-J6 / JFH-5C19Rluc2AUbi When HCV RNA obtained by in-vitro transcription of a plasmid with T7 RNA polymerase is transfected into Huh7.5 cell line of liver cancer cell line, Produces multiple proteins (polyproteins) through translation using the ribosome entry site (IRES). Among the multiple proteins produced, Renilla luciferase is separated from virus non-structural proteins with the aid of the Ubi sequence of the Salmonella virus 2A protein cut by itself, and the activity of the isolated Renilla luciferase is measured indirectly, It becomes possible to measure viral RNA genomic replication.

2-2. Measurement of hepatitis C virus genome replication inhibitory activity of the sulfonamide compound of formula (1)

Huh7.5 human liver cancer cells were trypsinized and resuspended in PBS solution to a cell density of 1.5X10 < ⁷ > cells / ml. A total of 5 μg of in vitro transfected FL-J6 / JFH-5C19Rluc2AUbi RNA was mixed with 400 μl PBS buffer containing Huh7.5 liver cancer cells and placed in a 2-mm-gap cuvette (BTX). The FL-J6 / JFH-5C19Rluc2AUbi RNA was transfected into Huh7.5 human liver cancer cells by applying a pulse of 5 times for 99ms at 0.82kV with a BTX-830 electric perforator. Six hours after the above-mentioned electroporation, the cell was treated with DMSO as a control group and a sulfonamide-based compound represented by Formulas 1-1 to 1-3 at various concentrations (0.00001 to 100 μM) Lt; / RTI > Then, renal luciferase assay was performed.

First, the culture medium of the cells treated with the compound was discarded, and the wells to which the cells were attached were washed with 20 μl of cell lysis buffer, and the cells were allowed to stand in ice for 20 minutes. A diluted solution obtained by diluting a Renilla luciferase substrate 100 times with Renilla luciferase buffer was prepared, and 100 μl of the diluted solution was added to each well. The luminescence of Renilla luciferase was measured by setting the integration time to 10 seconds. Relative luminescence at the time of treatment with the sulfonamide compound of the general formulas 1-1 to 1-3 is shown based on the degree of luminescence at the time of DMSO treatment as 100.

The results are shown in Table 2.

Genome copy inhibition activity of the sulfonamide compound of formula (1) Concentration (μM) Relative viral genome replication (%) (1-1) 1-2 1-3 100 - 0.47 25.39 10 38.0 6.59 39.82 8 - 12.50 - 4 - 47.96 - 2 - 68.52 - One - 233.87 68.64 0.1 - 220.02 94.36 0.01 - 121.66 98.48 0.001 - 106.55 99.36 0.0001 - 116.79 112.67 0.00001 - 104.49 114.77 The control (DMSO) 100.00 100.00 100.00

As shown in Table 2, the sulfonamide compound of Formula 1 of the present invention exhibits excellent activity of inhibiting replication of the HCV genome at a concentration of 1 to 100 μM, thereby showing excellent effect of inhibiting the proliferation of HCV .

Example 3. Estimation of Therapeutic Coefficient of the Sulfonamide Compound of the Present Invention

In order to confirm the effectiveness of the sulfonamide-based compound against hepatitis C, the cell survival rate and genome replication inhibition rate of sulfonamide-based compounds of Formulas 1-2 and 1-3 are shown in FIGS. 2 and 3, The treatment coefficients according to the following Equation 1 were calculated according to the concentrations of the sulfonamide-based compounds 1-1 to 1-3, and the results are shown in Table 3 below.

[Equation 1]

Therapeutic coefficient = cell viability / degree of genome replication (unit:% /%)

The therapeutic coefficient of the sulfonamide compound of formula (1) Concentration (μM) Treatment coefficient (1-1) 1-2 1-3 100 - 22.92 2.85 10 1.6 15.22 2.22 8 - 8.58 - 4 - 2.09 - 2 - 1.50 - One - 0.44 1.27 The control (DMSO) 1.00 1.00 1.00

As shown in Figs. 2, 3 and 3, when the sulfoneamide-based compounds of Formulas 1-1 to 1-3 were treated at various concentrations, the cell survival rate of hepatocytes was kept high, and the degree of genome replication was greatly decreased Respectively. From these results, it can be seen that the sulfonamide compound of the present invention has high safety and excellent effect of selectively inhibiting genome replication.

Examples of formulations for the composition of the present invention are illustrated below.

Formulation Example. Preparation of pharmaceutical preparations

1. Manufacturing of powder

100 mg of the sulfonamide compound of formula (1)

Lactose 200mg

The above components were mixed and packed in airtight bags to prepare powders.

2. Preparation of tablets

100 mg of the sulfonamide compound of formula (1)

100 mg of corn starch

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

100 mg of the sulfonamide compound of formula (1)

100 mg of corn starch

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

Claims (4)

A pharmaceutical composition for the prophylaxis or treatment of hepatitis C comprising as an active ingredient at least one sulfonamide compound selected from the group consisting of the compounds represented by the following Formulas 1-1 to 1-3 or a pharmaceutically acceptable salt thereof: Composition:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

delete delete The method according to claim 1, wherein the at least one sulfonamide compound selected from the group consisting of the compounds represented by Formulas 1-1 to 1-3 has a selective inhibitory activity of RNA genome replication of hepatitis C virus A pharmaceutical composition for the prevention or treatment of hepatitis C.

Images