KR20120071376A - Pharmaceutical composition for preventing or treating hepatitis c comprising sulfonamide compound - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing sulfonamide-based compounds is provided to prevent or treat hepatitis type C with low toxicity to hepatocytes. CONSTITUTION: A pharmaceutical composition for preventing or treating hepatitis type C contains sulfonamide-based compound or pharmaceutically acceptable salt thereof as an active ingredient. In chemical formula 1, R1 is H or halogen; R2 is C1-C10 alkyl or C1-C10 alkoxy; and R3 is -SO_2-NH-(C_6H_3)R4R5 or -CH_2-A-(C6-C20 aryl); and R4 and R5 are independently different or same halogen or CF3.

Description

Pharmaceutical composition for preventing or treating hepatitis C comprising sulfonamide compound}

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

Liver cancer and liver disease are among the top four diseases with the highest number of deaths per 100,000 Korean population, along with cerebrovascular disease, heart disease and respiratory disease. Hepatitis B and C viruses cause the liver cancer and liver disease in Korea. Hepatitis makes up 83% of the total. Among them, hepatitis C virus is a medically important pathogen with about 170 million infected people worldwide. Hepatitis C was classified as a non-A non-B post-transfusion associated hepatitis (NANB) until the mid-1980s, but it was not a new class until 1989. It has been found that the disease caused by the infection of the virus, and since then, active research and development has been progressed. Infection of hepatocytes by the hepatitis C virus is usually fatal because it develops chronic hepatitis and develops into chronic liver disease such as cirrhosis and liver cancer over a long period of 15 to 20 years. , Hepatitis B virus), and 8000 to 10,000 deaths in the United States alone are reported annually due to chronic hepatitis, cirrhosis and liver cancer. In particular, most patients with late hepatitis C die from waiting for liver transplantation without receiving a liver transplant. Statistically, 1.5% of the population in Korea is infected with hepatitis C virus. .

Interferon-alpha and ribavirin, which are currently used as treatments for hepatitis C, are indirect-acting antivirals (IAAs) that indirectly attack viruses through intrinsic immunity promotion and nucleic acid biosynthesis inhibition. However, even if the two drugs are used together, the sustained virologic response (SVR) after 52 weeks of intravenous injection and oral administration is less than 50% depending on the genotype of the virus. In addition, many hepatitis C patients give up treatment due to serious side effects and toxicity such as suicidal thoughts, depression and anemia. Therefore, there is an urgent need for a more effective and safe drug development to replace the antiviral drugs for the existing hepatitis C virus.

The inventors of the present invention, while researching to search for a substance having a prophylactic or therapeutic effect of hepatitis C, the sulfonamide-based compound is very excellent in selectively inhibiting the replication of the genome of HCV, while very low toxicity to hepatocytes It confirmed that it shows clearly, and completed this invention.

Accordingly, the present invention is to provide 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 the prevention or treatment of hepatitis C comprising a sulfonamide compound as an active ingredient.

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

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

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

[Formula 1]

In Formula 1 R ₁ is H or halogen, R ₂ is C ₁ ~ C ₁₀ Alkyl or C ₁ ~ C ₁₀ Alkoxy, R ₃ is -SO ₂ -NH- (C ₆ H ₃ ) R ₄ R ₅ or -CH ₂ -A- (aryl of C ₆ -C ₂₀ ), wherein R ₄ and R ₅ are independently the same 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 to a compound of Formula 1-3.

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

The sulfonamide compounds of the present invention can be used in the form of pharmaceutically acceptable salts such as metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids. 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 salts with suitable organic bases include, for example, trimethylamine,

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, and salts with suitable organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, Salts with 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, for example, salts with arginine, lysine, ornithine and the like, and suitable examples of salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid and the like. Particularly preferred salts include inorganic salts such as alkali metal salts (eg, sodium salts, potassium salts, etc.), alkaline earth metal salts (eg, calcium salts, magnesium salts, barium salts, etc.) when the compound has an acidic functional group therein, And organic salts such as ammonium salts, and when the compound has a basic functional group therein, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, Salts with organic acids such as 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 active ingredient of the composition of the present invention, has very low toxicity against hepatocytes, high safety, and has an excellent effect of inhibiting the replication of the genome of HCV.

The HCV is the only virus classified into the genus hepacivirus among the flaviviridae group, and has a single strand of RNA consisting of about 9600 nucleic acids as a viral genome. The RNA is translated into a polyprotein consisting of about 3000 amino acids in hepatocytes, and the translated polyprotein is a non-structural protein of signal peptidase and virus present in the endoplasmic reticulum of hepatocytes. It is expressed by ten different viral proteins by one NS3 protease. Among the expressed viral proteins, structural proteins such as envelope glycoproteins E1, E2 and capsid proteins core are used to make particles of the virus and NS2, NS3. Non-structural proteins, such as NS4A, NS4B, NS5A, and NS5B, are used to create the viral genome replication complex needed to replicate the virus's genome. HCV replicates the RNA genome of a virus by making a viral genomic clone in the membrane that develops in the endoplasmic reticulum. The viral genome clone maintains an invaginated form that is accumulated in the membrane and creates an independent structure separated from the external environment, expressing the nonstructural proteins of the virus in the structure, and the expressed nonstructural proteins are RNA RNA polymerase of the virus. Together with the NS5B protein, it acts as a genome replicator of the virus. However, it is not known how the virus genome clone is made and maintained. Among the nonstructural proteins of the virus, in particular the NS4B protein, has been found to play a decisive role in creating the membranous web, a multi-vesicular structure essential for the genome replication of the virus in hepatocytes. The structure called the membranous web made by NS4B is believed to provide the physical structure needed to make a viral genome clone. In addition, the NS5A protein assembles the viral genome replicator using non-structural proteins of other viruses, and transfers the resulting viral genome into a structure called a lipid droplet, where the core protein of the virus is combined with the viral genome. It is known that it plays an important role in making practical viral particles. In particular, the protein-protein interaction between the nonstructural proteins of the virus is known to play a decisive role in the production of viral genome clones, but the exact role of the molecule at the molecular level is still unknown.

Types of antiviral agents that can treat viral diseases include indirect-acting antivirals (IAAs) and indirectly attacking viruses, depending on the mechanism of action of the antiviral agents. , DAAs). Interferonalpha, which is secreted by the body when a pathogenic external microorganism such as a virus penetrates into the body, is a representative antiviral intrinsic immune enhancer that indirectly attacks the virus, rather than attacking the virus itself, rather than attacking the virus itself. Because it blocks intracellular proliferation of virus by increasing it, it is effective in many different kinds of viral diseases and shows antiviral activity by regulating the function of protein of host cell in which virus is parasitic. The chance of developing a mutant virus is relatively low. However, since the antiviral specificity, which can only display antiviral activity against a particular virus, is significantly lowered, in order to overcome this, a much larger amount of interferon than the amount of interferon naturally secreted in vivo is treated. In this case, serious side effects and toxicity, such as suicidal thoughts, depression, and anemia, will appear.

On the other hand, anti-viral agents that directly attack the virus, unlike interferon, selectively inhibits only the specific functions of the viral proteins essential in the life history of the virus, thereby exhibiting high anti-viral activity even at relatively low concentrations. In addition, compared to antiviral agents that indirectly attack the virus, it is characterized by low toxicity and high safety. Reverse transcriptase inhibitors and protease inhibitors of human immunodeficiency virus (HIV) are representative antiviral agents that directly attack the virus. It inhibits the proliferation of virus by inhibiting enzyme and protease. However, there is a relatively high possibility that the virus may exhibit resistance and resistance to existing antiviral agents by changing the amino acid sequence of the viral protein targeted by the antiviral agent, and in particular, RNA polymerase of hepatitis C virus ( polymerase has no 5'-3 'endonuclease activity that corrects nucleic acids that are misplaced during the replication of RNA genomes, so that mutant viruses are easily generated during RNA replication of the hepatitis C virus genome. It is easy to be resistant and resistant to antiviral agents that directly attack the virus.

Therefore, the sulfonamide compound, which is an active ingredient of the composition of the present invention, has an excellent effect of selectively blocking genome replication in the RNA genome replication step of the hepatitis C virus, thereby preventing the occurrence of mutant viruses that may exhibit resistance and resistance. While suppressing, it shows less side effects and toxicity. Therefore, the sulfonamide compound of the present invention can be usefully used as a drug for the prevention or treatment of hepatitis C, which is safe and has high specificity for HCV.

The composition of the present invention may include at least one known active ingredient having a prophylactic or therapeutic effect of hepatitis C with a sulfonamide compound as an active ingredient.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as an antioxidant, buffer And other conventional additives such as bacteriostatic agents can 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. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the sulfonamide compound is about 10-100 mg / kg, preferably divided once to several times daily.

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

Hereinafter, preferred examples are provided to aid in understanding 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 Hepatocellular Toxicity of Sulfonamide Compounds of the Present Invention

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

1-1. Hepatotoxicity of Sulfonamide Compounds of Formula 1

In a 96 well-plate, 1.7 × 10 ⁴ to 2.0 × 10 ⁴ Huh7.5 human liver cancer cells per well were added and incubated for 24 hours in a 37 ° C. incubator. As a control group, DMSO (dimethyl sulfoxide) was used as an experimental group, and sulfonamide compounds of Formulas 1-1 to 1-3 at various concentrations (0.00001-100 μM) were treated to the cells and incubated for 72 hours. Discard the culture medium, wash with PBS, and then add 100 μl of the 1/10 (v / v) dilution prepared by adding the cell culture solution to the EZ-Cytox reagent containing a water-soluble tetrazolium salt for 3 hours. Reacted. 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 compound treatment of Formulas 1-1 to 1-3 was shown based on the absorbance at the time of 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 rate (%) Formula 1-1 Formula 1-2 Formula 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 Control (DMSO) 100.00 100.00 100.00

As shown in Table 1, the sulfonamide-based compound of Formula 1 shows very good cell viability at a concentration of 0.00001 μM or more and less than 100 μM, thus showing little safety due to little toxicity to hepatocytes.

Example 2 Determination of Hepatitis C Virus Genome Replication Inhibition Activity of Sulfonamide Compounds 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 performed.

2-1. Preparation of Reporter Virus to Measure Genome Replication of Hepatitis C Virus

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

As shown in FIG. 1, the reporter virus contains the entire genome of hepatitis C virus belonging to genotype 2 in the cDNA state, and the internal ribosome entry site (IRES) of the virus and the virus. Between the core protein is attached a Ubi sequence of Renilla luciferase and a self cleaving foot and mouth disease virus 2A protein, which is FL-J6 / JFH-5C19Rluc2AUbi. When the plasmid was transfected into Huh7.5 cells, a liver cancer cell line, HCV RNA obtained by transcription in vitro using a T7 RNA polymerase was introduced into the RNA of the injected virus. Polyproteins are produced through translation using ribosomal entry sites (IRES). Renilla luciferase in the produced multiprotein is separated from viral nonstructural proteins with the help of the Ubi sequence of the foot disease virus 2A protein, which is cut off by itself, and indirectly in the hepatocyte cell by measuring the activity of the isolated lenilla luciferase. Viral RNA genome replication can be measured.

2-2. Measurement of Hepatitis C Virus Genome Replication Inhibitory Activity of the Sulfonamide Compounds of Formula 1

Huh7.5 human liver cancer cells were trypsinized and resuspended with PBS solution to have a cell density of 1.5 × 10 ⁷ / ml. A total of 5 μg of in vitro transcribed FL-J6 / JFH-5C19Rluc2AUbi RNA was mixed with 400 μl PBS buffer containing Huh7.5 liver cancer cells and placed in 2-mm-gap cuvette (BTX). The FL-J6 / JFH-5C19Rluc2AUbi RNA was transfected into Huh7.5 human liver cancer cells by applying pulses five times for 99 ms at 0.82 kV with a BTX-830 electroporator. After 6 hours of the electroporation, the cells were treated with DMSO, and with the experimental group, sulfonamide compounds of Formulas 1-1 to 1-3 at various concentrations (0.00001-100 μM). Incubated for hours. Then, a Renilla 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 with PBS were washed. Then, 20 μl of cell lysis buffer was added and allowed to stand in ice for 20 minutes. Dilutions of 100-fold dilution of the Renilla Luciferase substrate with Renilla Luciferase Buffer were prepared and 100 μl of the dilution was placed in each well. The luminescence of Renilla Luciferase was measured at an integration time of 10 seconds. The relative luminescence of the sulfonamide compound of Formulas 1-1 to 1-3 was shown based on the luminescence of DMSO treatment of 100.

The results are shown in Table 2.

Genomic Replication Inhibitory Activity of the Sulfonamide Compounds of Formula 1 Concentration (μM) Relative viral genome replication rate (%) Formula 1-1 Formula 1-2 Formula 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 Control (DMSO) 100.00 100.00 100.00

As shown in Table 2, the sulfonamide-based compound of formula 1 of the present invention exhibits an excellent activity of inhibiting the replication of the HCV genome at a concentration of 1 to 100 μM, which is excellent in inhibiting the proliferation of HCV. .

Example 3 Calculation of the Treatment Coefficient of Sulfonamide Compounds of the Present Invention

In order to confirm the effectiveness of the sulfonamide compound against hepatitis C, the cell survival rate and genome replication inhibition rate of the sulfonamide compound of Formulas 1-2 and 1-3 according to the concentration are shown in FIGS. 2 and 3, It is shown in Table 3 to calculate the treatment coefficient according to the formula (1) for each concentration of sulfonamide compounds of 1-1 to 1-3.

[Equation 1]

Treatment coefficient = cell viability / genomic replication degree (unit:% /%)

Therapeutic Factors of Sulfonamide Compounds of Formula 1 Concentration (μM) Treatment coefficient Formula 1-1 Formula 1-2 Formula 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 Control (DMSO) 1.00 1.00 1.00

As shown in Figures 2, 3 and Table 3, when the sulfonamide compounds of Formulas 1-1 to 1-3 are treated at various concentrations, the cell survival rate of hepatocytes is maintained high, and the degree of genome replication is greatly reduced. Check it. From these results, it can be seen that the sulfonamide compound of the present invention has a high safety and an effect of selectively inhibiting genome replication.

Examples of preparations for the compositions of the present invention are illustrated below.

Formulation Example Preparation of Pharmaceutical Formulations

1. Preparation of powder

100 mg of 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 sulfonamide compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

3. Preparation of Capsule

100 mg of sulfonamide compound of Formula 1

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Claims (4)

A pharmaceutical composition for preventing or treating hepatitis C, comprising a sulfonamide compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1, R ₁ is H or halogen,
R ₂ is C ₁ -C ₁₀ alkyl or C ₁ -C ₁₀ alkoxy,
R ₃ is -SO ₂ -NH- (C ₆ H ₃ ) R ₄ R ₅ or -CH ₂ -A- (aryl of C ₆ -C ₂₀ ),
Wherein R ₄ and R ₅ are independently the same or different and are halogen or CF ₃ ,
A is S, O, or NH.
The method according to claim 1, wherein 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, wherein the pharmaceutical composition for preventing or treating hepatitis C.
The method according to claim 1, wherein the sulfonamide compound of Formula 1 is at least one selected from the group consisting of compounds of Formula 1-1 to compounds of Formula 1-3, for the prevention or treatment of hepatitis C Pharmaceutical compositions.
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

According to claim 1, wherein the sulfonamide compound of formula 1 is characterized in that it has a selective inhibitory activity of RNA genome replication of hepatitis C virus, pharmaceutical composition for the prevention or treatment of hepatitis C.

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