KR100638709B1 - N-substituted sulfamoylbenzoic acid derivatives, preparation method thereof and pharmaceutical composition for antiviral comprising the same - Google Patents

Abstract

The present invention relates to an N -substituted-sulfamoylbenzoic acid derivative useful as an antiviral agent, and more particularly, a novel N represented by the following Chemical Formula 1 having an excellent antiproliferative effect of Hepatitis C virus (HCV). The present invention relates to a -substituted-sulfamoylbenzoic acid derivative, a pharmaceutically acceptable salt thereof, a preparation method thereof, and an antiviral pharmaceutical composition comprising the compound as an active ingredient. The novel N -substituted-sulfamoylbenzoic acid derivatives represented by the general formula (1) according to the present invention are excellent in inhibiting the proliferation of hepatitis C virus (HCV) and have low toxicity, thus preventing and treating hepatitis C. It can be usefully used as.

[Formula 1]

In Formula 1, R ₁ represents a hydroxyl group or a -NR ₂ R ₃ group.

In the substituent for R ₁ , R ₂ represents a hydrogen atom, a C ₁ -C ₄ straight or crushed alkyl group, or a-(CH ₂ ) _m -R ₄ group, and in the substituent for R ₂ , R ₄ represents a phenyl group or a heterocyclic ring, and m represents an integer of 0 to 4.

And, in the substituents representing R _1, R ₃ is C ₁ - C ₄ straight or branched chain of the alkyl group, or - (CH _{2) n} -R ₅ represents a group, in the substituent indicates the R _3, R ₅ is a C A dialkylamino group of ₂ -C ₆ , a heterocyclic ring, or a heterocyclic ring in which a straight or pulverized alkyl group of C ₁ -C ₄ is substituted, n represents an integer of 0 to 4.

Antiviral agents, N-substituted-sulfamoylbenzoic acid derivatives, hepatitis C virus, HCV, pharmaceutically acceptable salts, antivirals, pharmaceutical compositions, hepatitis C, prophylaxis, treatment

Description

N-substituted sulfamoylbenzoic acid derivatives, a method for preparing the same, and a pharmaceutical composition for antiviral comprising the same {N-Substituted-sulfamoylbenzoic acid derivatives, method for preparing background and antiviral pharmaceutical composition comprising the same}

The present invention relates to an N -substituted-sulfamoylbenzoic acid derivative useful as an antiviral agent, and more particularly, a novel N represented by the following Chemical Formula 1 having an excellent antiproliferative effect of Hepatitis C virus (HCV). The present invention relates to a -substituted-sulfamoylbenzoic acid derivative, a pharmaceutically acceptable salt thereof, a preparation method thereof, and an antiviral pharmaceutical composition comprising the compound as an active ingredient.

[Formula 1]

In Formula 1, R ₁ represents a hydroxyl group or a -NR ₂ R ₃ group.

In the substituent representing R ₁ , R ₂ represents a hydrogen atom, a C ₁ -C ₄ straight or crushed alkyl group, or a-(CH ₂ ) _m -R ₄ group, and in the substituent representing R ₂ , R ₄ represents a phenyl group or a heterocyclic ring, and m represents an integer of 0 to 4.

And, in the substituents representing R _1, R ₃ is C ₁ - C ₄ straight or branched chain of the alkyl group, or - (CH _{2) n} -R ₅ represents a group, in the substituent indicates the R _3, R ₅ is a C A dialkylamino group of ₂ -C ₆ , a heterocyclic ring, or a heterocyclic ring in which a straight or pulverized alkyl group of C ₁ -C ₄ is substituted, n represents an integer of 0 to 4.

Hepatitis C virus (HCV) (hereinafter referred to as “HCV”) is a major factor in non-A and non-B viral hepatitis, mainly transfusion and regionally specific infections. (community-acquired) When infected with HCV, about 80% of cases develop chronic hepatitis, and about 20% progress to acute hepatitis, which causes cirrhosis and metastasize to liver cancer. According to the study, more than 200 million people are infected with HCV worldwide, estimated at more than 4.5 million people in the United States (up to 15 million), and in Europe alone, more than 5 million people have HCV. Known.

HCV is a virus with a membrane belonging to another Flavivirus family. The membrane contains about 9.5 kb of (+)-RNA (single stranded positive-sense RNA) into the genome, and the RNA genome is divided into a long open frame (ORF) and a non-fractionated portion at the 5 'and 3' ends. Consists of. The ORF is expressed as a polyprotein consisting of 3,010 to 3,040 amino acids by enzymes of the host cell, and three structural proteins and six kinds of proteins by the host cell and the virus's own protease. It is divided into nonstructural proteins. Conserved regions exist at the 5 'and 3' end of the genome, respectively, which are thought to play an important role in viral protein formation and RNA replication.

One long ORF is expressed as a polyprotein and can be expressed through the transcriptional or posttranslational processing, which is the structural antigen core antigen protein (core), the surface antigen protein (E1, E2), and the nonstructural protein NS2 (protein). Degrading enzyme, protease), NS3 (serine protease serine protease, helicase helicase), NS4A (cofactor serine protease cofactor), NS4B (protease cofactor, resistance related), NS5A, NS5B (RNA polymerase RdRp, RNA dependent It is divided into RNA polymerase and the like and each acts on virus growth. In the case of structural proteins, they are divided into parts of core, E1, and E2 by signal peptidase of the host cell, and in the case of non-structural proteins, the virus's own serine protease (NS3) and cofactor (cofactor) , NS2, NS4A, NS4B). The central antigenic protein of the structural protein forms the capsid of the virus together with the surface antigenic protein. Non-structural proteins such as NS3 and NS5B play an important role in the replication of viral RNA (Bartenschager, R , 1997, Molecular targets in inhibition of hepatitis C virus replication, Antivir. Chem. Chemother. 8: 281-301).

The 5 'and 3' end portions of the viral RNA, like other flaviviruses, contain the same conserved untranslational region (UTR), which is known to play an important role in the growth of the virus. At the 5 'end, there is a 5'-UTR consisting of 341 nucleotides, which consists of four stem and loop (I, II, III, IV) structures, which are responsible for the translation process for protein expression. It acts as an internal ribosome entry site (IRES) necessary for. In particular, stem III, which has the largest and most stable structure and has a conserved sequence, has been reported as the most essential part for ribosomal attachment. In addition, the transcription process is initiated from the AUG present in the single RNA region of stem IV and the protein of the virus is known to be expressed (Stanley, M. Lemon and Masao Honda, 1997, Internal ribosome entry sites within the RNA genomes of hepatitis C). virus and other Flaviviruses, seminars in Virology 8: 274-288).

In addition, there is a 3'-UTR consisting of 318 nucleotides at the 3 'end, which is known to play an important role in initiating the binding of NS5B, an enzyme essential for RNA replication. This part can be divided into three different parts depending on the nucleotide sequence and the three-dimensional structure. That is, it is divided into -X-tail-5 'portion consisting of 98 nucleotides (98nt.) From the 5' end, -poly (U)-portion continuously presenting UTP, and three portions of 3'-UTR-. . X-tail-5 'is composed of 98 nucleotides with very conserved nucleotide sequence, and consists of three stem and loop structure and has a stable three-dimensional structure. In addition, the -poly (U)-moiety has been reported to facilitate the action of RNA polymerase by inducing pyrimidine tracks. In addition, the 3'-UTR part has a three-dimensional structure of the loop plays an important role in the attachment of NS5B, but the structure is not stable. This 3 'end region is known to form an essential structure for attachment of NS5B when RNA replication begins (Yamada et al ., 1996, Genetic organization and diversity of the hepatitis C virus genome, Virology 223: 255-281).

Among the various enzymes of HCV, NS5B is very important because it directly affects RNA replication. NS5B is an enzyme of 591 amino acids and has a molecular weight of 68 kDa. In the NS5B enzyme, there are two regions of the RBD (RNA-binding domain) 1 and 2 which are essential for the attachment of the RNA required for the reaction. RBD1 exists between amino acids 83 and 194 and RBD2 exists between amino acids 196 and 298. Essential amino acids essential for RNA attachment and activity are Asp 220, 283 Gly, 317 Gly, 318 Asp, 319 Asp, and 346 Lys. In addition, the enzyme can proceed polymerization without the presence of other primers in the presence of its own RNA template (Lohmann, V. et al ., 1997, Biochemical properties of hepatitis C virus NS5B RNA dependent RNA polymerase). and identification of amino acid sequence motifs essential for enzymatic activity, J. virol. 71: 8416-8428).

HCV was isolated from the RNA genome by molecular cloning in 1989 (Choo, QL, et al. , 1989, Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis) genome.Science 244: 359-362). Since then, molecular and biological research on HCV has been conducted, but there are limitations due to the lack of effective cell culture systems and animal models. Recently, however, hepatoma cell lines that can reliably replicate HCV have been developed to solve some of these problems (Lohmann, V., F. Korner, JO Koch, U. Herian, L. Theilmann, R. Bartenschlager). , 1999, Replication of subgenomic hepatitis c virus RNAs in a hepatoma cell line.Science 285: 110-113).

To date, no excellent vaccine or effective treatment for HCV exists. Therefore, many pharmaceutical companies and research institutes around the world are developing HCV therapy. HCV has a more even distribution worldwide than HBV, and the rate of liver cirrhosis and metastasis to liver cancer is much higher than that of HBV. And although the rate of progression to chronic hepatitis is high, studies on these infection mechanisms are still ongoing. In addition, hepatitis C can be infected not only by transfusion but also by intravenous drug use or tattooing, but mainly by direct blood contact. However, in 40% to 50% of cases, the path of infection is not related to the known factors, and when hepatitis is developed by HCV through these pathways, most patients progress to chronic and then develop cirrhosis, which has a great involvement in developing liver cancer. I think there will be. Therefore, the development of effective vaccines and treatments is urgently needed. HCV has various genotypes and mutations between strains.In case of chronic hepatitis caused by HCV, reinfection and coinfection are caused by genetic variants. You may have a back. As a result, the development of an effective vaccine for HCV was difficult. Although alpha-interferon is used as a treatment method, the effect varies depending on the genotype of HCV and in most cases recurs when discontinued, so it is an inhibitor that binds only to specific proteins of HCV to prevent HCV from replicating. Developing one could be one of the treatments. The best targets in these studies are HCV NS3 protease / helicase and NS5B RNA polymerase. These enzymes are essential for their proliferation but are not necessary for host cells, and thus are useful for the development of anti-HCV agents. Therefore, since NS5B (RNA dependent RNA polymerase (replicase)) of HCV is an enzyme of HCV essential for proliferation, it may be a good target for treatment to inhibit the growth of HCV.

Until now, it is difficult to prevent vaccines, and treatment with α-interferon and ribavirin has been carried out, but the effect is low because the treatment rate is low and side effects appear. In the case of interferon therapy, there is about 25% of cases that do not respond at all, and about 25% of cases temporarily relapse. In about 50% of patients, ALT levels remain normal and HCV RNA becomes negative until the end of treatment. Of these, about 50% relapse within 3-6 months after the end of treatment. It shows a sustained response where the therapeutic effect is maintained. In addition, there are six genotypes (subtypes) in the hepatitis C virus, the most type 1b in Korea does not respond better to the treatment of interferon than type 2,3. For this reason, the combination therapy with ribavirin has been shown to be twice as effective, but the use of ribavirin alone alone has little effect, and red blood cells are destroyed, resulting in side effects such as anemia. It is known to prescribe it when there is no response or relapse in treatment. Therefore, until now, antiviral drugs for treating hepatitis C by specifically acting on HCV and inhibiting proliferation have not been developed.

Accordingly, the present invention searches for a substance that inhibits the activity of recombinantly expressed HCV RNA polymerase (NS5B, RNA polymerase), thereby exhibiting excellent antiviral activity against HCV, and non-nucleoside compounds having low toxicity and side effects. The purpose was to develop.

The present inventors have found that a novel represented by the sought results, the formula (1) to develop a compound exhibiting excellent antiviral activity against HCV for the purpose of development of side effects and toxicity are less resistant virus emerged lower new hepatitis C therapeutic agent N A -substituted-sulfamoylbenzoic acid derivative was synthesized and the present invention was completed by revealing that the substance is excellent in inhibiting the proliferation of HCV.

It is an object of the present invention to provide novel N -substituted-sulfamoylbenzoic acid derivatives, pharmaceutically acceptable salts thereof and methods for their preparation.

Another object of the present invention is to provide a pharmaceutical composition for the treatment and prevention of hepatitis C, the compound as an active ingredient and less side effects and economical.

In order to achieve the above object, the present invention provides a novel N -substituted-sulfamoylbenzoic acid derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R ₁ represents a hydroxyl group or a -NR ₂ R ₃ group.

In the substituent for R ₁ , R ₂ represents a hydrogen atom, a C ₁ -C ₄ straight or crushed alkyl group, or a-(CH ₂ ) _m -R ₄ group, and in the substituent for R ₂ , R ₄ represents a phenyl group or a heterocyclic ring, and m represents an integer of 0 to 4.

And, in the substituents representing R _1, R ₃ is C ₁ - C ₄ straight or branched chain of the alkyl group, or - (CH _{2) n} -R ₅ represents a group, in the substituent indicates the R _3, R ₅ is a C A dialkylamino group of ₂ -C ₆ , a heterocyclic ring, or a heterocyclic ring in which a straight or pulverized alkyl group of C ₁ -C ₄ is substituted, n represents an integer of 0 to 4.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Compounds of formula (1) may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, benzenesulfonic acid, glutamic acid or aspartic acid Can be used.

In another aspect, the present invention provides a method for preparing an N -substituted-sulfamoylbenzoic acid derivative of Formula 1 represented by the following Scheme 1.

Scheme 1

In Reaction Scheme 1, R ₂ represents a hydrogen atom, a C ₁ -C ₄ linear or crushed alkyl group, or a-(CH ₂ ) _m -R ₄ group, and in the substituent representing R ₂ , R ₄ is a phenyl group, or Heterocyclic ring, m shows the integer of 0-4.

And R ₃ represents a C ₁ -C ₄ linear or crushed alkyl group, or a-(CH ₂ ) _n -R ₅ group, and in the substituent representing R ₃ , R ₅ is a C ₂ -C ₆ dialkylamino group , Heterocyclic ring, or a heterocyclic ring substituted with a C ₁ -C ₄ linear or crushed alkyl group, n represents an integer of 0 to 4.

In the preparation method of the present invention, as shown in Scheme 1,

(1) By reacting 4- (chlorosulfonyl) benzoic acid of Chemical Formula 2 with 4- (4-morpholino) aniline of Chemical Formula 3, 4- [ N- [4- (4-morpho) of Chemical Formula 4 Lino) phenyl] sulfamoyl] benzoic acid (a target compound of formula 1 when R ₁ is a hydroxyl group) (step 1); Wow

(2) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid of Formula 4 prepared in Step 1, using pivaloyl chloride, thionyl chloride, or 3,5-dichloro After activation using a general activation reagent such as 2-hydroxybenzenesulfonyl chloride and the like, and then reacted with an appropriate amine compound represented by HNR ₂ R ₃ of Chemical Formula 5, the chemical formula 6 (R ₁ = -NR ₂ R ₃ To prepare an N -substituted-sulfamoylbenzoic acid derivative of the compound of formula (1) in case of (step 2).

4- (chlorosulfonyl) benzoic acid and 4- (4-morpholino) aniline used as starting materials and reactants in Scheme 1 and an amine compound represented by HNR ₂ R ₃ of Formula 5, and Step 2 Pivaloyl chloride, thionyl chloride, or 3,5-dichloro-2-hydroxybenzenesulfonyl chloride and the like used as an activation reagent in the above can be easily purchased and used as a commercially available material.

In addition, the appropriate amine compound represented by HNR ₂ R ₃ of the general formula (5) used in step 2 is a substance for introducing a substituent to the target compound, it is possible to select a suitable reagent according to the type of substituent, Those skilled in the art can easily select and use.

In more detail the preparation method of step 1, pyridine, N, N -dimethylaniline, N, N -diethylaniline, 2,6-lutidine, 2,4,6-collidine, 2,6 Dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, N, N -dimethylacetamide, dimethyl sulfoxide, in the presence of relatively basic organic bases such as -di-tert-butyl-4-methylpyridine, In organic solvents such as 1-methyl-2-pyrrolidinone, ethyl ether, isopropyl ether, tetrahydrofuran, acetone and the like, they proceed well within 5 hours in the temperature range of 0 ° C-35 ° C.

The preparation method of step 2 will be described in more detail. Triethylamine, tribenzylamine, N, N -diisopropylethylamine, 4-methylmorpholine, 4- ethylmorpholine , 1-methylpiperidine of 1-ethyl piperidine, in the presence a strong basic organic base, such as 1,1,3,3-tetramethylguanidine, dichloromethane, chloroform, acetonitrile, N, N - dimethylformamide, N, N - dimethyl It proceeds well in single or mixed solvents of common organic solvents such as acetamide, dimethylsulfoxide, 1-methyl-2-pyrrolidinone, ethyl ether, isopropyl ether, tetrahydrofuran, acetone, and the like.

The reaction of step 2 proceeds well within 1 hour to 30 hours within the temperature range of 0 ° C-80 ° C, depending on the reactivity of the selected amine compound represented by HNR ₂ R ₃ of Formula 5 and the type of solvent used. do.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing hepatitis C comprising N -substituted-sulfamoylbenzoic acid derivative of Formula 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound of formula 1 of the present invention can be administered orally or parenterally during clinical administration as a therapeutic agent for hepatitis C, and can be used in the form of general pharmaceutical preparations. That is, the compound of formula 1 of the present invention may be administered in various dosage forms, orally and parenterally (preferably as an injection) during actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents. And diluents or excipients such as disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, or the like in one or more compounds of formula (I). Sucrose (Sucrose) or lactose (Lactose), gelatin and the like are mixed and prepared. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used.

The effective dose of the compound of formula 1 of the present invention may be appropriately selected in consideration of sex, age, patient's condition, etc., but is generally 10-1000 mg / day, preferably 20-500 mg / day in adults, It may be administered in divided doses 1-3 times a day.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl] benzoic acid Manufacturing.

4 g of 4- (4-morpholino) aniline and 5.45 ml of pyridine were added to 100 ml of dichloromethane, and the mixture was cooled to 0 ° C. 4.95 g of 4- (chlorosulfonyl) benzoic acid were added and reacted at 0 ° C-5 ° C for 2 hours, followed by further reaction at 20 ° C-30 ° C for 3 hours. The solvent was concentrated under reduced pressure, and the residue was crystallized from 80 ml of methanol. 120 ml of water were added and stirred at room temperature for 3 hours, followed by filtration and washing with 5 ml of methanol and 25 ml of water mixed solvent to separate the solid. In order to refine | purify the obtained solid, solid was put into the mixed solvent of 30 ml of methanol and 150 ml of water, 6.6 ml of 3N-sodium hydroxide aqueous solution was added, and it stirred for 2 hours. The insoluble solid was filtered and washed with 30 ml of water to remove impurities, and then crystals were precipitated by slowly adding 6.6 ml of 3N-hydrochloric acid to the purified filtrate. After stirring for 4 hours at room temperature, filtered and washed with 40 ml of water, the obtained crystals were vacuum dried at 40 ℃-50 ℃ to give 6.51 g (yield 80%) of the title compound.

m.p. : 256-258 ℃ (dec.)

¹ H-NMR (DMSO-d ₆ ), ppm: δ 3.00 (t, 4H), 3.68 (t, 4H), 6.78 (d, 2H), 6.85 (d, 2H), 7.77 (d, 2H), 8.04 (d, 2H), 9.98 (s, 1H)

Example 2 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′, N ′ -(Dimethyl) benzamide Manufacturing.

To 50 ml of dichloromethane, 1 g of 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid prepared in Example 1 and 0.43 ml of triethylamine were added successively, followed by cooling to 0 ° C. It was. 0.36 ml of pivaloyl chloride was slowly added at 0 ° C-5 ° C and reacted at 0 ° C-5 ° C for 30 minutes, followed by further reaction at 20 ° C-25 ° C for 2 hours. 0.25 g of dimethylamine hydrochloride and 0.85 ml of triethylamine were added sequentially at 0 ° C-5 ° C, and reacted at 0 ° C-5 ° C for 1 hour. After completion of the reaction, the solvent was concentrated under reduced pressure, and the residue was crystallized from 30 ml of methanol and 90 ml of water. After stirring for 2 hours at room temperature, the mixture was filtered, washed with 20 ml of water, and the obtained crystals were dried in vacuo at 35 ° C-45 ° C to obtain 0.83 g (yield 77%) of the title compound.

m.p. : 200-201 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.86 (s, 3H), 2.97-3.02 (m, 7H), 3.68 (t, 4H), 6.80 (d, 2H), 6.90 (d, 2H) , 7.51 (d, 2H), 7.69 (d, 2H), 9.92 (s, 1H)

Example 3 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -[2- (4-morpholino) ethyl] benzamide Manufacturing.

To 60 ml of dichloromethane, 1 g of 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid prepared in Example 1 and 0.85 ml of triethylamine were added sequentially to dissolve it. After adding 0.76 g of dichloro-2-hydroxybenzenesulfonyl chloride, the mixture was reacted at 20 ° C.-30 ° C. for 2 hours. 0.44 ml of 4- (2-aminoethyl) morpholine was added to the reaction solution, and further reacted at 20 ° C-30 ° C for 3 hours. The solvent was concentrated under reduced pressure, and the residue was crystallized from 20 ml of methanol and 80 ml of water. After stirring at room temperature for 4 hours, the mixture was filtered, washed with 30 ml of water, and the obtained crystals were dried in vacuo at 40 ° C-50 ° C to obtain 1.02 g (yield 78%) of the title compound.

m.p. : 208-210 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.45 (m, 6H), 2.99 (t, 4H), 3.38 (m, 2H), 3.56 (t, 4H), 3.67 (t, 4H), 6.78 (d, 2H), 6.89 (d, 2H), 7.73 (d, 2H), 7.90 (d, 2H), 8.58 (t, 1H), 9.92 (s, 1H)

Example 4 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -[(2-pyridyl) methyl] benzamide Manufacturing.

To a mixed solvent of 40 ml of chloroform and 40 ml of acetonitrile, 1.2 g of 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid prepared in Example 1 were sequentially added to 1.0 ml of triethylamine. The solution was dissolved, and 0.91 g of 3,5-dichloro-2-hydroxybenzenesulfonyl chloride was added thereto, followed by reaction at 20 ° C.-30 ° C. for 2 hours. 0.4 ml of 2- (aminomethyl) pyridine was added to the reaction solution, and the mixture was slowly heated to further react at 50 ° C-60 ° C for 24 hours. The solvent was concentrated under reduced pressure, and the residue was crystallized from 25 ml of methanol and 75 ml of water. After stirring for 5 hours at room temperature, filtered and washed with 30 ml of water, the obtained crystals were vacuum dried at 40 ℃-50 ℃ to give 1.28 g (yield 85%) of the title compound.

m.p. : 202-203 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 3.10 (t, 4H), 3.83 (t, 4H), 4.74 (d, 2H), 6.66 (s, 1H), 6.74 (d, 2H), 6.93 (d , 2H), 7.23-7.34 (m, 2H), 7.68-7.76 (m, 4H), 7.89 (d, 2H), 8.55 (m, 1H)

Example 5 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -[2- (3-pyridyl) ethyl] benzamide Manufacturing.

To 60 ml of dichloromethane, 1 g of 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid prepared in Example 1 and 0.85 ml of triethylamine were added sequentially to dissolve it. After adding 0.76 g of dichloro-2-hydroxybenzenesulfonyl chloride, the mixture was reacted at 20 ° C.-30 ° C. for 2 hours. 0.36 ml of 3- (2-aminoethyl) pyridine was added to the reaction solution, and further reacted at 20 ° C-30 ° C for 15 hours. The solvent was concentrated under reduced pressure, and the residue was crystallized from 20 ml of methanol and 50 ml of water. After stirring at room temperature for 4 hours, the mixture was filtered, washed with 20 ml of water, and the obtained crystals were dried in vacuo at 40 ° C-50 ° C to obtain 1.03 g (yield 80%) of the title compound.

m.p. : 204-206 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.83 (t, 2H), 2.99 (t, 4H), 3.47 (q, 2H), 3.68 (t, 4H), 6.78 (d, 2H), 6.89 (d, 2H), 7.29 (m, 1H), 7.64 (d, 1H), 7.72 (d, 2H), 7.86 (d, 2H), 8.40 (m, 2H), 8.74 (t, 1H), 9.92 ( s, 1 H)

Example 6 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -[2- (4-pyridyl) ethyl] benzamide Manufacturing.

The target compound was obtained in the same manner as in Example 5 using 4- (2-aminoethyl) pyridine instead of 3- (2-aminoethyl) pyridine.

Yield: 76%

m.p. : 222-225 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.86 (t, 2H), 2.99 (t, 4H), 3.49 (q, 2H), 3.68 (t, 4H), 6.78 (d, 2H), 6.89 (d, 2H), 7.25 (d, 2H), 7.72 (d, 2H), 7.86 (d, 2H), 8.45 (d, 2H), 8.75 (t, 1H), 9.93 (s, 1H)

Example 7 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -[(5-methylpyrazin-2-yl) methyl] benzamide Manufacturing.

2- (aminomethyl) -5-methylpyrazine was used in place of 2- (aminomethyl) pyridine to carry out the same procedure as in Example 4 to obtain the target compound.

Yield: 88%

m.p. : 227-230 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.46 (s, 3H), 2.99 (t, 4H), 3.67 (t, 4H), 4.54 (d, 2H), 6.78 (d, 2H), 6.89 (d, 2H), 7.74 (d, 2H), 7.96 (d, 2H), 8.47 (m, 2H), 9.31 (t, 1H), 9.93 (s, 1H)

Example 8 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′, N ′ -Bis [(2-pyridyl) methyl] benzamide Manufacturing.

To 60 ml of dichloromethane, 1 g of 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid prepared in Example 1 and 0.43 ml of triethylamine were added successively, followed by cooling to 0 ° C. It was. 0.36 ml of pivaloyl chloride was slowly added at 0 ° C-5 ° C and reacted at 0 ° C-5 ° C for 30 minutes, followed by further reaction at 20 ° C-25 ° C for 2 hours. After the reaction solution was cooled to 0 ° C., 0.55 ml of 2,2′-dipicolylamine and 0.43 ml of triethylamine were sequentially added at 0 ° C.-5 ° C., and reacted at 0 ° C.-5 ° C. for 30 minutes. The reaction was carried out at -25 ° C for 20 hours. After completion of the reaction, the reaction solution was washed twice with 60 ml of water, and the separated organic layer was concentrated under reduced pressure. The residue was dissolved in 5 ml of dichloromethane and crystallized by adding 25 ml of isopropyl ether and 25 ml of hexane. After stirring at room temperature for 3 hours, the mixture was filtered, washed with 10 ml of hexane, and the obtained crystals were dried in vacuo at 30 ° C-35 ° C to obtain 0.9 g (yield 60%) of the title compound.

m.p. : 84-87 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 3.08 (t, 4H), 3.82 (t, 4H), 4.57 (s, 2H), 4.84 (s, 2H), 6.71 (d, 2H), 6.83 (s , 1H), 6.89 (m, 2H), 7.06 (d, 1H), 7.18 (m, 2H), 7.37 (d, 1H), 7.64 (m, 6H), 8.52 (m, 2H)

Example 9 4- [ N -[4- (4-morpholino) phenyl] sulfamoyl]- N ′ -benzyl- N ′ -[2- (dimethylamino) ethyl] benzamide Manufacturing.

N' -benzyl- N, N -dimethylethylenediamine was used in place of 2,2'-dipicolylamine , and the target compound was obtained in the same manner as in Example 8.

Yield: 82%

m.p. : 108-110 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 1.79 (s, 4H), 2.15 (m, 3H), 2.43 (br s, 1H), 2.97 (br s, 4H), 3.09 (br s, 1H ), 3.43 (br s, 1H), 3.67 (t, 4H), 4.38 (br s, 1H), 4.69 (br s, 1H), 6.78 (m, 4H), 7.14 (br s, 1H), 7.29 ( m, 4H), 7.59 (m, 4H), 9.87 (s, 1H)

Experimental Example 1 In Vitro for HCV RNA Polymerase (RNA-dependent RNA Polymerase, NS5B) in vitro Inhibition activity investigation

In vitro experiments were performed to investigate the inhibitory effect of the compounds of the present invention on HCV RNA dependent RNA polymerase.

(Recombinant HCV RNA Polymerase Preparation)

HCV RNA polymerase was prepared by the following method. First, cDNA was obtained from the blood of HCV-1b hepatitis C virus patient, and then a recombinant transfer vector inserted into the baculovirus transition vector pVLHIS was prepared by amplifying the NS5B (1773bps) region by PCR. The prepared transfection vector was cotransfected with an insect cell (Sf 9 cell line) together with a wild-type AcNPV vector to produce a recombinant baculovirus with histidine tagged recombinant vector pVLHIS-NS5B. The recombinant baculovirus thus prepared was infected with sufficiently cultured insect cells, incubated for 3-4 days in Grace 'medium to which 10% FBS was added, and only infected cells were centrifuged. The obtained cells were washed three times with PBS, sonicated, and subjected to an affinity column chromatography using NI-NTA His bind resin (Novagen) to prepare pure NS5B protein.

(Preparation of RNA template comprising HCV 3 ′ end (3′-UTR))

RNA template containing HCV 3'-UTR was prepared by the following method. First, cDNA was obtained from the HCV RNA obtained from the blood of hepatitis C patients by PCR, and then DNA fragments containing the 3'-UTR region were obtained using restriction enzyme Eco Ri. RNA from the 3'-UTR was prepared from the DNA through in vitro transcription.

(In vitro Inhibitory Activity Determination of Compounds of the Invention on Recombinant HCV RNA Polymerase)

The method for measuring the in vitro inhibitory activity of the compounds of the present invention on the HCV RNA polymerase used in the present invention is as follows. Prepare wells coated with streptavidin for the sample to be measured, and firstly, in each well, 2X reaction buffer [50 mM Tris-Cl (pH 7.5), 100 mM NaCl, 10 mM MgCl ₂ , 20 mM KCl , 1mM EDTA, 1mM DTT] were added thereto, and 200ng of the purified HCV RNA polymerase was added 10µl each. Then, the final test substance was measured to have a final concentration of 10, 1, 0.1, 0.01µg / ml, respectively. 5 μl each was added, and finally a reaction mixture of DIG- (digoxigenin) -UTP, biotin-UTP, ATP, CTP, GTP, and UTP was mixed as a nucleotide for reaction with HCV 3'-UTR RNA, an RNA template. 10 μl each was added, followed by reaction at 22 ° C. for 60 minutes. The RNA is produced by the activity of HCV polymerase, which contains UTP with biotin attached, so the new RNA binds to the well-coated streptavidin. After the reaction, 200 μl of washing buffer (washing buffer, pH 7.0, Roche) was added to each well to remove impurities remaining unreacted, and then washed three times. Then, 100 μl of a secondary antibody, anti-DIG-POD (peroxidase, Roche) was added thereto, reacted at 37 ° C. for 1 hour, and each well was washed again with a washing buffer. Finally, 100 μl of ABTS ^R (Roche), which is a substrate of POD, was added and reacted for 15-30 minutes, and then the absorbance (OD value) was measured at 405 nm using an ELISA reader (Bio-Tek instrument). At this time, the inhibitory effect on the activity of HCV polymerase was calculated according to the difference in absorbance relative to the control without the sample (positive control). The results are shown in Table 1 below.

     compound       HCV RNA   % Inhibition of polymerase activity    10 μg / ml    1 μg / ml    0.1 μg / ml    0.01 µg / ml     Example 1       93       70       52      24     Example 2       92       74       53      20     Example 3       94       61       40      15     Example 4       98       76       65      50     Example 5       92       68       47      25     Example 6       90       58       39      12     Example 7       97       75       62      47     Example 8       90       68       42      18     Example 9       95       73       48      16

As described above, the compounds of the present invention have an excellent effect of inhibiting the activity of HCV RNA polymerase, which plays an important role in the replication of HCV. Therefore, the compounds of the present invention can suppress the proliferation of HCV and thus are useful as a prophylactic and therapeutic agent for hepatitis C. Can be used.

Experimental Example 2 Cytotoxicity Experiment

In order to determine whether the compounds of Formula 1 exhibit cytotoxicity (Cytotoxicity), in vitro experiments were carried out by MTT assay generally known using Hep G2 cells. As a result, the compounds used in the experiments all had a CC ₅₀ value of 100 µg / ml or more, and were found to be very low toxicity to cells.

As described above, the novel N -substituted-sulfamoylbenzoic acid derivative represented by Chemical Formula 1 according to the present invention has an excellent effect of inhibiting the proliferation of hepatitis C virus (HCV) and low toxicity, It can be usefully used as a prophylactic and therapeutic agent for hepatitis C.

Claims (4)

N -substituted-sulfamoylbenzoic acid derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof. [Formula 1]

In Formula 1, R ₁ represents a hydroxyl group or a -NR ₂ R ₃ group. In the substituent representing R ₁ , R ₂ represents a hydrogen atom, a C ₁ -C ₄ straight or crushed alkyl group, or a-(CH ₂ ) _m -R ₄ group, and in the substituent representing R ₂ , R ₄ represents a phenyl group or a pyridyl group, and m represents the integer of 0-4. And, in the substituents representing R _1, R ₃ is C ₁ - C ₄ straight or branched chain of the alkyl group, or - (CH _{2) n} -R ₅ represents a group, in the substituent indicates the R _3, R ₅ is a C A dialkylamino group of ₂ -C ₆ , a morpholino group, a pyridyl group, or a pyrazyl group in which a straight chain or pulverized alkyl group of C ₁ -C ₄ is substituted, n represents an integer of 0 to 4. The compound according to claim 1, wherein the N -substituted-sulfamoylbenzoic acid derivative of Formula 1 and a pharmaceutically acceptable salt thereof are selected from the following compounds (1) to (9) and pharmaceutically acceptable salts thereof salt. (1) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid (2) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ', N' -(dimethyl) benzamide (3) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ' -[2- (4-morpholino) ethyl] benzamide (4) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ' -[(2-pyridyl) methyl] benzamide (5) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ' -[2- (3-pyridyl) ethyl] benzamide (6) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ' -[2- (4-pyridyl) ethyl] benzamide (7) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ' -[(5-methylpyrazin-2-yl) methyl] benzamide (8) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] -N ', N' -bis [(2-pyridyl) methyl] benzamide (9) 4- [N - [ 4- (4- -morpholino) phenyl] sulfamoyl] - N '- benzyl - N' - [2- (dimethylamino) ethyl] benzamide As shown in Scheme 1 below (1) 4- [ N- [4- (4-morpholino) phenyl of formula 4 by reacting 4- (chlorosulfonyl) benzoic acid of formula 2 with 4- (4-morpholino) aniline of formula 3 ] Sulfamoyl] benzoic acid (the target compound of formula (1) when R ₁ is a hydroxyl group) (step 1); Wow (2) 4- [ N- [4- (4-morpholino) phenyl] sulfamoyl] benzoic acid of Formula 4 prepared in Step 1, using pivaloyl chloride, thionyl chloride, or 3,5-dichloro- After activation by using one of the activating reagents selected from 2-hydroxybenzenesulfonyl chloride, and reacted with an amine compound represented by HNR ₂ R ₃ of Formula 5 to formula (R ₁ = -NR ₂ R ₃ A process for preparing the N -substituted-sulfamoylbenzoic acid derivative of claim 1, comprising the step (step 2) of preparing an N -substituted-sulfamoylbenzoic acid derivative of the target compound of formula (1). Scheme 1

In Scheme 1, R ₂ represents a hydrogen atom, a C ₁ -C ₄ straight or crushed alkyl group, or-(CH ₂ ) _m -R ₄ group, in the substituent representing R ₂ , R ₄ is a phenyl group or a pyri A diyl group is shown and m shows the integer of 0-4. And R ₃ represents a C ₁ -C ₄ linear or crushed alkyl group, or a-(CH ₂ ) _n -R ₅ group, and in the substituent representing R ₃ , R ₅ is a C ₂ -C ₆ dialkylamino group , A morpholino group, a pyridyl group, or a pyrazyl group substituted with a C ₁ -C ₄ straight or crushed alkyl group, and n represents an integer of 0 to 4. A pharmaceutical composition for treating or preventing hepatitis C, comprising the N -substituted-sulfamoylbenzoic acid derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.