KR100490893B1 - 2-methoxy-1,3,5-triazine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a 2-methoxy-1,3,5-triazine derivative useful as an antiviral agent, and more particularly, to Chemical Formula 1 having an excellent antiproliferative effect of Hepatitis C virus (HCV). The present invention relates to a novel 2-methoxy-1,3,5-triazine derivative, a pharmaceutically acceptable salt thereof, a preparation method thereof and an antiviral pharmaceutical composition comprising the compound as an active ingredient. The novel 2-methoxy-1,3,5-triazine derivatives represented by the general formula (1) according to the present invention have excellent effects of inhibiting the proliferation of hepatitis C virus (HCV) and are less toxic. It can be usefully used as a prophylactic and therapeutic agent for hepatitis.

[Formula 1]

Wherein R represents a heterocyclic carbonyl group or a substituted phenyl group.

Description

2-methoxy-1,3,5-triazine derivative, preparation method thereof and pharmaceutical composition for antiviral comprising same {2-METHOXY-1,3,5-TRIAZINE DERIVATIVES COMPRISING THE SAME}

The present invention relates to a 2-methoxy-1,3,5-triazine derivative useful as an antiviral agent, and more particularly, to Chemical Formula 1 having an excellent antiproliferative effect of Hepatitis C virus (HCV). The present invention relates to a novel 2-methoxy-1,3,5-triazine derivative, a pharmaceutically acceptable salt thereof, a preparation method thereof and an antiviral pharmaceutical composition comprising the compound as an active ingredient.

[Formula 1]

Wherein R represents a heterocyclic carbonyl group or a substituted phenyl group. 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) In HCV infection, about 80% of the signs of the disease progress to chronic hepatitis and about 20% to acute hepatitis, which causes cirrhosis, and then to liver cancer. According to recent reports, more than 200 million people worldwide are infected with HCV, estimated at more than 4.5 million in the United States (up to 15 million), and more than 5 million in Europe alone. It is known to be a HCV patient.

HCV is a virus with a membrane belonging to a family of Flaviviruses different from HBV. 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, together with the surface antigenic protein, constitutes the capsid of the virus. 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).

At the 5 'and 3' end portions of the viral RNA, there is an untranslational region (UTR), which is conserved in the same way as other flaviviruses, and 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 portion of stem IV to express the protein of the virus (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 composed of 318 nucleotides at the 3 'end, which is known to play an important role in initiating 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 the attachment of NS5B when RNA replication begins (see 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 65 kDa. According to the amino acid sequence it can be divided into six genotypes, including 1a and 1b, and has been reported to have the activity of RNA polymerization and terminal transferase. 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 for RNA attachment and activity include asp 220, 282 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, a hepatoma cell line capable of stably replicating HCV has been developed to solve some of these problems (see 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. 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 recombinant expressed HCV RNA polymerase (NS5B, RNA polymerase), thereby exhibiting excellent antiviral activity against HCV, non-nuleoside with low toxicity and side effects ) To develop a compound.

Accordingly, the present inventors have tried to develop a compound exhibiting excellent antiviral activity against HCV for the purpose of developing a novel hepatitis C therapeutic agent with low side effects and toxicity, and low resistance virus appearance. -Methoxy-1,3,5-triazine derivatives were synthesized and the present invention was completed by revealing that the substance was excellent in inhibiting the proliferation of HCV.

It is an object of the present invention to provide novel 2-methoxy-1,3,5-triazine 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 2-methoxy-1,3,5-triazine derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

[Formula 1]

Wherein R represents a heterocyclic carbonyl group or a substituted phenyl group. Preferably, in Chemical Formula 1, R is a (3-pyridyl) carbonyl group, a (4-pyridyl) carbonyl group, a 2-hydroxyphenyl group, or a 4-hydroxyphenyl group. 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, glutamic acid or aspartic acid can be used. .

In the present invention, according to the kind of R which is a substituent of the 2-methoxy-1,3,5-triazine derivative of the general formula (1) represented by 2-methoxy-1,3, Provided is a method for preparing a 5-triazine derivative.

Scheme 1

In Scheme 1, R represents a heterocyclic carbonyl group.

Scheme 2

In Scheme 2, R represents a substituted phenyl group. In the method of the present invention, when R, which is a substituent of Formula 1, is a heterocyclic carbonyl group, as shown in Scheme 1, (1) 2-methoxy-6- [4- (4- Reaction of morpholino) anilino] -4-chloro-1,3,5-triazine with piperazine of Formula 3 to 2-methoxy-6- [4- (4-morpholino) ) Anilino] -4- (piperazin-1-yl) -1,3,5-triazine (step 1); And (2) reacting the synthetic intermediate of Chemical Formula 4 prepared in Step 1 with an appropriate heterocyclic carbonyl chloride compound to prepare 2-methoxy-1,3,5-triazine derivative of Chemical Formula 1 ( Step 2).

In more detail the preparation method of step 1, triethyl in a mixed solvent of organic solvents such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, acetone, etc. General organics such as amines, N, N -diisopropylethylamine, N -methylmorpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N -dimethylaniline and the like It is preferable to react in the presence of a base, and it progresses well within 1 to 3 hours within the temperature range of 0 degreeC-10 degreeC.

In more detail the preparation method of step 2, in an organic solvent such as dichloromethane, chloroform, acetonitrile, N, N- dimethylformamide, acetone, etc., triethylamine, N, N -diisopropyl The reaction is preferably carried out in the presence of a tertiary organic base such as ethylamine, N -methylmorpholine, 1-methylpiperidine, and the like, and proceeds well within 30 minutes to 2 hours within a temperature range of 0 ° C-10 ° C.

Piperazine and heterocyclic carbonyl chloride compounds used as reactants in Scheme 1 may be easily purchased and used as commercially available materials, and suitable heterocyclic carbonyl chloride compounds used in step 2 The reagent of is a substance for introducing a substituent to the target compound, it is possible to select a suitable reagent according to the type of the substituent, which can be easily selected and used by those of ordinary skill in the art.

In addition, another manufacturing method of the present invention, when R is a substituted phenyl group, the substituent of Formula 1, as shown in the above Scheme 2, 2-methoxy-6- [4- (4- 2-methoxy-1 of formula 1 by directly reacting with substituted phenylpiperazine of Formula 5 without undergoing another intermediate step from morpholino) anilino] -4-chloro-1,3,5-triazine Prepare 3,5-triazine derivatives.

In more detail, the preparation method of Scheme 2 may include triethylamine, N in an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, acetone, and the like. In the presence of common organic bases such as , N -diisopropylethylamine, N -methylmorpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N -dimethylaniline, etc. It is preferable to make it react, and it progresses well within 1 to 5 hours within the temperature range of 40 degreeC-80 degreeC.

The substituted phenylpiperazine compound used as the reactant in Scheme 2 is a substance for introducing a substituent to the target compound, it is possible to select a suitable commercially available reagent according to the type of substituent, which is usually Anyone with knowledge can easily choose and use it.

In addition, the present invention is used as a starting material in the reaction schemes 1 and 2, which is a method for preparing 2-methoxy-1,3,5-triazine derivative of the formula (1) which is the target compound of the present invention, 2 of the formula (2) To provide -methoxy-6- [4- (4-morpholino) anilino] -4-chloro-1,3,5-triazine is provided.

Scheme 3

In more detail, the preparation method of Scheme 3, in an organic solvent such as dichloromethane, chloroform, acetonitrile, acetone, and the like, triethylamine, N, N -diisopropylethylamine, N -methylmorpholine, It is preferable to react in the presence of general organic bases such as 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N -dimethylaniline, and the like, and in a temperature range of 0 ° C-10 ° C. In 2 hours it goes well.

2,4-dichloro-6-methoxy-1,3,5-triazine and 4- (4-morpholino) aniline used as starting materials and reactants in Scheme 3 are commercially available materials. It can be easily purchased and used.

In addition, the present invention provides a pharmaceutical composition for treating or preventing hepatitis C, comprising 2-methoxy-1,3,5-triazine derivative of Formula 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient. to provide.

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.

Preparation Example 1 Preparation of 2-methoxy-6- [4- (4-morpholino) anilino] -4-chloro-1,3,5-triazine.

5 g of 2,4-dichloro-6-methoxy-1,3,5-triazine was added and dissolved in 35 ml of dichloromethane, followed by cooling to 0 ° C. While maintaining the solution at 0 ° C.-5 ° C., a solution of 5.2 g of 4- (4-morpholino) aniline and 4.3 ml of triethylamine was gradually added to 40 ml of dichloromethane, gradually adding 1 to 0 ° C.-5 ° C. The reaction was carried out for a time. After completion of the reaction, dichloromethane as a solvent was concentrated under reduced pressure, crystallized with 60 ml of methanol, stirred for 1 hour, filtered, washed twice with 10 ml of methanol, and the crystals obtained were vacuum dried at 35 ° C.-45 ° C. to obtain 7.96 g ( Yield 89%) of the title compound.

  m.p. : 184-186 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 3.12 (q, 4H), 3.85 (m, 4H), 4.00 (s, 3H), 6.89 (d, 2H), 7.41 (m, 2H)

Preparation Example 2 Preparation of 2-methoxy-6- [4- (4-morpholino) anilino] -4- (piperazin-1-yl) -1,3,5-triazine.

12 g of anhydrous piperazine and 2.1 ml of triethylamine were added to 70 ml of methanol in order to dissolve, and it cooled to 0 degreeC. While maintaining the solution at 0 ° C-5 ° C, 2-methoxy-6- [4- (4-morpholino) anilino prepared in Preparation Example 1 in a mixed solvent of 50 ml of dichloromethane and 10 ml of methanol. ] The solution which melt | dissolved 4.5 g of 4-chloro- 1,3, 5- triazines previously was added gradually, and it reacted at 0 degreeC-5 degreeC for 1 hour. After completion of the reaction, a small amount of precipitated solid was filtered and washed twice with 10 ml of methanol to remove impurities. Dichloromethane and methanol as solvents were concentrated under reduced pressure, and 10 ml of methanol and 70 ml of water were added to the residue, followed by crystallization. The mixture was stirred at room temperature for 1 hour. The precipitated solid was filtered, washed twice with 10 ml of water, and then dried in vacuo at 40 ° C-50 ° C to obtain 4.9 g (yield 94%) of the title compound.

  m.p. : 175-178 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.96 (s, 2H), 2.87 (t, 4H), 3.10 (t, 4H), 3.79-3.91 (m, 9H), 6.88 (m, 2H), 6.99 (s, 1 H), 7.42 (m, 2 H)

Example 1 2-methoxy-6- [4- (4-morpholino) anilino] -4- [4-[(3-pyridyl) carbonyl] piperazin-1-yl] -1 , 3,5-triazine.

To 60 ml of dichloromethane, 2-methoxy-6- [4- (4-morpholino) anilino] -4- (piperazin-1-yl) -1,3,5- prepared in Preparation Example 2 1.5 g of triazine and 1.3 ml of triethylamine were added thereto and dissolved, followed by cooling to 0 ° C. 0.8 g of nicotinoyl chloride hydrochloride was slowly added at 0 ° C-5 ° C, and then reacted at 0 ° C-5 ° C for 30 minutes. After completion of the reaction, 25 ml of methanol and 100 ml of water were sequentially added to the solid obtained by concentrating the solvent, dichloromethane, under reduced pressure, stirred at room temperature for 1 hour, filtered, and washed twice with 10 ml of water. The solid was dried in vacuo at 40 ° C.-50 ° C. to obtain 1.7 g (yield 88%) of the title compound.

  m.p. : 201-203 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.77 (br s, 1H), 3.10 (t, 4H), 3.50 (br s, 1H), 3.84-3.92 (m, 13H), 6.88 (d, 3H) , 7.38 (m, 3H), 7.78 (m, 1H), 8.69 (m, 2H)

Example 2 2-methoxy-6- [4- (4-morpholino) anilino] -4- [4-[(4-pyridyl) carbonyl] piperazin-1-yl] -1 , 3,5-triazine.

Instead of nicotinoyl chloride hydrochloride, isonicotinoyl chloride hydrochloride was used in the same manner as in Example 1 to obtain the target compound.

  Yield: 93%

  m.p. : 176-177 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.70 (s, 2H), 3.11 (s, 4H), 3.40 (s, 2H), 3.85-3.91 (m, 11H), 6.88 (s, 3H), 7.29 (m, 2H), 7.40 (m, 2H), 8.71 (m, 2H)

Example 3 2-methoxy-6- [4- (4-morpholino) anilino] -4- [4- (2-hydroxyphenyl) piperazin-1-yl] -1,3, Preparation of 5-triazines.

To 60 ml of methanol, 1.5 g of 2-methoxy-6- [4- (4-morpholino) anilino] -4-chloro-1,3,5-triazine prepared in Preparation Example 1 and 2- ( 0.9 g of 1-piperazino) phenol and 1.1 ml of pyridine were added sequentially, and then heated to react at 55 ° C.-60 ° C. for 2 hours. After completion of the reaction, the reaction mixture was cooled to room temperature and 120 ml of water was slowly added to precipitate a solid, followed by stirring at 20 ° C-25 ° C for 1 hour. The solid which was filtered and washed twice with 10 ml of water was dried in vacuo at 40 ° C-50 ° C to obtain 1.82 g (yield 84%) of the title compound.

  m.p. : 123-125 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.79 (s, 1H), 2.88 (t, 4H), 3.10 (q, 4H), 3.84 (t, 4H), 3.93 (s, 3H), 3.98 (m , 3H), 6.84-7.13 (m, 7H), 7.41 (m, 2H)

Example 4 2-methoxy-6- [4- (4-morpholino) anilino] -4- [4- (4-hydroxyphenyl) piperazin-1-yl] -1,3, Preparation of 5-triazines.

The target compound was obtained in the same manner as in Example 3 using 4- (1-piperazino) phenol instead of 2- (1-piperazino) phenol.

  Yield: 90%

  m.p. : 113-115 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.76 (s, 2H), 3.05 (t, 3H), 3.11 (t, 4H), 3.85 (t, 4H), 3.93 (s, 3H), 3.96 (m , 3H), 6.76-6.91 (m, 7H), 7.40 (m, 3H)

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.

RNA template preparation comprising HCV 3 'terminus (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 Inhibition 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.

TABLE 1

 Test compound       Substituent (R) HCV RNA Activity inhibition rate of polymerase (%)  10 μg / ml  1 μg / ml  0.1 μg / ml 0.01 µg / ml   Example 1     (3-pyridyl) carbonyl    90    67    54    30   Example 2     (4-pyridyl) carbonyl    99    80    63    45   Example 3     2-hydroxyphenyl    99    75    62    38   Example 4     4-hydroxyphenyl    92    64    52    27

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 2-methoxy-1,3,5-triazine derivative represented by Chemical Formula 1 according to the present invention is excellent in inhibiting the proliferation of hepatitis C virus (HCV). Since it is less toxic, it can be usefully used as a prophylactic and therapeutic agent for hepatitis C.

Claims (5)

2-methoxy-1,3,5-triazine derivatives represented by Formula 1 and pharmaceutically acceptable salts thereof: [Formula 1] In the formula, R represents a (3-pyridyl) carbonyl group, a (4-pyridyl) carbonyl group, a 2-hydroxyphenyl group, or a 4-hydroxyphenyl group. delete As shown in Scheme 1 below, (1) 2-methoxy-6- [4- (4-morpholino) anilino] -4-chloro-1,3,5-triazine of the formula (2) Reaction with piperazine affords 2-methoxy-6- [4- (4-morpholino) anilino] -4- (piperazin-1-yl) -1,3,5-triazine of Formula 4 (Step 1); And (2) reacting the synthetic intermediate of Formula 4 prepared in Step 1 with nicotinoyl chloride hydrochloride or isicotinoyl chloride hydrochloride to prepare 2-methoxy-1,3,5-triazine derivative of formula 1. Method of preparing a 2-methoxy-1,3,5-triazine derivative of claim 1 consisting of a step (step 2): Scheme 1 In Scheme 1, R represents a (3-pyridyl) carbonyl group or a (4-pyridyl) carbonyl group. As shown in Scheme 2 below, without passing through another intermediate step from 2-methoxy-6- [4- (4-morpholino) anilino] -4-chloro-1,3,5-triazine of formula (2) First to prepare 2-methoxy-1,3,5-triazine derivative of Formula 1 by directly reacting with 2- (1-piperazino) phenol or 4- (1-piperazino) phenol of Formula 5 Method for producing 2-methoxy-1,3,5-triazine derivative of claim. Scheme 2 In Scheme 2, R represents a 2-hydroxyphenyl group or 4-hydroxyphenyl group. A pharmaceutical composition for the treatment or prevention of hepatitis C, containing 2-methoxy-1,3,5-triazine derivative represented by the following Chemical Formula 1 and a pharmaceutically acceptable salt thereof as an active ingredient: [Formula 1] In the formula, R represents a (3-pyridyl) carbonyl group, a (4-pyridyl) carbonyl group, a 2-hydroxyphenyl group, or a 4-hydroxyphenyl group.