KR100469085B1 - 3,4,5-Trifluoropyridine derivatives, its preparation and antiviral pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a 3,4,5-trifluoropyridine derivative useful as an antiviral agent, and a novel 3,4, represented by the following general formula (1) having an excellent antiproliferative effect of Hepatitis C virus (HCV): It relates to a 5-trifluoropyridine derivative, a pharmaceutically acceptable salt thereof, a preparation method thereof and an antiviral pharmaceutical composition comprising the compound as an active ingredient. The novel 3,4,5-trifluoropyridine derivative represented by Formula 1 according to the present invention is excellent in inhibiting the proliferation of hepatitis C virus (HCV) and less toxic, thus preventing and treating hepatitis C. It can be usefully used as.

[Formula 1]

In Chemical Formula 1, R represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group or substituted phenyl group.

Description

3,4,5-trifluoropyridine derivatives, preparation method thereof and pharmaceutical composition for antiviral comprising same {3,4,5-Trifluoropyridine derivatives, its preparation and antiviral pharmaceutical composition comprising the same}

The present invention relates to a 3,4,5-trifluoropyridine derivative useful as an antiviral agent, and more particularly, to a novel novel compound represented by the following Chemical Formula 1 having an excellent antiproliferative effect of Hepatitis C virus (HCV). It relates to a 3,4,5-trifluoropyridine derivative of the 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 represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group, or 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) 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 a recent report, more than 200 million people worldwide are infected with HCV, estimated to be more than 4.5 million in the United States (up to 15 million), and in Europe alone, more than 5 million people are infected with HCV. It is known.

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 regions 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, NS4A (cofactor serine protease cofactor), NS4B (protease cofactor, resistance related), NS5A, NS5B (RNA polymerization) It is divided into enzymes RdRp, RNA dependent RNA polymerase, etc., respectively, and acts on virus proliferation.Structural protein is divided into core, E1, and E2 by signal peptidase of host cell. The virus is isolated by its own serine protease (NS3) and cofactors (cofactors, NS2, NS4A, NS4B). Together with the original protein, the virus forms a capsid, and nonstructural 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 transcription 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 and it is known that the protein of the virus is expressed (Rem: 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, has been reported to have the activity of RNA polymerization and terminal transferase (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 (see 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 studies of HCV have been underway, 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 therapeutics is urgently needed. HCV has many genotypes and mutations between strains, and in cases of chronic hepatitis caused by HCV, reinfection and co-infection are caused by genetic variants. (coinfection) may occur. Because of this, the development of effective vaccines for HCV has been 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, hepatitis C virus has six subtypes (subtype), 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 a good antiviral activity against HCV, nontoxic compounds with low toxicity and side effects (nonnuleoside) The purpose was to develop.

Therefore, the present inventors have tried to develop a compound exhibiting excellent antiviral activity against HCV for the purpose of developing a new hepatitis C therapeutic agent with low side effects and toxicity and low resistance virus resistance. A, 4,5-trifluoropyridine derivative was 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 3,4,5-trifluoropyridine 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.

The present invention provides a 3,4,5-trifluoropyridine derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group, or substituted phenyl group. Preferably, in Chemical Formula 1, R is an isopropyl group, trifluoromethyl group, diethylamino group, 4-pyridyl group, or 3-nitrophenyl 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 another aspect, the present invention provides a method for preparing a 3,4,5-trifluoropyridine derivative represented by the formula (1).

Scheme 1

In Scheme 1, R represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group, or substituted phenyl group.

In the preparation method of the present invention, as shown in Scheme 1, (1) by reacting pentafluoropyridine of Formula 2 with 4- (2-aminoethyl) morpholine of Formula 3, 2- [ Preparing 2- (4-morpholino) ethylamino] -3,4,5,6-tetrafluoropyridine (step 1); (2) Reacting 2- [2- (4-morpholino) ethylamino] -3,4,5,6-tetrafluoropyridine of Chemical Formula 4 prepared in Step 1 with piperazine of Chemical Formula 5 To prepare 2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5, -trifluoropyridine of Chemical Formula 6 (Step 2 ); And (3) 2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5, -trifluoro of Chemical Formula 6 prepared in step 2; Ropyridine is reacted with a suitable acid chloride of Formula 7 or a suitable acid anhydride of Formula 8 to prepare a 3,4,5-trifluoropyridine derivative of Formula 1 (Step 3).

Pentafluoropyridine, 4- (2-aminoethyl) morpholine, piperazine, and acid chlorides, which are used as starting materials and reactants in Scheme 1, can be easily purchased and used as commercially available materials. Reagents such as acid chlorides or acid anhydrides used in 3 are substances for introducing a substituent into the target compound, and an appropriate reagent can be selected according to the type of the substituent, which can be easily obtained by those skilled in the art. You can choose to use it.

In more detail the production method of step 1, in the organic solvent or mixed solvents such as methanol, ethanol, dichloromethane, chloroform, acetonitrile, N, N- dimethylformamide, acetone, triethylamine, In the presence of general organic bases such as N, N -diisopropylethylamine, N -methylmorpholine, 1-methylpiperidine and the like, the process proceeds well within 3 to 15 hours in the temperature range of 0 ° C-30 ° C.

In more detail the preparation method of step 2 , triethylamine, N, In the presence of common organic bases such as N -diisopropylethylamine, N -methylmorpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N -dimethylaniline and the like, It proceeds well within 24 to 48 hours within the temperature range of 35 ° C-70 ° C.

In more detail, the preparation method of step 3, triethylamine, N, N -diisopropylethylamine, in an organic solvent such as dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, In the presence of general organic bases such as N -methylmorpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N -dimethylaniline and the like, a temperature range of 0 ° C-10 ° C Within 2 hours or well within 12 to 24 hours within a temperature range of 20 ° C.-50 ° C.

The present invention also provides a pharmaceutical composition for treating or preventing hepatitis C, comprising 3,4,5-trifluoropyridine 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 in clinical administration as a therapeutic agent for hepatitis C, and can be used in the form of a general pharmaceutical formulation. Shi can be administered in a variety of formulations, oral and parenteral (preferably injectables), when formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. It is prepared by. 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- [2- (4-morpholino) ethylamino] -3,4,5,6-tetrafluoropyridine.

10 g of pentafluoropyridine and 9.1 ml of triethylamine were sequentially added to 100 ml of methanol, and the mixture was cooled to 0 ° C. 8.54 ml of 4- (2-aminoethyl) morpholine was slowly added at 0 ° C-5 ° C and reacted at 0 ° C-5 ° C for 4 hours, and the temperature was gradually raised to be further reacted at 20 ° C-25 ° C for 2 hours. After completion of the reaction, methanol was concentrated under reduced pressure, 10 ml of methanol was added to the residue, and 200 ml of water was gradually added while stirring. The mixture was stirred at 20 ° C. for 1 hour, filtered, and washed with 30 ml of water to obtain a crystal obtained at 45 ° C.-55 ° C. Drying in vacuo gave 15.9 g (yield 96%) of the title compound.

m.p. : 63-64 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 2.48 (t, 4H), 2.63 (m, 2H), 3.59 (m, 2H), 3.72 (t, 4H), 5.46 (s, 1H)

Preparation Example 2 Preparation of 2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine.

To 100 ml of methanol, 10 g of 2- [2- (4-morpholino) ethylamino] -3,4,5,6-tetrafluoropyridine prepared in Preparation Example 1, 5.5 ml of triethylamine, and anhydrous Piperazine 30.9 g was added sequentially, heated, and reacted at 55 ° C-60 ° C for 36 hours. After completion of the reaction, methanol was concentrated under reduced pressure, and the residue was dissolved in 100 ml of dichloromethane and washed twice with 100 ml of water. The residue obtained by concentrating the organic layer under reduced pressure was purified by column chromatography (chloroform: methanol = 8: 1 (volume ratio)), and the resulting solid was concentrated under reduced pressure and dried under vacuum at 40 ° C-50 ° C to obtain 11.26 g (yield 91%). The compound was obtained.

m.p. : 67-69 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 2.46 (t, 4H), 2.59 (t, 2H), 2.95 (t, 4H), 3.27 (t, 4H), 3.46 (m, 2H), 3.71 (t , 4H), 5.06 (s, 1H)

Example 1 2- [2- (4-morpholino) ethylamino] -6- [4- (isopropylcarbonyl) piperazin-1-yl] -3,4,5-trifluoropyridine Preparation of Hydrochloride.

2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine 5 prepared in Preparation Example 2 in 60 ml of dichloromethane. g and 2.4 ml of triethylamine were added and cooled to 0 ° C. 1.7 ml of isobutyryl chloride was slowly added at 0 ° C-5 ° C and reacted at 0 ° C-5 ° C for 30 minutes. After completion of the reaction washed three times with 60 ml of water. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in 50 ml of methanol, and then 11.6 ml of 1.3 N methanolic acid hydrochloric acid was added thereto, followed by stirring at room temperature for 20 minutes. Methanol was concentrated again, and 60 ml of acetone was added to the residue to precipitate crystals. After stirring for 1 hour, the crystals obtained by filtration and washing with 20 ml of acetone were dried in vacuo at 35 ° C.-45 ° C. to obtain 5.76 g (yield 88%) of the title compound in the form of hydrochloride.

m.p. : 197-200 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.13 (d, 6H), 2.76 (m, 1H), 2.94 (m, 2H), 3.31 (m, 6H), 3.59 (m, 6H), 3.99 (m , 4H), 4.28 (t, 2H), 6.32 (s, 1H), 12.88 (s, 1H)

Example 2 2- [2- (4-morpholino) ethylamino] -6- [4- (trifluoroacetyl) piperazin-1-yl] -3,4,5-trifluoropyridine Preparation of Hydrochloride.

2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine 2 prepared in Preparation Example 2 in 40 ml of dichloromethane. g and 1.0 ml of triethylamine were added and cooled to 0 ° C. 0.9 ml of Trifluoroacetic anhydride was slowly added at 0 ° C-5 ° C and reacted at 0 ° C-5 ° C for 1 hour. After completion of the reaction washed three times with 40 ml of water. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was dissolved in 50 ml of methanol, and 4.5 ml of 1.3 N hydrochloric acid solution was added thereto, followed by stirring at room temperature for 20 minutes. The crystals obtained by concentrating methanol were dried in vacuo at 35 ° C.-45 ° C. to obtain 2.55 g (yield 92%) of the title compound in the form of hydrochloride.

m.p. : 185-187 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 2.96 (m, 2H), 3.29-3.42 (m, 6H), 3.61-3.77 (m, 6H), 3.99 (m, 4H), 4.28 (t, 2H) , 6.41 (s, 1H), 12.91 (s, 1H)

Example 3 6- [4- (diethylaminocarbonyl) piperazin-1-yl] -2- [2- (4-morpholino) ethylamino] -3,4,5-trifluoro Preparation of Pyridine.

2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine 2 prepared in Preparation Example 2 above in 60 ml of dichloromethane. g and 1.0 ml of triethylamine were added and cooled to 0 ° C. 0.81 ml of diethylcarbamyl chloride was slowly added at 0 ° C-5 ° C, and the temperature was slowly raised to react at 25 ° C-30 ° C for 15 hours. After completion of the reaction washed three times with 60 ml of water. The residue obtained by concentration of the organic layer under reduced pressure was purified by column chromatography (chloroform: methanol = 12: 1 (volume ratio)), and the residue obtained by concentration under reduced pressure was crystallized with pentane. (Yield 83%) of the title compound was obtained.

m.p. : 73-75 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.11 (t, 6H), 2.47 (t, 4H), 2.60 (t, 2H), 3.19-3.31 (m, 12H), 3.54 (m, 2H), 3.71 (t, 4H), 5.10 (s, 1H)

Example 4 2- [2- (4-morpholino) ethylamino] -6- [4-[(4-pyridyl) carbonyl] piperazin-1-yl] -3,4,5- Preparation of Trifluoropyridine Hydrochloride.

2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine 2 prepared in Preparation Example 2 above in 60 ml of dichloromethane. g and 2.1 ml of triethylamine were added and cooled to 0 ° C. 1.16 g of isicotinoyl chloride hydrochloride was slowly added at 0 ° C-5 ° C, and reacted at 0 ° C-5 ° C for 1 hour. After completion of the reaction washed twice with 40 ml of water. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography (chloroform: methanol = 16: 1 (volume ratio)). The residue obtained by concentrating under reduced pressure was dissolved in 50 ml of methanol, and 4.4 ml of 1.3 N hydrochloric acid solution was added thereto, followed by stirring at room temperature for 20 minutes. The crystals obtained by concentrating methanol were dried in vacuo at 35 ° C.-45 ° C. to obtain 2.17 g (yield 77%) of the title compound in the form of hydrochloride.

m.p. : 208-210 ℃

¹ H-NMR (CDCl ₃ + DMSO-d ₆ ), ppm: δ 3.19-4.16 (m, 20H), 6.74 (s, 1H), 7.65 (d, 2H), 8.83 (d, 2H), 11.89 (s , 1H)

Example 5 6- [4- (3-nitrobenzoyl) piperazin-1-yl] -2- [2- (4-morpholino) ethylamino] -3,4,5-trifluoropyridine Preparation of Hydrochloride.

2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5-trifluoropyridine 2 prepared in Preparation Example 2 above in 60 ml of dichloromethane. g and 1.0 ml of triethylamine were added and cooled to 0 ° C. 1.13 g of 3-nitrobenzoyl chloride was slowly added at 0 ° C-5 ° C, and then reacted at 0 ° C-5 ° C for 1 hour. After completion of the reaction washed twice with 60 ml of water. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The solid obtained was dissolved in 30 ml of methanol, and 4.4 ml of 1.3 N hydrochloric acid solution was added thereto, followed by stirring at room temperature for 20 minutes. The crystals obtained by concentrating methanol were dried in vacuo at 35 ° C.-45 ° C. to obtain 2.46 g (yield 80%) of the title compound in the form of hydrochloride.

m.p. : 227-230 ℃

¹ H-NMR (CDCl ₃ + DMSO-d ₆ ), ppm: δ 3.10 (m, 2H), 3.33-3.69 (m, 10H), 3.87-4.01 (m, 6H), 4.22 (t, 2H), 6.58 (s, 1H), 7.65 (t, 1H), 7.77 (m, 1H), 8.29-8.33 (m, 2H), 12.51 (d, 1H)

Experimental Example 1 In Vitro for HCV RNA Polymerase (RNA-dependent RNA Polymerase, NS5B)

( in vitro ) Inhibitory 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 transfer vector pVLHIS was prepared by amplifying the NS5B (1773bps) region using 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 the wells were washed again with 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 activity inhibitory effect on 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.

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 Isopropyl 93 62 40 18 Example 2 Trifluoromethyl 87 50 28 14 Example 3 Diethylamino 90 64 31 15 Example 4 4-pyridyl 92 60 42 13 Example 5 3-nitrophenyl 98 73 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 CC50 value of 100 µg / ml or more, and proved to be very low toxicity to cells.

As described above, the novel 3,4,5-trifluoropyridine derivative represented by Formula 1 according to the present invention has an excellent effect of inhibiting the proliferation of hepatitis C virus (HCV) and low toxicity. Therefore, it can be usefully used as a prophylactic and therapeutic agent for hepatitis C.

Claims (4)

3,4,5-trifluoropyridine derivatives represented by the following formula (1) and pharmaceutically acceptable salts thereof: [Formula 1] In Formula 1, R represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group, or 3-nitrophenyl group. The 3,4,5-trifluoropyridine derivative and pharmaceutical composition according to claim 1, wherein R is an isopropyl group, trifluoromethyl group, diethylamino group, 4-pyridyl group, or 3-nitrophenyl group. Its salts allowed. By reacting pentafluoropyridine of formula 2 with 4- (2-aminoethyl) morpholine of formula 3, 2- [2- (4-morpholino) ethylamino] -3,4, Preparing 5,6-tetrafluoropyridine (step 1); 2- [2- (4-morpholino) ethylamino] -3,4,5,6-tetrafluoropyridine of Formula 4 prepared in Step 1 was reacted with piperazine of Formula 5 Preparing 2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5, -trifluoropyridine of 6 (step 2); And 2- [2- (4-morpholino) ethylamino] -6- (piperazin-1-yl) -3,4,5, -trifluoropyridine of Formula 6 prepared in Step 2; 3,4 according to claim 1, comprising the step (step 3) of preparing a 3,4,5-trifluoropyridine derivative of formula 1 by reacting with an acid chloride of formula 7 or an acid anhydride of formula 8 Method for the preparation of, 5-trifluoropyridine derivative. Scheme 1 In Scheme 1, R represents a C ₁ -C ₄ straight or crushed alkyl group, C ₂ -C ₆ straight or crushed dialkylamino group, trifluoromethyl group, pyridyl group, or 3-nitrophenyl group. A pharmaceutical composition for treating or preventing hepatitis C, comprising the 3,4,5-trifluoropyridine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.