KR100502395B1 - 4-[4-(4-Morpholino)anilino]pyrimidine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a 4- [4- (4-morpholino) anilino] pyrimidine derivative useful as an antiviral agent. More particularly, the present invention has excellent antiproliferative effect of Hepatitis C virus (HCV). Novel 4- [4- (4-morpholino) anilino] pyrimidine derivatives represented by the following formula (1), pharmaceutically acceptable salts thereof, preparation methods thereof, and antiviral pharmaceuticals containing the compound as an active ingredient To a red composition. The novel 4- [4- (4-morpholino) anilino] pyrimidine derivatives represented by the general formula (1) according to the present invention are excellent in suppressing the proliferation of hepatitis C virus (HCV) and have low toxicity. Therefore, it can be usefully used as a prophylactic and therapeutic agent for hepatitis C.

[Formula 1]

In Formula 1, R ₁ represents a C ₁ -C ₄ straight or crushed alkoxy group, a -NR ₂ R ₃ group, or a 4- (R ₄ ) -piperazin-1-yl group.

Further, in the substituent group representing the R _1, R ₂ is hydrogen, or C ₁ - a C ₄ represents a straight or branched chain a group, R ₃ is C ₁ - straight or branched chain a group of C _4, C ₁ - C ₄ C ₁ -C ₄ linear or pulverized hydroxyalkyl group, C ₁ -C ₄ linear or pulverized alkyl group substituted with C ₂ -C ₆ linear or pulverized dialkylamino group, or C ₁ -C ₄ substituted with heterocyclic ring A straight chain or pulverized alkyl group of R ₄ represents a straight chain or pulverized alkoxycarbonylmethyl group, a carboxymethyl group, or a —CH ₂ —CO—NH—R ₅ group of C ₁ -C ₄ .

And, in a substituent indicates the R _4, R ₅ is C ₁ - C ₄ linear or grinding the alkyl group, C ₁ - a straight or branched chain of C ₄ hydroxy alkyl group, a C ₃ - a C ₆ cycloalkyl group, or a heteroaryl The cyclic ring represents a substituted C ₁ -C ₄ straight or crushed alkyl group.

Description

 4- [4- (4-morpholino) anilino] pyrimidine derivative, preparation method thereof, and antiviral pharmaceutical composition comprising the same. {4- [4- (4-Morpholino) anilino] pyrimidine derivatives, method for preparing honey and antiviral pharmaceutical composition comprising the same}

The present invention relates to a 4- [4- (4-morpholino) anilino] pyrimidine derivative useful as an antiviral agent. More particularly, the present invention has excellent antiproliferative effect of Hepatitis C virus (HCV). Novel 4- [4- (4-morpholino) anilino] pyrimidine derivatives represented by the following formula (1), pharmaceutically acceptable salts thereof, preparation methods thereof, and antiviral pharmaceuticals containing the compound as an active ingredient To a red composition.

[Formula 1]

In Formula 1, R ₁ represents a C ₁ -C ₄ straight or crushed alkoxy group, a -NR ₂ R ₃ group, or a 4- (R ₄ ) -piperazin-1-yl group.

Further, in the substituent group representing the R _1, R ₂ is hydrogen, or C ₁ - a C ₄ represents a straight or branched chain a group, R ₃ is C ₁ - straight or branched chain a group of C _4, C ₁ - C ₄ C ₁ -C ₄ linear or pulverized hydroxyalkyl group, C ₁ -C ₄ linear or pulverized alkyl group substituted with C ₂ -C ₆ linear or pulverized dialkylamino group, or C ₁ -C ₄ substituted with heterocyclic ring A straight chain or pulverized alkyl group of R ₄ represents a straight chain or pulverized alkoxycarbonylmethyl group, a carboxymethyl group, or a —CH ₂ —CO—NH—R ₅ group of C ₁ -C ₄ .

And, in a substituent indicates the R _4, R ₅ is C ₁ - C ₄ linear or grinding the alkyl group, C ₁ - a straight or branched chain of C ₄ hydroxy alkyl group, a C ₃ - a C ₆ cycloalkyl group, or a heteroaryl The cyclic ring represents a substituted C ₁ -C ₄ straight or crushed alkyl group.

Hepatitis C virus (HCV) (hereinafter referred to as “HCV”) is a major factor in non-A and non-B viral hepatitis, primarily 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 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 the 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, together with the surface antigenic protein, constitutes the capsid of the virus, 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 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, 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 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 highly conserved nucleotide sequences, and consists of three stem and loop structures 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. These 3 ′ end sites are known to form an essential structure for the 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 an effective cell culture system and an animal model. Recently, however, hepatoma cell lines have been developed that can reliably replicate HCV. Some problems have been addressed. 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, there is an urgent need for the development of effective vaccines and therapeutics. HCV has many genotypes and mutations between strains.In case 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 this study 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, NS5B (RNA dependent RNA polymerase (replicase)) of HCV is an enzyme of HCV essential for proliferation, and thus 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 and non-nucleoside compounds having low toxicity and side effects. The purpose was to develop.

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. A-[4- (4-morpholino) anilino] pyrimidine derivative was synthesized and the present invention was completed by revealing that the substance was excellent in inhibiting the proliferation of HCV.

It is therefore an object of the present invention to provide novel 4- [4- (4-morpholino) anilino] pyrimidine 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 technical problem of the present invention was achieved by synthesizing a 4- [4- (4-morpholino) anilino] pyrimidine derivative and measuring the inhibitory activity of the compound against recombinant HCV RNA polymerase.

The present invention provides a novel 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, R ₁ represents a C ₁ -C ₄ straight or crushed alkoxy group, a -NR ₂ R ₃ group, or a 4- (R ₄ ) -piperazin-1-yl group.

Further, in the substituent group representing the R _1, R ₂ is hydrogen, or C ₁ - a C ₄ represents a straight or branched chain a group, R ₃ is C ₁ - straight or branched chain a group of C _4, C ₁ - C ₄ C ₁ -C ₄ linear or pulverized hydroxyalkyl group, C ₁ -C ₄ linear or pulverized alkyl group substituted with C ₂ -C ₆ linear or pulverized dialkylamino group, or C ₁ -C ₄ substituted with heterocyclic ring A straight chain or pulverized alkyl group of R ₄ represents a straight chain or pulverized alkoxycarbonylmethyl group, a carboxymethyl group, or a —CH ₂ —CO—NH—R ₅ group of C ₁ -C ₄ .

And, in a substituent indicates the R _4, R ₅ is C ₁ - C ₄ linear or grinding the alkyl group, C ₁ - a straight or branched chain of C ₄ hydroxy alkyl group, a C ₃ - a C ₆ cycloalkyl group, or a heteroaryl The cyclic ring represents a substituted C ₁ -C ₄ straight or crushed alkyl 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 addition, according to the present invention, a method for preparing a 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by the following Scheme 1 and Scheme 2 according to the kind of R _{1 which} is a substituent of the general formula (1) to provide.

When R ₁ represents a —NR ₂ R ₃ group in Chemical Formula 1, a target compound is prepared according to the preparation method represented by the following Scheme 1.

In the above scheme 1, R ₂ is hydrogen, or C ₁ - C straight or denotes a pulverizing the group of _4, R ₃ is C ₁ - C ₄ linear or grinding the alkyl group, C ₁ - straight or branched chain of C ₄ a C ₁ -C ₄ straight or pulverized alkyl group substituted with a hydroxyalkyl group, C ₂ -C ₆ linear or pulverized dialkylamino group, or C ₁ -C ₄ linear or pulverized phase substituted with a heterocyclic ring An alkyl group is shown.

The preparation method of the present invention shown in Scheme 1,

1) 2-methylthio-4 of Chemical Formula 4 by reacting ethyl 4-chloro-2-methylthio-5-pyrimidine carboxylate of Chemical Formula 2 with 4- (4-morpholino) aniline of Chemical Formula 3 Preparing-[4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid ethyl ester (step 1);

(2) 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidine of formula 5 by hydrolyzing the ester derivative of formula 4 prepared in step 1 with an inorganic base Preparing a carboxylic acid (step 2);

(3) preparing a carboxylic acid chloride derivative of Chemical Formula 6 by reacting the carboxylic acid derivative of Chemical Formula 5 prepared in Step 2 with a halogen compound such as thionyl chloride (Step 3); And

(4) When the carboxylic acid chloride derivative of Formula 6 prepared in Step 3 is reacted with a suitable amine compound represented by HNR ₂ R ₃ of Formula 7 to formula 8 (R ₁ = -NR ₂ R ₃ It comprises the step (step 4) of preparing a 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by the target compound of formula 1).

Ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate, 4- (4-morpholino) aniline, thionyl chloride, and amine compounds used as starting materials and reactants in Scheme 1 are commercially available. A commercially available material can be easily purchased and used, and the reagent of the amine compound represented by HNR ₂ R ₃ of Chemical Formula 7 used in Step 4 is a substance for introducing a substituent into the target compound, depending on the type of substituent Appropriate reagents can be selected, which can be readily selected and used by those of ordinary skill in the art.

The preparation method of step 1 will be described in more detail. In an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, acetone, and the like, 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, etc. It proceeds well within 2 hours in the temperature range of 10 ℃ -30 ℃.

In more detail, the production method of step 2, in the mixed solvent of alcohol solvent and water such as methanol, ethanol, isopropanol and the like, in the presence of inorganic base such as sodium hydroxide, potassium hydroxide and the like temperature of 30 ℃ -55 ℃ Quantitatively within 3 hours within the range.

In more detail, the preparation method of step 3 is carried out using a halogen compound such as thionyl chloride in an organic solvent such as dichloromethane, chloroform, acetonitrile, N, N -dimethylformamide, and the like. It proceeds well within 10 hours within the temperature range of 40 ° C., and it is usually preferred to separate the carboxylic acid chloride in the form of hydrochloride.

In more detail the preparation method of step 4, triethylamine, N, N -diisopropylethylamine in an organic solvent such as dichloromethane, chloroform, acetonitrile, N, N- dimethylformamide, etc. , N - methyl morpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N - under normal organic presence of a base such as dimethylaniline, according to the type of the amine compound 0 It is usually completed within 1 hour in the temperature range of < RTI ID = 0.0 >

Steps 1, 2, and 3 of the scheme 1 of the scheme 1, the present inventors have already selected a patent filed "4- (4-substituted-anilino) pyrimidine derivative, a method for preparing the same and the same It is described in detail in the invention specification of "Pharmaceutical composition for viruses" (Korean Patent Application No. 2002-0018396, filed April 4, 2002).

In addition, in Formula 1, when R ₁ represents a C ₁ -C ₄ linear or pulverized alkoxy group, the reaction is carried out using an appropriate alcohol compound instead of the amine compound of Formula 7 or the ester compound of Formula 4 The desired compound can also be prepared by directly reacting with an excess alcohol compound in the presence of a base such as sodium methoxide.

When R ₁ represents a 4- (R ₄ ) -piperazin-1-yl group in Chemical Formula 1, a target compound is prepared according to the preparation method represented by the following Scheme 2.

In Scheme 2, R ₅ is a C ₁ -C ₄ linear or crushed alkyl group, C ₁ -C ₄ linear or pulverized hydroxyalkyl group, C ₃ -C ₆ cycloalkyl group, or a heterocyclic ring is substituted C ₁ -C ₄ linear or pulverized alkyl group.

The production method of the present invention shown in Scheme 2,

(1) preparing an ester derivative of Chemical Formula 10 by reacting the carboxylic acid chloride derivative of Chemical Formula 6, which is a synthetic intermediate prepared in Scheme 1, with 1- (ethoxycarbonylmethyl) piperazine of Chemical Formula 9 (step) One) ;

(2) preparing a carboxylic acid derivative of Formula 11 by hydrolyzing the ester derivative of Formula 10 prepared in Step 1 with an inorganic base (Step 2); And

(3) After activating the carboxylic acid derivative of Formula 11 prepared in Step 2 with a carboxylic acid anhydride or carboxylic acid chloride, etc. using a general activation reagent such as pivaloyl chloride or thionyl chloride, H ₂ NR of Formula 12 The purpose of formula 1 when reacting with an appropriate amine compound represented by ₅ to formula 13 (R ₁ = 4- (R ₄ ) -piperazin-1-yl group, R ₄ = CH ₂ -CO-NH-R ₅ ) It comprises the step (step 3) of preparing a 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by the compound).

In addition, the ester derivative of Formula 10, which is a synthetic intermediate prepared in Step 1 of Scheme 2, is a compound of Formula 1 when R1 = 4- (R ₄ ) -piperazin-1-yl group and R ₄ = alkoxycarbonylmethyl group The carboxylic acid derivative of Chemical Formula 11, which is a target compound and is a synthetic intermediate prepared in Step 2 of Scheme 2, has a formula of R ₁ = 4- (R ₄ ) -piperazin-1-yl group and R ₄ = carboxymethyl group It becomes the target compound of 1.

Activation reagents such as 1- (ethoxycarbonylmethyl) piperazine of Chemical Formula 9 used as a reactant in Scheme 2 and pivaloyl chloride or thionyl chloride used in Step 3 and H of Chemical Formula 12 A suitable amine compound represented by ₂ NR ₅ , etc. may be easily purchased and used as a commercially available material, and the reagent of the amine compound represented by H ₂ NR ₅ of Formula 12 used in Step 3 may be added to the target compound. As a substance for introducing a substituent, an appropriate reagent can be selected according to the type of the substituent, which can be easily selected and used by those skilled in the art.

The more specific preparation method of step 1 uses the same technique as the preparation method of step 4 of Scheme 1, the hydrolysis reaction of step 2 uses the same production method as the hydrolysis reaction of step 2 of Scheme 1 It goes well.

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, N - manufactured by the above formula under the generic organic base such as dimethylaniline exist, the step 2-methyl morpholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, N, N The carboxylic acid derivative of 11 is first reacted with a general activation reagent such as pivaloyl chloride to be activated with carboxylic anhydride or the like, and then reacted with an appropriate amine compound represented by H ₂ NR ₅ of Formula 12 to react the target compound of Formula 13. In the preparation, this reaction usually proceeds well within 1 to 3 hours in the temperature range of -15 ° C to + 20 ° C.

In the case of preparing the target compound in which R ₅ is a methyl group in Chemical Formula 13, by directly reacting with an ester derivative of Chemical Formula 10 prepared in Step 1 of Scheme 2 using an alcohol solution of industrially commercial methylamine It can manufacture.

The reaction proceeds well within 10 to 20 hours in the temperature range of 30 ° C.-50 ° C. without using other bases in alcohol solvents such as methanol and ethanol.

In addition, the present invention comprises a 4- [4- (4-morpholino) anilino] pyrimidine derivative of Formula 1 and / or a pharmaceutically acceptable salt thereof as an active ingredient, for the treatment or prevention of hepatitis C It provides a pharmaceutical composition.

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-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid ethyl ester

To 60 ml of methanol, 3 g of ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate and 2.2 ml of triethylamine were added and dissolved, and then 4- (4-morpholino) aniline 2.4 at 20 ° C-25 ° C. g was slowly added and reacted for 1 hour. The precipitated solid was filtered and washed with 10 ml of methanol to obtain 4.1 g (yield 85%) of the title compound by vacuum drying the obtained crystals at 30 ° C-40 ° C.

.p. : 130-132 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.37 (m, 3H), 2.51 (d, 3H), 3.14 (t, 4H), 3.86 (t, 4H), 4.34 (m, 2H), 6.90 ( d, 2H), 7.55 (d, 2H), 8.73 (d, 1H), 10.21 (s, 1H)

Preparation Example 2 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidine carboxylic acid.

2.5 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid ethyl ester prepared in Preparation Example 1 was added to 30 ml of methanol, and 20 ml of water and 3 N were added. -6.7 ml of aqueous sodium hydroxide solution was slowly added, followed by heating to perform a hydrolysis reaction at 40 ° C-50 ° C for 2 hours. Cooling to 20 ° C.-25 ° C. and slowly adding 3N hydrochloric acid to adjust the pH to 6.0 to precipitate a solid. After stirring for 1 hour, the crystals obtained by filtration and washing with 10 ml of water were vacuum-dried at 40 ℃-50 ℃ to give 2.2 g (yield 95%) of the title compound.

m.p. : 256-258 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.47 (s, 3H), 3.07 (t, 4H), 3.72 (t, 4H), 6.94 (d, 2H), 7.51 (s, 1H), 7.53 (d, 1H), 8.65 (s, 1H), 10.32 (s, 1H)

Preparation Example 3 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarbonyl chloride hydrochloride.

1.3 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid prepared in Preparation Example 2 was added to 30 ml of dichloromethane, and 0.41 ml of thionyl chloride was gradually added. After the addition, the reaction was carried out at 20 ° C.-30 ° C. for 7 hours. As the reaction proceeded, crystals in the form of hydrochloride that slowly precipitated were filtered and washed with 5 ml of dichloromethane to obtain 1.24 g (yield 82%) of the title compound as a hydrochloride by vacuum drying the obtained crystals at 30 ° C-40 ° C.

m.p. : 170-173 ℃ (dec.)

¹ H-NMR (DMSO-d ₆ ), ppm: δ2.53 (s, 3H), 3.47 (t, 4H), 4.00 (t, 4H), 7.65 (d, 2H), 7.79 (d, 2H), 8.73 (s, 1H), 10.66 (s, 1H)

Example 1 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid methyl ester.

To 50 ml of methanol was added 2 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid ethyl ester prepared in Preparation Example 1, and sodium methoxide (25% Methanol solution, d = 0.945) 1.2 ml was added and then reacted at 25 ° C-30 ° C for 1 hour. After completion of the reaction, 0.3 ml of acetic acid and 50 ml of water were slowly added and stirred for 2 hours. The precipitated solid was filtered and washed with 10 ml of water to obtain 1.75 g (91% yield) of the title compound by vacuum drying the obtained crystals at 40 ℃-50 ℃.

m.p. : 140-142 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ2.51 (s, 3H), 3.13 (m, 4H), 3.85 (t, 4H), 3.91 (s, 3H), 6.88 (m, 2H), 7.53 ( m, 2H), 8.72 (s, 1H), 10.17 (s, 1H)

Example 2 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5- [ N- (2-hydroxyethyl) aminocarbonyl] pyrimidine.

0.1 ml of ethanolamine and 0.5 ml of triethylamine were added to 25 ml of dichloromethane, and it cooled to 0. 0.6 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarbonyl chloride hydrochloride prepared in Preparation Example 3 was added thereto and reacted at 0 ° C-5 ° C for 1 hour. . After completion of the reaction, the precipitated solid was filtered and washed with 5 ml of dichloromethane. The solid was vacuum dried at 30 ° C.-40 ° C. to obtain 0.48 g (yield 82%) of the title compound.

m.p. : 258-260 ℃

¹ H-NMR (DMSO-d ₆ + CDCl ₃ ), ppm: δ2.51 (s, 3H), 3.12 (t, 4H), 3.49 (t, 2H), 3.71 (t, 2H), 3.83 (t, 4H), 4.50 (t, 1H), 6.88 (d, 2H), 7.55 (d, 2H), 8.23 (m, 1H), 8.60 (s, 1H), 11.02 (s, 1H)

Example 3 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5- [ N- [2- (diisopropylamino) ethyl] aminocarbonyl] pyrimidine .

0.29 ml of N, N -diisopropylethylenediamine and 0.5 ml of triethylamine were added to 30 ml of dichloromethane, and it cooled to 0 degreeC. 0.6 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarbonyl chloride hydrochloride prepared in Preparation Example 3 was added thereto and reacted at 0 ° C-5 ° C for 30 minutes. . After completion of the reaction, dichloromethane was concentrated under reduced pressure, and the residue was crystallized from 5 ml of methanol and 30 ml of water and stirred at room temperature for 2 hours. The precipitated solid was filtered off and washed with 5 ml of water. The solid was vacuum dried at 40 占 폚 to 50 占 폚 to obtain 0.55 g (yield 78%) of the title compound.

m.p. : 177-179 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.03 (d, 12H), 2.52 (s, 3H), 2.70 (t, 2H), 2.99-3.16 (m, 6H), 3.37 (m, 2H), 3.85 (t, 4H), 6.88 (d, 2H), 7.11 (br s, 1H), 7.57 (d, 2H), 8.28 (s, 1H), 10.99 (s, 1H)

Example 4 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5- [ N -ethyl- N- (4-pyridyl) methylaminocarbonyl] pyrimidine .

To 25 ml of dichloromethane, 0.23 ml of N- (4-pyridylmethyl) ethylamine and 0.5 ml of triethylamine were added and cooled to 0. 0.6 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarbonyl chloride hydrochloride prepared in Preparation Example 3 was added thereto and reacted at 0 ° C-5 ° C for 1 hour. . After completion of the reaction, the mixture was washed twice with 25 ml of water and the organic layer was concentrated under reduced pressure. The residue was purified by column chromatography (chloroform: methanol = 16: 1 (volume ratio)). The residue obtained by concentrating the purified fractions under reduced pressure was crystallized with 3 ml of acetone and 30 ml of isopropyl ether, stirred for 2 hours, filtered, washed with 3 ml of isopropyl ether, and the resulting solid was vacuum dried at 30 ° C-40 ° C. To 0.45 g (yield 65%) of the title compound.

m.p. : 137-139 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.25 (t, 3H), 2.50 (s, 3H), 3.13 (t, 4H), 3.49 (q, 2H), 3.86 (t, 3H), 4.71 ( s, 2H), 6.89 (d, 2H), 7.19 (d, 2H), 7.49 (d, 2H), 8.11 (s, 1H), 8.59 (m, 2H), 9.08 (s, 1H)

Example 5 of 2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (ethoxycarbonylmethyl) piperazin-1-ylcarbonyl] pyrimidine Produce.

4.25 g of 1- (ethoxycarbonylmethyl) piperazine and 6.9 ml of triethylamine were added to 100 ml of dichloromethane, and the mixture was cooled to 0 ° C. 9.0 g of 2-methylthio-4- [4- (4-morpholino) anilino] -5-pyrimidinecarbonyl chloride hydrochloride prepared in Preparation Example 3 was slowly added and reacted at 0 ° C-5 ° C for 30 minutes. I was. After completion of the reaction, dichloromethane was concentrated under reduced pressure, and the residue was crystallized with 50 ml of methanol, and then 25 ml of water was slowly added thereto and stirred at room temperature for 3 hours. The precipitated solid was filtered, washed with 20 ml of a 1: 1 mixed solvent of methanol and water, and the resulting solid was vacuum dried at 40 ° C.-50 ° C. to obtain 10.22 g (yield 91%) of the title compound.

m.p. : 127-129 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 1.25 (t, 3H), 2.51 (s, 3H), 2.65 (t, 4H), 3.13 (t, 4H), 3.27 (s, 2H), 3.73 ( t, 4H), 3.85 (t, 4H), 4.16 (q, 2H), 6.88 (m, 2H), 7.51 (m, 2H), 8.06 (s, 1H), 9.04 (s, 1H)

Example 6 Preparation of 2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (carboxymethyl) piperazin-1-ylcarbonyl] pyrimidine.

2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (ethoxycarbonylmethyl) piperazin-1-yl prepared in Example 5 in 30 ml of methanol 3.4 g of carbonyl] pyrimidine were added, and 20 ml of water and 6.8 ml of 3 N-sodium hydroxide aqueous solution were slowly added thereto, followed by heating to undergo a hydrolysis reaction at 50 ° C-55 ° C for 1 hour. The reaction solution was cooled to 10 ° C-15 ° C, and 6.8 ml of 3N hydrochloric acid was slowly added to precipitate a solid. 70 ml of water was added thereto, stirred at room temperature for 2 hours, filtered and washed with 20 ml of water to obtain 2.73 g (yield 85%) of the title compound by vacuum drying the obtained crystal at 40 ° C-50 ° C.

m.p. : 165-168 ℃

¹ H-NMR (DMSO-d ₆ + CDCl ₃ ), ppm: δ2.51 (s, 3H), 2.67 (t, 4H), 3.12 (t, 4H), 3.25 (s, 2H), 3.76 (t, 4H), 3.84 (t, 4H), 6.88 (m, 2H), 7.50 (m, 2H), 8.06 (s, 1H), 9.01 (s, 1H), 10.59 (s, 1H)

Example 7 2-Methylthio-4- [4- (4-morpholino) anilino] -5- [4-[( N -methylcarbamoyl) methyl] piperazin-1-ylcarbonyl] Preparation of pyrimidine.

2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (ethoxycarbonylmethyl) piperazin-1-yl prepared in Example 5 in 10 ml of methanol. 0.8 g of carbonyl] pyrimidine was added, and 10 ml of methylamine methanol solution (40%) was added slowly, followed by heating for 10 hours at 40 ° C-45 ° C. After completion of the reaction, the residue obtained by concentration under reduced pressure was purified by column chromatography (chloroform: methanol = 12: 1 (volume ratio)). The purified fractions were concentrated under reduced pressure, and the residue was crystallized from 2 ml of dichloromethane and 20 ml of isopropyl ether. After stirring for 1 hour at room temperature, the mixture was filtered and washed with 3 ml of isopropyl ether, and the crystals were dried in vacuo at 30 ° C-40 ° C to obtain 0.62 g (yield 80%) of the title compound.

m.p. : 173-175 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ2.51 (s, 3H), 2.58 (t, 4H), 2.86 (d, 3H), 3.07 (s, 2H), 3.13 (t, 4H), 3.73 ( t, 4H), 3.85 (t, 4H), 6.88 (m, 2H), 7.00 (m, 1H), 7.50 (m, 2H), 8.05 (s, 1H), 9.06 (s, 1H)

Example 8 2-Methylthio-4- [4- (4-morpholino) anilino] -5- [4-[[ N- (isobutyl) carbamoyl] methyl] piperazin-1-yl Preparation of Carbonyl] pyrimidine.

To 20 ml of dichloromethane, 2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (carboxymethyl) piperazin-1-ylcarbo prepared in Example 6 above. 0.15 g of nil] pyrimidine was added, 0.1 ml of triethylamine was added, it melt | dissolved, and it cooled to 10 degreeC. 0.043 ml of pivaloyl chloride was slowly added and reacted at 10 ° C-20 ° C for 30 minutes. The reaction solution was cooled again to 0 ° C., and 0.04 ml of isobutylamine was slowly added, followed by reaction at 0 ° C.-5 ° C. for 1 hour. After completion of the reaction, 20 ml of water was added thereto, stirred vigorously for 10 minutes, the organic layer was separated, washed once more with 20 ml of water, and the organic layer was dehydrated with anhydrous magnesium sulfate, and then dichloromethane was concentrated under reduced pressure. The residue was crystallized from 1 ml of dichloromethane and 15 ml of ethyl ether. After stirring for 2 hours at room temperature, the mixture was filtered and washed with 3 ml of ethyl ether to obtain 0.14 g (yield 83%) of the title compound by vacuum drying at 30 ° C-40 ° C.

m.p. : 129-131 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 0.92 (d, 6H), 1.76 (m, 1H), 2.51 (s, 3H), 2.60 (t, 4H), 3.08 (s, 2H), 3.13 ( m, 6H), 3.72 (t, 4H), 3.85 (t, 4H), 6.88 (m, 2H), 7.07 (t, 1H), 7.50 (m, 2H), 8.06 (s, 1H), 9.05 (s , 1H)

Example 9 2-Methylthio-4- [4- (4-morpholino) anilino] -5- [4-[[ N- (2-hydroxyethyl) carbamoyl] methyl] piperazine- Preparation of 1-ylcarbonyl] pyrimidine.

To 20 ml of dichloromethane, 2-methylthio-4- [4- (4-morpholino) anilino] -5- [4- (carboxymethyl) piperazin-1-ylcarbo prepared in Example 6 above. 0.15 g of nil] pyrimidine was added, 0.1 ml of triethylamine was added, it melt | dissolved, and it cooled to 10 degreeC. 0.043 ml of pivaloyl chloride was slowly added and reacted at 10 ° C-20 ° C for 30 minutes. The reaction solution was cooled again to 0 ° C., and 0.021 ml of ethanolamine was slowly added thereto, followed by reaction at 0 ° C.-5 ° C. for 1 hour. After completion of the reaction, 20 ml of water was added thereto, stirred vigorously for 10 minutes, the organic layer was separated, washed once more with 20 ml of water, and the organic layer was dehydrated with anhydrous magnesium sulfate, and then dichloromethane was concentrated under reduced pressure. The residue was crystallized from 1 ml of acetone and 10 ml of isopropyl ether. After stirring for 1 hour at room temperature, the mixture was filtered and washed with 3 ml of isopropyl ether, and the crystals were dried in vacuo at 30 ° C-40 ° C to obtain 0.13 g (yield 79%) of the title compound.

m.p. : 136-138 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ2.51 (s, 3H), 2.60 (t, 4H), 3.10 (m, 6H), 3.45 (q, 2H), 3.74 (m, 6H), 3.85 ( t, 4H), 6.88 (d, 2H), 7.42 (m, 1H), 7.50 (d, 2H), 8.05 (s, 1H), 9.05 (s, 1H)

Example 10 2-Methylthio-4- [4- (4-morpholino) anilino] -5- [4-[[ N- (cyclopropyl) carbamoyl] methyl] piperazin-1-yl Preparation of Carbonyl] pyrimidine.

Cyclopropylamine was used instead of isobutylamine to carry out the same method as in Example 8 to obtain the target compound.

Yield: 80%

m.p. : 147-149 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ 0.51 (m, 2H), 0.79 (m, 2H), 2.51 (s, 3H), 2.55 (t, 4H), 2.71 (m, 1H), 3.04 ( s, 2H), 3.13 (t, 4H), 3.70 (t, 4H), 3.85 (t, 4H), 6.88 (m, 2H), 7.00 (br s, 1H), 7.50 (m, 2H), 8.04 ( s, 1H), 9.05 (s, 1H)

Example 11 2-Methylthio-4- [4- (4-morpholino) anilino] -5- [4-[[ N- (2-pyridyl) methylcarbamoyl] methyl] piperazin- Preparation of 1-ylcarbonyl] pyrimidine.

2-aminomethylpyridine was used instead of isobutylamine to carry out the same method as in Example 8 to obtain the target compound.

Yield: 86%

m.p. : 109-111 ℃

¹ H-NMR (CDCl ₃ ), ppm: δ2.51 (s, 3H), 2.63 (t, 4H), 3.13 (m, 6H), 3.76 (br s, 4H), 3.85 (t, 4H), 4.60 (d, 2H), 6.88 (d, 2H), 7.19 (m, 2H), 7.51 (d, 2H), 7.64 (m, 1H), 8.06 (s, 1H), 8.15 (m, 1H), 8.54 ( d, 1H), 9.06 (s, 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.

1. Preparation of Recombinant HCV RNA Polymerase

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.

2. Preparation of RNA Template Containing 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.

3. Measurement of In Vitro Inhibitory Activity 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 with washing buffer again. 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 Substituent (R ₁ ) Inhibition Rate of HCV RNA Polymerase (%) 10 μg / ml 1 µg / ml 0.1 µg / ml 0.01 µg / ml Example 1 Methoxy 95 73 61 35 Example 2 (2-hydroxyethyl) amino 91 64 40 18 Example 3 2- (diisopropylamino) ethylamino 94 70 53 21 Example 4 N- ethyl- N- (4-pyridyl) methylamino 96 69 55 23 Example 5 4- (ethoxycarbonylmethyl) piperazin-1-yl 94 71 62 40 Example 6 4- (carboxymethyl) piperazin-1-yl 82 58 42 15 Example 7 4-[( N -methylcarbamoyl) methyl] piperazin-1-yl 95 76 62 44 Example 8 4-[[ N- (isobutyl) carbamoyl] methyl] piperazin-1-yl 92 68 52 27 Example 9 4-[[ N- (2-hydroxyethyl) carbamoyl] methyl] piperazin-1-yl 89 63 49 16 Example 10 4-[[ N- (cyclopropyl) carbamoyl] methyl] piperazin-1-yl 93 65 51 25 Example 11 4-[[ N- (2-pyridyl) methylcarbamoyl] methyl] piperazin-1-yl 88 60 38 15

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 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by Formula 1 according to the present invention inhibits the proliferation of hepatitis C virus (HCV). Since the effect is excellent and the toxicity is low, it can be usefully used as a prophylactic and therapeutic agent for hepatitis C.

Claims (5)

4- [4- (4-morpholino) anilino] pyrimidine derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.        Formula 1 In Formula 1, R ₁ represents a C ₁ -C ₄ straight or crushed alkoxy group, a -NR ₂ R ₃ group, or a 4- (R ₄ ) -piperazin-1-yl group. Further, in the substituent group representing the R _1, R ₂ is hydrogen, or C ₁ - a C ₄ represents a straight or branched chain a group, R ₃ is C ₁ - straight or branched chain a group of C _4, C ₁ - C ₄ a straight or branched chain a hydroxy group, a C ₂ - straight-chain of C ₄ - C ₆ linear or grinding phase with a dialkylamino group substituted with C ₁ - a linear or branched a an alkyl group, or pyridyl in the C ₄ group is substituted C ₁ Or a pulverized alkyl group, R ₄ represents a C ₁ -C ₄ straight or pulverized alkoxycarbonylmethyl group, a carboxymethyl group, or a -CH ₂ -CO-NH-R ₅ group. And, in a substituent indicates the R _4, R ₅ is C ₁ - C ₄ straight or branched chain the alkyl group, C ₁ - a straight or branched chain of C ₄ hydroxyalkyl group, C ₃ - a cycloalkyl group of C _6, or flutes It represents a straight or branched chain alkyl group of a C ₄ - pyridyl groups; substituted C _1. The compound of claim 1, wherein R ₁ is a methoxy group, (2-hydroxyethyl) amino group, 2- (diisopropylamino) ethylamino group, N -ethyl- N- (4-pyridyl) methylamino group, 4- ( Ethoxycarbonylmethyl) piperazin-1-yl group, 4- (carboxymethyl) piperazin-1-yl group, 4-[( N -methylcarbamoyl) methyl] piperazin-1-yl group, 4-[[ N -(Isobutyl) carbamoyl] methyl] piperazin-1-yl group, 4-[[ N- (2-hydroxyethyl) carbamoyl] methyl] piperazin-1-yl group, 4-[[ N- (cyclo Propyl) carbamoyl] methyl] piperazin-1-yl group or 4-[[ N- (2-pyridyl) methylcarbamoyl] methyl] piperazin-1-yl group. 4-morpholino) anilino] pyrimidine derivatives and pharmaceutically acceptable salts thereof. As shown in Scheme 1 below (1) Reaction of ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate of formula 2 with 4- (4-morpholino) aniline of formula 3 to react 2-methylthio-4- [ Preparing 4- (4-morpholino) anilino] -5-pyrimidinecarboxylic acid ethyl ester (step 1); (2) 2-methylthio-4- [4- (4-morpholino) anilino] -5- of formula (5) by hydrolysis of the ester derivative of formula (4) prepared in step 1 with sodium hydroxide or carium hydroxide Preparing pyrimidine carboxylic acid (step 2); (3) reacting the carboxylic acid derivative of formula 5 prepared in step 2 with a halogen compound such as thionyl chloride to prepare a carboxylic acid chloride derivative of formula 6 (step 3); And (4) when the carboxylic acid chloride derivative of formula 6 prepared in step 3 is reacted with a suitable amine compound represented by HNR ₂ R ₃ of formula 7 to formula 8 (R ₁ = -NR ₂ R ₃ The compound of claim 1, comprising the step (step 4) of preparing a 4- [4- (4-morpholino) anilino] pyrimidine derivative represented by Method for preparing morpholino) anilino] pyrimidine derivatives. Scheme 1 In the above scheme 1, R ₂ is hydrogen, or C ₁ - C straight or denotes a pulverizing the group of _4, R ₃ is C ₁ - C ₄ linear or grinding the alkyl group, C ₁ - straight or branched chain of C ₄ a C ₁ -C ₄ straight or pulverized alkyl group substituted with a hydroxyalkyl group, C ₂ -C ₆ linear or pulverized dialkylamino group, or C ₁ -C ₄ linear or pulverized alkyl group substituted with a pyridyl group Indicates. As shown in Scheme 2 below (1) preparing an ester derivative of formula 10 by reacting a carboxylic acid chloride derivative of formula 6, which is a synthetic intermediate prepared in Scheme 1, with 1- (ethoxycarbonylmethyl) piperazine of formula 9 (step 1 ); (2) preparing a carboxylic acid derivative of Formula 11 by hydrolyzing the ester derivative of Formula 10 prepared in Step 1 with sodium hydroxide or potassium hydroxide (Step 2); And (3) after activating the carboxylic acid derivative of Formula 11 prepared in step 2 with carboxylic anhydride or carboxylic acid chloride using pivaloyl chloride or thionyl chloride, and the amine compound represented by H ₂ NR ₅ of Formula 12 4- [ ₄ ] represented by the formula 13 (R ₁ = 4- (R ₄ ) -piperazin-1-yl group, and R ₄ = CH ₂ -CO-NH-R ₅ as a target compound). A method of preparing a 4- (4-morpholino) anilino] pyrimidine derivative (step 3), wherein the 4- [4- (4-morpholino) anilino] pyrimidine derivative is used. Manufacturing method. Scheme 2 In Scheme 2, R ₅ is C ₁ -C ₄ linear or pulverized alkyl group, C ₁ -C ₄ linear or pulverized hydroxyalkyl group, C ₃ -C ₆ cycloalkyl group, or pyridyl group substituted C ₁ - represents a C ₄ straight or branched chain of the alkyl group. A pharmaceutical composition for treating or preventing hepatitis C, comprising the 4- [4- (4-morpholino) anilino] pyrimidine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.