WO2004108068A2

WO2004108068A2 - 4-[4-(4-morpholino)anilino]pyrimidine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same

Info

Publication number: WO2004108068A2
Application number: PCT/KR2004/001297
Authority: WO
Inventors: Jong-Woo Kim; Sang-Wook Lee; Geun-Hyung Lee; Jae-Jin Han; Sang-Jin Park; Eul-Yong Park; Joong-Chul Shin
Original assignee: B & C Biopharm Co., Ltd.
Priority date: 2003-06-04
Filing date: 2004-06-01
Publication date: 2004-12-16
Also published as: WO2004108068A3; KR20040104823A; KR100502395B1

Abstract

The present invention relates to 4-[4-(4-morpholino)anilino]pyrimidine derivatives useful as an antiviral agent, and more particularly novel 4-[4-(4-morpholino)anilino]pyrimidine derivatives having an excellent inhibitory effect on replication of Hepatitis C virus (HCV), represented by the following formula I: in which R1 represents C1-C4 straight or branched alkoxy group, -NR2R3 group or 4-(R4)-piperazin-l-yl group; R2 represents hydrogen or C1-C4 straight or branched alkyl group; R3 represents C1-C4 straight or branched alkyl group, C1-C4 straight or branched hydroxyalkyl group, C1-C4 straight or branched alkyl group substituted by C2-C6 straight or branched dialkylamino group or C1-C4 straight or branched alkyl group substituted by heterocyclic ring; R4 represents C1-C4 straight or branched alkoxycarbonylmethyl group, carboxymethyl group or -CH2-CO-NH-R5 group; and R5 represents C1-C4 straight or branched alkyl group, C1-C4 straight or branched hydroxyalkyl group, C3-C6 cycloalkyl group or C1-C4 straight or branched alkyl group substituted by heterocyclic ring; and pharmaceutically acceptable salts thereof, a preparation method thereof, and an antiviral pharmaceutical composition comprising the compound as an effective component. The 4-[4-(4-morpholino)anilino]pyrimidine derivatives according to the present invention have an excellent inhibitory effect on replication of Hepatitis C virus (HCV) and thus can be advantageously used as a therapeutic or prophylactic agent of hepatitis C.

Description

4-[4-(4-MORPHOLINO)ANI INO]PYRIMEDINE DERIVATIVES,

METHOD FOR PREPARING THEREOF AND ANTIVIRAL PHARMACEUTICAL COMPOSITION COMPRISING THE SAME

Field of the Invention

The present invention relates to 4-[4-(4-morpholino)anilino]pyrimidine derivatives useful as an antiviral agent, and more particularly novel 4-[4-(4- mo holino)anilino]pyrimidine derivatives having an excellent inhibitory effect on replication of Hepatitis C virus (HCV), represented by the following formula I:

... (I) or pharmaceutically acceptable salts thereof in which R\ represents C₁-C₄ straight or branched alkoxy group, -NR₂R₃ group or 4-(R₄)-piperazin-l-yl group; R₂ represents hydrogen or C₁-C₄ straight or branched alkyl group; R₃ represents Ci- C₄ straight or branched alkyl group, C₁-C₄ straight or branched hydroxyalkyl group, C₁-C₄ straight or branched alkyl group substituted by C₂-C₆ straight or branched dialkylamino group or C₁-C₄ straight or branched alkyl group substituted by heterocyclic ring; R₄ represents C₁-C₄ straight or branched alkoxycarbonylmethyl group, carboxymethyl group or -CH₂-CO-NH-R₅ group; and R₅ represents C₁-C₄ straight or branched alkyl group, C₁-C₄ straight or branched hydroxyalkyl group, C₃-C₆ cycloalkyl group or C₁-C4 straight or branched alkyl group substituted by heterocyclic ring, to a method for preparing the compounds, and to an antiviral pharmaceutical composition comprising the same as an active ingredient.

Description of the Related Art Hepatitis C virus (HCV) is the major etiological agent of non-A and non- 13 viral hepatitis, mainly being post-transfusion and community-acquired. Once infected with HCN, approximately 80% of infected people, given its symptom is manifested, progress to chronic hepatitis, and the rest 20% of infected people progress to acute hepatitis causing hepatic cirrhosis, which is eventually transferred to liver cancer. According to a recently published report, more than 200 million worldwide are infected with HCN. For instance, more than 4.5 million Americans are infected with the same virus (The number is likely to be 15 million in maximum.) and more than 5 million Europeans are HCN patients.

HCN is a member of the Flaviviridae family. More specifically, HCN has about 9.5kb sized (+)- RΝA (single stranded positive-sense RΝA) genome inside its envelope. The RΝA genome consists of an untranslational region at 5 'and 3 'ends (UTR) and a long open reading frame (ORF). This ORF is expressed as a polyprotein including 3,010 to 3,040 amino acids by host cell enzymes and divided into 3 structural proteins and 6 nonstructural proteins by host cell enzymes and its own protease. Also, there is a uniformly conserved region in the 5 'and the 3 'end of the genome, respectively. This region is believed to play an important role for protein expression and RΝA replication of the virus.

The long ORF is expressed as a polyprotein, and through co-translational or post-translational processing, it is processed into structural proteins, i.e. core antigen protein (core) and surface antigen protein (El, E2), and nonstructural proteins, NS2 (protease), NS3 (serine protease, helicase), NS4A (serine protease cofactor), NS4B (protease cofactor, involved in resistance), NS5A, and NS5B (RNA dependent RNA polymerase, RdRp), each contributing to replication of virus. The structural proteins are divided into core, El, and E2 by signal peptidase of the host cell. Meanwhile, the nonstructural proteins are processed by serine protease (NS3) and cofactor (NS2, NS4A, and NS4B) of the virus. The core antigen protein together with surface antigen protein of the structural protein compose a capsid of the virus, and the nonstructural proteins like NS3 and NS5B play an important role of the RNA replication of the virus (Reference: Bartenschager, R., 1997, Molecular targets in inhibition of hepatitis C virus replication, Antivir. Chem. Chemother. 8: 281-301).

Similar to other Flaviviruses, the 5' and 3' ends of the virus RNA has a uniformly conserved untranslational region (UTR). Generally, this region is known to play a very important role in replication of the virus. The 5 'end has 5 ' -UTR composed of 341 nucleotides, and this part has the structure of 4 stem and loop (I, II, III, and IV). Actually, this functions as an internal ribosome entry site (IRES) necessary for translation processing to express protein. Particularly, the stem El, which has the biggest and the most stable structure with a conserved sequence, has been reported to play the most essential part for ribosome binding. In addition, a recent study tells that the virus proteins are expressed by initiation of translaional processing from AUG that exists in the single RNA of the stem IV (Reference: 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). Moreover, the 3'end has 3'-UTR composed of 318 nucleotides. This part is known to play a very important role in the initiation step of binding of NS5B, an essential enzyme of RNA replication. The 3'-UTR, according to the sequence and tertiary structure, is composed of three different parts: -X-tail- 5 'starting from the 5 'end to 98th nucleotide (98nt), -poly (U)- having UTP consecutively, and the rest of 3'-UTR-. More specifically, X-tail-5'part consists of 98 nucleotides having a very conserved sequence, and has three stem and loop structures, thereby forming a very stable tertiary structure. Probably, this is why X-tail-5'part is considered very essential of NS5B binding. Also, it is reported that -poly (U)- part induces a pyrimidine track, facilitating RNA polymerase effect. Lastly, the rest part of 3' -UTR has the tertiary structure of loop and plays an important role in NS5B binding. However, its structure is known somewhat unstable. Overall, the 3 'end region of HCV RNA is known to have an essential structure in NS5B binding when the RNA replication starts (Reference: Yamada et al, 1996, Genetic organization and diversity of the hepatitis C virus genome, Virology 223:255-281).

Among other enzymes of HCV, NS5B is the one that is directly involved in RNA replication and thus it is very important. NS5B is an enzyme consisting of 591 amino acids having the molecular weight of about 68kDa. There are two RNA-binding domains, i.e. RBD1 and RBD2, in the NS5B enzyme. RBDl exists between the amino acid numbers 83 and 194, and RBD2 exists between the amino acid numbers 196 and 298. Meanwhile, essential motif amino acids for RNA binding and activity are 'Asp' (amino acid number 220), 'Gly' (amino acid number 283), 'Gly' (amino acid number 317), 'Asp' (amino acid number 318), 'Asp' (amino acid number 319), and 'Lys' (amino acid number 346). Further, provided that there exists a RNA template of the virus itself, this enzyme can lead a polymerization reaction without another primer (Reference: 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 viral. 71:8416-8428).

RNA genome of HCV was isolated in 1989 by molecular cloning (Reference: Choo, Q-L, et ah, 1989, Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359-362). Although there have been a number of molecular biological researches on HCV from that point, there were always limitations due to lack of more effective cell culture systems and animal models. Fortunately, the above problem has been somewhat resolved by the introduction of a hepatoma cell line which made it possible to replicate HCV more stably (Reference: Lohmann, V, F. Korner, J-O Koch, U. Herian, L. Theilmann, R. Bartenschlarger, 1999, Replication of subgenomic hepatitis c virus RNAs in a hepatoma cell line. Science 285:110-113). So far, no one has actually found vaccine or therapeutics that is very effective for HCV. Hence, many pharmaceutical companies and institutes around the world are now trying to develop therapeutics and prevention of hepatitis C. HCV patients are prevalent in the world, and its frequency to be progressed to hepatic cirrhosis and/or liver cancer is much higher than HBV Also, despite its high frequency to be progressed to chronic hepatitis, the research on infection mechanism of the virus is still under progress. People are infected with HCV through blood transfusion or medication via phleboclysis or tattooing, but most of cases HCV infection takes place through a direct blood contact. However, 40-50% of the HCV patients still do not exactly know how they became infected. In view of this situation, it is a very urgent matter to develop a new vaccine and therapeutics to treat the diseases. In general, HCV exist as diverse genotypes between strains and mutation. Once a person is progressed to chronic hepatitis from HCN it is not hard to see reinfection or coinfection owing to genetic variants. Because of this, few succeeded to develop an effective vaccine for HCV. Another example of HCV treatments is using alpha interferon (α - interferon). However, this approach proved to be not that good because the effects of alpha interferon on different HCV genotypes were very diverse and when its administration was discontinued, patients relapsed into hepatitis C in most of cases. Hence it will be important to develop an inhibitor that binds only to a particular HCV protein in order to control HCV replication. The best targets of such research are NS3 protease/helicase and NS5B RNA polymerase of HCV. These enzymes are very useful for developing anti-HCV agent since these types of enzyme is not necessary for the host cell but essential for its own replication. In other words, NS5B of HCV (RNA dependent RNA polymerase) is an essential enzyme for HCN and this makes the enzyme a good target for suppressing the replication of HCV.

Now that HCV is not easily treated by vaccine, a new therapy using - interferon and Ribavirin was introduced. But this, too, caused side effects and was not effective for treating hepatitis C. For example, about 25% of HCV patients showed no reaction to the interferon therapy, and about 25% reacted to it only for temporarily and relapsed into hepatitis C. The rest 50% of the patients maintained ALT at a normal level after the treatment was completed and their HCV RNA became negative. However, 50% of them relapsed into hepatitis C within 3-6 months. In short, only 25% of the HCV patients showed sustained response for more than 6 months. Meanwhile, the most HCV subtype found in patient world wide is 1 (la, lb) that is not easily treated by interferon, compared to 2 and 3 subtypes. In case of combination therapy with interferon and ribavirin, the treatment effect was doubled. What is known about ribavirin is that when it was used alone, it showed little effect on HCV and rather, caused side effects like erythroclastic anemia. Thus ribavirin was prescribed only when the interferon therapy was no good or relapsed into hepatitis C again. So far, no one actually developed an antiviral agent for treating hepatitis C by suppressing the replication of HCV. The present invention, therefore, is directed to develop a nonnucleoside small molecule having low toxicity and side effect but manifesting excellent antiviral activity against HCN by studying any possible compound that inhibits the activity of the recombinant HCV RΝA polymerase (ΝS5B, RNA polymerase). After making so much efforts for developing a compound with excellent antiviral activity against HCV as an attempt to develop a new HCV therapeutics having low toxicity and side effect, the inventors finally succeeded to synthesize a new 4-[4-(4-morpholino)anilino]pyrimidine derivatives represented by the above chemical formula I and proved that these compounds are indeed very effective for inhibiting the replication of HCV.

Disclosure of Invention

It is, therefore, an object of the present invention to provide 4-[4-(4- moφholino)anilino]pyrimidine derivatives and pharmaceutically acceptable salts thereof, and method for preparing the compounds.

Another object of the present invention is to provide a pharmaceutical composition comprising the above compound as an effective component, which has little side effect and is economical, for prevention and treatment of hepatitis C. To achieve the above objects, the present invention provides novel 4-[4-

(4-morpholino)anilino]pyrimidine derivatives, represented by the following formula I:

or pharmaceutically acceptable salts thereof in which R_t represents C₁-C₄ straight or branched alkoxy group, -NR₂R₃ group or 4-(R₄)-piperazin-l-yl group; R₂ represents hydrogen or C₁-C₄ straight or branched alkyl group; R₃ represents - C₄ straight or branched alkyl group, C₁-C₄ straight or branched hydroxyalkyl group, C₁-C₄ straight or branched alkyl group substituted by C₂-C₆ straight or branched dialkylamino group or C₁-C₄ straight or branched alkyl group substituted by heterocyclic ring; R₄ represents C₁-C₄ straight or branched alkoxycarbonylmethyl group, carboxymethyl group or -CH2-CO-NH-R₅ group; and R₅ represents C₁-C₄ straight or branched alkyl group, C1-C₄ straight or branched hydroxyalkyl group, C₃-C₆ cycloalkyl group or C₁-C₄ straight or branched alkyl group substituted by heterocyclic ring.

As aforementioned, the above compounds can be used in form of pharmaceutically acceptable salts. As for that salts, an acid addition salts that are prepared by pharmaceutically acceptable free acids are available. The compounds with the chemical formula I can make pharmaceutically acceptable acid addition salts following the conventional method in the related art. As for free acids, both organic acids and inorganic acids can be used. For instance, inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid. Organic acids include 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. In another aspect, the present invention provides a method for preparing

4-[4-(4-morpholino)anilino]pyrimidine derivatives, according to R_t of formula I, represented by the following scheme I and II.

In case R_t of formula I represents -NR₂R₃ group, 4-[4-(4- moφholino)anilino]pyrimidine derivatives are prepared by the following scheme(I):

in which R₂ represents hydrogen or C₁-C₄ straight or branched alkyl group; R₃ represents C₁-C₄ straight or branched alkyl group, C1-C₄ straight or branched hydroxyalkyl group, C₁-C₄ straight or branched alkyl group substituted by C₂-C₆ straight or branched dialkylamino group or -C₄ straight or branched alkyl group substituted by heterocyclic ring.

As shown in the above scheme (I), the method for preparing a 4-[4-(4- moφholino)anilino]pyrimidine derivatives according to the present invention comprises the steps of: (i) reacting ethyl 4-chloro-2-methylthio-5- pyrimidinecarboxylate of Formula 2 with 4-(4-moφholino)aniline of Formula 3 to form an intermediate of 2-methylthio-4-[4-(4-moφholino)anilino]-5- pyrimidinecarboxylic acid ethyl ester of Formula 4 ; (ii) hydrolyzing the intermediate of Formula 4 prepared in the step (i) with inorganic base to form an intermediate of 2-methylthio-4-[4-(4-moφholino)anilino]-5-pyrimidinecarboxylic acid of Formula 5 ; (iii) reacting the intermediate of Formula 5 prepared in the step (ii) with appropriate halogen compounds such as thionyl chloride to form an intermediate of carboxylic acid chloride derivative of Formula 6 ; and (iv) reacting the intermediate of Formula 6 prepared in the step (iii) with appropriate amine compounds represented by HNR₂R₃ of Formula 7 to form 4-[4-(4- moφholino)anilino]pyrimidine derivatives of Formula 8 (the desired compounds of the present invention, in case R_t = -NR₂R_3;). Ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate, 4-(4- moφholino)aniline, thionyl chloride and amine compounds used as starting materials and reactants in the scheme (I) are commercially available. The amine compounds represented by HNR₂R₃ of Formula 7 used in the step (iv) are appropriate reagents to introduce substituents to target compounds and can be suitably selected, depending on substituents to be introduced, by a person possessing ordinary knowledge in the art.

To give more details on the step (i) of the preparation method described above, the reactions are performed in an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, NN-dimethylformamide, acetone and the like, and in the presence of an organic base such as triethylamine, NN-diisopropylethylamine, N-methylmoφholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, NN-dimethylaniline and the like. The reaction is completed within 2 hours at a temperature in the range of 10 - 30 ^°C .

To give more details on the step (ii) of the preparation method described above, the reactions are performed in a mixed solvent of water and alcohol such as methanol, ethanol, isopropanol and the like, and in the presence of an inorganic base such as sodium hydroxide, potassium hydroxide and the like. The reaction is completed within 3 hours at a temperature in the range of 30 - 55 ^°C .

To give more details on the step (iii) of the preparation method described above, the reaction is performed in an organic solvent such as dichloromethane, chloroform, acetonitrile, NN-dimethylformamide and the like, and by using a halogen compound such as thionyl chloride. The reaction is completed within 10 hours at a temperature in the range of 20 - 40 ^°C, and carboxylic acid chloride is preferably separated in the form of HC1 salt.

To give more details on the steps (iv) of the preparation method described above, the reactions are performed in an organic solvent such as dichloromethane, chloroform, acetonitrile, NN-dimethylformamide and the like, and in the presence of an organic base such as triethylamme, NN-diisopropylethylamine, N- methylmoφholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4- dimethylaminopyridine, NN-dimethylaniline and the like. The reaction is completed within 1 hour at a temperature in the range of 0 - 10°C, depending on kinds of amine compounds. More details on the steps (i),(ii) and (iii) in the scheme (I) of the preparation method described above, have been disclosed in patent application in Korea(Νo. 2002-0018396, application date: 2002.4.4.) "4-(4-substituted- anilino)pyrimidine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same.". And in case Rι of formula I represents C₁-C₄ straight or branched alkoxy group, the desired compound can be prepared by reacting appropriate alcohol compounds instead of amine compounds of Formula 7, or by reacting ester compounds of Formula 4 with an excess of alcohol compounds in the presence of a base such as sodium methoxide. In case R] of formula I represents 4-(R₄)-piperazin-l-yl group, 4-[4-(4- moφholino)anilino]pyrimidine derivatives are prepared by the following scheme

(π) ;

10

(1:1₄= »lkox carboιι lιn«ftι l group)

φR₄= øart»oxyu«tlι l group) (lιR₄= -CBj-CO-NH-Rj)

...(H)

in which R₅ represents C_t-C₄ straight or branched alkyl group, d-C₄ straight or branched hydroxyalkyl group, C₃-C₆ cycloalkyl group or C₁-C₄ straight or branched alkyl group substituted by heterocyclic ring.

As shown in the above scheme (II), another method for preparing a 4-[4- (4-moφholino)anilino]pyrimidine derivatives according to the present invention comprises the steps of: (i) reacting the intermediate of carboxylic acid chloride derivative of Formula 6 prepared in the above scheme (I) with 1- (ethoxycarbonylmethyl)piperazine of Formula 9 to form an intermediate of Formula 10 ; (ii) hydrolyzing the intermediate of Formula 10 prepared in the step (i) with inorganic base to form an intermediate of Formula 11 ; and (iii) after activating the intermediate of Formula 11 prepared in the step (ii) with general activating reagents such as pivaloyl chloride and thionyl chloride to form carboxylic acid anhydride or carboxylic acid chloride derivative, reacting with appropriate amine compounds represented by H₂N-R₅ of Formula 12 to form 4-[4- (4-moφholino)anilino]pyrimidine derivatives of Formula 13 (the desired compounds of the present invention, in case R₁=4-(R )-piperazin-l-yl group and R₄=-CH₂-CO-NH-R₅).

The intermediate of Formula 10 prepared in the step (ii) of the scheme (II) is the desired compound of Formula I, in case R₁=4-(R₄)-piperazin-l-yl group and j =alkoxycarbonylmethyl group, and the intermediate of Formula 11 prepared in the step (ii) of scheme (II) is the desired compound of Formula I, in case R₁=4-(R₄)-piperazin- 1 -yl group and R₄ =carboxymethyl group. l-(Ethoxycarbonylmethyl)piperazine, pivaloyl chloride, thionyl chloride and amine compounds used as reactants and activating reagents in the scheme (II) are commercially available. The amine compounds represented by H₂N-R₅ of Formula 12 used in the step (iii) is appropriate reagents to introduce substituents to target compounds and can be suitably selected, depending on substituents to be introduced, by a person possessing ordinary knowledge in the art.

To give more details on the steps (i) and (ii) of the preparation method described above, the reactions of step (i) are performed by using the same method as the step (iv) of the scheme (I), and the hydrolysis reactions of step (ii) are performed by using the same method as the step (ii) of the scheme (I) described above.

To give more details on the step (iii) of the preparation method described above, the reaction is performed in an organic solvent such as dichloromethane, chloroform, acetonitrile, NN-dimethylformamide and the like, and in the presence of an organic base such as triethylamine, NN-diisopropylethylamine, N- methylmoφholine, 1-methylpiperidine, pyridine, 2,6-lutidine, 4- dimethylaminopyridine, NN-dimethylaniline and the like. The intermediate of Formula 11 prepared in the step (ii) is activated with general activating reagents such as pivaloyl chloride to form carboxylic acid anhydride derivative, and then reacted with appropriate amine compounds represented by H₂N-R₅ of Formula 12 to form the desired compound of Formula 13, in the step (iii). The reaction is completed within 1 - 3 hours at a temperature in the range of -15 ~ 20 ^°C . And in case the desired compound of Formula 13 in which R₅ is methyl group is prepared, it can be prepared by reacting directly ester derivative of Formula 10 prepared in the step (i) of scheme (II) with alcohol solution of methyl amine which is commercially available. The reaction is completed within 10 ~ 20 hours at a temperature in the range of 30 ~ 50 ^°C in an alcohol solvent such as methanol and ethanol without other base.

The present invention also provides the pharmaceutical compositions for treatment and prevention of hepatitis C, which contains the 4-[4-(4- moφholino)anilino]pyrimidine derivatives represented by the chemical formula I and/or its pharmaceutically acceptable salts as an active ingredient. The compounds of the chemical formula I as the therapeutics for hepatitis

C may be administered orally as well as through other routes in clinical uses, and can be used in the form of general drugs. If it needs to be prepared, a generally used diluent including filler, builder, binder, humectant, dis-integration agent or surfactant, or excipient can be employed. In the meantime, the solid preparation for oral administration includes tablets, pills, powder, granules or capsules. This solid preparation involves more than one compound of the chemical formula I and more than one excipient, for example, starch, calcium carbonate, sucrose or lactose, or gelatin. As for the liquid preparation for oral administration, suspension, solution, oily medicine or syrup can be used, but it can also employ a simple diluent, namely water, liquid paraffin, or other kinds of excipient, e.g. humectant, sweetening agent, odorant, or preservative. As for liquid preparation for non-oral administration, sterilized water solution, non-aqueous solvent, suspension or oily medicine. Preferably used non-aqueous solvent and suspension is propylene glycol, polyethylene glycol, vegetable oil like olive oil, and injectable esters like ethyl oleate.

The effective dose of the compounds of the chemical formula I is controlled depending on the patient's sex, age and condition. In general, it can be dosed to adults 10 to lOOOmg/day, more preferably 20 to 500mg/day, or one to three times dividedly per day.

Detailed description of the preferred embodiment

Now, the present invention is explained in detail by the following examples. However, the examples are provided for illustration of the present invention not for limitation thereof.

Preparation 1: Preparation of 2-methylthio-4-[4-(4-moφholino anilino]- 5-pyrimidinecarboxylic acid ethyl ester 3 g of ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate and 2.2 ml of triethylamme were added to 60 ml of methanol, and then 2.4 g of 4-(4- moφholino)aniline was added slowly in 20 - 25 ^°C and the mixture was stirred for 1 hour. The precipitated solid was filtered and washed with 10 ml of methanol to give a crystalline product. The product was dried in vacuo at 30 - 40 ^°C to give 4.1 g of the desired compound (85% yield). m.p.: 130-132 ^°C

1H-NMR (CDC1₃), 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, IH), 10.21 (s, IH)

Preparation 2 : Preparation of 2-methylthio-4-[4-(4-moφholino)anilino]-

5-pyrimidinecarboxylic acid

2.5 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5- pyrimidinecarboxylic acid ethyl ester, prepared in Preparation 1, was added to 30 ml of methanol and then 20 ml of water and 6.7 ml of 3N-sodium hydroxide were added slowly and hydrolysis reaction was performed at 40 - 50 ^°C for 2 hours. The reaction mixture was cooled to 20 - 25 ^°C , adjusted to pH 6.0 by adding 3N- hydrochloric acid, stirred for 1 hour, filtered and washed with 10 ml of water to give a crystalline product. The product was dried in vacuo at 40 - 50 ^°C to give 2.2 g of the desired compound (95% yield). m.p.: 256-258 ^°C

1H-NMR (DMSO-d₆), ppm: δ 2.47 (s, 3H), 3.07 (t, 4H), 3.72 (t, 4H), 6.94 (d, 2H), 7.51 (s, IH), 7.53 (d, IH), 8.65 (s, IH), 10.32 (s, IH)

Preparation 3: Preparation of 2-methylthio-4-[4-(4-moφholino anilino^~|- 5-pyrimidinecarbonyl chloride hydrochloride

1.3 g of 2-(methylthio)-4-[4-(4-moφholino)anilino]-5- pyrimidinecarboxylic acid, prepared in Preparation 2, was added to 30 ml of dichloromethane, and then 0.41 ml of thionyl chloride was added slowly and the mixture was stirred at 20 - 30 ^°C for 7 hours. The precipitated solid (hydrochloride salt) was filtered and washed with 5 ml of dichloromethane to give a crystalline product. The product was dried in vacuo at 30 - 40 ^°C to give 1.24 g of the desired compound (82% yield). m.p.: 170-173 ^°C (dec.)

1H-NMR (DMSO-d₆), ppm: δ 2.53 (s, 3H), 3.47 (t, 4H), 4.00 (t, 4H), 7.65 (d, 2H), 7.79 (d, 2H), 8.73 (s, IH), 10.66 (s, IH)

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

2 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5-pyrimidinecarboxylic acid ethyl ester, prepared in Preparation 1, was added to 50 ml of methanol, and 1.2 ml of sodium methoxide (25% methanol solution, d=0.945) was added and the mixture was stirred at 25 - 30 ^°C for 1 hour. After the reaction was completed, 0.3 ml of acetic acid and 50 ml of water were added slowly, stirred for 2 hours and filtered to give a product. The product was washed with 10 ml of water and dried in vacuo at 40 - 50 ^°C to give 1.75 g of the desired compound (91% yield). m.p.: 140-142 ^°C

1H-NMR (CDC ), ppm: δ 2.51 (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, IH), 10.17 (s, IH)

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

0.1 ml of ethanolamine and 0.5 ml of triethylamme were added to 25 ml of dichloromethane and cooled to 0^°C . 0.6 g of 2-methylthio-4-[4-(4- moφholino)anilino]-5-pyrimidinecarbonyl chloride hydrochloride, prepared in Preparation 3, was added, and the mixture was stirred at 0 - 5 ^°C for 1 hour and filtered to give a solid product. The product was washed with 5 ml of dichloromethane and dried in vacuo at 30 - 40 ^°C to give 0.48 g of the desired compound (82% yield). m.p.: 258-260 ^°C

1H-NMR (DMSO-d₆ + CDC1₃ ), ppm: δ 2.51 (s, 3H), 3.12 (t, 4H), 3.49 (t, 2H), 3.71 (t, 2H), 3.83 (t, 4H), 4.50 (t, IH), 6.88 (d, 2H), 7.55 (d, 2H), 8.23 (m, IH), 8.60 (s, IH), 11.02 (s, IH)

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

0.29 ml of NN-diisopropylethylenediamine and 0.5 ml of triethylamme were added to 30 ml of dichloromethane and cooled to 0 ^°C . Then, 0.6 g of 2- methylthio-4-[4-(4-moφholino)anilino]-5-pyrimidinecarbonyl chloride hydrochloride, prepared in Preparation 3, was added and the mixture was stirred at 0 - 5 ^°C for 30 minutes. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was crystallized by 5 ml of methanol and 30 ml of water, stirred at room temperature for 2 hours, filtered, and washed with 5 ml of water to give a product. The product was dried in vacuo at 40 - 50 ^°C to give 0.55 g of the desired compound (78% yield). m.p.: 177-179 ^°C

1H-NMR (CDC1₃), 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, IH), 7.57 (d, 2H), 8.28 (s, IH), 10.99 (s, IH)

Example 4 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[N-ethyl-N-(4- pyridyl)methylaminocarbonyl]pyrimidine 0.23 ml of N-(4-pyridylmethyl)ethylamine and 0.5 ml of triethylamme were added to 25 ml of dichloromethane and cooled to 0 ^°C . Then, 0.6 g of 2- methylthio-4-[4-(4-moφholino)anilino]-5-pyrimidinecarbonyl chloride hydrochloride, prepared in Preparation 3, was added and the mixture was stirred at 0 - 5 ^°C for 1 hour. After the reaction was completed, the reaction mixture was washed 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 purified fraction was concentrated under reduced pressure and the residue was crystallized by 3 ml of acetone and 30 ml of isopropyl ether, stirred for 2 hours, filtered, and washed with 3 ml of isopropyl ether to give a product. The product was dried in vacuo at 30 - 40 ^°C to give 0.45 g of the desired compound (65% yield). m.p.: 137-139 ^°C 1H-ΝMR (CDC1₃), 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, IH), 8.59 (m, 2H), 9.08 (s, IH)

Example 5 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-(ethoxycarbonylmethyl)piperazin-l- ylcarbonyl]pyrimidine

4.25 g of l-(ethoxycarbonylmethyl)piperazine and 6.9 ml of triethylamme were added to 100 ml of dichloromethane and cooled to 0 ^°C . Then, 9.0 g of 2-methylthio-4-[4-(4-moφholino)anilino]r5-pyrimidinecarbonyl chloride hydrochloride, prepared in Preparation 3, was added slowly and the mixture was stirred at 0 - 5 °C for 30 minutes. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was crystallized by 50 ml of methanol and then 25 ml of water was added. The solution was stirred at room temperature for 3 hours, filtered, and washed with 20 ml of mixed solvent of methanol and water(l : 1) to give a product. The product was dried in vacuo at 40 - 50 ^°C to give 10.22 g of the desired compound (91% yield). m.p.: 127-129 ^°C

1H-NMR (CDC1₃), 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, IH), 9.04 (s, IH)

Example 6 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-(carboxymethyl)piperazin-l-ylcarbonyl]pyrimidine 3.4 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5-[4-

(ethoxycarbonylmethyl)piperazin-l-ylcarbonyl]pyrimidine, prepared in Example 5, was added to 30 ml of methanol and then 20 ml of water and 6.8 ml of 3N- sodium hydroxide solution were added slowly and hydrolysis reaction was performed at 50 - 55 ^°C for 1 hour. The reaction mixture was cooled to 10 - 15 ^°C, and 3N - hydrochloric acid was added slowly to precipitate. 70 ml of water was added, stirred at room temperature for 2 hours, filtered, and washed with 20 ml of water to give a product. The product was dried in vacuo at 40 - 50 ^°C to give 2.73 g of the desired compound (85% yield). m.p.: 165-168 ^°C

1H-NMR (DMSO-d₆ + CDC1₃ ), ppm: δ 2.51 (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, IH), 9.01 (s, IH), 10.59 (s, IH)

Example 7 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-[(N-methylcarbamoyl)methyl]piperazin-l- ylcarbonyljpyrimidine

0.8 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5-[4- (ethoxycarbonylmethyl)piperazin-l-ylcarbonyl]pyrimidine, prepared in Example

5, was added to 10 ml of methanol and then 10 ml of methylamine methanol solution(40%) was added slowly and the mixture was stirred at 40 - 45 ^°C for 10 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography(chloroform : methanol = 12 : 1 (volume ratio)). The purified fraction was concentrated under reduced pressure. And the residue was crystallized by 2 ml of dichloromethane and 20 ml of isopropyl ether, stirred at room temperature for 1 hour, filtered, and washed with 3 ml of isopropyl ether to give a product. The product was dried in vacuo at 30 - 40 ^°C to give 0.62 g of the desired compound (80% yield). m.p.: 173-175 ^°C

1H-NMR (CDC1₃), ppm: δ 2.51 (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, IH), 7.50 (m, 2H), 8.05 (s, IH), 9.06 (s, IH)

Example 8 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-[[iV-(isobutyl)carbamoyl]methyl]piperazin-l- ylcarbonyLJpyrimidine

0.15 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5-[4-

(carboxymethyl)piperazin-l-ylcarbonyl]pyrimidine, prepared in Example 6, was added to 20 ml of dichloromethane and then 0.1 ml of triethylamme was added to dissolve the solid, and cooled to 10 ^°C . 0.043 ml of pivaloyl chloride was added slowly and the mixture was stirred at 10 - 20 ^°C for 30 minutes. The reaction mixture was cooled again to 0 ^°C and 0.04 ml of isobutylamine was added slowly and the mixture was stirred at 0 - 5 ^°C for 1 hour. After the reaction was completed, 20 ml of water was added and the mixture was stirred vigorously for 10 minutes. The organic layer was separated, washed with 20 ml of water, dehydrated by anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crystallized by 1 ml of dichloromethane and 15 ml of ethyl ether, stirred at room temperature for 2 hours, filtered, and washed with 3 ml of ethyl ether to give a product. The product was dried in vacuo at 30 - 40 ^°C to give 0.14 g of the desired compound (83% yield). m.p.: 129-131 ^°C

1H-NMR (CDC1₃), ppm: δ 0.92 (d, 6H), 1.76 (m, IH), 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, IH), 7.50 (m, 2H), 8.06 (s, IH), 9.05 (s, IH)

Example 9 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-[[iV-(2-hydroxyethyl)carbamoyl]methyl]piperazin- 1-ylcarbonyrjpyrimidine

0.15 g of 2-methylthio-4-[4-(4-moφholino)anilino]-5-[4- (carboxymethyl)piperazin-l-ylcarbonyl]pyrimidine, prepared in Example 6, was added to 20 ml of dichloromethane and then 0.1 ml of triethylamine was added to dissolve the solid, and cooled to 10 ^°C . 0.043 ml of pivaloyl chloride was added slowly and the mixture was stirred at 10 - 20 ^°C for 30 minutes. The reaction mixture was cooled again to 0 °C and 0.021 ml of ethanolamine was added slowly and the mixture was stirred at 0 - 5 ^°C for 1 hour. After the reaction was completed, 20 ml of water was added and the mixture was stirred vigorously for 10 minutes. The organic layer was separated, washed with 20 ml of water, dehydrated by anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was crystallized by 1 ml of acetone and 10 ml of isopropyl ether, stirred at room temperature for 1 hour, filtered, and washed with 3 ml of isopropyl ether to give a product. The product was dried in vacuo at 30 - 40 ^°C to give 0.13 g of the desired compound (79% yield). m.p.: 136-138 ^°C 1H-NMR (CDC1₃), ppm: δ 2.51 (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, IH), 7.50 (d, 2H), 8.05 (s, IH), 9.05 (s, IH)

Example 10 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-[[N-(cyclopropyl)carbamoyl]methyl]piperazin-l- ylcarbonyl]pyrimidine

The desired compound was prepared by following the same procedure with Example 8, except that cyclopropylamine was substituted for isobutylamine.

Yield: 80% m.p.: 147-149 ^°C

1H-NMR (CDC1₃), ppm: δ 0.51 (m, 2H), 0.79 (m, 2H), 2.51 (s, 3H), 2.55 (t, 4H), 2.71 (m, IH), 3.04 (s, 2H), 3.13 (t, 4H), 3.70 (t, 4H), 3.85 (t, 4H), 6.88 (m, 2H), 7.00 (br s, IH), 7.50 (m, 2H), 8.04 (s, IH), 9.05 (s, IH) Example 11 : Preparation of 2-methylthio-4-[4-(4- morpholino)anilino]-5-[4-[[N-(2-pyridyl)methlycarbamoyl]methyl]piperazin- 1-ylcarbonyrjpyrimidine

The desired compound was prepared by following the same procedure with Example 8, except that 2-aminomethylpyridine was substituted for isobutylamine.

Yield: 86% m.p.: 109-111 ^°C

1H-NMR (CDC1₃), ppm: δ 2.51 (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, IH), 8.06 (s, IH), 8.15 (m, IH), 8.54 (d, IH), 9.06 (s, IH)

Experimental example 1 : Test of inhibitory effect on activity of HCV RNA Polymerase (RNA dependent RNA polymerase, NS5B in vitro The following in vitro experiments were conducted to examine the inhibitory effect of the compounds according to the present invention on the activity of HCV RNA dependent RNA Polymerase.

Construct of recombinant HCV RNA polymerase HCV RNA polymerase was prepared as follows.

HCV cDNA was obtained from the blood of HCV-lb type HCV patient and NS5B region (1773bps) was amplified by PCR and cloned into pVLHIS, a baculovirus transfer vector, to prepare a recombinant transfer vector. The prepared transfer vector and the wild-type AcNPV vector were cotransfected into Sf 9 insect cell line to yield a recombinant baculovirus with the histidine-tagged recombinant vector pVLHIS-NS5B. Sufficiently cultured insect cells were infected with the resulting recombinant baculovirus and cultured in Grace's medium containing 10% FBS for 3 to 4 days. The culture broth was centrifuged to obtain only the infected cells. The cells were washed three times with PBS and resuspended in binding buffer [50mM Na-phosphate (pH 8.0), 30mM NaCl, lOmM imidazole, ImM DTT, 10% glycerol, 1% NP-40], sonicated and the clearized lysate was obtained. Recombinant NS5B was purified by affinity column chrorhatography using a Ni-NTA His bind resin (Novagen) to produce pure NS5B protein. The (His)₆-tagged NS5B was bound to Ni-NTA resin and washed with the binding buffer containing 50mM imidazole. The bound NS5B was eluted with the binding buffer containing imidazole in a step-gradient manner (100 - 300mM). The NS5B protein fractions were dialyzed against buffer [50mM Tris-HCl, 50mM NaCl, ImM DTT, 5mg MgCl₂, 10% glycerol], followed by at - 70°C in a small aliquot.

Construct of RNA template containing HCV 3' end (3'-UTR) The RNA template containing HCV 3' end (3' -UTR) was prepared as follows.

The 3 'UTR cDNA (220bp) of HCV was obtained from lb HCV RNA of the blood of a hepatitis C patient by PCR and cloned into pcDNA3 vector.

Linearized DNA fragment containing the 3' -UTR was prepared using the restriction enzyme, Eco RI and used as a template for in vitro transcription using T7 RNA ploymerase to prepare RNA fragment containing 3 ' -UTR.

Measurement of inhibitory activity of compounds of the present invention on recombinant HCV RNA polymerase in vitro

In vitro inhibitory activity of the compounds of the present invention against recombinant HCV RNA polymerase was measured as follows.

A streptavidin-coated well plate was prepared suitable for the sample to be examined. 25 l& of 2X assay buffer [50mM Tris-Cl (pH 7.5), lOOmM NaCl, lOmM MgCl₂, 20mM KC1, ImM EDTA, ImM DTT] and 10 ≠ of purified HCV RNA polymerase 200ng and 3'-UTR template RNA were added to each well. Then, 5 β& of the sample to be examined was added to have final concentrations of 10, 1, 0.1 and O.Oiμg/ml . Finally, 10 ≠ of a reactant solution containing DIG-(digoxigenin)-UTP, biotin-UTP, ATP, CTP, GTP, and UTP as a nucleotide for the ploymerase reaction with the RNA template of HCV 3 '-UTR RNA was added to each well. The reaction mixture was incubated at 22°C for 60 minutes. By the action of HCV polymerase, newly generated RNAs including UTP conjugated with biotin and DIG were copied and these new RNAs could bind to streptavidin coated on the well by biotin-conjugated UTP. After completion of the reaction, the plate was washed three times with 200 β& of a washing buffer (pH 7.0, Roche Co.) to remove unreacted substances and impurities. Then, 100 β& of the secondary antibody anti-DIG-POD (peroxidase, Roche Co.) was added to each well and incubated at 37°C for 1 hour. Again, the well plate was washed with the washing buffer. Finally, 100 ≠ of ABTS^R (Roche Co.) as a POD substrate was added to each well and reacted for 15 to 30 minutes. The optical density (OD) was measured using an ELISA reader (Bio-Tek instrument Co.) at 405nm. The inhibitory effect on the activity of HCV polymerase was calculated by subtracting the OD of the positive control without the sample. The results are shown in Table 1 below.

[Table 1]

As can be seen from the above table, it is proved that the compounds according to the present invention show excellent inhibitory effects on activity of HCV RΝA polymerase which plays an important role in reproduction of HCN thereby inhibiting replication of HCV by this property. Also, the compounds according to the present invention can be advantageously used as a therapeutic or prophylactic agent of hepatitis C.

Experimental example 2 : Cytotoxicity assay The cytotoxicity of the compounds of Formula I was examined by the

MTT assay, one of well-known in vitro toxicology assay methods, using Hep G2 cells. As a result, all the compounds used in the experiment were found to have

CC₅₀ of greater than 100 g/ml, indicating that they are safe compounds with extremely low cytotoxicity.

Industrial Applicability

As described above, the novel 4-[4-(4-moφholino)anilino]pyrimidine derivatives according to the present invention represented by the Formula I have excellent inhibitory effect on replication of hepatitis C virus and low cytotoxicity.

Therefore, they can be advantageously used as a therapeutic or prophylactic agent of hepatitis C.

Claims

What Is Claimed Is:

1. A 4-[4-(4-moφholino)anilino]pyrimidine derivatives, represented by the following formula I:

• • • (I) or pharmaceutically acceptable salts thereof in which Rι represents C C₄ straight or branched alkoxy group, -NR R₃ group or 4-(R₄)-piperazin-l-yl group; R₂ represents hydrogen or C₁-C₄ straight or branched alkyl group; R represents C₁-C₄ sfraight or branched alkyl group, C C₄ straight or branched hydroxyalkyl group, CrC₄ straight or branched alkyl group substituted by C₂-C₆ straight or branched dialkylamino group or d-C^ straight or branched alkyl group substituted by heterocyclic ring; R₄ represents C C₄ straight or branched alkoxycarbonylmethyl group, carboxymethyl group or -CH₂-CO-NH-R₅ group; and R₅ represents -C₄ straight or branched alkyl group, C C₄ straight or branched hydroxyalkyl group, C₃-C₆ cycloalkyl group or CrC₄ straight or branched alkyl group substituted by heterocyclic ring.

2. The compounds according to claim 1 or pharmaceutically acceptable salts thereof in which R\ is one selected from the group consisting of 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- (cyclopropyl)carbamoyl]methyl]piperazin- 1 -yl group and 4-[[N-(2- pyridyl)methylcarbamoyl]methyl]piperazin-l -yl group.

3. A method for preparing 4-[4-(4-moφholino)anilino]pyrimidine derivatives, the method comprising the steps of:

(i) reacting ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate of Formula 2 with 4-(4-moφholino)aniline of Formula 3 to form an intermediate of 2-methylthio-4-[4-(4-moφholino)anilino]-5-pyrimidinecarboxylic acid ethyl ester of Formula 4; (ii) hydrolyzing the intermediate of Formula 4 prepared in the step (i) with inorganic base to form an intermediate of 2-methylthio-4-[4-(4- moφholino)anilino]-5-pyrimidinecarboxylic acid of Formula 5;

(iii) reacting the intermediate of Formula 5 prepared in the step (ii) with appropriate halogen compounds such as thionyl chloride to form an intermediate of carboxylic acid chloride derivative of Formula 6; and

(iv) reacting the intermediate of Formula 6 prepared in the step (iii) with appropriate amine compounds represented by HΝR₂R₃ of Formula 7 to form 4-[4- (4-moφholino)anilino]pyrimidine derivatives of Formula 8 (the desired compounds of Formula I, in case R-[ = -NR₂R₃₎):

in which R represents hydrogen or C₁-C₄ straight or branched alkyl group; R₃ represents C₁-C₄ straight or branched alkyl group, -C₄ straight or branched hydroxyalkyl group, -C₄ straight or branched alkyl group substituted by C₂-C₆ straight or branched dialkylamino group or C₁-C₄ straight or branched alkyl group substituted by heterocyclic ring.

4. A method for preparing 4-[4-(4-moφholino)anilino]pyrimidine derivatives, the method comprising the steps of: (i) reacting the intermediate of carboxylic acid chloride derivative of

Formula 6 prepared in the above scheme (I) with 1- (ethoxycarbonylmethyl)piperazine of Formula 9 to form an intermediate of Formula 10;

(ii) hydrolyzing the intermediate of Formula 10 prepared in the step (i) with inorganic base to form an intermediate of Formula 11 ; and

(iii) after activating the intermediate of Formula 11 prepared in the step (ii) with general activating reagents such as pivaloyl chloride and thionyl chloride to form carboxylic acid anhydride or carboxylic acid chloride derivative, reacting with appropriate amine compounds represented by H₂N-R₅ of Formula 12 to form 4-[4-(4-moφholino)anilino]pyrimidine derivatives of Formula 13 (the desired compound of Formula I, in case R₁=4-(R₄)-piperazin-l-yl group and R₄ =-CH₂- CO-NH-R₅):

(1:^4= a&osycaAouyli rtiyi group)

(J:R₄

-GH₂-CO-NH-E₅)

■ •(H)

in which R₅ represents C₁-C₄ straight or branched alkyl group, C1-C₄ straight or branched hydroxyalkyl group, C₃-C₆ cycloalkyl group or d-C₄ straight or branched alkyl group substituted by heterocyclic ring.

5. A pharmaceutical composition for treatment and prevention of C type hepatitis comprising 4-[4-(4-moφholino)anilino]pyrimidine derivatives or pharmaceutically acceptable salts thereof according to claim 1 as an active ingredient.