WO2004108719A1 - 2-[2-(4-morpholino)ethylamino]pyridine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same - Google Patents

2-[2-(4-morpholino)ethylamino]pyridine derivatives, method for preparing thereof and antiviral pharmaceutical composition comprising the same Download PDF

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WO2004108719A1
WO2004108719A1 PCT/KR2004/001298 KR2004001298W WO2004108719A1 WO 2004108719 A1 WO2004108719 A1 WO 2004108719A1 KR 2004001298 W KR2004001298 W KR 2004001298W WO 2004108719 A1 WO2004108719 A1 WO 2004108719A1
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ethylamino
group
formula
hcv
pyridine derivatives
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PCT/KR2004/001298
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WO2004108719A9 (en
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Jong-Woo Kim
Sang-Wook Lee
Geun-Hyung Lee
Jae-Jin Han
Sang-Jin Park
Eul-Yong Park
Joong-Chul Shin
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B & C Biopharm Co., Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/74—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals

Definitions

  • the present invention relates to 2-[2-(4-morpholino)ethylamino]pyridine derivatives useful as an antiviral agent, and more particularly novel 2-[2-(4- mo holino)ethylamino]pyridine derivatives having an excellent inhibitory effect on replication of Hepatitis C virus (HCV), represented by the following formula I:
  • R ⁇ and R 2 represent respectively C 1 -C 4 straight or branched alkyl group, C ⁇ -C 4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom, to a method for preparing the compounds, and to an antiviral pharmaceutical composition comprising the same as an active ingredient.
  • Hepatitis C virus is the major etiological agent of non- A and non- B viral hepatitis, mainly being post-transfusion and community-acquired.
  • HCN Hepatitis C virus
  • HCV is a member of the Flaviviridae family. More specifically, HCV has about 9.5kb sized (+)- RNA (single stranded positive-sense RNA) genome inside its envelope.
  • the RNA 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 RNA replication of the virus.
  • the long ORF is expressed as a polyprotein, and through co-franslational or post-franslational 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.
  • nonstructural proteins are processed by serine protease (NS3) and cofactor (NS2, NS4A, and NS4B) of the virus.
  • NS3 and NS5B serine protease
  • 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).
  • 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.
  • IRS internal ribosome entry site
  • the stem LU 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.
  • the 3'-UTR 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.
  • 3'-UTR has the tertiary structure of loop and plays an important role in NS5B binding.
  • its structure is known somewhat unstable.
  • 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 ah, 1996, Genetic organization and diversity of the hepatitis C virus genome, Virology 223:255-281).
  • NS5B is an enzyme consisting of 591 amino acids having the molecular weight of about 68kDa.
  • RBD1 and RBD2 are two RNA-binding domains, i.e. RBD1 and RBD2, in the NS5B enzyme.
  • RBD1 exists between the amino acid numbers 83 and 194, and RBD2 exists between the amino acid numbers 196 and 298.
  • 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).
  • this enzyme can lead a polymerization reaction without another primer (Reference: Lohmann, V. et ah, 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.
  • HCV alpha interferon
  • HCV is not easily freated 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.
  • 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 HCN R A became negative.
  • 50% of them relapsed into hepatitis C within 3-6 months.
  • only 25% of the HCV patients showed sustained response for more than 6 months.
  • HCV subtype 1 la, lb
  • 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.
  • the treatment effect was doubled.
  • ribavirin is that when it was used alone, it showed little effect on HCV and rather, caused side effects like erythroclastic anemia.
  • 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 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 HCN R ⁇ A polymerase ( ⁇ S5B, RNA polymerase).
  • 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.
  • the present invention provides novel 2-[2- (4-morpholino)ethylamino]pyridine derivatives, represented by the following formula I:
  • Ri and R 2 represent respectively C ⁇ -C 4 straight or branched alkyl group, C C 4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
  • Ri and R 2 in the Formula I is respectively methyl group, methoxy group, fluoro group, chloro group or hydrogen atom.
  • the above compounds can be used in form of pharmaceutically acceptable 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.
  • free acids both organic acids and inorganic acids can be used.
  • 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.
  • Another aspect of the present invention provides a method for preparing 2-[2-(4-morpholino)ethylamino]pyridine derivatives, represented by the following scheme.
  • Ri and R 2 represent respectively C ⁇ -C 4 straight or branched alkyl group, C 1 -C 4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
  • the method for preparing 2-[2-(4- morpholino)ethylamino]pyridine derivatives comprises the steps of: (i) reacting 2,6-dichloro-3-nifropyridine of Formula 2 with 4-(2-aminoethyl)mo ⁇ holine of Formula 3 to form an intermediate of 3-nitro-2-[2- (4-mo ⁇ holino)ethylamino]-6-chloropyridine of Formula 4; (ii) reacting the intermediate of Formula 4 prepared in the step (i) with piperazine of Formula 5 to form an intermediate of 3-nitro-2-[2-(4-mo ⁇ holino)ethylamino]-6-(piperazin-l- yl)pyridine of Formula 6; and (iii) reacting the intermediate of Formula 6 prepared in the step (ii) with isocyanate or isothiocyanate compounds of Formula 7 to form 2-[2-(4-mo ⁇ holino)ethylamino]
  • 2,6-Dichloro-3-nitropyridine, 4-(2-aminoethyl)mo ⁇ holine, piperazine and isocyanate or isothiocyanate compounds used as starting materials and reactants in the scheme (I) are commercially available.
  • the isocyanate or isothiocyanate compounds used in the step (iii) 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.
  • the reaction is 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-methylmo ⁇ holine, 1-methylpiperidine and the like.
  • an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetonitrile, NN-dimethylformamide, acetone and the like
  • an organic base such as triethylamine, NN-diisopropylethylamine, N-methylmo ⁇ holine, 1-methylpiperidine and the like.
  • the reaction is completed within 2 to 5 hours at a temperature in the range of 0 - 10 TJ .
  • the reaction is performed by using excess piperazine in an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetone and the like, or in a mixed solvent, and in the presence of an organic base such as triethylamine, NN-diisopropylethylamine, N-methylmo ⁇ holine, 1- methylpiperidine and the like.
  • an organic base such as triethylamine, NN-diisopropylethylamine, N-methylmo ⁇ holine, 1- methylpiperidine and the like.
  • the reaction is completed within 1 hour to 3 hours at a temperature in the range of 0 - 10 ° C .
  • the reaction is performed in an organic solvent such as dichloromethane, chloroform and the like.
  • the reaction is completed within 30 minutes to 3 hours at a temperature in the range of 10 - 30 °C .
  • the present invention also provides the pharmaceutical compositions for treatment and prevention of hepatitis C, which contains the 2-[2-(4- mo ⁇ holino)ethylamino]pyridine 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 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.
  • 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.
  • 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.
  • a simple diluent namely water, liquid paraffin, or other kinds of excipient, e.g. humectant, sweetening agent, odorant, or preservative.
  • liquid preparation for non-oral administration sterilized water solution, non-aqueous solvent, suspension or oily medicine.
  • 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.
  • Preparation 2 Preparation of 3-nifro-2- 2-(4-mo ⁇ holino ethylamino]-6- (piperazin- 1 -yPpyridine 24 g of anhydrous piperazine and 4.3 ml of triethylamine were added in sequence to 80 ml of methanol, stirred to dissolve and cooled to 0 ° C .
  • Example 2 Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]- 6-[4-[iV-(3-methylphenyl)thiocarbamoyl]piperazin-l-yl]pyridine 5.2 g of 3-nitro-2-[2-(4-mo ⁇ holino)ethylamino]-6-(piperazin-l- yl)pyridine, prepared in Preparation 2, was added to 70 ml of dichloromethane and stirred to dissolve. Then, 2.2 ml of 3-methylphenyl isothiocyanate was added slowly at 20 ° C and the mixture was stirred at 20 - 30 ° C for 3 hours.
  • the desired compound was prepared by following the same procedure with Example 1, except that 2-methoxyphenyl isothiocyanate was substituted for 3-methoxyphenyl isocyanate. Yield: 86% m.p.: 174-176 ° C
  • the desired compound was prepared by following the same procedure with Example 1, except that 2-fluorophenyl isothiocyanate was substituted for 3- methoxyphenyl isocyanate.
  • the desired compound was prepared by following the same procedure with Example 1, except that 4-fluorophenyl isothiocyanate was substituted for 3- methoxyphenyl isocyanate.
  • the desired compound was prepared by following the same procedure with Example 1, except that 3,4-dichlorophenyl isothiocyanate was substituted for 3-methoxyphenyl isocyanate. Yield: 95% m.p.: 187-189 ° C
  • Experimental example 1 Test of inhibitory effect on activity of HCN R ⁇ A Polymerase ⁇ R ⁇ A dependent R ⁇ A polymerase, ⁇ S5B 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.
  • HCV 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 cofransfected 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 chromatography 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 MgCl 2 , 10% glycerol], followed by at - 70°C in a small aliquot.
  • 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.
  • a sfreptavidin-coated well plate was prepared suitable for the sample to be examined.
  • 25 ⁇ of 2X assay buffer [50mM Tris-Cl (pH 7.5), lOOmM ⁇ aCl, lOmM MgCl 2 , 20mM KC1, ImM EDTA, ImM DTT] and 10 l& of purified HCV R ⁇ A polymerase 200ng and 3' -UTR template R ⁇ A were added to each well. Then, 5 l ⁇ of the sample to be examined was added to have final concentrations of 10, 1, 0.1 and 0.01 tg/ml .
  • the compounds according to the present invention show excellent inhibitory effects on activity of HCV RNA 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.
  • 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
  • CC 50 of greater than 100 tg/ml indicating that they are safe compounds with extremely low cytotoxicity.
  • the novel 2-[2-(4-mo ⁇ holino)ethylamino]pyridine derivatives according to the present invention represented by the chemical 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.

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Abstract

The present invention relates to 2-[2-(4-morpholino)ethylamino]pyridine derivatives useful as an antiviral agent, and more particularly novel 2-[2-(4-morpholino)ethylamino]pyridine derivatives having an excellent inhibitory effect on replication of Hepatitis C virus (HCV), represented by the following formula (I) in which R1 and R2 represent respectively C1-C4 straight or branched alkyl group, C1-C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom; and pharmaceutically acceptable salts thereof, a preparation method thereof, and an antiviral pharmaceutical composition comprising the compound as an effective component. The 2-[2-(4-morpholino)ethylamino]pyridine 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

2-[2-(4-MORPHOLINO)ETHYLAMINO]PYRIDINE DERIVATIVES,
METHOD FOR PREPARING THEREOF AND ANTIVIRAL PHARMACEUTICAL COMPOSITION COMPRISING THE SAME
Field of the Invention
The present invention relates to 2-[2-(4-morpholino)ethylamino]pyridine derivatives useful as an antiviral agent, and more particularly novel 2-[2-(4- mo holino)ethylamino]pyridine derivatives having an excellent inhibitory effect on replication of Hepatitis C virus (HCV), represented by the following formula I:
Figure imgf000002_0001
... © or pharmaceutically acceptable salts thereof in which Rι and R 2 represent respectively C1-C4 straight or branched alkyl group, Cι-C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom, 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- B 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 HCV. 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 HCV patients.
HCV is a member of the Flaviviridae family. More specifically, HCV has about 9.5kb sized (+)- RNA (single stranded positive-sense RNA) genome inside its envelope. The RNA 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 RNA replication of the virus.
The long ORF is expressed as a polyprotein, and through co-franslational or post-franslational 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 LU, 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 translational 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 ah, 1996, Genetic organization and diversity of the hepatitis C virus genome, Virology 223:255-281).
Among other enzymes of HCN 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. RBD1 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 ah, 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, hi 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 ( - interferon). However, this approach proved to be not that good because the effects of alpha interferon on different HCN 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 HCN protein in order to confrol HCV replication. The best targets of such research are ΝS3 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 HCV, and this makes the enzyme a good target for suppressing the replication of HCV.
Now that HCV is not easily freated 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 HCN R A 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 HCN 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 2-[2-(4-morpholino)ethylamino]pyridine 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 2-[2-(4- moφholino)ethylamino]pyridine 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 2-[2- (4-morpholino)ethylamino]pyridine derivatives, represented by the following formula I:
Figure imgf000008_0001
... (I) or pharmaceutically acceptable salts thereof in which Ri and R 2 represent respectively Cι-C4 straight or branched alkyl group, C C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
Preferably, Ri and R 2 in the Formula I is respectively methyl group, methoxy group, fluoro group, chloro group or hydrogen atom.
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.
Another aspect of the present invention provides a method for preparing 2-[2-(4-morpholino)ethylamino]pyridine derivatives, represented by the following scheme.
Figure imgf000009_0001
2 3 4
Figure imgf000009_0002
Figure imgf000009_0003
in which Ri and R2 represent respectively Cι-C4 straight or branched alkyl group, C1-C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
As shown in the above scheme, the method for preparing 2-[2-(4- morpholino)ethylamino]pyridine derivatives according to the present invention comprises the steps of: (i) reacting 2,6-dichloro-3-nifropyridine of Formula 2 with 4-(2-aminoethyl)moφholine of Formula 3 to form an intermediate of 3-nitro-2-[2- (4-moφholino)ethylamino]-6-chloropyridine of Formula 4; (ii) reacting the intermediate of Formula 4 prepared in the step (i) with piperazine of Formula 5 to form an intermediate of 3-nitro-2-[2-(4-moφholino)ethylamino]-6-(piperazin-l- yl)pyridine of Formula 6; and (iii) reacting the intermediate of Formula 6 prepared in the step (ii) with isocyanate or isothiocyanate compounds of Formula 7 to form 2-[2-(4-moφholino)ethylamino]pyridine derivatives of Formula I of the present invention. 2,6-Dichloro-3-nitropyridine, 4-(2-aminoethyl)moφholine, piperazine and isocyanate or isothiocyanate compounds used as starting materials and reactants in the scheme (I) are commercially available. The isocyanate or isothiocyanate compounds used in the step (iii) 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 reaction is 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 and the like. The reaction is completed within 2 to 5 hours at a temperature in the range of 0 - 10 TJ .
To give more details on the step (ii) of the preparation method described above, the reaction is performed by using excess piperazine in an organic solvent such as methanol, ethanol, isopropanol, dichloromethane, chloroform, acetone and the like, or in a mixed solvent, and in the presence of an organic base such as triethylamine, NN-diisopropylethylamine, N-methylmoφholine, 1- methylpiperidine and the like. The reaction is completed within 1 hour to 3 hours at a temperature in the range of 0 - 10 °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 and the like. The reaction is completed within 30 minutes to 3 hours at a temperature in the range of 10 - 30 °C .
The present invention also provides the pharmaceutical compositions for treatment and prevention of hepatitis C, which contains the 2-[2-(4- moφholino)ethylamino]pyridine 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 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 3-nitro-2-r2-(4-moφholino ethylamino1-6- chloropyridine
8 g of 2,6-dichloro-3-nitropyridine and 6.4 ml of triethylamine were added in sequence to 70 ml of methanol and cooled to 0°C. Then, 5.44 ml of 4-(2- aminoethyl)moφholine was added slowly at 0 - 5 °C , and the mixture was stirred at 0 - 5 °C for 4 hours. After the reaction was completed, 35 ml of water was added, stirred at 20 °C for 1 hour, filtered and washed with mixed solvent of 10 ml of methanol and 10 ml of water, to give a crystalline product. The product was dried in vacuo at 35 - 45 °C to give 9.5 g of the desired compound (80% yield). m.p.: 76-78 °C 1H-NMR (CDC ), ppm: δ 2.54 (br s, 4H), 2.64 (t, 2H), 3.71 (m, 6H),
6.58 (dd, 1H), 8.33 (dd, 1H), 8.82 (s, 1H)
Preparation 2 : Preparation of 3-nifro-2- 2-(4-moφholino ethylamino]-6- (piperazin- 1 -yPpyridine 24 g of anhydrous piperazine and 4.3 ml of triethylamine were added in sequence to 80 ml of methanol, stirred to dissolve and cooled to 0°C . 30 ml of dichloromethane solution wherein 8 g of 3-nitro-2-[2-(4-moφholino)ethylamino]- 6-chloropyridine, prepared in Preparation 1 was dissolved, was dropped slowly for 30 minutes to the mixture at 0 - 5 °C , and stirred at 0 - 5 °C for 1 hour. After the reaction was completed, the reaction mixture was concenfrated under reduced pressure, and after adding 30 ml of methanol and 100 ml of water, stirred at 20 °C for 2 hours. The precipitated solid was filtered for removing impurities, and methanol was concenfrated under reduced pressure and extracted with 150 ml of dichloromethane. The organic layer was separated, washed 3 times with 80 ml of water, and concentrated under reduced pressure. The residue was crystallized by 10 ml of dichloromethane and 90 ml of isopropyl ether. The mixture was stirred at room temperature for 3 hours, filtered, washed with 20 ml of isopropyl ether and dried in vacuo at 35 - 45 °C to give 8.45 g of the desired compound (90% yield). m.p.: 127-129 °C
1H-NMR (CDC13), ppm: δ 1.95 (s, 1H), 2.50 (t, 4H), 2.62 (t, 2H), 2.94 (t, 4H), 3.61 (q, 2H), 3.73 (m, 8H), 5.99 (m, 1H), 8.18 (d, 1H), 9.05 (br s, 1H)
Example 1 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]- 6-[4-[iV-(3-methoxyphenyl)carbamoyl]piperazin-l-yl]pyridine
1.5 g of 3-nitro-2-[2-(4-moφholino)ethylamino]-6-(piperazin-l- yl)pyridine, prepared in Preparation 2, was added to 50 ml of dichloromethane and stirred to dissolve. Then, 0.64 ml of 3-methoxyphenyl isocyanate was added slowly at 20 °C and the mixture was stirred at 20 - 25 °C for 1 hour.
After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was crystallized by 4 ml of dichloromethane and
50 ml of ethyl ether, stirred at 20 °C for 2 hours, filtered, and washed with 8 ml of ethyl ether to give a product. The product was dried in vacuo at 35 - 45 °C to give 2.0 g of the desired compound (92% yield). m.p.: 178-180 °C
1H-NMR (CDC13), ppm: δ 2.51 (t, 4H), 2.64 (t, 2H), 3.64 (m, 6H), 3.74 (t, 4H), 3.80 (s, 3H), 3.84 (m, 4H), 5.98 (d, 1H), 6.42 (s, 1H), 6.60 (m, 1H), 6.83 (m, 1H), 7.11 (t, 1H), 7.16 (t, 1H), 8.22 (d, 1H), 9.04 (br s, 1H)
Example 2 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]- 6-[4-[iV-(3-methylphenyl)thiocarbamoyl]piperazin-l-yl]pyridine 5.2 g of 3-nitro-2-[2-(4-moφholino)ethylamino]-6-(piperazin-l- yl)pyridine, prepared in Preparation 2, was added to 70 ml of dichloromethane and stirred to dissolve. Then, 2.2 ml of 3-methylphenyl isothiocyanate was added slowly at 20 °C and the mixture was stirred at 20 - 30 °C for 3 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure. The residue was crystallized by 10 ml of dichloromethane and 70 ml of methanol, stirred at 20 °C for 3 hours, filtered, and washed with 20 ml of methanol to give a product. The product was dried in vacuo at 35 - 45 °C to give 7.0 g of the desired compound (93% yield). m.p.: 115-117 °C
1H-NMR (CDC13), ppm: δ 2.34 (s, 3H), 2.53 (br s, 4H), 2.63 (t, 2H), 3.60 (q, 2H), 3.74 (t, 4H), 3.87 (br s, 4H), 3.98 (m, 4H), 5.94 (d, 1H), 6.97 (m, 3H), 7.21 (m, 2H), 8.22 (d, 1H), 9.02 (br s, 1H)
Example 3 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]-
6- [4- [7Y-(2-methoxyphenyl)thiocarb amoyl] piperazin-1-yl] py ridine
The desired compound was prepared by following the same procedure with Example 1, except that 2-methoxyphenyl isothiocyanate was substituted for 3-methoxyphenyl isocyanate. Yield: 86% m.p.: 174-176 °C
1H-NMR (CDC13), ppm: δ 2.52 (br s, 4H), 2.63 (t, 2H), 3.61 (q, 2H), 3.73 (t, 4H), 3.88 (s, 3H), 3.92 (br s, 4H), 4.07 (m, 4H), 5.95 (d, 1H), 6.94 (m, 2H), 7.12 (m, 1H), 7.48 (s, 1H), 7.86 (m, 1H), 8.23 (d, 1H), 9.03 (br s, 1H)
Example 4 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]- 6-[4-[iV-(2-fluorophenyl)thiocarbamoyl]piperazin-l-yl]pyridine
The desired compound was prepared by following the same procedure with Example 1, except that 2-fluorophenyl isothiocyanate was substituted for 3- methoxyphenyl isocyanate.
Yield: 92% m.p.: 195-198 °C 1H-NMR (CDC13), ppm: δ 2.52 (t, 4H), 2.63 (t, 2H), 3.60 (q, 2H), 3.73 (t,
4H), 3.94 (m, 4H), 4.10 (m, 4H), 5.95 (d, IH), 7.07 - 7.22 (m, 4H), 7.67 (m, IH), 8.24 (d, IH), 9.04 (br s, IH)
Example 5 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]- 6-[4-[iV-(4-fluorophenyl)thiocarbamoyl]piperazin-l-yl]pyridine
The desired compound was prepared by following the same procedure with Example 1, except that 4-fluorophenyl isothiocyanate was substituted for 3- methoxyphenyl isocyanate.
Yield: 89% m.p.: 163-165 °C
1H-NMR (CDC13), ppm: δ 2.50 (t, 4H), 2.62 (t, 2H), 3.59 (q, 2H), 3.72 (t, 4H), 3.91 (m, 4H), 4.05 (m, 4H), 5.94 (d, IH), 7.05 (m, 2H), 7.20 (m, 3H), 8.22 (d, IH), 9.03 (br s, IH)
Example 6 : Preparation of 3-nitro-2-[2-(4-morpholino)ethylamino]-
6-[4-[7Y-(3,4-dichlorophenyl)thiocarbamoyl]piperazin-l-yl]pyridine
The desired compound was prepared by following the same procedure with Example 1, except that 3,4-dichlorophenyl isothiocyanate was substituted for 3-methoxyphenyl isocyanate. Yield: 95% m.p.: 187-189 °C
1H-NMR (CDC13), ppm: δ 2.50 (t, 4H), 2.62 (t, 2H), 3.60 (q, 2H), 3.72 (t, 4H), 3.92 (m, 4H), 4.05 (m, 4H), 5.95 (d, IH), 7.10 (dd, IH), 7.29 (s, IH), 7.36 (d, IH), 7.39 (d, IH), 8.22 (d, IH), 9.04 (t, IH)
Experimental example 1 : Test of inhibitory effect on activity of HCN RΝA Polymerase ΪRΝA dependent RΝA polymerase, ΝS5B 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 cofransfected 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 chromatography 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 HCN RΝA polymerase was measured as follows.
A sfreptavidin-coated well plate was prepared suitable for the sample to be examined. 25 ≠ of 2X assay buffer [50mM Tris-Cl (pH 7.5), lOOmM ΝaCl, lOmM MgCl2, 20mM KC1, ImM EDTA, ImM DTT] and 10 l& of purified HCV RΝA polymerase 200ng and 3' -UTR template RΝA were added to each well. Then, 5 lΛ of the sample to be examined was added to have final concentrations of 10, 1, 0.1 and 0.01 tg/ml . Finally, 10 >Λ of a reactant solution containing DIG-(digoxigenin)-UTP, biotin-UTP, ATP, CTP, GTP, and UTP as a nucleotide for the ploymerase reaction with the RΝA template of HCV 3' -UTR RΝA was added to each well. The reaction mixture was incubated at 22°C for 60 minutes. By the action of HCV polymerase, newly generated RΝAs including UTP conjugated with biotin and DIG were copied and these new RΝAs could bind to sfreptavidin 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 & 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 l of ABTSR (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]
Figure imgf000018_0001
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 RNA 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 : cytotoxicitv 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 tg/ml, indicating that they are safe compounds with extremely low cytotoxicity.
Industrial Applicability
As described above, the novel 2-[2-(4-moφholino)ethylamino]pyridine derivatives according to the present invention represented by the chemical 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 2-[2-(4-moφholino)ethylamino]pyridine derivatives, represented by the following formula I:
Figure imgf000020_0001
... (I) or pharmaceutically acceptable salts thereof in which Ri and R 2 represent respectively Cι-C4 straight or branched alkyl group, C1-C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
2. The compounds according to claim 1 or pharmaceutically acceptable salts thereof in which Ri is 3 -methyl group, 2-methoxy group, 3-methoxy group, 2-fluoro group, 4-fluoro group or 3-chloro group, and R 2 is 4-chloro group or hydrogen atom, and X is oxygen atom or sulfur atom.
3. A method for preparing 2-[2-(4-moφholino)ethylamino]pyridine derivatives, the method comprising the steps of: (i) reacting 2,6-dichloro-3-nifropyridine of Formula 2 with 4-(2- aminoethyl)moφholine of Formula 3 to form an intermediate of 3-nitro-2-[2-(4- moφholino)ethylamino]-6-chloropyridine of Formula 4;
(ii) reacting the intermediate of Formula 4 prepared in the step (i) with piperazine of Formula 5 to form an intermediate of 3-nitro-2-[2-(4- moφholino)ethylamino]-6-(piperazin-l-yl)pyridine of Formula 6; and
(iii) reacting the intermediate of Formula 6 prepared in the step (ii) with isocyanate or isothiocyanate compounds of Formula 7 to form 2-[2-(4- moφholino)ethylamino]pyridine derivatives of Formula I:
Figure imgf000021_0001
Figure imgf000021_0002
Figure imgf000021_0003
in which Ri and R2 represent respectively C1-C4 sfraight or branched alkyl group, C1-C4 straight or branched alkoxy group, methylthio group, trifluoromethyl group, halogen atom, or hydrogen atom, and X represents oxygen atom or sulfur atom.
4. A pharmaceutical composition for treatment and prevention of C type hepatitis comprising 2-[2-(4-moφholino)ethylamino]pyridme derivatives or pharmaceutically acceptable salts thereof according to claim 1 as an active ingredient.
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CN117105928A (en) * 2023-08-22 2023-11-24 上海蓝木化工有限公司 Protease inhibitor and preparation method thereof

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