NOVEL 3-NITROPYRIDINE DERIVATIVES AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING SAID DERIVATIVES

Abstract: The present invention relates to novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives, and more specifically, to 3-nitropyridine derivatives and their pharmaceutically acceptable salts, the process for preparing them, and the pharmaceutical compositions containing said compounds as active ingredients. In particular, said 3-nitropyridine derivatives of the present invention, due to their inhibitory activity against the proliferation of human immunodeficiency virus (HIV) as well as hepatitis B virus (HBV), can be used as a therapeutic agent as well as a preventive agent for hepatitis B and acquired immune deficiency syndrome (AIDS).

Description: NOVEL 3-NITROPYRIDINE DERIVATIVES AND THE PHARMACEUTICAL COMPOSITIONS CONTANING SAID DERIVATIVES TECHNICAL FIELD The present invention relates to novel 3-nιtropyrιdme derivatives and the pharmaceutical compositions containing said derivatives. More specifically, the present invention relates to 3-nιtropyrιdme derivatives and their pharmaceutically acceptable salts, represented by the following formula 1, which effectively inhibit proliferation of hepatitis B virus and human immunodeficiency virus. This invention also relates to the process for preparing 3-nιtropyrιdme derivatives and to the pharmaceutical compositions containing said derivatives as effective ingredients against viruses. Formula 1 iJ - - R ■ K . lerein R I-CH 2 - Ri is methoxy or R_ . R 3 is h, hydroxy, dialkylammo group with C 2 ~~C 6 , straight or branched hyαroxyalkyl group with C 2 ~C 6 , straight or branched dihydroxyalkyi group with C 3 ~C 6 , alkoxyalkyl group with C 3 ~C 6 , or saturated or unsaturated 5 or 6 memberedheterocyclic compounds containing 1 to 3 heteroatoms selected from N, 0, and S, which may be unsubstituted or substituted with alkyl group of Cι~C 3 ; R 3 may or may not contain asymmetrical carbons; R is H, straight or branched alkyl group with Cι~C 4 , or cycloalkyl group with C 3 ~C 6 ; R 3 and R 4 both may consist of 5 or 6 membered heterocyclic ring containing 1~3 heteroatoms selected from , 0, and S, which is unsubstituted or substituted with straight or branched alkyl group with Cι~Cs, straight or branched hydroxyalkyl group with C 2 ~C 5 , or hydroxy; R 2 is mdazol-5-yl, or indazol-6-yl ; n is an integer between 0 and 3. BACKGROUND ART Hepatitis B virus (HBV; referred as "HBV" hereinafter) causes acute or chronic hepatitis, which may progress to liver cirrhosis and liver cancer. It is estimated that three hundred million people are infected with HBV in the world (Tiollais & Buendia, Sci . Am . , 264, 48, 1991) . There has been much research about the molecular biological characteristics of HBV and their relationship to liver diseases in order to find ways to prevent and treat hepatitis B. Various vaccines and αiagnostic drugs have been developed and much effort is being channeled into research to find treatment for hepatitis B. HBV genome consists of genes for polymerase (P) , surface protein (pre-Sl, pre-S2 and S) , core protein (pre-C and C) , and X protein. Of these proteins expressed from HBV genes, polymerase, surface protein, and core protein are structural proteins and X protein has a regulatory function. The gene for HBV polymerase comprises 80% of the whole virus genome and produces a protein of 94kD size with 845 amino acids, which has several functions in the replication of virus genome. This polypeptide includes sequences responsible for activities of protein primer, RNA dependent DNA polymerase, DNA dependent DNA polymerase, andRNaseH. Kaplan and his coworkers first discovered reverse transcriptase activities of polymerase, which led to much research in replicating mechanism of HBV. HBV enters liver when antigenic protein on virion surface is recognized by hepatic cell-specific receptor. Inside the liver cell, DNAs are synthesized by HBV polymerase action, attached to short chain to form complete double helix for HBV genome. Completed double helical DNA genome of HBV produces pre-genomic mRNA and mRNAs of core protein, surface protein, and regulatory protein by the action of RNA polymerase. Using these mRNAs, virus proteins are synthesized. Polymerase has an important function in the production of virus genome, forming a structure called replicasome with core protein and pre-genomic mRNA. This process is called encapsidation. Polymerase has repeated units of glutamic acid at the 3' -end with high affinity for nucleic acids, which is responsible for facile encapsidation. When replicasome is formed, (-) DNA strand is synthesized by reverse transcribing action of HBV polymerase and ( + ) DNA strand is made through the action of DNA dependent DNA polymerase, which in turn produces pre-genomic mRNAs. The whole process is repeated until the pool of more than 200 to 300 genomes is maintained (Tiollais and Buendia, Sci en tifi c American , 264: 48-54, 1991) . Although HBV and HIV are different viruses, tne replication mechanisms during their proliferation have some common steps, namely , the reverse transcription of virus RNA to form DNA ana the removal of RNA strand from subsequently formed RNA-DNA hybrid . Recently, nucleoside compounds such as lamivudme and famvir have been reported to be useful inhibitors of HBV proliferation, although they have been originally developeα as therapeutics for the treatment of acquired immune deficiency syndrome (AIDS; referred as "AIDS" hereinafter ) and herpes zoster infection (Germ, J. L, Hepa tology, 14: 198-199, 1991; Lok, A. S. P., J. Viral Hepa ti ti s, 1: 105-124, 1994; Dienstag, J. L. et al . , New England Journal of Medi cine, 333: 1657-1661, 1995). However, these nucleoside compounds are considered a poor cnoice for treatment of hepatitis B because of their high cost ana side effects such as toxicity, development of resistant virus and recurrence of the disease after stopping treatment. Effort to find therapeutics for hepatitis B among non-nucleoside compounds has been continued and antiviral effects against HBV have been reported for qumoione compounds (EP0563732, EP0563734) , lridos compounds (KR 94-1886) , and terephthalic amide derivatives (KR 96-72384, KR 97-36589, KR 99-5100). In spite of much effort, however, effective drugs for treating hepatitis B have net been developed yet and tnerapeutic methoα mainly depends on symptomatic treatment. AIDS is a disease inducing dramatic decrease in immune function in the body cells and causing various symptoms of infection rarely seen m normal human beings, which spread to the whole body. Human immunodeficiency virus (HIV; referred as "HIV" hereinafter) responsible for AIDS is known to mainly attack helper T cells, which is one of the T cells with regulatory function in the immune system. When helper T cells are infected with HIV virus and undergo necrosis, human immune system cannot function properly. Impairment m immune function subsequently results m fatal infection and development of malignant tumor. Since AIDS patient has been found in USA m 1981 for the first time, the number increased to more than 850,000 patients m 187 countries m 1993 (WHO 1993 report) . WHO predicted that 30 to 40 million more people would be infected with HIV by tne year 2000 and 10 to 20 million of them would develop the disease. At the present time, drugs controlling proliferation of HIV have been most widely used for the treatment of AIDS. Of these, Zidovudme, which had been named Azidothymidme previously, is a drug developed m 1987. Didanosme was developed in 1991 as an alternative medicine for AIDS patients when Zidovudine was either ineffective or could not be used due to side effects. In addition, Zalcitabme was approved for concurrent use with Zidovudine m 1992. These drugs alleviate symptoms, slow down progression of the disease in the infected individuals to full-blown AIDS, and somewhat extend life span in the patients. These drugs, however, are not able to cure the patients completely and often develop problems such as resistance and side effects. In light of these problems, we, inventors of the present invention, tried to develop therapeutics to treat hepatitis B with little chance of toxicity, side effects, and development of resistant viral strains. We found non-nucleoside compounαs with excellent antiviral effect against HBV; synthesized novel 3-nitropyridine derivatives represented in formula 1 and completed the invention by showing their dramatic inhibitory effect on proliferation of HIV as well as of HBV. DISCLOSURE OF INVENTION The present invention provides novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives. More specifically, the present invention provides 3-nitropyridine derivatives and their pharmaceutically acceptable salts, the process for their preparation and the pharmaceutical compositions containing said derivatives as effective ingredient. 3-nitropyridine derivatives of the present invention inhibit proliferation of hepatitis B virus as well as of human immunodeficiency virus and may be effectively used for prevention and treatment of hepatitis B and AIDS. In order to accomplish the aforementioned goal, the present invention provides novel 3-nitropyridine derivatives represented below in formula 1 and their pharmaceutically acceptable salts. Formula 1 Wherein , Ri is methoxy or R 3 is H, hydroxy, dialkylamino group with C 2 ~C 6 , straight or branched hydroxyalkyl group with C 2 ~ -C 6 , straight or branched dihydroxyalkyl group with C 3 ~C 6 , alkoxyalkyl group with C 3 ~C 6 , or saturated or unsaturated 5 or δmemberedheterocyclic compounds containing 1 to 3 heteroatoms selected from N, 0, and S, which may be unsubstituted or substituted with alkyl group with Cι~C 3 ; R 3 may or may not have asymmetrical carbons; R 4 is H, straight or branched alkyl group with Cι~C 4 , or cycloalkyl group with C 3 ~C 6 ; R 3 and R both may consist of 5 or 6 membered heterocyclic ring with 1 to 3 heteroatoms selected from N, 0, and S, which is either unsubstituted or substituted with straight or branched alkyl group with Cι~C 5 , straight or branched hydroxyalkyl group with C 2 ~C 5 , or hydroxy; R 2 is indazol-5-yl, or indazol-6-yl ; n is an integer between 0 and 3 When both R 3 and R 4 are represented as a 5 or 6 me oered heterocyclic compounds with 1 to 3 heteroatoms selected from N, C, and S, n equals 0. This heterocyclic ring may be unsubstituted or substituted with straight or branched alkyl group with Cι~C 5 , straight or branched hydroxyalkyl group with C 2 ~C 5 , or hydroxy group; When compounds of formula 1 have asymmetrical carbons, they may exist as either R or S optical isomer and the present invention covers both optical isomers and the racemic mixture as well. Indazol-5-yl and ιndazol-6-yl groups for R 2 in the present invention are represented in formula 2 and 3 respectively. Formula 2 Formula 3 Compounds of formula 1 in the present invention may be utilized m the form of salts and the acid addition salts prepared by adding pharmaceutically acceptable free acids are useful. Compounds of formula 1 may be changed to the corresponding acid addition salts according to the general practices m this field. Both inorganic and organic acids may be used as free acids in this case. Among inorganic acids, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acidmay be used. Among organic acids, 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, succimc acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid and aspartic acid may be used. The present invention also provides the process for preparing 3-nιtropyrιdme derivatives of formula 1 as repre sented m s cheme 1 S cheme 1 herein , X is Cl or OCH 3 ; R 2 , R 3 , R 4 and n are as defined m formula 1 The process of preparation in the present invention comprises the following steps: (Step 1) Synthesis of 3-nιtropyrιdme derivatives of formula 6 by reacting 2-chloro-3-nιtropyrιdιne derivatives of formula 4 with 5-amιnomdazole or 6-amιnoιndazole of formula 5 in a proper solvent under a basic condition at an appropriate temperature; (Step 2) Synthesis of 3-nιtropyrιdme derivatives of formula 1 by reacting 3-nιtropyrιdme derivatives of formula 6 prepared m step 1 with appropriate amine compounds of formula 7 m a proper solvent under a oasic condition at an appropriate temperature . When Ri m the compound of formula 1 is a metnoxy group, step 1 completes the synthesis of desired compounα (X=OCH 3 ) . In this case, the present invention includes the method of preparing β-methoxy-3-nιtropyndιne derivatives of formula 6 by reacting 2-chloro-6-methoxy-3-nιtropyrιdιne of formula 4 with 5-amιnomdazole or 6-amιnomdazole of formula 5 in the presence of a base. Scheme 2 Compound of formula 4 in the first step of scneme 1 is 2-chloro-6-methoxy-3-nιtropyrιdιne or 2, 6-dιchloro-3-nιtropyrιdme . Chemical reagents used in the first and the second steps of scheme 1, namely, 2-chloro-3-nιtropyrιdme derivatives of formula 4, 5-amιnomdazole or 6-amιnoιndazole of formula 5, and amme compounds of formula 7, are commercially available and may be purchased. Compound of formula 7 in the step 2 above is used to introduce a substituent (R 3 - (CH 2 ) n -NR 4 -) into the compound of formula 1 and an appropriate amme compound should be selected depending on the substituent desired, which can be easily done by one with general knowledge in the technical field. To give more specific details about step 1 m the synthetic process, an organic base may be used and common tertiary amines such as triethylamme, N, N-dιιsopropylethylamιne , N-methylmorpholme , Λ7-methylpιperιdme, 4-dιmethylammopyπdme, N, N-dιmethylanιlιne , 2,6-lutιdme, pyridme are preferable. Preferable reaction time and temperature are 4~15 hrs and 20~60 ° C. Preferable for the reaction is a single or a mixture of solvents selected from chloroform, methylene chloride, acetonitπle and alcohols such as methanol and ethanol . Of 3-mtropyπdιne derivatives of formula 6 produced in the reaction of step 1, one with chloro group at 6 position is used m tne following reaction of step 2 Tne reaction m step 2 is described more detail. Preferable solvent is a single or a mixture of solvents selected from acetonitπle, chloroform, methylene chloride, tetranydrofuran, Α N-dimethyIformamide, Λf-methylpyrrolidmone, pyridme, water and alcoholic solvents such as methanol, ethanol, and isopropanol. It is preferable to use excess amount of amme compound (formula 7 ) to increase tne efficiency of the reaction . Solvents used in the previous step 1 for the synthesis of 3-nιtropyπdme derivatives of formula 6 are preferable. Reaction temperature of 25~80 ° C is preferable although it depends on the kind of amme compound used. In another aspect of this invention, also provideα are the pharmaceutical compositions of therapeutics for preventing and treating hepatitis B, which contain 3-nιtropyπdme derivatives of formula 1 and their pharmaceutically acceptable salts as effective ingredients. The present invention also provides the pnarmaceutical compositions of therapeutics for preventing and treating AIDS, which contain 3-nιtropyrιdme derivatives and their pharmaceutically acceptable salts of formula 1 as effective ingredients . 3-nιtropyrιdme derivatives of formula 1 m this invention have inhibitory effect on proliferation of both HIV and HBV because they interfere with removal of RNA strand from RNA-DNA hybrid formed during the reverse transcription of viral RNA to DNA, which is a common step in the replication mechanism of the two viruses. Compounds of formula 1 may be taken orally as well as through other routes in clinical uses; for example, it may be administered intravenously, subcutaneously, mtrapeπtoneally, or locally and used m the form of general drugs. For clinical use of drugs with the pharmaceutical compositions of the present invention, compounds of formula 1 may be mixed with pharmaceutically acceptable excipients and made into various pharmaceutically acceptable forms ; for example, tablets, capsules, trochese, solutions, suspensions for oral administration; and injection solutions, suspensions, or dried powder to be mixed with distilled water for the formulation of instant injection solution. Effective dosage for compound of formula 1 ^ ε general.-v 10—500 mg/kg, preferably 50—300 mg/kg for adults, which may be divided into several doses, preferably into 1~ 6 doses per day if deemed appropriate by a doctor or a pharmacist. BEST MODE FOR CARRYING OUT THE INVENTION Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples . However, it will be appreciated that those skilled m the art, on consideration of this disclosure, may make modifications and improvements withm the spirit and scope of the present invention. <Preparation Example 1> Preparation of 6-chloro-2- (1H-5- indazolylamino) -3-n_.tropyrid_.n . To the solution of 2, 6-dιchloro-3-nιtropyrιdme (4g) and 5-ammomdazole (2.8 g) m acetonitrile (50 ml) was added triethylamme (3.2 ml), and then the solution was reacted at 20-25 ° C for 12 hr . The reaction mixture was cooled at room temperature, added H 2 0 30 ml slowly, and tnen reacted at 20°C for 1 hr . The reaction mixture was filtered, washed with the mixture solution (15 ml) of acetonitrile : H 2 0 = 1: 1 (volume ratio) and dried at 50 ° C in va cuo to obtain the desired compound (4.7 g, 78%) . m.p. : 233 ° C (dec. ) 2 H-NMR (DMSO-de) , ppm : δ 6.94(d, 1H), 7.44(d, 1H), 7.56 (d, 1H) , 7.91 (s, 1H) , 8.08 (s, 1H) , 8.53 (d, 1H) , 10.20(s, 1H) , 13.12 (br s, 1H) <Preparation Example 2> Preparation of 6-chloro-2- (lff-6- indazolylamino) -3-nitropyridine . To the solution of 2 , 6-dιchloro-3-nιtropyπdme (4 g) and 6-ammomdazole (2.8 g) in acetonitrile (50 ml) was added triethylamme (3.2 ml), and then the solution was reacted at 35-40 ° C for 12 hr . The reaction mixture was cooled at room temperature with slowly adding H 2 0 (20 ml) , and then reacted at 20-25 ° C for 1 hr. The reaction mixture was filtered, washed with acetonitrile (6ml) and H 2 0 (15 ml), and then dried at 50 ° C in vacuo to obtain the desired compound (4.4 g, 73%) . m. p . : 234 ° C (dec . ) AANMR (DMSO-de) , ppm : δ 7.02 (d, 1H; , 7.20 (α, 1H) , 7.73 (d, 1H) , 7.99(d, 2H) , 8.55(d, 1H) , 10.26(s, 1H) , 13.09(s, 1H) <Example 1> Preparation of 2- (lff-5-indazolylamino) -6-methoxy -3-nitropyridine . To the solution of 2-chloro-6-methoxy-3-nitropyridine (5 g) and 5-aminoindazole (3.7 g) in methanol (60 ml) was added triethylamine (4.1 ml), and then the solution was reacted at 25-30 ° C for 5 hr. The reaction mixture was cooled at room temperature with slowly adding H 2 0 (30 ml) , and then stirred for 0.5 hr. The reaction mixture was filtered, washed with methanol (10 ml), obtained a solid product. The solid product was dried at 50 ° C in vacuo to obtain the αesired compound (6.5 g, 86%) . m.p. : 206-208 ° C ^-NMR (DMSO-d 6 ) , ppm : δ 3.80 (s, 3H) , 6.32 (d, 1H) , 7.55 (m, 2H), 8.06(s, 2H), 8.43(d, 1H) , 10.52(s, 1H), 13.09(br s, 1H) <Example 2> Preparation of 2- (lH-6-indazolylamino) -6-methoxy -3-nitropyridine . To the solution of 2-chloro-6-methoxy-3-nιtropyrιdme (5 g) and 6-ammomdazole (3.9 g) in methanol (60 ml) was added tπetnylamme (4.1 ml), and then the solution was reacted at 55-60°C for 14 hr. The reaction mixture was cooled at room temperature, added H 2 0 30 ml slowly at 25 ° C , and then stirred for 0.5 hr. The reaction mixture was filtered, washed with 50% aqueous methanol solution (15 ml) and obtained a solid product. The solid product was dried at 50 ° C m va cuo to obtain the desired compound (6.8 g, 90%). m.p. : 261-264 ° C ^- MR (DMSO-d 6 ) , ppm : δ 3.94 (s, 3H) , 6.39 (d, 1H) , 7.24 ( , 1H), 7.71(d, 1H), 8.01(s, 1H) , 8.19(s, 1H) , 8.44(d, 1H) , 10.62(s, 1H) , 13.0 (br s, 1H) <Example 3> Preparation of 2- (lH-5-indazolylam_.no) -6-methyl amino-3-nitropyridine . To the solution of methanol with 40% methylamme (20 ml) was added 2- ( lH-5-mdazolylamino) -6-methoxy-3-nιtropyπdme (1 g) obtained by the example 1, and the solution was reacted at25 ° C forlhr. The reaction mixture was added H 2 0 (20ml) slowly and stirred for 1 hr. The reaction mixture was filtered, washed with 30% aqueous methanol solution (5 ml) and then obtained a solid product. The solid product was dried at 50-60 ° C m va cuo to obtain the desired compound (0.82 g, 82%). m.p. : 238-240 ° C ^-NMR (DMSO-d 6 ) , ppm : 6 2.86 (d, 3H) , 6.09(d, IH) , 7.51 (d, IH) , 7.57 (d, IH) , 8.05(t, 2H) , 8.24 (d, 2H) , 10.97 (s, IH) , 13.05 (br s, IH) <Example 4> Preparation of 2- (lH-6-indazolylamino) -6-methyl amino-3-nitropyridine. To the solution of methanol with 40% methylamine (20 ml) was added 2- (lH-6-indazolylamino) -6-methoxy-3-nitropyridine (1 g) obtained by the example 2, and the solution was reacted at 25-30 * 0 for 2 hr. The reaction mixture was cooled and stirred at 20 ° C for 0.5 hr. The reaction mixture was filtered, washed with methanol (4 ml) and then obtained a solid product . The solid product was dried at 40 ° C in va cuo to obtain the desired compound (0.79 g, 79%) . m.p. : > 270 ° C ^-NMR (DMSO-d 6 ) , ppm : δ 2.98 (d, 3H) , 6.15 (d, IH) , 7.18 (d, IH) , 7.69(d, IH) , 7.99(s, IH) , 8.09(d, IH) , 8.35 (brs, IH) , 8.44 (s, IH ; 11 . 14 ( s , IH ) , 13 . 03 ( br s , IH ) <Example 5> Preparation of 2- (lH-5-indazolylamino) -6-isopropyl amino-3-nitropyridine . To the solution of 2- (lH-5-indazolylamino) -6-methoxy-3-nitropyridine (1 g) obtained by the example 1 in methanol (20 ml) was added isopropylamine (20 ml) slowly and reacted at 45 ° C for 20 hr. The reaction mixture was cooled, added H 2 0 (60 ml) at 25°C and then stirred for 1 hr . The reaction mixture was filtered, washed with 20 % aqueous methanol solution (5 ml) and then obtained a solid product . The solid product was dried at 50 — 60 ° C in vacuo to obtain the desired compound (1.05 g, 96%). m.p. : 233 — 235 ° C 2 H-NMR (DMSO-d 6 ) , ppm : δ 1.15 (d, 6H) , 4.03 (m, IH) , 6.06 (d, IH) , 7.50 (d, 2H) , 8.05(m, 2H) , 8.15(t, 2H) , 10.97 (s, IH) ,13.06 (br s, IH) <Example 6> Preparation of 2- (lH-6-indazolylamino) -6-isopropyl amino-3-nitropyridine . To the solution of 2- ( lH-6-ιndazolylamιno) -6-methoxy-3-nιtropyπdme (1 g) obtained by the example 2 in methano_ (20 ml) was addeα lsopropylamine (20 ml) slowly and reacteα at 45 ° C for 45 hr. The reaction mixture was cooled and stirred at 25 ° C for 1 hr. The reaction mixture was filtered, washed with methanol (5 ml) and then obtained a solid product. The solid product was dried at 40 ~ 50 ° C in va cuo to obtain the desired compound (0.95g, 87%) . m.p. : > 270 ° C X H-NMR (DMSO-d 6 ) , ppm : δ 1.23 (d, 6H) , 4.17 (m, IH) , 6.12 (d, IH), 7.15(d, IH), 7.68(d, IH) , 8.00(s, IH) , 8.09(d, IH) , 8.28(d, IH), 8.35(s, IH) , 11.12(s, IH) , 13.08(br s, IH) <Example 7> Preparation of 2- (lff-5-indazolylamino) -6-isobuthyl amino-3-nitropyridιne . To the solution of 2- ( l -5-mdazolylammo) -6-methoxy-3-nιtropyπdme (1 g) obtained by the example 1 m methanol (20 ml) was added lsobuthylamme (15 ml) slowly and reacted at 45 — 50 ° C for 20 hr. The reaction mixture was cooled, added H 2 0 (40 ml) slowly and then stirred at 25 ° C forlhr. The reaction mixture was filtered, washed with 30% aqueous methanol solution (5 ml) , obtained a solid proαuct . T e solid product was drieα at 50 — 60 ° C m va cuo to obtain the desired compound (0.95 g, 83%). m.p. : 230-232 ° C A-NMR (DMS0-d 6 ) , ppm : δ 0.83 (d, 6H), 1.83 ( , IH), 3.11 (d, 2H) , 6.11(d, IH) , 7.50(s, 2H) , 7.99(s, IH), 8.06(α, IH) , 8.19(d, IH) , 8.39(t, IH), 10.91(s, IH) , 13.07(br s, IH) <Example 8> Preparation of 6-cyclopropylamino-2- (lH-5-indazol ylamino) -3-nitropyridine . To the solution of 2- (lfl-5-indazolylamino) -6-methoxy-3-nitropyridine (1 g) obtained by the example 1 in methanol (20 ml) was added cyclopropylamine (10 ml) slowly, heated and reacted at 40 — 5 ° C for 25 hr. The reaction mixture was cooled with adding H 2 0 (40 ml) slowly and stirred at 25 ° C for 1 hr. The reaction mixture was filtered, washed with 30% aqueous methanol solution (5 ml) , obtained a solid product. The solid product was dried at 50 ° C in va cuo to obtain the desired compound (0.82 g, 75%) . m.p. : 237-240 ° C ^-NMR (DMSO-d 6 ) , ppm : δ 0.56(m, 2H) , 0.81 ( , 2H) , 2.81 (br S,1H) , 6.06(d, IH) , 7.50 (d, IH) , 7.62 (d, IH) , 8.02 (s, IH) , 8.09(d, IH; 46(s ; .57(s, IH; .1.02(s, IH), 13.04(br IH; <Example 9> Preparation of 6-ammo-2- (lff-5-ιndazolylamιno) - 3-nιtropyrιdme . To the solution of 6-chloro-2- (l--5-mdazolylammo) -3-nιtropyπdme (1 g) obtained by the preparation example 1 chloroform (20 ml was added 7 N ammonia solution in methanol (30 ml) and reacted at 35 — 40 ° C for 15 hr. The reaction mixture was cooled, concentrated under reduced pressure at 25 ° C and then precipitated with treatment of methanol (10 ml) . The reaction mixture was filtered, which was recrystallised with methanol : methylene chloride = 4: 1 to obtain the desired compound (0.63 g, 67%) m.p. : 263 ° C (dec. ) ^-N R (DMSO-d 6 ) , ppm : δ 6.05 (d, IH) , 7.48 (m, 2H) , A56(br s, IH), 7.65(br s, IH) , 8.01(s, IH) , 8.12(d, IH) , 8.28(s, IH) , 10.81 (s, IH) , 13.06(br s, IH) <Example 10> Preparation of 6- (2-hydroxyethyl)methylamιno-2- (lH-5-ιndazolylamιno) -3-nιtropyπdιne . To the solution of 6-chloro-2- ( lH-5-ιndazolylamιno) -3-nιtropyπdme (1 g) obtained by the preparation example 1 in acetonitrile (20 ml) was added 2- (methylammo) ethanol (1.4ml) and triethylamme (0.6 ml) and then refluxed for 12 hr . The reaction mixture was cooled, precipitated with addmgexcess H 2 Oat 20 — 25 ° C , filtered to obtain solid. The above obtained solid was washed with water, dried at 50 ° C m va cuo and recrystallised with chloroform : ether = 1: 3 to obtain the desired compound (0.7 g, 62%) . m.p. : 172-174 ° C ^-NMR (DMSO-d 6 ) , ppm : δ 3.14 (s, 3H), 3.64 (m, 4H), 4.80(d, IH) , 6.36(d, IH) , 7.50 (s, 2H) , 8.02 (s, IH) , 8.15(m, 2H) , 10.71 (d, IH) , 13.03(br s, IH) It was prepared compounds m prepared example 11 ~ 30 as the same method used for the example 10. It is shown in Table 1 that the compound name, yield, recrystallizmg solution, melting point of compounds m prepared example 11—30 and 3-nιtropyπdme derivatives (_6) and amme compound (7_) as starting materials. It is shown in Table 2 that 1H -NMR of compounds in prepared example 11—30. TABLE la TABLE lb TABLE 2a TABLE 2b <Experiment 1> Inhibitory effect on the in vitro activities of HBV polymerase in reverse transcription The following in vitro experiment was performed to determine the effect of the compounds of formula 1 on the activity of HBV polymerase during reverse transcription. The present inventors submitted application for a patent concerning HB\ poiymerase genetically expresseα m and separated from E . coli , the process of its preparation, and the method to measure the enzyme activities (KR 94-3916, KR 96-33998,. In the present experiments HBV polymerase was useα which had been expressed m E . coli as stated above. The method used m the present invention to measure in vitro reverse transcribing activities of HBV polymerase is as follows. Basic principles are the same as for ELISA. Nucleotides with biotin or digoxigenm group attached are included as substrates and anti-DIG antibodies attached to peroxidase enzyme recognize the polymerized substrates. To the wells coated with streptavidm, 20//C of HBV polymerase, 20 μJ of reaction mixture (10 μM each of DIG-UTP and Biotm-UTP, 46 mM Tπs-HCl, 266 mM KC1, 27.5 mM MgCl 2 , 9.2 mM DTT substrate/primer hybrid) , and 20 J. of test compound (added to 1, 0.1, and 0.C1 ) were added ana allowed to react at 22 ° C for 15 hrs. During this reaction, HBV polymerase catalyzes DNA synthesis and digoxigenm and biotm attached to nucleotides form bonds with streptavidm coated on the bottom of wells. When the reaction was done, each well was washed with 250 μl of cleaning buffer (pH 7.0) for 30 seconds, which was repeated five times to remove remaining impurities . 200,0 of anti-DIG-POD antibody was added to each well and allowed to react for 1 hr at 37 ° C, and the wells were washed with cleaning buffer to remove impurities. 200//I each of ABTS™, a substrate of peroxidase, was then added and allowed to react at room temperature for 30 mm. Absorbance was measured at 405 nm using ELISA reader. The percentage of reduction in HBV polymerase activities for reverse transcription was calculated using the group without test compound as a control and the results are shown in Table TABLE 3a Effect on the HBV polymerase activities in reverse transription TABLE 3b. Effect on the HBV polymerase activities in revers; transπptior. As shown in Table 3, compounds of tne present inventio: have excellent inhibitory effec Ae HBV polymerase activities with more than 90% inhibition at the concentraticr. of 1 μg/ml. Moreover, compounds of tne present invention are not expected to have problems such as toxicity and development of resistant viruses as observed in the use of nucleosides and maybe applied together with nucleoside compounds due to different mechanisms of action. In summary, compounds of the present invention effectively reduce the activities of HBV polymerase, inhibit replication and proliferation of HBV and may be useful as therapeutics for prevention and treatment of hepatitis B. <Experiment 2> Inhibitory effect on the proliferation of HBV in HBV producing cell line The following experiment was performed to determine inhibitory effects of compounds of formula 1 on the proliferation of HBV producing cell line. To test for antiviral effect, replication andproliferation of HBV were measured m HepG 2.2.15, a human liver cancer cell line. The cell concentration was adjusted to 1 10 5 cells mi and 1 mi was added to each well of a 24-well cell culture plate, which was then kept m a culture box for 3 - 4 days at 37 ° C under 5% C0 2 until cells grew sufficiently, changing culture medium everyday. When the cells matured sufficiently, the test compounds were adoeα to the final concentrations of 0.01, 0.1, and 1 ug/mi . Ore week after the addition of test compounds, the culture solution was centrifugeα at 5,000 rpm for 10 mm. 25 μi of supernatant was transferred to a nev. tube and 5 μi of lysis solution [0.54N NaOH, 0.06% NP40]was added to each tube. After keeping the tube at 37 ° C for 1 hr, 30 μi of neutralizing solution [0.09N HCl, 0. IMTπs-HCi, pH 7.4 ] was added as a reaction solution for competitive polymerase chain reaction (PCR) . PCR was performed using genetic sequence of HBV core protein as a matrix. PCR reaction was carried out by adding 1 unit of Taq polymerase enzyme to 25 pmol of each primer, 250 μM dNTP, 5 μi of PCR reaction solution [0.54NNaOH, 0.06% NP40, 0.09NHC1, DNA polymerized by PCR was electrophoreseα on Agarose gel and quantitatively analyzed using an image analyzer (Gel Doc 1000, Bio-Rad) in order to evaluate the effect of compounds of the present invention on the reduction of HBV proliferation. 3TC (lamivudme) was used as a positive control at the same concentrations as those of the test compounds. The percentage of reduction in HBV proliferation was calculated using the group without test compound as a control and the results are represented in Table 4 TABI Innibitorv effect en the HBV proliferaation As shown above in Table 4, non-nucleoside compounds of the present invention have excellent inhibitory effect on the HBV polymerase activities in reverse transcription with more than 80% reduction of HBV proliferation at the concentration of 1 μglmi . Moreover, compounds of the present invention, beinσ non-nucleosides, may not have problems such as toxicity and early development of resistant virus strains ooserved in the use of nucieosioe substances. It is also expected that compounds of the present invention may be used m parallel with nucleoside compounds since the former act on allosteric binding pockets while the latter act in the domain of polymerase activities. As described above, compounds of the present invention have excellent inhibitory effect on the HBV polymerase activities important reverse transcription step of HBV replication. Based on the mechanism, these compounds are able to effectively control HBV proliferation and may be useful as therapeutics for prevention and treatment of hepatitis B. <Expeπment 3> Inhibitory effect on the in vitro HIV enzyme activities in reverse transcription The following in vitro experiments were done to determine the effect of compounds of formula 1 on the reduction of HIV enzyme activities m reverse transcriotion . Non-radioactive reverse transcπptase assay kit (Boehringer Mannheim) was used in the measurement of in vitro transcriptase activities. 20 μi (40 ng) of HIV transcπptase and 20 μi of reaction mixture containing matrix-primer hybrid poly (A) oligo (dT) i 5 , DIG (digoxigenm) -dUTP, biot -dUTP, and TTP were added to wells coated with streptavidm. Test compounds were also added at the final concentrations of 0.1 ana 1 μg/mi and allowed to react at 37 ° C for 1 hr . At this time, DNA is formed from RNA by the action of HIV reverse transcriptase, forming bonds with streptavidm coated on the bottom of wells oecause of digoxigenm and biotm moieties attached to nucleotides. When the reaction was completed, each well was washed with 250 μi of cleaning buffer (pH 7.0) for 30 sec. five times to remove remaining impurities . 200 μi of anti-DIG-POD antigen was added to each well, allowed to react at 37 ° C for 1 hr an washed as above to remove impurities. 200 μi of ABTS™, a substrate for peroxidase, was added to each well and allowed to react at room temperature for 30mm. Absorbance at 405 nm was then read for each solution using ELISA reader and used for quantitative determination of inhibitory effect on the HIV transcriptase activities. The percentage of reduction m the activities of HIV reverse transcriptase was calculated using the group without test compound as control and the results are represented in Table 5. TABLE 5 Inhibitory effect on the activities of HIV reverse transcriptase As shown above m Table 5, compounds of the present invention have excellent inhibitory effect on the activities of HIV reverse transcriptase, having more than 70% reduction at the concentration of 1 μg/mi . Moreover, it is expected that compounds of the present invention, being non-nucleosidic, do not have problems such as toxicity and early development of resistant virus strains observed the use of nucleoside substances. Furthermore, compounds of the present invention may be used together with nucleoside compounds since the former act on allosteric binding pockets while the latter act m the domain of polymerase activities. As described above, compounds of the present invention have excellent inhibitory effect on the HIV enzyme activities in reverse transcription, which is a step in HIV replication. Based on the mechanism, these compounds are able to effectively control HIV proliferation and may be useful as therapeutics for prevention and treatment of AIDS. <Experiment 4> Cytotoxicity test To determine if compounds of formula 1 exhibit cytotoxicity, in vitro tests were carried out using HepG2 cells withMTT analysis method as generally known and the results are in Table 6 shown below. TABLE 6 Cytotoxicity tests on HepG2 cells As shown above in Table 6, compounds used in the experiments have higher than 100 μg/ i for IC 50 and are considered to have little cvtotoxicitv . <Experiment 5> Acute toxicity in rats tested via oral administration The following experiments were performed to see if compounds of formula 1 have acute toxicity m rats . 6-week old SPF SD line rats were used m the tests for acute toxicity. Compounds in the examples of 1 — 22 were suspended in 0.5% methylcellulose solution and orally administered once to 6 rats per group at the dosage of 2 glkg/ lδ i . Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen checked with eyes during autopsy . The results showed that the test compounds did not cause any specific clinical symptoms , weight change, or death rats . No change was observed m hematological tests, biocnemical tests of blood, and autopsy . The compounds used m this experiment are evaluated to be safe substances since they do not cause any toxic change m rats up to the level of 2 g/kg and their estimated LD 50 values are much greater than 2 g/kg in rats INDUSTRIAL APPLICABILITY As described above, novel 3-nιtropyπdme derivatives of formula 1 in the present invention have dramatic inhibitory effect on proliferation of HBV and of HIV with little side effect and may be useful as therapeutic agents for prevention and treatment of hepatitis B and AIDS. Moreover, it is expected that compounds of the present invention, being non-nucleosidic, do not have problems such as toxicity and early development of resistant virus strains observed the use of nucleoside substances. Furthermore, compounds of the present invention may be used together with nucleoside compounds since the former seem to act on allosteric binding pockets while the latter work in the domain of polymerase activities .

Claims: WHAT IS CLAIMED IS; 1. 3-Nιtropyrιdme derivatives and their pharmaceutically acceptable salts as represented by formula 1. Formula 1 Wh erein Ri is methoxy or ; R 3 is H, hydroxy, dialkyla ino group with C 2 ~C 6 , straight or branched hydroxyalkyl group with C 2 ~C 6 , straight or branched dihydroxyalkyl group with C 3 ~C 6 , alkoxyalkyl group with C 3 -~C 6 , or saturated or unsaturated heterocyclic compounds containing 1 to 3 heteroatoms selected from N, 0, and S, which may be unsubstituted or substituted with alkyl group of Cι~C 3 ; R 3 may or may not contain asymmetrical carbons; R is H, straight or branched alkyl group with Cι~C 4 , or cycloalkyl group with C 3 ~-C 6 ; R 3 and R 4 both may consist of 5 or 6 membered heterocyclic ring containing 1~3 heteroatoms selected fromN, 0, and S, which is unsubstituted or substituted with straight or branched alkyl group with Cι~C 5 , straight or branched hydroxyalkyl group with C 2 ~C 5 , or hydroxy; R 2 is mdazol-5-yl, or mdazol-6-yl ; n is an integer between 0 and 3. 2. Process for the preparation of 3-nιtropyπdme derivatives of formula 1 comprising the following two steps as represented n scheme 1 : Stepl. Synthesis of 3-nιtropyrιdme derivatives of formula 6 by reacting 2-chloro-3-nιtropyrιdme derivatives of formula 4 with 5-ammoindazole or 6-ammoιndazole of formula 5 in the presence of a base; Step 2. Preparation of 3-nιtropyrιdιne derivatives of formula 1 by reacting 3-nιtropyπdme derivatives of formula 6 synthesized in step 1 with amme compounds of formula 7 Scheme 1 1 Wherein, X is chloro or methoxy group; R 2 , R 3 , R 4 and n are as defined in formula 1 3. Method for preparation of 6-methoxy-3-nitropyridine derivatives of formula 6 by reacting 2-chloro-6-methoxy-3-nitropyridine derivatives of formula 4 with 5-aminoindazole or 6-aminoindazole of formula 5 in the presence of a base as in scheme 2. Scheme 2 Wherein, R 2 is as defined in formula 1. 4. Therapeutic agent and a preventive agent for hepatisis B with 3-mtropyrιdme derivatives and their pharmaceutically acceptable salts in claim 1 as effective ingredient. 5. Therapeutic agent and a preventive agent for acquired immune deficiency syndrome (AIDS) with 3-nitropyπdme derivatives and their pharmaceutically acceptable salts m claim 1 as effective ingredient.

Inventors: KIM JONG WOO, KIM NAM DOO, LEE GEUN HYUNG, LEE SANG WOOK, PARK HEE JEOUNG, PARK SANG JIN, PARK YONG KYUN, SHIN DONG HYUK, YOON SUNG JUNE, KIM, JONG, WOO, KIM, NAM, DOO, LEE, GEUN, HYUNG, LEE, SANG, WOOK, PARK, HEE, JEOUNG, PARK, SANG, JIN, PARK, YONG, KYUN, SHIN, DONG, HYUK, YOON, SUNG, JUNE
Applicants: DONG WHA PHARM IND CO LTD, KIM JONG WOO, KIM NAM DOO, LEE GEUN HYUNG, LEE SANG WOOK, PARK HEE JEOUNG, PARK SANG JIN, PARK YONG KYUN, SHIN DONG HYUK, YOON SUNG JUNE, DONG WHA PHARM. IND. CO., LTD, KIM, JONG, WOO, KIM, NAM, DOO, LEE, GEUN, HYUNG, LEE, SANG, WOOK, PARK, HEE, JEOUNG, PARK, SANG, JIN, PARK, YONG, KYUN, SHIN, DONG, HYUK, YOON, SUNG, JUNE
Application year: 2000
Main classification: C07D213
IPC codes: C07D401