KR100566193B1 - Novel 3-nitropyridine derivatives and pharmaceutical compositions thereof - Google Patents

Abstract

The present invention relates to a novel 3-nitropyridine derivative, a method for preparing the same, and a pharmaceutical composition comprising the same. Specifically, the 3-nitropyridine derivative represented by the following Chemical Formula 1, which is a non-nucleic acid compound, is used to proliferate HBV (Hepatitis B Virus). In addition, since it exhibits an effect of inhibiting HIV (Human Immunodeficiency Virus) proliferation, it can be usefully used as a therapeutic and preventive agent for hepatitis B and AIDS.

Wherein R and R ₂ are as described in the specification.

Description

Novel 3-nitropyridine derivatives and pharmaceutical compositions

The present invention relates to a novel 3-nitropyridine derivative, and more particularly, the novel 3-nitropyridine derivative and its pharmaceutically acceptable salts, which are excellent in inhibiting the proliferation of HBV and HIV, A method for producing the same and an antiviral pharmaceutical composition comprising the compound as an active ingredient.

Formula 1

Wherein R is a C ₁ -C ₄ straight or crushed alkylamino group, a C ₃ -C ₆ cycloalkylamino group or a methoxy group, and R ₂ is indazol-5-yl or indazol-6-yl.

Hepatitis B virus (HBV; hereinafter referred to as "HBV") is a pathogen that causes chronic or acute hepatitis and worsens liver cirrhosis and liver cancer. It is estimated (Tiollais & Buendia, Sci. Am. , 264, 48, 1991). Therefore, for the purpose of treatment and prevention of hepatitis B, many studies have been conducted on the relationship between HBV and liver disease, including molecular biological characteristics of HBV, and various vaccines and diagnostic reagents for hepatitis B have been developed. In addition, efforts are underway to develop treatments for hepatitis B.

The genome of HBV is polymerase gene (P; polymerase), surface antigen gene (S; surface protein; pre-S1, pre-S2, S), central antigen gene (C; core protein; pre-C, C), X It consists of four genes, including the protein gene. Among them, polymerase, surface antigen, and central antigen genes express structural proteins, and X protein genes are known to express regulatory proteins.

The HBV polymerase gene accounts for 80% of the entire viral genome and produces a 94kD protein consisting of 845 amino acids, which contains a set of functions necessary for the replication of the viral genome. Ie) protein primer, ii) RNA dependent DNA polymerase (RT), iii) DNA dependent DNA polymerase (DDDP), and Enzyme (RNase H) function and the like are present in one polypeptide. Kaplan et al. Described the reverse transcription activity of polymerase protein for the first time and many studies on the replication mechanism of HBV have been made.

In HBV, surface antigen proteins outside the virion are recognized by hepatocyte-specific receptors and enter into the hepatocytes. At this time, the rest of the incomplete double-stranded DNA is synthesized by HBV polymerase activity, and the HBV DNA genome is synthesized. Is completed. The completed HBV DNA genome produces mRNAs such as pre-genomic mRNAs, central antigens (C), surface antigens (S), and regulatory proteins (X) by intracellular RNA polymerase activity. Viral proteins are made from these mRNAs, and in particular, polymerase proteins play a role in synthesizing the viral genome and form structures called replicatics with central antigen proteins and genome mRNAs. This is called encapsidation, and the polymerase protein is easily encapsidated by nucleic acid affinity at a site where glutamic acid is repeated at the 3′-end. When a replicasome is formed, (-) DNA chain is synthesized by reverse transcription activity of HBV polymerase protein, and (+) DNA chain is synthesized by DNA dependent DNA polymerase activity, which in turn produces genome mRNAs. This repetition maintains a pool of 200-300 or more genomic DNA in the cell (Tiollais and Buendia, Scientific American , 264: 48-54, 1991).

HBV and HIV are different kinds of viruses, but they have a common replication process. That is, the reverse transcription process in which transcription from viral RNA to DNA is performed and the process of decomposing and erasing the RNA portion of the RNA-DNA hybrid generated by reverse transcription is common.

HBV inhibitors of nucleic acid-based compounds, such as lamivudine and famvir, which have recently been developed as treatments for Acquired Immune Deficiency Syndrome (AIDS) or shingles infection As usefulness (Gerin J. L, Hepatology , 14: 198-199, 1991; Lok A. .SP, J. Viral Hepatitis , 1: 105-124, 1994; Dienstag, JL et al ., New England Journal of Medicine , 333: 1657-1661, 1995). However, these nucleic acid-based compounds are very expensive and put a high economic burden on patients, and furthermore, since nucleic acid-based compounds inherently have serious problems in side effects such as toxicity, emergence of resistant virus and relapse after discontinuation of drug administration. It is known that it is unsuitable as a hepatitis agent. Therefore, efforts have been made to develop a hepatitis B therapeutic agent among non-nucleosides compounds, and quinolone compounds having antiviral activity against HBV (European Patent Nos. 563732 and 563734), iridoid compounds (Korean Patent Publication No. 94-1886) and terephthalic acid amide derivatives (Korean Patent Application Nos. 96-72384, 97-36589, 99-5100) and the like have been reported. However, despite many efforts, no clear treatment for hepatitis B has been developed so far, and the treatment of hepatitis B is mainly dependent on symptomatic therapy.

On the other hand, AIDS is a disease that causes a wide variety of rare infectious diseases that cannot be seen in ordinary people due to its markedly diminished cellular immune function, which is the main cause of HIV (Human Immunodeficiency Virus, or "HIV"). The target of attack is known to be a helper T cell, one of the T cells that regulate immune function. When secondary T cells become infected by HIV and cause necrosis, the body's immune function is not functioning properly, resulting in an immunodeficiency condition, which causes fatal infections and malignant tumors. Since it was first discovered in the United States in 1981, more than 850,000 people have been reported in 187 countries in 1993 (the World Health Organization's late 1993 report). Furthermore, the World Health Organization reports that by 2000, 30 million to 40 million more will be infected and 10 million to 20 million of them will develop.

Drugs that inhibit the proliferation process of HIV are currently the most widely used in the treatment of AIDS, including Zidovudine (ZDV), formerly named Azidothymidine (AZT), which was first developed in 1987, and have side effects or treatment effects Alternative medicines include Didanosine (ddI), developed in 1991, and Zalcitabine (ddC), licensed for use in combination with zidovudine in 1992. These drugs have the effect of alleviating the symptoms of AIDS patients, slowing the transition of HIV infected people to AIDS, or slightly extending their survival. However, there is no cure and resistance and side effects are a problem.

Accordingly, the present inventors have endeavored to develop a non-nucleic acid compound exhibiting excellent antiviral activity against HBV for the purpose of developing a new hepatitis B drug having low side effects and toxicity, and low resistance virus appearance. A novel 3-nitropyridine derivative was synthesized and the present invention was completed by revealing that the substance is excellent in inhibiting HBV growth as well as in inhibiting HIV growth.

An object of the present invention is to provide a novel 3-nitropyridine derivative, a pharmaceutically acceptable salt thereof, and a method for preparing the same, which are excellent in inhibiting the growth of HBV and HIV.

It is also an object of the present invention to provide a pharmaceutical composition for the treatment and prevention of hepatitis B or AIDS, comprising the compound as an active ingredient and having low side effects and economical.

In order to achieve the above object, the present invention provides a novel 3-nitropyridine derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof.

Formula 1

Wherein R is a C ₁ -C ₄ straight or crushed alkylamino group, a C ₃ -C ₆ cycloalkylamino group or a methoxy group, and R ₂ is indazol-5-yl or indazol-6-yl.

Indazol-5-yl and indazol-6-yl in the present invention are represented by the following formula (2) and (3).

Preferably, in Formula 1, R is a methoxy, methylamino, ethylamino, isopropylamino, isobutylamino or cyclopropylamino group, and R ₂ is indazol-5-yl or indazol-6-yl.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Compounds of formula (1) may form pharmaceutically acceptable acid addition salts according to methods conventional in the art. Organic acids and inorganic acids may be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

In another aspect, the present invention provides a method for preparing a 3-nitropyridine derivative represented by the following formula (1).

In Scheme 1, R ₁ is a C ₁ -C ₄ straight or crushed alkyl group or C ₃ -C ₆ cycloalkyl group, R ₂ is indazol-5-yl or indazol-6-yl.

The manufacturing method of the present invention

1) 2-nitropyridine derivative by reacting 2-chloro-6-methoxy-3-nitropyridine ( 2 ) with 5-aminoindazole or 6-aminoindazole ( 3 ) in a suitable temperature and a suitable solvent in the presence of a base Preparing ( 1a ) (step 1); And

2) reacting the 3-nitropyridine derivative ( 1a ) prepared in Step 1 with a suitable amine compound ( 4 ) in a suitable temperature and a suitable solvent to prepare 3-nitropyridine derivative ( 1b ) (step 2). .

Meanwhile, when R in the compound of Formula 1 is a methoxy group, it is prepared through Step 1.

2-chloro-6-methoxy-3-nitropyridine ( 2 ), 5-aminoindazole or 6-aminoindazole ( 3 ) and amine compounds (used as starting materials and reactants in steps 1 and 2 ) 4 ) is readily available as a commercially available material. In particular, the amine compound ( 4 ) is inexpensive.

In addition, the amine compound ( 4 ) of step 2 is a substance for introducing a substituent R into the compound of Formula 1, it is possible to select an appropriate amine compound ( 4 ) according to the type of the substituent. Such amine compounds ( 4 ) include, for example, methylamine methanol solution, ethylamine aqueous solution, isopropylamine, isobutylamine, cyclopropylamine and the like, all of which are commercially available materials.

In more detail the step 1, an organic base may be used as a base, for example triethylamine, N, N -diisopropylethylamine, N -methylmorpholine, N -methylpiperidine Preference is given to using common tertiary organic bases such as, 4-dimethylaminopyridine, N, N -dimethylaniline, 2,6-lutidine, pyridine and the like.

It is preferable that reaction temperature is 20-60 degreeC, and it is preferable to set reaction time as 4-15 hours.

As the reaction solvent, it is preferable to use a single solvent or a mixed solvent selected from alcohols such as methanol and ethanol, chloroform, methylene chloride, acetonitrile and the like.

In addition, when the step 2 is described in more detail, it is preferable to use a single solvent or a mixed solvent selected from alcohols such as water, methanol, ethanol and isopropanol, acetonitrile, chloroform, methylene chloride, and the like as the reaction solvent.

The reaction temperature may vary depending on the type of amine compound ( 4 ), and is preferably 25 to 60 ° C.

It is preferable to use an amine compound ( 4 ) in excess with respect to 3-nitropyridine derivative ( 1a ).

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing hepatitis B comprising as an active ingredient a 3-nitropyridine derivative of formula (1) and / or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of acquired immunodeficiency syndrome, comprising as an active ingredient a 3-nitropyridine derivative of formula (1) and / or a pharmaceutically acceptable salt thereof.

The 3-nitropyridine derivative represented by the general formula (1) of the present invention is a replication process that is common in the proliferation process of HBV and HIV, that is, a process of reverse transcription from viral RNA to DNA and degradation of an RNA portion in the generated RAN-DNA hybrid. It has a mechanism of action that inhibits and has proliferation inhibitory activity against HIV as well as HBV.

The compound of formula 1 may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration and may be used in the form of general pharmaceutical formulations.

Clinical use of the pharmaceutical composition of the present invention may be combined with a conventional carrier in the pharmaceutical field for oral administration of a conventional agent in the pharmaceutical field, for example, tablets, capsules, troches, solutions, suspensions, and the like. Formulations; It may be formulated into a variety of preparations, such as injectable solutions or suspensions, or injectable preparations in the form of ready-to-use injectable dry powders that can be prepared and used as injectable distilled water at the time of injection.

The effective dose of the compound of formula 1 is generally 10-500 mg / kg in adults, preferably 50-300 mg / kg, several times a day at a predetermined time interval, preferably one day, at the discretion of the doctor or pharmacist. It may be administered in 1 to 6 divided doses.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 2- (1 H Preparation of -5-indazolylamino) -6-methoxy-3-nitropyridine

To 60 ml of methanol, 5 g of 2-chloro-6-methoxy-3-nitropyridine, 3.7 g of 5-aminoindazole and 4.1 ml of triethylamine were added, followed by reaction at 25 to 30 ° C. for 5 hours. 30 ml of water was added to the reaction mixture, stirred for 30 minutes, filtered, washed with 10 ml of methanol to obtain crystals, which were dried in vacuo at 50 ° C. to obtain 6.5 g (yield 86%) of the title compound.

m.p. : 206-208 ℃

¹ H-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, 1 H), 13.09 (br s, 1 H)

Example 2 2- (1 H Preparation of -6-indazolylamino) -6-methoxy-3-nitropyridine

To 60 ml of methanol, 5 g of 2-chloro-6-methoxy-3-nitropyridine, 3.9 g of 6-aminoindazole and 4.1 ml of triethylamine were added, followed by reaction at 55 to 60 ° C. for 14 hours. The reaction mixture was cooled, added 30 ml of water at 25 ° C., stirred for 30 minutes, filtered and washed with 15 ml of 50% methanol to obtain crystals, which were then dried under vacuum at 50 ° C. to yield 6.8 g (90% yield) of the target. The compound was obtained.

m.p. : 261-264 degreeC

¹ H-NMR (DMSO-d ₆ ), ppm: δ 3.94 (s, 3H), 6.39 (d, 1H), 7.24 (m, 1H), 7.71 (d, 1H), 8.01 (s, 1H), 8.19 (s, 1 H), 8.44 (d, 1 H), 10.62 (s, 1 H), 13.04 (br s, 1 H)

Example 3 2- (1 H Preparation of -5-indazolylamino) -6-methylamino-3-nitropyridine

1 g of 2- ( 1H -5-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 1 was added to 20 ml of methylamine 40% methanol solution and reacted at 25 ° C for 1 hour. 20 ml of water was slowly added to the reaction mixture, stirred for 1 hour, filtered, washed with 5 ml of 30% methanol to obtain crystals, which were then dried under vacuum at 50-60 ° C. to yield 0.82 g (yield 82%) of the title compound. Got it.

m.p. : 238-240 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.86 (d, 3H), 6.09 (d, 1H), 7.51 (d, 1H), 7.57 (d, 1H), 8.05 (t, 2H), 8.24 (d, 2H), 10.97 (s, 1H), 13.05 (br s, 1H)

Example 4 2- (1 H Preparation of -6-indazolylamino) -6-methylamino-3-nitropyridine

To 20 ml of methylamine 40% methanol solution, 1 g of 2- ( 1H -6-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 2 was added and reacted at 25 to 30 ° C. for 2 hours. I was. The reaction mixture was cooled slightly and stirred at 20 ° C. for 30 minutes, filtered and washed with 4 ml of methanol to give crystals which were then dried in vacuo at 40 ° C. to yield 0.79 g (79% yield) of the title compound.

m.p. :> 270 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 2.98 (d, 3H), 6.15 (d, 1H), 7.18 (d, 1H), 7.69 (d, 1H), 7.99 (s, 1H), 8.09 (d, 1H), 8.35 (br s, 1H), 8.44 (s, 1H), 11.14 (s, 1H), 13.03 (br s, 1H)

Example 5 2- (1 H Preparation of -5-indazolylamino) -6-isopropylamino-3-nitropyridine

1 g of 2- ( 1H -5-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 1 was added to 20 ml of methanol, and 20 ml of isopropylamine was added slowly, followed by heating at 45 ° C. The reaction was carried out for 20 hours. The reaction mixture was cooled, slowly added 60 ml of water at 25 ° C., stirred for 1 hour, filtered and washed with 5 ml of 20% methanol to obtain crystals, which were dried under vacuum at 50-60 ° C. to yield 1.05 g (yield 96 %) Of the title compound.

m.p. : 233-235 ° C

¹ H-NMR (DMSO-d ₆ ), ppm: δ 1.15 (d, 6H), 4.03 (m, 1H), 6.06 (d, 1H), 7.50 (d, 2H), 8.05 (m, 2H), 8.15 (t, 2H), 10.97 (s, 1H), 13.06 (br s, 1H)

Example 6 2- (1 H Preparation of -6-indazolylamino) -6-isopropylamino-3-nitropyridine

1 g of 2- ( 1H -6-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 2 was added to 20 ml of methanol, and 20 ml of isopropylamine was added slowly, followed by heating at 45 ° C. The reaction was carried out for 45 hours. The reaction mixture was cooled, stirred at 25 ° C. for 1 hour, filtered and washed with 5 ml of methanol to obtain crystals, which were then vacuum dried at 40-50 ° C. to yield 0.95 g (yield 87%) of the title compound.

m.p. :> 270 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ1.23 (d, 6H), 4.17 (m, 1H), 6.12 (d, 1H), 7.15 (d, 1H), 7.68 (d, 1H), 8.00 (s, 1H), 8.09 (d, 1H), 8.28 (d, 1H), 8.35 (s, 1H), 11.12 (s, 1H), 13.08 (br s, 1H)

Example 7 2- (1 H Preparation of -5-indazolylamino) -6-isobutylamino-3-nitropyridine

1 g of 2- ( 1H -5-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 1 was added to 20 ml of methanol, 15 ml of isobutylamine was added slowly, followed by heating to 45-50 The reaction was carried out at 20 ° C. for 20 hours. The reaction mixture was cooled, slowly added 40 ml of water at 25 ° C., stirred for 1 hour, filtered, washed with 5 ml of 30% methanol to obtain crystals, which were then dried under vacuum at 50-60 ° C. to 0.95 g (yield 83%). ) To obtain the target compound.

m.p. : 230 ~ 232 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ 0.83 (d, 6H), 1.83 (m, 1H), 3.11 (d, 2H), 6.11 (d, 1H), 7.50 (s, 2H), 7.99 (s, 1H), 8.06 (d, 1H), 8.19 (d, 1H), 8.39 (t, 1H), 10.91 (s, 1H), 13.07 (br s, 1H)

Example 8 6-cyclopropylamino-2- (1 H Preparation of -5-indazolylamino) -3-nitropyridine

1 g of 2- ( 1H -5-indazolylamino) -6-methoxy-3-nitropyridine prepared in Example 1 was added to 20 ml of methanol, and 10 ml of cyclopropylamine was added slowly, followed by heating to 40 to The reaction was carried out at 45 ° C. for 25 hours. The reaction mixture was cooled, slowly added 40 ml of water at 25 ° C., stirred for 1 hour, filtered and washed with 5 ml of 30% methanol to obtain crystals, which were then vacuum dried at 50 ° C. to yield 0.82 g (yield 75%) of The desired compound was obtained.

m.p. : 237 ~ 240 ℃

¹ H-NMR (DMSO-d ₆ ), ppm: δ0.56 (m, 2H), 0.81 (m, 2H), 2.81 (br s, 1H), 6.06 (d, 1H), 7.50 (d, 1H) , 7.62 (d, 1H), 8.02 (s, 1H), 8.09 (d, 1H), 8.46 (s, 1H), 8.57 (s, 1H), 11.02 (s, 1H), 13.04 (br s, 1H)

Compounds of Formula 1 prepared through Examples 1 to 8 are shown in Table 1 below.

Example R R ₂ One Methoxy Indazole-5-day 2 Methoxy Indazole-6-day 3 Methylamino Indazole-5-day 4 Methylamino Indazole-6-day 5 Isopropylamino Indazole-5-day 6 Isopropylamino Indazole-6-day 7 Isobutylamino Indazole-5-day 8 Cyclopropylamino Indazole-5-day

Experimental Example 1 Inhibitory Effect of In Vitro Reverse Transcription Activity on HBV Polymerase

In order to determine the effect of the compounds of Formula 1 inhibit the reverse transcription activity of HBV polymerase, the following in vitro experiment was performed.

The present inventors have already filed a patent for a recombinant polymerase protein of HBV expressed in E. coli, a method for preparing the same, and a method for measuring the enzyme activity thereof (Korean Patent Application Nos. 94-3918 and 96-33998). In this experiment, HBV polymerase expressed in E. coli was used as described above.

The method for measuring reverse transcriptase activity of hepatitis B virus polymerase in vitro used in the present invention is as follows. The basic principle is the same as the enzyme-immunological method (ELISA), in which nucleotides modified with biotin- and DIG- (digoxigenin-) are reacted with the substrate, and then the polymerized substrate is attached to the antiperoxidant The method of recognizing with -DIG antibody was used.

20 μl of HBV polymerase was added to a well coated with streptavidin and the reaction mixture [10 μM of DIG-UTP, Biotin-UTP, 46 mM Tris-HCl, 266 mM KCl, 27.5 mM MgCl 2, 9.2 mM DTT, respectively. Substrate / Primer Hybrid] 20 µl and 20 µl of the test substance (added so that the concentrations are 1, 0.1, and 0.01 µg / ml, respectively) were reacted at 22 ° C for 15 hours. The DNA is produced by the action of HBV polymerase and contains digoxigenin and biotin-attached nucleotides, which binds to streptavidin coated on the bottom of the well. After the reaction was completed, washed 5 times 30 seconds with 250 μl of wash buffer (pH 7.0) per well to remove the remaining impurities. After 200 μl of anti-DIG-POD antibody was added to each well and reacted at 37 ° C. for 1 hour, each well was washed with a washing buffer to remove impurities. Thereafter, 200 μl of ABTS ™, a substrate of POD (peroxidase), was added thereto, and the mixture was reacted at room temperature for 30 minutes, and the absorbance at 405 nm was measured using an ELISA reader.

Inhibition rate of the reverse transcription activity of the HBV polymerase was calculated based on the control without the test compound, the results are shown in Table 2 below.

Inhibitory Effect on Reverse Transcriptase Activity of HBV Polymerase Test compound % Inhibition of activity of HBV-RT 1 μg / ml 0.1 μg / ml 0.01 μg / ml Example 1 85 54 30 Example 2 76 50 12 Example 3 58 47 20 Example 4 60 51 26 Example 5 96 87 53 Example 6 91 76 49 Example 7 95 80 47 Example 8 72 52 38

As can be seen in Table 2 , the compounds of the present invention has an activity inhibition rate of 90% or more for HBV polymerase at a concentration of 1 ㎍ / ㎖, it is very excellent in inhibiting the activity of HBV polymerase. In addition, since the compounds of the present invention are non-nucleic acid-based materials, it is expected to solve problems such as the early appearance of toxic and resistant viruses possessed by nucleic acid-based materials, and they may be used in combination with nucleic acid-based materials because they have different mechanisms of action. It can also be used as a therapy.

As described above, the compounds of the present invention have an excellent effect of inhibiting the activity of HBV polymerase, which plays an important role in the replication of HBV. Therefore, the compounds of the present invention can inhibit the proliferation of HBV. Can be used.

Experimental Example 2 Effect of HBV Proliferation Inhibitory Activity Using HBV Producing Cell Line

In order to determine the effects of the compounds of Formula 1 inhibit the proliferation of HBV producing cell line, the following experiment was carried out.

To detect antiviral activity, HepG 2.2.15, a human liver cancer cell line, was used to measure the extent of HBV replication and proliferation.

The cell concentration was adjusted to 1 × 10 ⁵ cells / ml and then dispensed in 1 ml / well into 24-well cell culture plates. This was incubated in a 5% CO ₂ incubator at 37 ° C., the medium was changed daily and incubated for 3-4 days until the cells were sufficiently grown (confluent). After sufficient growth of the cells, test compounds were added so that the final concentrations were 0.01, 0.1, and 1 μg / ml, respectively. One week after the addition of the test compound, the culture medium was taken and centrifuged at 5,000 rpm for 10 minutes. 25 μl of supernatant was transferred to a new tube, and 5 μl of lysis solution [0.54N NaOH, 0.06% NP40] was added and incubated at 37 ° C. for 1 hour. After incubation, 30 μl of neutralization solution [0.09N HCl, 0.1M Tris-HCl, pH 7.4] was added and used as a reaction solution for competitive PCR (Competitive Polymerase Chain Reaction).

PCR was performed using the core gene sequence of HBV as a template. 1 unit of Taq polymerase was added to 25 pmol of each primer, 250 μM dNTP, 5 μl of the above PCR reaction solution [0.54N NaOH, 0.06% NP40, 0.09N HCl, 0.1M Tris-HCl, pH 7.4]. PCR reaction was performed.

DNA amplified by PCR was subjected to agarose gel electrophoresis, and then quantitative analysis of HBV DNA using an image analyzer (Gel Doc 1000, BIO-RAD) to evaluate the HBV growth inhibitory activity of the compounds of the present invention.

3TC (lamivudine) was used as a positive control and treated at the same concentration as the test compound. HBV proliferation inhibitory activity rate was calculated based on the control without the test compound, the results are shown in Table 3 below.

HBV proliferation inhibitory activity Test compound HBV proliferation inhibitory activity rate (%) 1 μg / ml 0.1 μg / ml 0.01 μg / ml Example 1 82 45 20 Example 2 72 40 - Example 3 53 35 - Example 4 58 40 - Example 5 94 81 41 Example 6 90 72 33 Example 7 93 75 30 Example 8 65 46 25 3TC 99 80 48

As can be seen in Table 3 , the compounds of the present invention is a non-nucleic acid-based substance having a proliferation inhibitory activity against HBV of 90% or more at a concentration of 1 ㎍ / ㎖, very excellent effect of inhibiting reverse transcription activity of HBV polymerase Do. In particular, since the compounds of the present invention are non-nucleic acid-based materials, it is expected to solve problems such as the early appearance of toxic and resistant viruses possessed by nucleic acid-based materials. In addition, since the nucleic acid-based materials act on the active domain of the polymerase, and the compounds of the present invention are expected to act on the allosteric binding pocket, the compounds of the present invention may be used as a combination therapy with the nucleic acid-based materials. Can be used.

As described above, the compounds of the present invention have an excellent effect of inhibiting the reverse transcription activity of HBV polymerase, which plays an important role in the reverse transcription phase of HBV replication. And as therapeutic agents.

Experimental Example 3 In Vitro Inhibitory Effect on HIV Reverse Transcriptase

In order to determine the effect of the compounds of Formula 1 inhibit the activity of HIV reverse transcriptase, the following in vitro experiments were carried out.

In vitro inhibitory activity was measured using a non-radioactive reverse transcriptase assay kit (Boehringer Mannheim).

20 μl (40 ng) of HIV reverse transcriptase was added to a well coated with streptavidin, and the template- primer hybrid poly (A) oligo (dT) ₁₅ and DIG- (digoxigenin) -dUTP, biotin- After 20 µl of a reaction mixture containing dUTP and TTP was added, the test compound was added thereto to a final concentration of 0.1 and 1 µg / ml, and reacted at 37 ° C for 1 hour. At this time, DNA is made from RNA by the action of HIV reverse transcriptase, and since digoxigenin and biotin are labeled together, streptavidin is coated on the bottom of the well. Will be combined.

After completion of the reaction, washing was performed 5 times for 30 seconds with 250 μl of washing buffer solution (pH 7.0) per well to remove remaining impurities. After washing, 200 μl of anti-DIG-POD antibody was added thereto and reacted at 37 ° C. for 1 hour, and the resultant was washed with the same buffer solution for washing to remove impurities. After washing, 200 μl of ABTS ™, a substrate of POD (peroxidase), was added to each well and allowed to react at room temperature for 30 minutes. After completion of the reaction, the degree of inhibition of reverse transcriptase activity of HIV was quantified by measuring the absorbance at 405 nm of each solution using an ELISA reader. Inhibition rate for HIV reverse transcriptase activity was calculated based on the control without the test compound, the results are shown in Table 4 below.

Inhibitory Effect on HIV Reverse Transcriptase Activity Test compound HIV-RT activity inhibition rate (%) 1 μg / ml 0.1 μg / ml Example 1 75 35 Example 4 70 51 Example 5 55 20 Example 6 69 50 Example 7 72 45

As can be seen in Table 4 , the compounds of the present invention has an activity inhibition rate of 70% or more against HIV-RT (HIV reverse transcriptase) at a concentration of 1 ㎍ / ㎖, very excellent effect of inhibiting the activity of HIV reverse transcriptase Do. In particular, since the compounds of the present invention are non-nucleic acid-based materials, it is expected to solve problems such as the early appearance of toxic and resistant viruses possessed by nucleic acid-based materials. In addition, since the nucleic acid-based substances act on the active domain of the polymerase, the compounds of the present invention are expected to act on the allosteric binding pocket, so the compounds of the present invention may be used as a combination therapy with nucleic acid-based substances. Can be used.

As described above, the compounds of the present invention have an excellent effect of inhibiting the activity of HIV reverse transcriptase, which plays an important role in the reverse transcription phase of HIV replication. Therefore, the compounds of the present invention can suppress the proliferation of HIV, thus preventing and preventing AIDS. It can be usefully used as a therapeutic agent.

Experimental Example 4 Cytotoxicity Test

In order to determine whether the compounds of Formula 1 exhibit cytotoxicity, in vitro experiments were carried out using HepG2 cells in a generally known MTT assay method.

As a result, all of the compounds used in the experiment were found to have a CC ₅₀ of 100 µg / ml or more, which is a very low toxicity to cells.

Experimental Example 5 Oral Acute Toxicity in Rats

In order to determine the acute toxicity of the compound of Formula 1 was performed the following experiment.

Acute toxicity experiments were performed using 6-week-old SPF SD rats. Six animals per group were suspended orally at a dose of 2 g / kg / 15 ml, each of the compounds of Examples 1-8 suspended in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and blood biochemical tests were performed. Necropsy was performed to visually check for abdominal and thoracic organ abnormalities. As a result, all animals treated with test substance showed no clinical symptoms and no dead animals, and no toxic changes were observed in weight change, blood test, blood biochemistry, autopsy findings. As a result, all of the tested compounds did not show toxic changes up to 2 g / kg in rats, and the minimum lethal dose (LD ₅₀ ) for oral administration was determined to be a safe substance of 2 g / kg or more.

As described above, the novel 3-nitropyridine derivative represented by Formula 1 according to the present invention has an excellent effect of inhibiting the proliferation of HBV and HIV, and has fewer side effects, thereby preventing hepatitis B and AIDS. It can be usefully used as a therapeutic agent. In particular, since the compounds of the present invention are non-nucleic acid-based materials, it is expected to solve problems such as early appearance of toxic and resistant viruses possessed by nucleic acid-based materials. In addition, since the nucleic acid compounds act on the active domain of the polymerase while the compounds of the present invention are expected to act on the allosteric binding pocket, the compounds of the present invention may be used as a combination therapy with nucleic acid compounds. have.

Claims (5)

3-nitropyridine derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof: Formula 1

(In Formula 1, R is a C _{1 ~} C ₄ linear or crushed alkylamino group, C ₃ ~ C ₆ cycloalkylamino group or methoxy group; R ₂ is indazol-5-yl or indazole-6- Work). The 3-nitropyridine according to claim 1, wherein R is a methoxy, methylamino, isopropylamino, isobutylamino or cyclopropylamino group and R ₂ is indazol-5-yl or indazol-6-yl. Derivatives or pharmaceutically acceptable salts thereof. 1) 2-chloro-6-methoxy-3-nitropyridine ( 2 ) and 5-aminoindazole or 6-aminoindazole ( 3 ) to triethylamine, N, N -diisopropylethylamine, N- 1 type selected from the group consisting of methyl morpholine, N -methylpiperidine, 4-dimethylaminopyridine, N, N -dimethylaniline, 2,6-lutidine and pyridine Preparing 3-nitropyridine derivative ( 1a ) by reacting in a single solvent or at least one mixed solvent selected from the group consisting of methanol, ethanol, chloroform, methylene chloride and acetonitrile in the presence of a base (step 1); And 2) The 3-nitropyridine derivative ( 1a ) and the appropriate amine compound ( 4 ) prepared in step 1 are selected from the group consisting of water, lower alcohols having 1 to 3 carbon atoms, acetonitrile, chloroform and methylene chloride. A process for preparing the 3-nitropyridine derivative of claim 1, comprising the step (step 2) of preparing 3-nitropyridine derivative ( 1b ) by reacting in a single solvent or one or more mixed solvents: Scheme 1

(In Scheme 1, R ₁ is a C ₁ -C ₄ straight or crushed alkyl group or C ₃ -C ₆ cycloalkyl group; R ₂ is indazol-5-yl or indazol-6-yl). A pharmaceutical composition for treating or preventing hepatitis B, comprising the 3-nitropyridine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for treating or preventing AIDS, comprising the 3-nitropyridine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.