3-nitropyridine derivaives and the pharmaceutical compositions containing said derivatives

TECHNICAL FIELD

The present invention relates to novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives. More specifically, the present invention relates to 3-nitropyridine 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-nitropyridine derivatives and to the pharmaceutical compositions containing said derivatives as effective ingredients against viruses.

Wherein,

R

1

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 5or 6 membered heterocyclic compounds containing 1 to 3 heteroatoms selected from N, O, and S, which may be unsubstituted or substituted with alkyl group of C

1

˜C

3

; R

3

may or may not contain asymmetrical carbons;

R

4

is H, straight or branched alkyl group with C

1

˜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 N, O, and S, which is unsubstituted or substituted with straight or branched alkyl group with C

1

˜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.

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 diagnostic 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-S1, 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 94 kD 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, and RNase H. 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,

Scientific American

, 264: 48-54, 1991).

Although HBV and HIV are different viruses, the replication mechanisms during their proliferation have some common steps, namely, the reverse transcription of virus RNA to form DNA and the removal of RNA strand from subsequently formed RNA-DNA hybrid.

Recently, nucleoside compounds such as lamivudine and famvir have been reported to be useful inhibitors of HBV proliferation, although they have been originally developed as therapeutics for the treatment of acquired immune deficiency syndrome (AIDS; referred as “AIDS” hereinafter) and herpes zoster infection (Gerin, J. L,

Hepatology

, 14: 198-199, 1991; Lok, A. S. P., J.

Viral Hepatitis

, 1: 105-124, 1994; Dienstag, J. L. et al.,

New England Journal of Medicine

, 333: 1657-1661, 1995). However, these nucleoside compounds are considered a poor choice for treatment of hepatitis B because of their high cost and 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 quinolone compounds (EPO0563732, EPO0563734), iridos 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 not been developed yet and therapeutic method 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 in 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 in immune function subsequently results in fatal infection and development of malignant tumor. Since AIDS patient has been found in USA in 1981 for the first time, the number increased to more than 850,000 patients in 187 countries in 1993 (WHO 1993 report). WHO predicted that 30 to 40 million more people would be infected with HIV by the 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, Zidovudine, which had been named Azidothymidine previously, is a drug developed in 1987. Didanosine 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, Zalcitabine was approved for concurrent use with Zidovudine in 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 compounds 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.

Wherein,

R

1

is methoxy or

Compounds of formula 1 in the present invention may be utilized in 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 in 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 acid may 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, succinic 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-nitropyridine derivatives of formula 1 as represented in scheme 1.

Wherein, X is Cl or OCH

3

; R

2

R

3

, R

4

and n are as defined in formula 1.

The process of preparation in the present invention comprises the following steps:

(Step 1) Synthesis of 3-nitropyridine derivatives of formula 6 by reacting 2-chloro-3-nitropyridine derivatives of formula 4 with 5-aminoindazole or 6-aminoindazole of formula 5 in a proper solvent under a basic condition at an appropriate temperature;

(Step 2) Synthesis of 3-nitropyridine derivatives of formula 1 by reacting 3-nitropyridine derivatives of formula 6 prepared in step 1 with appropriate amine compounds of formula 7 in a proper solvent under a basic condition at an appropriate temperature.

When R

1

in the compound of formula 1 is a methoxy group, step 1 completes the synthesis of desired compound (X=OCH

3

). In this case, the present invention includes the method of preparing 6-methoxy-3-nitropyridine derivatives of formula 6 by reacting 2-chloro-6-methoxy-3-nitropyridine of formula 4 with 5-aminoindazole or 6-aminoindazole of formula 5 in the presence of a base.

Compound of formula 4 in the first step of scheme 1 is 2-chloro-6-methoxy-3-nitropyridine or 2,6-dichloro-3-nitropyridine.

Chemical reagents used in the first and the second steps of scheme 1, namely, 2-chloro-3-nitropyridine derivatives of formula 4, 5-aminoindazole or 6-aminoindazole of formula 5, and amine 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 amine 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 in the synthetic process, an organic base may be used and common tertiary amines such as triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, N-methylpiperidine, 4-dimethylaminopyridine, N,N-dimethylaniline, 2,6-lutidine, pyridine 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, acetonitrile and alcohols such as methanol and ethanol.

Of 3-nitropyridine derivatives of formula 6 produced in the reaction of step 1, one with chloro group at 6 position is used in the following reaction of step 2.

The reaction in step 2 is described in more detail. Preferable solvent is a single or a mixture of solvents selected from acetonitrile, chloroform, methylene chloride, tetrahydrofuran, N,N-dimethylformamide, N-methylpyrrolidinone, pyridine, water and alcoholic solvents such as methanol, ethanol, and isopropanol.

It is preferable to use excess amount of amine compound (formula7) to increase the efficiency of the reaction. Solvents used in the previous step 1 for the synthesis of 3-nitropyridine derivatives of formula 6 are preferable. Reaction temperature of 25˜80° C. is preferable although it depends on the kind of amine compound used.

In another aspect of this invention, also provided are the pharmaceutical compositions of therapeutics for preventing and treating hepatitis B, which contain 3-nitropyridine derivatives of formula 1 and their pharmaceutically acceptable salts as effective ingredients.

The present invention also provides the pharmaceutical compositions of therapeutics for preventing and treating AIDS, which contain 3-nitropyridine derivatives and their pharmaceutically acceptable salts of formula 1 as effective ingredients.

3-nitropyridine derivatives of formula 1 in 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, intraperitoneally, or locally and used in 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 is generally 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 in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

PREPARATION EXAMPLE 1

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

To the solution of 2,6-dichloro-3-nitropyridine (4 g) and 5-aminoindazole (2.8 g) in acetonitrile (50 ml) was added triethylamine (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

O 30 ml slowly, and then reacted at 20° C. for 1 hr. The reaction mixture was filtered, washed with the mixture solution(15 ml) of acetonitrile: H

2

O=1:1 (volume ratio) and dried at 50° C. in vacuo to obtain the desired compound (4.7 g, 78%).

m.p.: 233° C. (dec.)

1

H-NMR (DMSO-d

6

), 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-(1H-6-indazolylamino)-3-nitropyridine

To the solution of 2,6-dichloro-3-nitropyridine (4 g) and 6-aminoindazole (2.8 g) in acetonitrile (50 ml) was added triethylamine (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

O (20 ml), and then reacted at 20-25° C. for 1 hr. The reaction mixture was filtered, washed with acetonitrile (6 ml) and H

2

O (15 ml), and then dried at 50° C. in vacuo to obtain the desired compound (4.4 g, 73%).

m.p.: 234° C. (dec.)

1

H-NMR (DMSO-d

6

), ppm: δ 7.02(d, 1H), 7.20(d, 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-(1H-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

O (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 desired compound (6.5 g, 86%).

m.p.: 206˜208° C.

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, 1H), 13.09(br s, 1H)

EXAMPLE 2

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

To the solution of 2-chloro-6-methoxy-3-nitropyridine (5 g) and 6-aminoindazole (3.9 g) in methanol (60 ml) was added triethylamine (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

O 30 mi 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. in vacuo to obtain the desired compound (6.8 g, 90%).

m.p.: 261˜264° C.

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, 1H), 8.44(d, 1H), 10.62(s, 1H), 13.04(br s, 1H)

EXAMPLE 3

Preparation of 2-(1H-5-indazolylamino)-6-methyl amino-3-nitropyridine

To the solution of methanol with 40% methylamine (20 ml) was added 2-(1H-5-indazolylamino)-6-methoxy-3-nitropyridine (1 g) obtained by the example 1, and the solution was reacted at 25° C. for 1 hr. The reaction mixture was added H

2

O (20 ml) 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. in vacuo to obtain the desired compound (0.82 g, 82%).

m.p.: 238˜240° C.

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

Preparation of 2-(1H-6-indazolylamino)-6-methyl amino-3-nitropyridine

To the solution of methanol with 40% methylamine (20 ml) was added 2-(1H-6-indazolylamino)-6-methoxy-3-nitropyridine (1 g) obtained by the example 2, and the solution was reacted at 25-30° C. 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 vacuo to obtain the desired compound (0.79 g, 79%).

m.p.: >270° C.

1

H-NMR (DMSO-d

6

), ppm: δ 2.98(d, 3H), 6.15(d, 1H), 7.18(d, 1H), 7.69(d, 1), 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

Preparation of 2-(1H-5-indazolylamino)-6-isopropyl amino-3-nitropyridine

To the solution of 2-(1H-5-indazolylamino)-6-methoxy-3-nitropyridine (1 q) 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

O (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.

1H-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

Preparation of 2-(1H-6-indazolylamino)-6-isopropyl amino-3-nitropyridine

To the solution of 2-(1H-6-indazolylamino)-6-methoxy-3-nitropyridine (1 g) obtained by the example 2 in methanol (20 ml) was added isopropylamine (20 ml) slowly and reacted 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 vacuo to obtain the desired compound (0.95 g, 87%).

m.p.: >270° C.

1

H-NMR (DMSO-d

6

), ppm: δ 1.23(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

Preparation of 2-(1H-5-indazolylamino)-6-isobuthyl amino-3-nitropyridine

To the solution of 2-(1H-5-indazolylamino)-6-methoxy-3-nitropyridine (1 g) obtained by the example 1 in methanol (20 ml) was added isobuthylamine (15 ml) slowly and reacted at 45˜50° C. for 20 hr. The reaction mixture was cooled, added H

2

O (40 ml) slowly and then 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 sold product was dried at 50˜60° C. in vacuo to obtain the desired compound (0.95 g, 83%).

m.p.: 230˜232° C.

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

Preparation of 6-cyclopropylamino-2-(1H-5-indazol ylamino)-3-nitropyridine

To the solution of 2-(1H-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˜45° C. for 25 hr. The reaction mixture was cooled with adding H

2

O (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 vacuo to obtain the desired compound (0.82 g, 75%).

m.p.: 237˜240° C.

1

H-NMR (DMSO-d

6

), ppm: δ 0.56(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)

EXAMPLE 9

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

To the solution of 6-chloro-2-(1H-5-indazolylamino)-3-nitropyridine (1 g) obtained by the preparation example 1 in 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.)

1

H-NMR (DMSO-d

6

), ppm: δ 6.05(d, 1H), 7.48(m, 2H), 7.56(br s, 1H), 7.65(br s, 1H), 8.01 (s, 1H), 8.12 (d, 1H), 8.28 (s, 1H), 10.81(s, 1H), 13.06(br s, 1H)

EXAMPLE 10

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

To the solution of 6-chloro-2-(1H-5-indazolylamino)-3-nitropyridine (1 g) obtained by the preparation example 1 in acetonitrile (20 ml) was added 2-(methylamino)ethanol (1.4ml) and triethylamine (0.6 ml) and then refluxed for 12 hr. The reaction mixture was cooled, precipitated with adding excess H

2

O at 20˜25° C., filtered to obtain solid. The above obtained solid was washed with water, dried at 50° C. in vacuo and recrystallised with chloroform:ether=1:3 to obtain the desired compound (0.7 g, 62%).

m.p.: 172˜174° C.

1

H-NMR (DMSO-d6), ppm: δ 3.14(s, 3H), 3.64(m, 4H), 4.80(d, 1H), 6.36(d, 1H), 7.50(s, 2H), 8.02(s, 1H), 8.15(m, 2H), 10.71(d, 1H), 13.03(br s, 1H)

It was prepared compounds in prepared example 11˜30 as the same method used for the example 10. It is shown in Table 1 that the compound name, yield, recrystallizing solution, melting point of compounds in prepared example 11˜30 and 3-nitropyridine derivatives (6) and amine compound (7) as starting materials. It is shown in Table 2 that

1H

-NMR of compounds in prepared example 11˜30.

TABLE 1a

Compounds

Example

3-nitropyridine derivatives (

6

)

amine compound (

7

)

Recrystallising soln

Yield (%)

m.p. (° C.)

11

6-ethyl-(2-hydroxyethyl)amino-2-(1H-5-indazolylamino)-3-nitropyridine

Preparation example 1

2-(ethylamino)ethanol

methanol/ether (1:2)

52

164˜166

12

6-[(1S)-1-(hydroxyethyl)ethylamino]-2-(1H-5-indazolylamino)-3-nitropyridine

Preparation example 1

(S)-2-amino-1-propanol

ethanol

75

>270

13

6-[(1S)-1-(hydroxyethyl)ethylamino]-2-(1H-6-indazolylamino)-3-nitropyridine

Preparation example 2

(S)-2-amino-1-propanol

ethanol

69

264˜265

14

6-[bis(hydroxymethyl)methylamino)-2-(1H-5-indazolylamino)-3-nitropyridine

Preparation example 1

2-amino-1,3-propanediol

methanol

80

>270

15

2-(1H-5-indazolylamino)-6-(2-methoxy-1-methyl)ethylamino-3-nitropyridine

Preparation example 1

2-amino-1-methoxypropane

chloroform/ether (1:5)

58

138˜142

16

6-[2-(dimethylamino)ethylamino]-2-(1H-6-indazolylamino)-3-nitropyridine

Preparation example 2

N,N-dimethylethylenediamine

methanol/H

2

O (1:2)

84

219˜221

17

2-(1H-5-indazolylamino)-6-(4-methyl-1-piperazinyl)amino-3-nitropyridine

Preparation example 1

1-amino-4-methylpiperazine

mehanol/ether (1:4)

78

170˜174

18

2-(1H-6-indazolylamino)-6-(4-methyl-1-piperazinyl)amino-3-nitropyridine

Preparation example 2

1-amino-4-methylpiperazine

methanol

56

260 (dec.)

19

2-(1H-5-indazolylamino)-3-nitro-6-(3-pyridyl)methylaminopyridine

Preparation example 1

3-(aminomethyl)pyridine

methanol/H

2

O (1:1)

81

250˜253

20

2-(1H-6-indazolylamino)-3-nitro-6-(3-pyridyl)methylaminopyridine

Preparation example 2

3-(aminomethyl)pyridine

ethanol

77

>270

21

2-(1H-5-indazolylamino)-3-nitro-6-(4-pyridyl)methylaminopyridine

Preparation example 1

4-(aminomethyl)pyridine

ethanol/H

2

O (2:1)

75

219˜221

22

2-(1H-6-indazolylamino)-3-nitro-6-(2-pyridyl)methylaminopyridine

Preparation example 2

2-(aminomethyl)pyridine

methanol/ethanol (1:1)

72

256 (dec.)

23

2-(1H-5-indazolylamino)-3-nitro-6-(1-piperazinyl)pyridine

Preparation example 1

piperazine

ethanol

78

270 (dec.)

TABLE 1b

Compounds

Example

3-nitropyridine derivatives (

6

)

amine compound (

7

)

Recrystallising soln

Yield (%)

m.p. (° C.)

24

2-(1H-6-indazolylamino)-3-nitro-6-(1-piperazinyl)pyridine

Preparation example 2

piperazine

ethanol

88

268 (dec.)

25

2-(1H-5-indazolylamino)-6-(4-methyl-1-piperazinyl)-3-nitropyridine

Preparation example 1

1-methylpiperazine

methylene chloride/

63

116˜120

isopropyl ether (1:5)

26

2-(1H-6-indazolylamino)-6-(4-methyl-1-piperazinyl)-3-nitropyridine

Preparation example 2

1-methylpiperazine

ethanol/H

2

O (1:1)

60

253 (dec.)

27

6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-(1H-6-indazolylamino)-3-nitropyridine

Preparation example 2

N-(2-hydroxyethyl)piperazine

ethanol

82

215˜217

28

6-(4-hydroxy-1-piperidinyl)-2-(1H-5-indazolylamino)-3-nitropyridine

Preparation example 1

4-hydroxypiperidine

ethanol

76

235˜239

29

6-(4-hydroxy-1-piperidinyl)-2-(1H-6-indazolylamino)-3-nitropyridine

Preparation example 2

4-hydroxypiperidine

methanol

80

270˜272

30

2-(1H-5-indazolylamino)-6-(4-morpholinyl)amino-3-nitropyridine

Preparation example 1

4-aminomorpholine

methanol

64

>270

TABLE 2a

NMR

Example

solution

1

H-NMR data (ppm)

11

DMSO-d

6

δ1.09(d, 3H), 3.56(s, 6H), 4.80(d, 1H),

6.35(d, 1H), 7.49(s, 2H), 8.01(s, 1H),

8.15(m, 2H), 10.68(s, 1H), 13.03(br s, 1H)

12

DMSO-d

6

δ1.13(d, 3H), 3.43(t, 2H), 4.01(br s, 1H),

4.81(t, 1H), 6.14(d, 1H), 7.51(t, 2H),

8.01(s, 1H), 8.06(d, 1H), 8.13(d, 1H),

8.20(s, 1H), 10.92(s, 1H), 13.03(br s, 1H)

13

DMSO-d

6

δ1.18(d, 3H), 3.51(m, 2H), 4.13(br s, 1H),

4.81(d, 1H), 6.19(d, 1H), 7.20(d, 1H),

7.69(d, 1H), 8.00(s, 1H), 8.08(d, 1H),

8.25(d, 2H), 11.09(br s, 1H), 13.03(br s, 1H)

14

DMSO-d

6

δ3.54(s, 4H), 4.00(s, 1H), 4.76(s, 2H),

6.21(d, 1H), 7.51(m, 2H), 8.00(s, 1H),

8.06(d, 1H), 8.12(d, 1H), 8.23(s, 1H),

10.93(s, 1H), 13.02(br s, 1H)

15

DMSO-d

6

δ1.12(d, 3H), 3.19(s, 3H), 3.26(m, 1H),

3.37(m, 1H), 4.13(br s, 1H), 6.11(d, 1H),

7.50(s, 2H), 8.00(s, 1H), 8.07 (d, 1H),

8.16(t, 2H), 10.86(s, 1H), 13.06(br s, 1H)

16

DMSO-d

6

δ2.11(s, 6H), 2.45(s, 2H), 3.49(s, 2H),

6.21(d, 1H), 7.23(d, 1H), 7.69(d, 1H),

8.00(s, 1H), 8.09(d, 1H), 8.17(s, 1H),

8.28(br s 1H), 11.04(s, 1H), 13.01(br s, 1H)

17

DMSO-d

6

δ2.30(s, 3H), 2.83(br s, 6H), 3.16(s, 2H),

6.49(d, 1H), 7.50(t, 2H), 8.03(s, 1H),

8.25(t, 2H), 9.15(s, 1H), 10.69(s, 1H),

13.06(s, 1H)

18

DMSO-d

6

+

δ2.80(s, 3H), 2.99(br s, 2H), 3.06(br s, 2H),

TFA-d

1

3.23(br s, 2H), 3.46(br s, 2H), 6.54(br s, 1H),

7.27(d, 1H), 7.71(d, 1H), 8.14(s, 1H),

8.26(br s, 2H)

19

DMSO-d

6

δ4.51(s, 2H), 6.18(m, 1H), 7.26(br s, 1H),

7.38(d, 1H), 7.43(d, 1H), 7.51(d, 1H),

7.93(s, 2H), 8.14(d, 1H), 8.42(s, 2H),

8.77(br s, 1H), 10.80(s, 1H), 13.03(br s, 1H)

20

DMSO-d

6

δ4.62(s, 2H), 6.22(d, 1H), 7.15(d, 1H),

7.27(br s, 1H), 7.64(d, 2H), 8.00(s, 1H),

8.15(s, 2H), 8.42(s, 1H), 8.50(s, 1H),

8.80(br s, 1H), 10.99(s, 1H), 13.01(br s, 1H)

21

DMSO-d

6

δ4.51(s, 2H), 6.23(d, 1H), 7.15(s, 2H),

7.32(m, 2H), 7.85(d, 2H), 8.16(d, 1H),

8.43(m, 2H), 8.79(t, 1H), 10.77(s, 1H),

13.01(br s, 1H)

22

DMSO-d

6

δ4.70(s, 2H), 6.30(d, 1H), 7.15(d, 1H),

7.25(m, 2H), 7.58(d, 1H), 7.66(t, 1H),

7.99(s, 1H), 8.06(s, 1H), 8.15(d, 1H),

8.52(d, 1H), 8.85(br s, 1H), 10.98(s, 1H)

12.98(br s, 1H)

DMSO: dimethylsulfoxide, TFA: trifluoroacetic acid

TABLE 2b

NMR

Example

solution

1

H-NMR data (ppm)

23

DMSO-d

6

δ2.70(s, 4H), 3.59(s, 4H), 6.46(d, 1H),

7.51(s, 2H), 7.96(s, 1H), 8.03(s, 1H),

8.16(d, 1H), 10.55(s, 1H), 13.07(br s, 1H)

24

DMSO-d

6

δ2.76(s, 4H), 3.66(s, 4H), 6.51(d, 1H),

7.16(d, 1H), 7.69(d, 1H), 8.00(s, 1H),

8.07(d, 1H), 8.20(d, 1H), 10.82(s, 1H),

13.07(br s, 1H)

25

DMSO-d

6

δ2.17(s, 3H), 2.34(s, 4H), 3.65(s, 4H),

6.48(d, 1H), 7.51(t, 2H), 7.96(d, 1H),

8.04(d, 1H), 8.18(d, 1H), 10.59(s, 1H), 13.07

(br s, 1H)

26

DMSO-d

6

δ2.21(s, 3H), 2.40(s, 4H), 3.74(br s, 4H),

6.54(d, 1H), 7.17(d, 1H), 7.70(d, 1H),

8.00(s, 1H), 8.07(d, 1H), 8.22(d, 1H),

10.78(s, 1H), 13.06(br s, 1H)

27

DMSO-d

6

+

δ3.19(t, 4H), 3.59(m, 4H), 3.74(t, 2H),

TFA-d

1

4.51(br s, 2H), 6.50(d, 1H), 7.17(d, 1H),

7.69(d, 1H), 8.08(d, 2H), 8.28(d, 1H)

28

DMSO-d

6

δ1.35(m, 2H), 1.76(d, 2H), 3.37(d, 2H),

3.75(br s, 1H), 3.99(br s, 2H), 4.78(s, 1H),

6.50(d, 1H), 7.51(s, 2H), 7.98(d, 1H),

8.02(s, 1H), 8.17(d, 1H), 10.61(s, 1H),

13.05(br s, 1H)

29

DMSO-d

6

δ1.42(m, 2H), 1.82(d, 2H), 3.45(t, 2H),

3.79(br s, 1H), 4.06(br s, 2H), 4.82(s, 1H),

6.55(d, 1H), 7.18(d, 1H), 7.69(d, 1H),

8.00(s, 1H), 8.05(s, 1H), 8.19(d, 1H),

10.81(s, 1H), 13.06(s, 1H)

30

DMSO-d

6

δ2.77(s, 4H), 3.68(s, 4H), 6.55(s, 1H),

7.50(t, 2H), 8.02(s, 1H), 8.25(t, 2H),

9.12(s, 1H), 10.67(s, 1H), 13.04(s, 1H)

DMSO: dimethylsulfoxide, TFA: trifluoroacetic acid

<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 HBV polymerase genetically expressed in and separated from

E.coli

, the process of its preparation, and the method to measure the enzyme activities (KR 94-3918, KR 96-33998). In the present experiments HBV polymerase was used which had been expressed in

E. coli

as stated above.

The method used in 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 digoxigenin group attached are included as substrates and anti-DIG antibodies attached to peroxidase enzyme recognize the polymerized substrates.

To the wells coated with streptavidin, 20 μl of HBV polymerase, 20 μl of reaction mixture (10 μM each of DIG-UTP and Biotin-UTP, 46 mM Tris-HCl, 266 mM KCl, 27.5 mM MgCl

2

, 9.2 mM DTT substrate/primer nybrid), and 20 μl of test compound (added to 1, 0.1, and 0.01 μg/ml) were added and allowed to react at 22° C. for 15 hrs. During this reaction, HBV polymerase catalyzes DNA synthesis and digoxigenin and biotin attached to nucleotides form bonds with streptavidin 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 μl 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 μl each of ABTS™, a substrate of peroxidase, was then added and allowed to react at room temperature for 30 min. 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 3.

TABLE 3a

Effect on the HBV polymerase activities in reverse

transription

Inhibition activity on HBV-RT (%)

Compound

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

TABLE 3b

Effect on the HBV polymerase activities in reverse

transription

Inhibition activity on HBV-RT (%)

Compound

1 μg/ml

0.1 μg/ml

0.01 μg/ml

Example 9

80

59

40

Example 10

78

58

50

Example 11

85

60

35

Example 12

90

56

38

Example 13

66

35

10

Example 14

85

49

30

Example 15

97

68

47

Example 16

80

55

38

Example 17

75

52

41

Example 18

70

43

25

Example 19

91

58

42

Example 20

66

40

21

Example 21

94

65

45

Example 22

81

60

49

Example 23

96

66

51

Example 24

62

43

20

Example 25

88

54

35

Example 26

63

40

18

Example 27

60

38

15

Example 28

55

34

20

Example 29

52

35

10

Example 30

71

49

32

As shown in Table 3, compound of the present invention have excellent inhibitory effects on the HBV polymerase activities with more than 90% inhibition at the concentration of 1 μg/ml. Moreover, compounds of the 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 and proliferation of HBV were measured in HepG 2.2.15, a human liver cancer cell line.

The cell concentration was adjusted to 1×10

5

cells/ml and 1 ml was added to each well of a 24-well cell culture plate, which was then kept in a culture box for 3-4 days at 37° C. under 5% CO

2

until cells grew sufficiently, changing culture medium everyday. When the cells matured sufficiently, the test compounds were added to the final concentrations of 0.01, 0.1, and 1 μg/ml. One week after the addition of test compounds, the culture solution was centrifuged at 5,000 rpm for 10 min. 25 μl of supernatant was transferred to a new tube and 5 μl 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 μl of neutralizing solution [0.09N HCl 0.1MTris-HCl, pH7.4] 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 μl of PCR reaction solution [0.54N NaOH, 0. 06% NP40, 0.09N HCl, 0.1M Tris-HCl, pH 7.4].

DNA polymerized by PCR was electrophoresed 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 (lamivudine) 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.

TABLE 4

Inhibitory effect on the HBV proliferation

Inhibition activity on HBV-RT (%)

Compound

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

Example 11

83

45

20

Example 12

85

48

29

Example 14

80

41

22

Example 15

95

60

36

Example 19

85

51

30

Example 21

90

50

33

Example 23

92

55

35

Example 25

83

46

25

3TC

99

80

48

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 μg/ml. Moreover, compounds of the present invention, being non-nucleosides, may not have problems such as toxicity and early development of resistant virus strains observed in the use of nucleoside substances. It is also expected that compounds of the present invention may be used in 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 in 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.

<Experiment 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 in reverse transcription.

Non-radioactive reverse transcriptase assay kit (Boehringer Mannheim) was used in the measurement of in vitro transcriptase activities. 20 μl (40 ng) of HIV transcriptase and 20 μl of reaction mixture containing matrix-primer hybrid poly(A)oligo(dT)

15

, DIG(digoxigenin)-dUTP, biotin-dUTP, and TTP were added to wells coated with streptavidin. Test compounds were also added at the final concentrations of 0.1 and 1 μg/ml 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 streptavidin coated on the bottom of wells because of digoxigenin and biotin moieties attached to nucleotides.

When the reaction was completed, each well was washed with 250 μl of cleaning buffer (pH7.0) for 30sec. five times to remove remaining impurities. 200 μl of anti-DIG-POD antigen was added to each well, allowed to react at 37° C. for 1 hr and washed as above to remove impurities. 200 μl of ABTS™, a substrate for peroxidase, was added to each well and allowed to react at room temperature for 30 min. 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 in 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

Inhibition

activity on HBV-RT (%)

Compound

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

Example 11

67

46

Example 12

64

40

Example 14

59

44

Example 16

75

51

Example 20

84

53

Example 21

72

39

Example 27

81

45

As shown above in 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/ml. 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 in 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 in 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, invitro tests were carried out using HepG2 cells with MTT analysis method as generally known and the results are in Table 6 shown below.

TABLE 6

Cytotoxicity tests on HepG2 cells

Cytotoxicity on HepG2

Compound

IC

50

1)

MCD

2)

Example 2

>100

100

Example 6

>100

100

Example 11

>100

100

1)

IC

50

: 50% Inhibitory Concentration (μg/ml)

2)

MCD: Minimal Cytotoxic Concentration (μg/ml)

As shown above in Table 6, compounds used in the experiments have higher than 100 μg/ml for IC

50

and are considered to have little cytotoxicity.

<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 in rats.

6-week old SPF SD line rats were used in 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 g/kg/15 ml. 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 in rats. No chance was observed in hematological tests, biochemical tests of blood, and autopsy. The compounds used in this experiment are evaluated to be safe substances since they do not cause any toxic change in 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-nitropyridine 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 in 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.

Number	Date	Country	Kind
1999-53295	Nov 1999	KR
1999-64403	Dec 1999	KR

Number	Date	Country
WO 98541410	Dec 1998	WO
WO 9958526	Nov 1999	WO

3-nitropyridine derivaives and the pharmaceutical compositions containing said derivatives

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

Priority Claims (2)

Parent Case Info

PCT Information

Foreign Referenced Citations (2)