WO2001038306A9 - Novel 3-nitropyridine derivatives and the pharmaceutical compositions containing said derivatives - Google Patents

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-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.

Formula 1

Wherein,

Ri is methoxy or

R₃ is H, hydroxy, dialkylamino group with C2~C₆, straight

or branched hydroxyalkyl group with C₂~C₆, straight or branched

dihydroxyalkyl group with C₃~C6, alkoxyalkyl group with C₃~C6,

or saturatedor unsaturated 5 or 6memberedheterocyclic compounds

containing 1 to 3 heteroatoms selected from N, 0, and S, which

may be unsubstituted or substituted with alkyl group of Cι~C₃;

R₃ may or may not contain asymmetrical carbons;

R₄ is H, straight or branched alkyl group with Cι~C₄, or

cycloalkyl group with C₃~C₆;

R₃ and R₄ 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~C₅, straight or branched hydroxyalkyl group with

C₂~C₅, or hydroxy;

R₂ 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-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, Scien tific 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 RNAstrand fromsubsequently formedRNA-DNAhybrid.

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; referredas "AIDS" hereinafter) and herpes zoster

infection (Gerin, J. L, Hepatology, 14: 198-199, 1991; Lok, A.

S. P., J. Viral Hepa ti tis, 1: 105-124, 1994; Dienstag, J. L.

et al . _f 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 (EP0563732, EP0563734), 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.

Formula 1

Wherein,

Ri is methoxy or

^•

R₃ is H, hydroxy, dialkylamino group with C₂~C₆, straight

or branched hydroxyalkyl group with C₂~C₆, straight or branched

dihydroxyalkyl group with C ~C6, alkoxyalkyl group with C₃~C₆,

or saturatedorunsaturated5 or 6memberedheterocyclic compounds

containing 1 to 3 heteroatoms selected from N, 0, and S, which

may be unsubstituted or substituted with alkyl group with Cι~C₃;

R₃ may or may not have asymmetrical carbons;

R₄ is H, straight or branched alkyl group with Cι~C₄, or

cycloalkyl group with C₃~C₆;

R₃ and R₄ 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 Ci~C₅, straight or branched hydroxyalkyl group

with C₂~C5, or hydroxy;

R₂ is indazol-5-yl, or indazol-6-yl; n is an integer between 0 and 3.

When both R₃ and R₄ are represented as a 5 or 6 membered

heterocyclic compounds with 1 to 3 heteroatoms selected from

N, 0, and S, n equals 0. This heterocyclic ring may be

unsubstituted or substituted with straight or branched alkyl

group with Ci~C₅, straight or branched hydroxyalkyl group with

C₂~C₅, 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 indazol-6-yl groups for R₂ 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 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.

Scheme 1

1

Wherein, X is Cl or OCH₃; R₂, R₃, R₄ 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 Ri in the compound of formula 1 is a methoxy group,

step 1 completes the synthesis of desired compound (X=OCH₃) .

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.

Scheme 2

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 (R₃- (CH₂) _n-NR₄-) 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, IV,Λ/-diisopropylethylamine,

W-methylmorpholine , N-methylpiperidine ,

4-dimethylaminopyridine, NVΛJ-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_^JV-dimethylformamide,

I\7-methylpyrrolidinone, pyridine, water and alcoholic solvents

such as methanol, ethanol, and isopropanol.

It is preferable to use excess amount of amine compound

(formula 7) 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 H₂030 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 : H₂0 = 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. )

¹H-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- (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 H₂0 (20 ml) , and then reacted

at 20-25^°C for 1 hr . The reaction mixture was filtered, washed

with acetonitrile (6ml) and H₂0 (15ml), and then dried at 50 ^°C

in vacuo to obtain the desired compound (4.4 g, 73%). m. p . : 234 ^°C ( dec . )

^XH-NMR (DMSO-d₅) , 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- (lH-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 H₂0 (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

¹H-NMR (DMSO-d⁶) , ppm : δ 3.80 (s, 3H) , 6.32 (d, 1H) , 7.55 ( ,

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-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 H₂030 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 in vacuo to obtain the desired

compound (6.8 g, 90%).

m.p. : 261-264 ^°C

^XH-NMR (DMSO-d₆) , 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- (lH-5-indazolylamino) -6-methyl

amino-3-nitropyridine .

To the solution of methanol with 40% methyla ine (20 ml)

was added 2- (liϊ-5-indazolylamino) -6-methoxy-3-nitropyridine

(1 g) obtained by the example 1, and the solution was reacted

at25^°C forlhr. The reaction mixture was added H₂0 (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 in vacuo

to obtain the desired compound (0.82 g, 82%).

m.p. : 238-240 ^°C

¹H-NMR (DMS0-d₅) , ppm : 82.86(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- (lH-6-indazolylamino) -6-methyl

amino-3-nitropyridine.

To the solution of methanol with 40% methylamine (20 ml)

was added 2- (l#-6-indazolylamino) -6-methoxy-3-nitropyridine

(1 g) obtained by the example 2, and the solution was reacted

at 25-30 ^°C for 2hr. 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

^XH-NMR (DMS0-d₆) , 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 (brs, 1H) , 8.44(s, 1H) , 11 . 14 ( s , 1H) , 13 . 03 (br s , 1H)

<Example 5> Preparation of 2- (lH-5-indazolylamino) -6-isopropyl

amino-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

isopropylamine (20 ml) slowly and reacted at 45^°C for 20 hr.

The reaction mixture was cooled, added H₂0 (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

^XH-NMR (DMSO-d₆) , ppm : δ 1.15 (d, 6H) , 4.03 (m, 1H) , 6.06 (d,

1H) , 7.50 (d, 2H) , 8.05 ( , 2H) , 8.15(t, 2H) , 10.97 (s, 1H) ,13.06 (br

s, 1H)

<Example 6> Preparation of 2- (lH-6-indazolylamino) -6-isopropyl

amino-3-nitropyridine.

To the solution of 2- (liϊ-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

^XH-NMR (DMSO-d₆) , 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- (lff-5-indazolylamino) -6-isobuthyl

amino-3-nitropyridine .

To the solution of

2- (liϊ-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 H₂0 (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 product. The solid product was dried at 50 — 60^°C in va cuo

to obtain the desired compound (0.95 g, 83%).

m.p. : 230-232 ^°C

^XH-NMR (DMS0-d₆) , 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- (lH-5-indazol

ylamino) -3-nitropyridine .

To the solution of

2- (liϊ-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 H₂0 (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

^XH-NMR (DMSO-d₆) , ppm : δ 0.56(m, 2H) , 0.81(m, 2H) , 2.81(br

s,lH) , 6.06 (d, 1H) , 7.50 (d, 1H) , 7.62 (d, 1H) , 8.02 (s, 1H) , 8.09 (d, 1H) 46(s, 1H) 57(s, 1H) , 11.02(s, 1H) , 13.04(br s, 1H)

<Example 9> Preparation of 6-amino-2- (lff-5-indazolylamino) -

3-nitropyridine .

To the solution of

6-chloro-2- (lif-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.)

^XH-NMR (DMSO-d₆) , 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-

(lff-5-indazolylamino) -3-nitropyridine . To the solution of

6-chloro-2- (liϊ-5-indazolylamino) -3-nitropyridine (1 g)

obtained by the preparation example 1 in acetonitrile (20 ml)

was added 2- (methylamino) ethanol (1.4 ml) and triethylamine (0.6

ml) and then refluxed for 12 hr . The reaction mixture was cooled,

precipitatedwith adding excess H₂Oat 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

^Η-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 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 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, 20μi 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 MgCl₂, 9.2 mM

DTT substrate/primer hybrid) , and 20 μi of test compound (added

to 1, 0.1, and 0.01 g/m£) were added and allowed to react at

22 °C forl5hrs. 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 μi of cleaning

buffer (pH 7.0) for 30 seconds, which was repeated five times

to remove remaining impurities . 200 μi 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. 200μi 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

TABLE 3a Effect on the HBV polymerase activities in reverse

transription

TABLE 3b. Effect on the HBV polymerase activities in reverse

transription

As shown in Table 3, compounds 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 withnucleoside 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 in HepG 2.2.15, a human liver cancer cell

line .

The cell concentration was adjusted to l^χl0⁵ cells/m£ and

1 mi 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%

C0₂ 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 . One week after the addition of test compounds, the

culture solution was centrifuged at 5,000 rpm for 10 min. 25

μi of supernatant was transferred to a new 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

[0.09 HC1, 0.1M Tris-HCl, pH 7.4 ] was added as a reaction solution

for competitive polymerase chain reaction (PCR) .

PCRwas 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,

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 HBVproliferationwas 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

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 βg/mi . 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 HBVpolymerase 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 μi (40 ng) of HIV transcriptase

and 20 μi of reaction mixture containing matrix-primer hybrid poly (A) oligo (dT) is, 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 βg/vai

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 μi of cleaning buffer (pH7.0) for 30 sec. five times to remove

remaining impurities . 200 μi of anti-DIG-POD antigenwas added

to each well, allowed to react at 37^°C for 1 hr and 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 30min. 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

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/mt . 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,

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 βg/mi for IC₅₀ 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/15m# .

Death, clinical symptoms, andweight 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 checkedwith eyes during autopsy. The results showed

that the test compounds did not cause any specific clinical

symptoms, weight change, or death in rats . No change 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 LD₅₀ 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 inventionhave dramatic inhibitoryeffect

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 .

Claims

WHAT IS CLAIMED IS;

1. 3-Nitropyridine derivatives and their pharmaceutically

acceptable salts as represented by formula 1.

Formula 1

Wherein,

^-^2-7^-

Ri is methoxy or ^R* ;

R₃ is H, hydroxy, dialkylamino group with C₂~C₆, straight

or branched hydroxyalkyl group with C₂~C6, straight or branched

dihydroxyalkyl group with C₃~C_δ, alkoxyalkyl group with C₃~C₆,

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 Cχ~C₃; R₃ may

or may not contain asymmetrical carbons;

R₄ is H, straight or branched alkyl group with C]_.~C₄, or

cycloalkyl group with C₃~C₆;

R₃ and R₄ both may consist of 5 or 6 membered heterocyclic

ring containing 1~3 heteroatoms selected from N, 0, and S, which is unsubstituted or substituted with straight or branched alkyl

group with Cι~C₅, straight or branched hydroxyalkyl group with

C₂~C₅, or hydroxy;

R₂ is indazol-5-yl, or indazol-β-yl;

n is an integer between 0 and 3.

2. Process for the preparation of 3-nitropyridine

derivatives of formula 1 comprising the following two steps as

represented in scheme 1:

Stepl. 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 the

presence of a base;

Step 2. Preparation of 3-nitropyridine derivatives of

formula 1 by reacting 3-nitropyridine derivatives of formula

6 synthesized in step 1 with amine compounds of formula 7

Scheme 1

R₃- H_{f lr}NH

Wherein, X is chloro or methoxy group;

R₂, R₃, R₄ 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, R₂ is as defined in formula 1.

4. Therapeutic agent and a preventive agent for hepatisis

B with 3-nitropyridine 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-nitropyridine

derivatives and their pharmaceutically acceptable salts in claim

1 as effective ingredient.