WO2001038308A1

WO2001038308A1 - Novel 5-pyrimidinecarboxamide derivatives and the pharmaceutical compositions containing said derivatives

Info

Publication number: WO2001038308A1
Application number: PCT/KR2000/001364
Authority: WO
Inventors: Sung June Yoon; Sang Wook Lee; Nam Doo Kim; Yong Kyun Park; Geun Hyung Lee; Jong Woo Kim; Sang Jin Park; Hee Jeoung Park; Hwan Bong Jang
Original assignee: Dong Wha Pharm. Ind. Co., Ltd
Priority date: 1999-11-27
Filing date: 2000-11-27
Publication date: 2001-05-31
Also published as: EP1235806A4; EP1235806A1; AU1899401A

Abstract

The present invention relates to novel 5-pyrimidinecarboxamide derivatives and the pharmaceutical compositions containing said derivatives, and more specifically, to 5-pyrimidinecarboxamide derivatives and their pharmaceutically acceptable salts, the process for preparing them, and the pharmaceutical compositions containing said compounds as active ingredients. In particular, said 5-pyrimidinecarboxamide 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 5-PYRIM DINECARBOXAMIDE DERIVATIVES AND THE

PHARMACEUTICAL COMPOSITIONS CONTAINING SAID DERIVATIVES

TECHNICAL FIELD

The present invention relates to novel

5-pyrimidmecarboxamide derivatives and the pharmaceutical

compositions containing said derivatives. More specifically,

the present invention relates to novel 5-pyrιmιdιnecarboxamιde

derivatives and their pharmaceutically acceptable salts

represented below in formula 1, which have excellent inhibitory

effect on proliferation of hepatitis B virus and of human

immunodeficiency virus. The present invention also relates to

the process for preparing compounds of formula 1 and to the

pharmaceutical compositions containing said derivatives as

effective ingredients against viruses.

Formula 1

Wherein,

Ri is H, hydroxy, straight or branched al yl group with Cι~ C₅, straight or branched alkoxy group with Cι~C₅, straight

or branched hydroxyalkyl group with C₂~C₆, dialkylammo group

with C₂~C₆, straight or branched alkyl group with C->~C_e

substituted with hydroxy or alkoxycarbonyl group with C₂~C₅,

cycloalkyl group with C₃~C₆, or saturated or unsaturated 5 or

6 membered heterocyclic compounds containing 1 to 3 heteroatoms

selected from N, 0, and S, which may be unsubstituted or

substituted with alkyl group of Cι~C₃; Ri may or may not contain

asymmetrical carbons;

R₂ is H or Straight or branched alkyl group with Cι~C₄;

Or both R_x and R₂ consist of 5 or 6 membered saturated

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~Cs or straight or branched

hydroxyalkyl group with C₂~Cs;

n is an integer between 0 and 4;

R₃ is mdazol-5-yl , or indazol-β-yl .

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 m 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 ammo

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 viπon 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 lamivudme 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) andherpes zoster

infection (Germ, J. L, Hepa tology, 14: 198-199, 1991; Lok, A.

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

et al . , New Engl and Journa l of Medi cine , 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) , lridos

compounds (KR 94-1886) , and terephthalic amide derivatives (KR

96-72384, KR 97-36589, KR 99-5100). In spite of much effort,

however, effective drugs for treating hepatitis B have not been

developed yet and therapeutic method mamly depends on

symptomatic treatment.

AIDS is a disease inducing dramatic decrease in immune

function 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 mamly

attack helper T cells, which is one of the T cells with regulatory

function n 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 1981 for the first

time, the number increased to more than 850, 000 patients 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 Azidothymidme

previously, is a drug developed in 1987. Didanosme was

developed in 1991 as an alternative medicine for AIDS patients

when Zidovudme was either ineffective or could not be used due

to side effects. In addition, Zalcitabine was approved for

concurrent use wrth 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 the compounds with

excellent antiviral effect against HBV; synthesized novel 5-pyrιmιdmecarboxamιde 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

5-pyπmιdmecarboxamιde derivatives and the pharmaceutical

compositions containing said derivatives. More specifically,

the present invention provides 5-pyrιmιdmecarboxamιde

derivatives and their pharmaceutically acceptable salts, the

process for their preparation and the pharmaceutical

compositions containing said derivatives as effective

ingredient .5-pyrιmιdmecarboxamιde 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 5-pyrιmιdmecarboxamιde derivatives

represented below in formula 1 and their pharmaceutically

acceptable salts. Formula 1

Wherein,

Ri is H, hydroxy, straight or branched alkyl group with

Cι~C₅, straight or branched alkoxy group with Cι~C₃, straight

or branched hydroxyalkyl group with C₂~C₆, dialkylammo group

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

substituted with hydroxy or alkoxycarbonyl group with C₂~C₅,

cycloalkyl group with C₃~C₆, or saturated or unsaturated 5 or

6 membered heterocyclic compounds containing 1 to 3 heteroatoms

selected from N, 0, and S, which may be unsubstituted or

substituted with alkyl group of Cι~C₃; Ri may or may not contain

asymmetrical carbons;

R₂ is H or straight or branched alkyl group with Cι"-C₄;

Or both Ri and R₂ consist of 5 or 6 membered saturated

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~-Cs or straight or branched hydroxyalkyl group with C₂~C₅;

R₃ is indazol-5-yl or indazol-6-yl ;

n is an integer between 0 and .

When both Rx and R₂ 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 Cι~Cs, straight or branched hydroxyalkyl group with

C₂~C₅, or hydroxy group;

When Ri in formula 1 contains asymmetrical carbons, they

may exist as eitheri 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 R₃ 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 acidmay be used.

Among organic acids, citric acid, acetic acid, lactic acid,

tartaric acid, maleic acid, fumaric acid, formic acid, propionic

acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic

acid, methanesulfonic acid, glycolic acid, succinic acid,

4-toluenesulfonic acid, galacturonic acid, embonic acid,

glutamic acid and aspartic acid may be used.

Furthermore, the present invention provides a process for preparing 5-pyrimidinecarboxamide derivatives represented

below in scheme 1.

Scheme 1

4

R₃-NH-

Wherein, Ri, R₂, R₃ and n are as defined in formula 1.

The process of preparing compounds of formula 1 in the

present invention comprises two steps as in the following:

1) Preparation of 5-pyrimidinecarboxylic acid ethyl

ester derivatives of formula by reacting

4-chloro-2-methylthio-5-pyrimidinecarboxylic acid ethyl ester of formula 4 with 5-ammomdazole or 6-ammomdazole of formula

5 ( 5 represents compounds of both formula 2 and formula 3; referred

as formula 5 hereinafter) m a proper solvent under a basic

condition at an appropriate temperature (step 1 ) ;

2-A) Preparation of 5-pyrιmιdmecarboxamιde derivatives

of formula 1 by reacting the compound of formula 6 prepared in

step 1 with amme compound of formula 7 in an appropriate solvent

at a proper temperature, or

2-B) Preparation of 5-pyrιmιdmecarboxamιde derivatives

of formula 1 by first hydrolyzmg the compound of formula 6

synthesized m step 1 to form 5-pyrιmιdmecarboxylιc acid

derivatives of formula 8, then activating to Vilsmeier

intermediate mthepresenceofN, N-dimethyIformamide and S0C1₂,

and allowing it to react with amme compound of formula 7.

Chemical agents used scheme 1, namely,

4-chloro-2-methylthιo-5-pyπmιdmecarboxylιc acid ethyl ester

of formula 4, 5-ammomdazole or 6-ammomdazole of formula 5,

and amme compound of formula 7 are commercially available and

may be purchased easily.

The process of preparing compounds of formula 1 is described in more detail in the following.

For the reaction of

4-chloro-2-methylthio-5-pyrimidinecarboxylic acidethyl ester

of formula 4 with 5-aminoindazole or 6-aminoindazole of formula

5 to synthesize 5-pyrimidinecarboxylic acid ethyl ester

derivatives of formula 6, organic compound may be used as a base .

It is preferable to use tertiary amine such as triethylamine,

N, N-diisopropylethylamine, N-methylmorpholine ,

ΪV-methylpiperidine, 4-dimethylaminopyridine,

N,N-dimethylaniline , 2, 6-lutidine, pyridine.

Preferable reaction temperature is20~40^°C andpreferable

reaction time is 1~6 hrs.

Preferable is a single solvent or a mixture of solvents

selected from alcohol such as methanol and ethanol, chloroform,

methylene chloride, and acetonitrile .

Preparation of 5-pyrimidinecarboxamide derivatives of

formula 1 by reacting 5-pyrimidinecarboxylic acid ethyl ester

derivatives of formula 6 synthesized in step 1 with amine compound

of formula 7 may be carried out using one of two methods.

As in step 2-A, an appropriate amine compound of formula

7 may be used to give good yield of 5-pyrimidinecarboxamide derivatives of formula 1 without first going through the

hydrolysis of aminoindazole 5-pyrimidinecarboxylic acid ethyl

ester of formula 6.

In this case, amine compound of formula 7 is used to

introduce substituents Ri and R₂ and an appropriate amine may

be selected depending on the kind of substituents desired. For

amine compound, methanolic ammonia solution, methanolic

methylamine solution, aqueous ethylamine solution,

isopropylamine, cyclopropylamine, ethanolamine, or

propanolamine are used all of which are commercially available.

For a base, organic base such as used in preparing compounds

of formula 6 is used preferably in excess amount compared with

that of intermediate of formula 7 in order to increase the reaction

efficiency.

For a solvent, a single or a mixture of solvents selected

from alcohol such as H₂0, methanol, ethanol, and isopropanol

or chloroform, methylene chloride, and acetonitrile is

preferable .

The reaction temperature is preferably 25~60 ^°C and may

depend on the amine compound used. As for the reaction in step 2-B, alkali compound used for

hydrolyzmg the compound of formula 6 is preferably sodium

hydroxide, potassium hydroxide, sodium carbonate, or potassium

carbonate . 5-Pyrιmidinecarboxylic acid derivatives are formed

almost quantitatively in the hydrolysis reaction.

For a solvent in this reaction, a mixture of water and

alcohol such as methanol or ethanol is preferably used.

Preferable reaction temperature and time are 30 — 60 ^°C and

0.5-3 hrs.

5-pyrimidinecarboxylic acid derivatives are activated

using Vilsmeier reagent formed by heating N, N-dimethylformamide

and thionyl chloride at 30~50 ^°C and used in the reaction with

an appropriate amme compound of formula 7 at 0~20 ^°C to prepare

5-pyπmιdinecarboxamide derivatives of formula 1, target

compound of the present invention.

For a solvent in this reaction, aprotic solvent is

preferable such as chloroform, methylene chloride, acetonitrile,

tetrahydrofuran, or ether.

Furthermore, the present invention provides the

pharmaceutical compositions of therapeutics containing 5-pynmιdmecarboxamιde derivatives and their pharmaceutically

acceptable salts of formula 1 as effective ingredients to prevent

and treat hepatitis B.

The present invention also provides the pharmaceutical

compositions of therapeutics with 5-pyπmιdmecarboxamιde

derivatives and their pharmaceutically acceptable salts of

formula 1 as effective ingredients to prevent and treat AIDS.

5-pyπmιdmecarboxamιde derivatives of formula 1 m this

invention are effective inhibitors proliferation of HIV as

well as of HBV, since they interfere with removal of RNA strand

from RNA-DNA hybrid formed during reverse transcription of viral

RNA to DNA which is common the replication processes for HBV

and HIV.

Compounds of formula 1 maybe takenorallyas well as through

other routes in clinical uses ; forexample, it may be administered

intravenously, subcutaneously, mtraperitoneally, or locally

and used in the form of general drugs.

For the 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, trochise, 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 compounds 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 and administered 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 4- (lH-5-indazolylamino) -2-methylt ιo-5-pyrιmidιnecarbox_Ll:_.c

acid ethyl ester .

To the solution of 4-chloro-2-methylthιo-5-pyrιmιdme

carboxylic acid ethyl ester (5 g) and 5-ammomdazole (3.15 g)

in methanol (70 ml) was added tπethylamme (3.5 l), and then

the solution was reacted at 30^°C for 3 hr. The reaction mixture

was cooled at room temperature, stirred at 20^°C for 1 hr . Then

the reaction mixture was filtered and washed with methanol (20

ml) . The obtained solid was dried at 40-50^°C in vacuo to obtain

the desired compound (6.15 g, 87%).

m.p. : 199-201 ^°C

^-NMR (DMSO-de) , ppm : δ 1.34 (t, 3H) , 2.42 (s, 3H) , 4.34 (m,

2H), 7.48(d, 1H), 7.53 (d, 1H), 8.06(d, 2H), 8.70 (s, 1H), 10.18 (s,

1H) , 13.10 (br s, 1H)

<Preparation Example 2> Preparation of 4- (lH-6-xndazolylamιno)

-2-methylthio-5-pyrimidinecarboxylic acid ethyl ester

To the solution of 4-chloro-2-methylthιo-5-pyrιmιdme

carboxylic acid ethyl ester (5 g) and 6-ammomdazole (3.15 g)

in methanol (70 ml) was added N, N-dnsopropylethylamme (4.2

ml) , and then the solution was reacted at 30-35^°C for 4 hr. The reaction mixture was cooled, and then stirred at 20 ^°C for 1

hr . The reaction mixture was filtered, washed with methanol (20

ml) and dried at 40-50^°C m va cuo to obtain the desired compound

(5.8 g, 82%) .

m.p. : 212-214 ^°C

^-NMR (DMSO-de) , ppm :δ 1.33 (t, 3H), 2.53 (s, 3H), 4.33 (m,

2H) , 7.10 (d, 1H) , 7.70(d, 1H) , 8.00(s, 1H) , 8.22(s, 1H), 8.72(s,

1H) , 10.40(s, 1H), 13.09(br s, 1H)

<Preparation Example 3> Preparation of 4- (lH-5-__.ndazolylamino)

-2-methylthio-5-pyrimidinecarboxylic acid

To the solution of methanol (80 ml) was added the

4- (lH-5-mdazolylammo) -2-methylthιo-5-pyrιmιdmecarboxylιc

acid ethyl ester (5 g) obtained by the preparation example 1,

and the solution was hydrolyzed at 40-50^°C for 1 hr, adding H₂0

(30 ml) and 3N aq. NaOH (15 ml) . The reaction mixture was cooled,

measured pH 5 at 20 ^°C , slowly adding 3 N aq. HCl. And then

the reaction mixture was slowly added H₂0 (100 ml), stirred at

20 ^°C for 1 hr, filtered, washed with H₂0 (30 ml) , and obtained

a solid product. The solid product was dried at 50 ^°C m vacuo

to obtain the desired compound (4.44 g, 97%). m.p. : > 270 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2.45 (s, 3H), 7.49 (d, 1H), 7.53 (d,

1H) , 8.05(s, 1H) , 8.10(s, 1H), 8.68(s, 1H) , 10.50(s, 1H),

13.09(br s, 1H)

<Preparation Example 4> Preparation of 4- (lH-6-indazolylamino)

-2-methylthio-5-pyrimidinecarbox lic acid

The desired compound (95 %) was obtained the same method

used for the preparation of example 3, except using

4- ( lH-5-indazolylamino) -2-methylthιo-5-pyrimidinecarboxylic

acid ethyl ester (5 g) prepared from preparation example 2 as

a starting.

m.p. : > 270 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2.56 (s, 3H) , 7.10 (d, 1H) , 7.72 (d,

1H) , 8.01(s, 1H) , 8.25(s, 1H) , 8.73(s, 1H) , 10.81(br s, 1H) ,

13.06(br s,lH)

<Example 1> Preparation of 4- (lH-5-indazolylamino) -2-methyl

thio-5-pyrimidinecarboxamide

To the solution of 4- ( lH-5-indazolylamino) -2-methylthio

-5-pyrimidinecarboxilic acid ethylester (1 g) prepared from preparation example 1 in chloroform (10 ml) was added 10% ammonia

methanol solution (20 ml), the solution was slowly heated and

then reacted at 40^°C for 2 days. The reaction mixture was

concentrated by evaporation under pressure crystallized by

adding methanol (15 ml) . The reaction mixture containing solid

product was stirred at 20^°C, filteres and then seperated a solid

product. The solid product was recrystallized

chloroform: ethanol = 1: 1 (v/v) and dried m va cuo to obtain the

desired compound (0.62 g, 68%).

m.p. : > 270 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2.49 (s, 3H) , 7.47 (d, 1H) , 7.55 (d,

1H),7.73 (br s,lH), 8.07(s, 1H) , 8.15(s, 1H) , 8.28(br s, 1H) ,

8.71(s, 1H) , 11.46(s, 1H) , 13.07(br s, 1H)

<Example 2> Preparation of 4- (lH-6-indazolylamino) -2-

methylthio-5-pyrimidinecarboxamide

The desired compound (62 %) was obtained the same method

used for the example 1, except using

4- ( lH-6-mdazolylammo) -2-methylthιo-5-pyrιmιdιnecarboxylιc

acid ethyl ester prepared from preparation example 2 as a

starting. ^XH-NMR (DMSO-d₆) , ppm : δ 2.56 (s, 3H) , 7.06(d, 1H) , 7.70 (d,

1H) , 7.81 (br s, 1H) , 7.98 (s, 1H) , 8.28(s, 1H) , 8.32(br s, 1H) ,

8.73 (s, 1H) , 11.75(s, 1H) , 13.02 (br s, 1H)

<Example 3> Preparation of 4- (lH-5-indazolylamino) -N-methyl-

2-methylthio-5-pyrimidine carboxamide

To the solution of methanol with 40% methylamine (30 ml)

was added 4- ( lH-5-indazolylamino) -2-methylthio-5-pyrimidine

carboxylic acid ethyl ester (1.5 g) obtained by by preparation

example 1, and the solution was reacted at 25-30^°C for 1 hr.

The reaction mixture was cooled at 20-25^°C, added H₂0 (90 ml)

and stirred for 0.5 hr . The reactionmixture was filtered, washed

with 25% aqueous methanol solution (10 ml), obtained a solid

product . The solidproduct was dried in vacuo to obtain the desired

compound (1.26 g, 88%).

m.p. : 259-261 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2. 6(s, 3H), 2.80 (d, 3H), 7.45 (d,

1H) , 7.52(d, 1H) , 8.03(s, 1H) , 8.13(s, 1H) , 8.61(s, 1H) , 8.75(br

s, 1H), 11.28(s, 1H) , 13.06(br s, 1H)

<Example4> Preparation of 4- (lH-6-indazolylamino) -N-methyl-2- methylthio-5-pyrimidinecarboxamide

The desired compound (92 %) was obtained the same method

used for the example 3, except using

4- ( lH-6-indazolylamino) -2-methylthio-5-pyrimidinecarboxylic

acid ethyl ester prepared from preparation example 2 as a

starting .

m.p. : 263 ~ 265 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2.56(s, 3H), 2.81 (d, 3H), 7.05 (d,

IH) , 7.69(d, IH) , 7.98(s, IH) , 8.28(s, IH) , 8.67(s, IH) , 8.79(br

s, IH) , 11.57 (s,- IH) , 13.02(br s, IH)

<Example 5> Preparation of N-ethyl-4- (lH-5-indazolylamino) -2-

methylthio-5-pyrimidinecarboxamide

To the solution of methanol (20 ml) was added

4- ( liϊ-5-indazolylamino) -2-methylthio-5-pyrimidinecarboxylic

acid ethyl ester (1 g) prepared from preparation example 1,

added 70 % aq. ethylamine (25 ml) at room temperatute, and reacted

at 30-35^°C for 3 hr . The reaction mixture was cooled and slowly

added H₂0 (30 ml) and stirred for 0.5 hr. The reaction mixture

was filtered, washed with 30% aqueous methanol solution (10 ml) ,

obtained the desired compound (0.8 g, 80%) m.p. : 252-254 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 1.17 (t, 3H), 2.49 (s, 3H), 3.33 (m,

2H), 7.48(d, IH) , 7.55(d, IH) , 8.07(s, IH), 8.15(s, IH) , 8.65(s,

IH) , 8.78(br s, IH) , 11.29(s, IH) , 13.09(br s, IH)

<Example 6> Preparation of N-cyclopropyl-4- (lH-6-indazolyl

amino) -2-methylthio-5-pyrimidinecarboxamide

To the solution of methanol (20 ml) was added

4- ( lJf-6-indazolylamino) -2-methylthio-5-pyrimidinecarboxylic

acid ethyl ester (lg) prepared from preparation example 2 , added

cyclopropylamine (7 ml) at room temperature, and reacted at 50^°C

for 6 hr. The reaction mixture was cooled, and stirred at 25 ^°C

for 0.5 hr with slowly adding H₂0 (20 ml) . The reaction mixture

was filtered, washed with 30% aqueous methanol solution (10 ml) ,

obtained the desired compound (0.67 g, 65%)

m.p : > 270 ^°C

^XH-NMR (DMSO-de) , ppm : δ 0.54 (m, 2H) , 0.67 (m, 2H) , 2.49 (s,

3H) , 2.78 (m, lH) ,7.00(d, IH) , 7.63(d, IH) , 7.92(s, IH) , 8.21(s,

IH) , 8.58 (s, IH) ,8.69(br s,lH) , 11.46(s, IH) , 12.97 (br s , IH)

<Example 7> Preparation of N- (2-hydroxyethyl) -4- (lff- 5-indazolylamino) -2-methylthio-5-pyrimidinecarboxamide

To the solution of methanol (20 ml) was added

4- ( lH-5-mdazolylammo) -2-methylthιo-5-pyπmιdιnecarboxylιc

acid ethyl ester (lg) prepared from preparation example 1, added

ethanol amme (7 ml) at room temperature, and refluxed for 4

hr. The reaction mixture was cooled and slowly added H₂0 (40

ml) at 20 ^°C and stirred for 0.5 hr . The reaction mixture was

filtered, washed with 25% aqueous methanol solution (10 ml),

obtained the desired compound (0.75 g, 72%)

m.p : > 270 ^°C

^- MR (DMS0-d₆) , ppm : δ 2.45 (s, 3H) , 3.36(m, 2H) , 3.55 (m,

2H), 4.81 (m, IH) , 7.47(d, IH) , 7.54(d, IH) , 8.06(s, IH) , 8.15(s,

IH) , 8.71(s,lH), 8.77(br s,lH), 11.25(s,lH), 13.07(br s,lH)

<Example 8> Preparation of N- (2-hydroxyethyl) -4- (1H-6-

indazolylamino) -2-methylthio-5-pyrimidιne carboxamide

The desired compound (76 %) was obtained the same method

used for the example 7, except using

4- (liY-6-mdazolylammo) -2-methylthιo-5-pyπmιdmecarboxylιc

acid ethyl ester prepared from preparation example 2.

m.p : 269-270 ^°C ^XH-NMR (DMS0-d₆) , ppm : δ 2.56(s, 3H) , 3.33 (m, 2H) , 3.54 (m,

2H) , 4.79(m, IH) , 7.04 (d, IH) , 7.69(d, IH) , 7.98 (s, IH) , 8.27(s,

IH) , 8.72 (s, IH) , 8.81(br s, IH) , 11.52(s, IH) , 13.03(br s, IH)

<Example 9> Preparation of N-hydroxyethyl-N-methyl-4-

(lH-5-indazolylamino) -2-methylthio-5-pyrimidine carboxamide

To the solution of methylene chloride (120 ml) was added

N, N-dimethylformamide (1.1 ml) and thionyl chloride (1.2 ml)

and refluxed for 2 hr. The solution was added

4- (lH-5-ιndazolylaraιno) -2-methylthιo-5-pyπmιdmecarboxylιc

acid ethyl ester (3 g) prepared from preparation example 3, and

refluxed for 10 hr . The reaction mixture was cooled and slowly

added 2- (methylammo) ethanol (4 ml) at 0~5 ^°Cand stirred for

1 hr. The reaction mixture was added methanol (80 ml) , stirred

5 mm. and then filtered for removing impurity. The filtrate

was concentration by evaporation under pressure and it was

obtained a solid product. The solid product methanol : H₂0

= 1: 1 was added aq. 3N NaOH (3 ml) , stirred for 1 hr, filtered

and washed with H₂0. The desired compound (1.61 g. 45%) was

obtained by recrystallization with methylene chloride :

isopropyl ether =1 : 4. m.p : 106-114 ^°C

^XH-NMR (DMSO-d₆) , ppm : δ 2.40 (s,3H) , 2.99(s,3H), 3.43(br

s,2H) ,3.56(br s,2H) , 7.44(d, IH) , 7.50(d, IH) , 7.93 (br s , IH) ,

8.03(s, IH) , 8.14(s, IH) , 8.93 (br s, IH) , 13.03(br s IH)

It was prepared compounds in prepared example 10—35 as

the same method used for the example 9. It is shown in Table

1 that the compound name, yield, recrystallizmg solution,

melting point of compounds in prepared example 10—35 and

5-pyrimidinecarboxylic acid derivatives (8_) and amine compound

( ) as starting materials. It is shown in Table 2 that ^1H-NMR

of compounds in prepared example 10—35.

TABLE la

TABLE lb

TABLE lc

TABLE 2a

TABLE 2b

1 <Ξxperiment 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/-? of HBV

polymerase, 20 μi of reaction mixture (10 μM each of DIG-UTP

and Biotin-UTP, 46 mM Tris-HCl, 266 mM KCl, 27.5 mMMgCl₂, 9.2

mM DTT substrate/primer hybrid) , and 20 μl of test compound (added to 1, 0.1, and 0.01 .g/m-?) were added and allowed to react at

22^°C for 15 hrs. During this reaction, HBV polymerase catalyzes

DNA synthesis and digoxigenm and biotin attached to nucleotides

form bonds with streptavid coated on the bottom of wells . When

the reaction was done, each well was washed with 250 μJl of cleaning

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

to remove remaining impurities . 200 ^ 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//-? each of ABTS™, a substrate of peroxidase,

was then added and allowed to react at room temperature for 30

mm. Absorbance was measured at 405 nm using ELISA reader.

The percentage of reduction m 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 70%, up to max. 98% 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 the use of

nucleosides andmay be appliedtogether 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><10⁵ cells/m? and

1 ill? 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 βg/ml . One week after the addition of test compounds, the

culture solution was centπfuged at 5,000 rpm for 10 mm. 25

μl 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 μ& of neutralizing solution

[0.09NHC1, 0. lMTris-HCi, pH 7.4 ] was added as a reaction solution

for competitive polymerase chain reaction (PCR).

PCR was performed using genetic sequence of HBV core protein as a matrix. PCR reaction was carried out by adding 1 unit of

Taq polymerase enzyme to 25 pmol of each primer, 250 μM dNTP,

5 μ of PCR reaction solution [0.54N NaOH, 0.06% NP40, 0.09N

HCl, 0.1M Tπs-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 (lamivudme) was used as a positive control at the

same concentrations as those of the test compounds. The

percentage of reduction mHBVproliferation was calculatedusmg

the group without test compound as a control and the results

are represented m 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%, up to max. 97% reduction of HBV proliferation at the

concentration of 1 //g/m-?, . Moreover, compounds of the present

invention, being non-nucleosides, may not have problems such

as toxicity and early development of resistant virus strains

observed n the use of nucleoside substances . It is also expected

that compounds of the present invention may be used parallel with nucleoside compounds since the former act on allosteπc

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 transcπptase assay kit

(Boehπnger Mannheim) was used in the measurement of in vitro

transcπptase activities. 20 μi (40 ng) of HIV transcπptase

and 20 μ of reaction mixture containing matrix-primer hybrid

poly (A) oligo (dT) 15, DIG (digoxigenm) -dUTP, biotm-dUTP, and

TTP were added to wells coated with streptavidm. Test compounds were also added at the final concentrations of 0.1 and 1 g/ml'

and allowed to react at 37^°C for 1 hr . At this time, DNA is

formed from RNA by the action of HIV transcriptase, forming bonds

with streptavid coated on the bottom of wells because of

digoxigenm and biotm moieties attached to nucleotides.

When the reaction was completed, each well was washed with

250 μl of cleaning buffer (pH 7.0) for 30 sec. five times to

remove remaining impurities. 200 μi of anti-DIG-POD antigen

was added to each well, allowed to react at 37^°C for 1 hr 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 30mm. Absorbance at 405 nm was then read

for each solution using ELISA reader and used for quantitative

determination of inhibitory effect on the HIV transcriptase

activities. The percentage of reduction m the activities of

HIV reverse transcriptase was calculated using the group without

test compound as control and the results are represented m Table TABLE 5 Inhibitory effect on the activities of HIV reverse

transcriptase

As shown above m Table 5, compounds of the present invention

have excellent inhibitory effect on the activities of HIV reverse

transcriptase, having more than 80%, up to max. 89% 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 the use of nucleoside

substances. Furthermore, compounds of the present invention

may be used together with nucleoside compounds since the former

act on allosteric binding pockets while the latter act m the of polymerase activities.

As described above, compounds of the present invention

have excellent inhibitory effect on the HIV enzyme activities

in reverse transcription, which is a step in HIV replication.

Based on the mechanism, these compounds are able to effectively

control HIV proliferation and may be useful as therapeutics for

prevention and treatment of AIDS

<Experiment 4> Cytotoxicity test

To determine if compounds of formula 1 exhibit cytotoxicity,

in vitro tests were carried out using HepG2 cells withMTT analysis

method as generally known and the results are in Table 6 shown

below .

TABLE 6 Cytotoxicity tests on HepG2 cells

As shown above in Table 6, compounds used in the experiments

have higher than 100 μg/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 m the examples of 1~35 were

suspended n 0.5% methylcellulose solution and orally

administered once to 6 rats per group at the dosage of 4 g/kg/15m-?.

Death, clinical symptoms , and weight change m 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 m 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 rats up to the level of 4 g/kg and their estimated LD₅₀ values are much

greater than 4 g/kg in rats.

INDUSTRIAL APPLICABILITY

As described above, novel 5-pyrimidinecarboxamide

derivatives of formula 1 m the present invention have dramatic

inhibitory effect on proliferation of HBV and of HIV with little

side effect andmay 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. 5-Pyrimidιnecarboxamide derivatives and their

pharmaceutically acceptable salts represented below in formula

Formula 1

Wherein,

Ri is H, hydroxy, straight or branched alkyl group with

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

or branched hydroxyalkyl group with C₂~C₆, dialkylammo group

with 2~6 carbons, straight or branched alkyl group with C₂~C6

substituted with hydroxy or alkoxycarbonyl group with C₂ ^~~C₅,

cycloalkyl group with C₃~C₆, or saturated or unsaturated 5 or

6 membered heterocyclic compounds containing 1 to 3 heteroatoms

selected from N, 0, and S, which may be unsubstituted or

substituted with alkyl group of Cι~C₃; Ri may or may not contain

asymmetrical carbons; R₂ is H or straight or branched alkyl group with Cι~C₄;

Or both Ri and R₂ consist of 5 or 6 membered saturated

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 or straight or branched

hydroxyalkyl group with C₂ - C ;

R is mdazol-5-yl or indazol-6-yl;

n is an integer between 0 and 4.

2. Process for preparing 5-pyrimidinecarboxamide

derivatives of claim 1, which comprises the following steps.

1) Preparation of 5-pyrimidinecarboxylic acid ethyl

ester derivatives of formula 6 by reacting

4-chloro-2-methylthio-5-pyrimιdinecarboxylιc acid ethyl ester

of formula 4 with 5-ammomdazole or 6-ammomdazole of formula

5 under a basic condition (step 1);

2-A) Preparation of 5-pyrimidinecarboxamide derivatives

of formula 1 by reacting the compound of formula 6 with amme

compound of formula 7, or

2-B) Preparation of 5-pyrimidinecarboxamide derivatives

of formula 1 by first hydrolyzing the compound of formula 6 to form 5-pyrιmιdmecarboxylιc acid derivatives of formula 8, and

then activating to Vilsmeier intermediate m the presence of

N, N-dimethylformamide and S0C1₂, and allowing it to react with

amme compound of formula 7 (step 2

Scheme 1

R,-NH₂

Wherein, Ri, R₂, R₃ and n are as defined in claim 1

3. Therapeutic agent and a preventive agent for hepatisis

B with 5-pyπmιdmecarboxamιde derivatives and their

pharmaceutically acceptable salts in claim 1 as effective ingredient .

4. Therapeutic agent ana a preventive agent for acquired

immune deficiency syndrome (AIDS) with 5-pyrιmιdmecarboxamιde

derivatives and their pharmaceutically acceptable salts in claim

1 as effective ingredient.