WO1999007352A2

WO1999007352A2 - Method for promoting expression of ldl receptor gene

Info

Publication number: WO1999007352A2
Application number: PCT/JP1998/003595
Authority: WO
Inventors: Yoshihide Ueno; Koji Morishita; Masami Muraoka; Naohito Ohashi; Bei Shan
Original assignee: Sumitomo Pharmaceuticals Co Ltd; Tularik Inc
Current assignee: Sumitomo Pharma Co Ltd; Tularik Inc
Priority date: 1997-08-12
Filing date: 1998-08-12
Publication date: 1999-02-18
Anticipated expiration: 2000-02-12
Also published as: WO1999007352A3; AU8647698A; JPH11106378A

Abstract

Method of promoting expression of LDL receptor gene and an agent therefor, which comprises as an active ingredient a 1,2,4-triazin-5-one derivative of formula (I), wherein R1 is unsubstituted or substituted phenyl, unsubstituted or substituted heterocycle; R2 is unsubstituted or substituted phenyl, unsubstituted or substituted naphthyl, unsubstituted or substituted aralkyl, unsubstituted or substituted 5- to 6-membered aromatic heteromonocycle, unsubstituted or substituted C¿1?-C6alkyl, or unsubstituted or substituted C2-C6alkenyl, or pharmaceutically acceptable salt thereof, said active compound (I) increasing the LDL receptor amount by promoting expression of LDL receptor gene (amount of mRNA), by which showing activity of reducing cholesterol level in the blood, and therefore being useful in the prophylaxis or treatment of hyperlipidemia.

Description

DESCRIPTION

METHOD OF PROMOTING EXPRESSION OF LDL RECEPTOR GENE

TECHNICAL FIELD

The present invention relates to a method of promoting expression of LDL receptor gene, and an agent therefor, which are useful in the prophylaxis or treatment of hyperlipidemia, especially hypercholesterolemia. More particularly, the present invention relates to a novel l,2,4-triazin-5-one derivative having an activity for promoting expression of LDL receptor gene.

BACKGROUND ART

It has been known that some of l,2,4-triazin-5-one derivatives show antifungal activity, as described in USP 4343801.

It has been also known that low density lipoprotein (LDL) receptor in hepatic cells plays an important role in the regulation of cholesterol level in the blood. That is, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor inhibits the synthesis of cholesterol in hepatic cells, by which the expression of LDL receptor is indirectly promoted. As a result, the uptake of LDL by LDL receptor from the blood is increased, and then the cholesterol level in the blood is reduced.

HMG-CoA reductase inhibitors have been highly valued in clinical field as an agent for reducing the blood cholesterol level. However, HMG-CoA reductase inhibitors cannot sufficiently reduce the blood cholesterol level to the desired lower level in the patients with familial hypercholesterolemia or of coronary artery diseases. Under these circumstances, it is desired to develop an antihyperlipidemic agent showing more effective reducing activity of LDL level in the blood in such patients.

While HMG-CoA reductase inhibitors indirectly promote the synthesis of LDL receptor by inhibition of cholesterol synthesis, an agent for promoting expression of LDL receptor gene can be expected to show more potent reducing activity of LDL level in the blood by promoting more directly the synthesis of LDL receptor.

In the upstream of each gene of LDL receptor, there is a sequence named SRE (Sterol Regulatory Element), of which transcription is regulated by a free cholesterol in the cells (cf., Goldstein, Brown, Nature, vol. 343, p. 425, 1990).

Recently, SREBPs (SRE binding proteins) were identified as substances binding to SRE of LDL receptor gene (cf., Goldstein, Brown, et al., The Journal of Biological Chemistry, vol. 268, p. 14497, 1993), and it has been reported that the transcription of LDL receptor gene is activated by the SREBP's binding to SRE of LDL receptor gene (cf., Goldstein, Brown, et al, Cell, vol. 75, p. 187, 1993 and

Proc. Natl. Acad. Sci. USA, vol. 90, p. 11603, 1993). Based on these findings, it is possible to develop a medicament showing an activity of reducing the cholesterol level in the blood with a completely new mechanism, by selectively activating the expression of LDL receptor. DISCLOSURE OF INVENTION

The present invention provides a method of promoting expression of LDL receptor gene which either directly or indirectly regulates the synthesis of LDL receptor on a level with gene transcription, and is useful for the treatment of hypercholesterolemia, and also provides an agent for promoting expression of LDL receptor gene, useful for the prophylaxis or treatment of hyperlipidemia, especially hypercholesterolemia. Moreover, the present invention provides a novel compound which is useful in the regulation of the synthesis of LDL receptor, in the reduction of LDL cholesterol level in the blood, and in the treatment or prevention of arteriosclerosis. That is, the present invention relates to a method of promoting expression

of LDL receptor gene which comprises administering to a mammalian a pharmaceutically effective amount of a l,2,4-triazin-5-one derivative of the formula (I):

wherein R¹ is an unsubstituted or substituted phenyl group, or an unsubstituted

or substituted heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted 5- to 6-membered aromatic heteromonocyclic group, an unsubstituted or substituted Ci-Cg alkyl

group, or an unsubstituted or substituted C₂-C₆ alkenyl group, or a

pharmaceutically acceptable salt thereof.

The present invention also relates to an agent for promoting expression of LDL receptor gene, particularly for the prophylaxis or treatment of hyperlipidemia, which comprises as an active ingredient a l,2,4-triazin-5-one derivative of the above formula (I), or a pharmaceutically acceptable salt thereof.

The present invention further relates to a novel l,2,4-triazin-5-one derivative of the formula (I) wherein R¹ is an unsubstituted or substituted aromatic heterocyclic group having two nitrogen atoms, and R² is an

unsubstituted or substituted phenyl group, or R¹ is an unsubstituted or

substituted pyridyl group, and R² is an unsubstituted or substituted benzyl group or an unsubstituted or substituted furyl group, or a pharmaceutically acceptable salt thereof.

The terms used in the present specification are explained below.

The "substituted phenyl group" and the "substituted naphthyl group" have one or more substituents, for example, 1 to 5 substituents, and the substituent is, for example, a halogen atom, a C_rC alkyl group, a C₂-C₄ alkenyl

group, a -C₄ alkoxy group, a hydroxy group, a nitro group, a cyano group, a

trifluoromethyl group, a mercapto group, -S(O)_n(C₁-C alkyl) group, a carboxyl

group, an ester group, an unsubstituted or substituted amino group, a phenyl group, or a phenyloxy group (these phenyl or phenyloxy group may be optionally substituted by a group selected from the above halogen atom, a C1-C₄

alkyl group, a C₂-C₄ alkenyl group, a C1-C₄ alkoxy group, a hydroxy group, a

nitro group, a cyano group, a trifluoromethyl group, a mercapto group, -S(O)_n(C_r

C alkyl) group, a carboxyl group, an ester group, an unsubstituted or substituted

amino group), etc. (in the above formulae, n is 0, 1 or 2, hereinafter, the same). The aryl moiety of the "aralkyl group" is, for example, an aryl group having 6 to 10 carbon atoms such as phenyl, naphthyl, etc., and the alkyl moiety of the aralkyl group is, for example, an alkyl group having 1 to 3 carbon atoms such as methyl, ethyl, propyl, etc. The representative aralkyl group is, for example, benzyl group.

The "substituted aralkyl group" and the "substituted benzyl group" has one or more substituents at the aryl (or phenyl) moiety and/or the alkyl moiety, for example, 1 to 7 substituents, and the substituent is, for example, a halogen atom, a C1-C₄ haloalkyl group, a C1-C₄ alkyl group, a C₂-C alkenyl group, a C1-C₄

alkoxy group, a hydroxy group, a nitro group, a mercapto group, -S(O)_n(C_rC₄

alkyl) group, a carboxyl group, an ester group, an unsubstituted or substituted amino group, a substituted phenyloxy group (the substituents are a halogen atom, a C1-C₄ alkyl group, a C₂-C₄ alkenyl group, a C1-C₄ alkoxy group, a hydroxy

group, a nitro group, a cyano group, a trifluoromethyl group, a mercapto group, -S(O)_n(Cι-C alkyl) group, a carboxyl group, an ester group, an unsubstituted or

substituted amino group), etc.

The "5- to 6-membered aromatic heteromonocyclic group" is, for example, a 5- to 6-membered aromatic heteromonocyclic group consisting of one heteroatom selected from an oxygen atom, a sulfur atom, a nitrogen atom, etc., and carbon atoms, such as furyl, thienyl, pyrrolyl, pyridyl, etc. The "substituted 5- to 6-membered aromatic heterocyclic group" has one or more substituents, for example, 1 to 3 substituents, and the substituent is, for example, a halogen atom, a -C₄ alkyl group, a C₂-C₄ alkenyl group, a C₁-C₄

alkoxy group, a hydroxy group, a mercapto group, -S(O)_n(C_rC alkyl) group, a

carboxyl group, an ester group, an unsubstituted or substituted amino group, an unsubstituted or substituted phenyloxy group (the substituents are a halogen atom, a C1-C₄ alkyl group, a C₂-C alkenyl group, a C1-C₄ alkoxy group, a

hydroxy group, a nitro group, a cyano group, a trifluoromethyl group, a mercapto group, -S(O)_n(Cι-C₄ alkyl) group, a carboxyl group, an ester group, an

unsubstituted or substituted amino group), etc. The "alkyl" is a straight chain or branched chain alkyl group having carbon atoms of the defined number unless specified otherwise, for example, a straight chain or branched chain C₁-C₄ alkyl group such as methyl, ethyl, n-

propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, or a straight chain or branched chain Cι-C₆ alkyl group including n-pentyl, n-hexyl, 2-methylpentyl in

addition to the above-mentioned alkyl groups. The "substituted alkyl group" has one or more substituents, for example, 1 to 5 substituents, and the substituent is, for example, a halogen atom, a C_rC₄ alkoxy group, a hydroxy group, a

mercapto group, -S(O)_n(C_rC alkyl) group, an unsubstituted or substituted

amino group, etc.

The "alkenyl group" is a straight chain or branched chain C₂-C₄ alkenyl

group such as vinyl, 1-propenyl, allyl, isopropenyl, many butenyl isomers (e.g., 1-, 2- or 3-butenyl, isobutenyl), and a straight chain or branched chain C₂-C₆

alkenyl group including 1-, 2-, 3- or 4-pentenyl, 1-, 2-, 3-, 4- or 5-hexenyl, in addition to the above-mentioned groups. The "substituted alkenyl group" has one or more substituents, for example, 1 to 5 substituents, and the substituent is, for example, a halogen atom, a C1-C₄ alkoxy group, a hydroxy group, a mercapto

group, -S(O)_n(Cι-C alkyl) group, an unsubstituted or substituted amino group,

etc. The "halogen atom" or the halogen atom in the "haloalkyl group" is chlorine atom, bromine atom, fluorine atom or iodine atom.

The "substituted amino group" means an amino group wherein one or both hydrogen atoms of the amino group are independently substituted, for example, by a halogen atom, a C1-C₄ alkyl group, a C₂-C₄ alkenyl group, a C₁-C₄ alkoxy group, a hydroxy group, a mercapto group, or -S(O)_n(C_rC₄ alkyl) group.

The "heterocyclic group" may be an aromatic heterocyclic group or a saturated or unsaturated aliphatic heterocyclic group, for example, a 5- to 6- membered heterocyclic group consisting of 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom and a sulfur atom, and carbon atoms, such as piperidinyl, piperazinyl, 2-oxopiperidinyl, 2-oxopiperazinyl, 2-oxopyrrolidinyl, 2- oxazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyyrolidinyl, pyrazolyl, pyrazolidin- yl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, moφholinyl, thiazol- yl, thiazolidinyl, isothiazolyl, isothiazolidinyl, thiadiazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, thiamoφholinyl, thiamoφholinyl sulfoxide, thiamoφholinylsulfone, oxadiazolyl, and triazolyl. The substituent is 1 to 2 groups independently selected from a halogen atom, a C1-C₄ alkyl group, a C₂-C₄

alkenyl group, a C_rC₄ alkoxy group, a hydroxy group, a mercapto group,

-S(O)_n(C₁-C alkyl) group, a carboxyl group, an ester group, an substituted or

unsubstituted amino group, -CONR⁴R⁵, etc.

Among the above heterocyclic groups, particularly preferred heterocyclic group is a "nitrogen-containing aromatic heterocyclic group", for example, a 6- membered nitrogen-containing aromatic heterocyclic group consisting of one or two nitrogen atoms and carbon atoms, such as pyridyl, pyridazyl, pyrimidyl, and pyrazyl, and the substituent is 1 to 2 groups independently selected from a halogen atom, a C1-C₄ alkyl group, a C₂-C₄ alkenyl group, a C₁-C₄ alkoxy group,

a hydroxy group, a mercapto group, -S(O)_n(Cι-C alkyl) group, a carboxyl group,

an ester group, an unsubstituted or substituted amino group, -CONR⁴R⁵, etc. The "nitrogen-containing aromatic heterocyclic group having two nitrogen atoms" includes a 6-membered nitrogen containing aromatic heterocyclic group consisting of two nitrogen atoms and carbon atoms, such as pyridazyl, pyrimidyl, pyrazyl, etc. and the substituent is 1 to 2 groups independently selected from a halogen atom, a -C₄ alkyl group, a C₂-C₄ alkenyl

group, a C_rC₄ alkoxy group, a hydroxy group, a mercapto group, -S(O)_n(Cι-C₄

alkyl) group, a carboxyl group, an ester group, an unsubstituted or substituted amino group, -CONR⁴R⁵, etc.

R⁴ and R⁵ in the group -CONR⁴R⁵ are independently a hydrogen atom, an unsubstituted or substituted C₁-C₄ alkyl group, a C₂-C₄ alkenyl group, an

unsubstituted or substituted aralkyl group, an unsubstituted or substituted phenyl group, or a C -C₇ cycloalkyl group, or R⁴ and R⁵ may combine to make an

alkylene chain, and combine together with the adjacent nitrogen atom to which they bond to form a 5- to 7-membered ring (e.g., piperazinyl). The C₄-C₇

cycloalkyl group is, for example, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.

The "ester group" means an esterified carboxyl group, and includes a (C

Cg alkoxy)carbonyl group, a (C₆-C₁Q aryloxy)carbonyl group, a (C₇-C₁₃ aralkyl-

oxy)carbonyl group, etc. The examples of the ester group are methoxycarbonyl, ethoxycarbonyl, phenoxycarbonyl, benzyloxycarbonyl, phenethyloxycarbonyl, etc.

The "haloalkyl" is a straight chain or branched chain alkyl group substituted by one or more halogen atoms, for example, a straight chain or branched chain C₁-C₄ alkyl group unless specified otherwise and substituted by 1 to 9 halogen atoms. The representative haloalkyl group is, for example, trifluoromethyl and trifluoroethyl.

The preferable compound for an active ingredient of the present invention for promoting expression of LDL receptor gene is compounds of the above formula (I) wherein R¹ is an unsubstituted or substituted nitrogen-containing

aromatic heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted benzyl group, an unsubstituted or substituted furyl group, an unsubstituted or substituted thienyl group, an unsubstituted or substituted pyrrolyl group, an unsubstituted or substituted C_rC₆ alkyl group, or an unsubstituted or substituted

C₂-C₆ alkenyl group.

The more preferable compound for an active ingredient of the present invention for promoting expression of LDL receptor gene is compounds of the above formula (I) wherein R¹ is an unsubstituted or substituted pyridyl group (the substituent therein is selected from a carboxyl group, an ester group, a substituted amino group, or -CONR⁴R⁵); R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted benzyl group, an unsubstituted or substituted furyl group, an unsubstituted or substituted thienyl group, an unsubstituted or substituted pyrrolyl group, an unsubstituted or substituted C1-C₆ alkyl group, or an unsubstituted or substituted

C₂-C₆ alkenyl group.

The representative compounds being useful as an active ingredient of the present invention are listed below.

6-(l-naphthyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-naphthyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-phenoxyphenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(4-phenoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -((l , 1 ' -biphenyl)-4-yl)-3-(2-pyridyl)-l ,2,4-triazin-5-one, -phenyl-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-aminophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-hydroxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3,4-dimethoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-dimethoxyphenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(4-(dimethylamino)phenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-cy anophenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(2-pyridyl)-6-(4-(trifluoromethyl)phenyl)-l,2,4-triazin-5-one, -(2-pyridyl)-6-(3-(trifluoromethyl)phenyl)-l,2,4-triazin-5-one, -(4-nitrophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-nitrophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-(methylsulfonyl)phenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-(methylthio)phenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-methoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-methylphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-chloro-6-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-difluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3,4-dichlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-dichlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-iodophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-bromophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-chlorophenyl)-3-(2-pyridyl)-l ,2,4-triazin-5-one, -(2-chlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(3-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(4-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(2-pyrazyl)-l,2,4-triazin-5-one, -phenyl-3-(4-pyridyl)- 1 ,2,4-triazin-5-one, -phenyl-3-(2-pyrimidyl)- 1 ,2,4-triazin-5-one, -phenyl-3-(4-pyrimidyl)-l,2,4-triazin-5-one, -phenyl-3-(5-pyrimidyl)-l,2,4-triazin-5-one, -phenyl-3-(4-pyridazyl)-l,2,4-triazin-5-one, -benzyl-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-hydroxybenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-dichlorobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, - (4-aminobenzy 1) -3 - (2-py ridyl) - 1 , 2,4-triazin-5-one, -(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-furyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-thienyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-(6-chloro)thienyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(2-(3-bromo)thienyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(2-(l-methyl)pyrrolyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-isobutyl-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(sec-butyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-isopropyl-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(N,N-dimethylaminophenyl)-3-phenyl-l,2,4-triazin-5-one. As explained above, the active ingredient of the present invention includes a pharmaceutically acceptable salt of the compound of the formula (I) as well. The present compounds may be converted into a pharmaceutically acceptable salt thereof by reacting with various inorganic acids, organic acids, or inorganic bases. The acid for forming an acid addition salt is, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. The organic carboxylic acid includes, for example, formic acid, acetic acid, fumaric acid, maleic acid, malic acid, tartaric acid, aspartic acid, glutamic aid, etc. The sulfonic acid includes, for example, methanesulfonic acid, benzenesulfonic acid, p-toluene- sulfonic acid, hydroxybenzenesulfonic acid, dihydroxybenzenesulfonic acid, etc. The salt with a base includes ones derived from inorganic bases such as hydroxides, carbonates, hydrogen carbonates of ammonium, alkali metals or alkaline earth metals (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydroxide, calcium carbonate, etc.), and potassium and sodium are especially preferable bases.

Moreover, the above compounds or a salt thereof, which is an active ingredient of the present invention, may be in the form of either anhydride, a hydrate (e.g., monohydrate, dihydrate) or a solvate.

The l,2,4-triazin-5-one derivatives of the above formula (I), an active ingredient of the present invention, can be prepared by methods which are known in this field. For example, according to the method disclosed in USP 4343801, the compound (I) can be prepared by condensing a ketocarboxylic acid derivative of the formula (II):

R²-COCOOR³ (II)

wherein R² is the same as defined above, and R³ is a hydrogen atom or a lower alkyl group, with an amidrazone derivative of the formula (III):

wherein R¹ is the same as defined above, to form a Schiff base of d e formula (IV):

wherein R¹, R² and R³ are the same as defined above, followed by subjecting the compound (IV) to cyclization reaction. The above process for preparing the compound (I) is explained in more detail. The ketocarboxylic acid (II) and the amidrazone (III) are used each in equimolar amount, and condensed in a solvent at room temperature to give the Schiff base (IV). The Schiff base (IV) thus obtained is heated in a solvent to give the desired compound (I). In the process, it is not necessary to isolate the Schiff base (IV), and the ketocarboxylic acid derivative (II) and the amidrazone derivative (III) are heated in a suitable solvent to directly give the compound (I).

The solvent may be alcohols (e.g., methanol, ethanol), ethers (e.g., tetra- hydrofuran, dioxane), halogenated hydrocarbons (e.g., carbon tetrachloride, methylene chloride), and dimethylformamide, but it is not limited thereto and is inclusive any solvent which does not disturb the reaction. The reaction of the compound (II) and the compound (III) is usually carried out at room temperature, and the step of converting the compound (IV) into the compound (I) by cyclization reaction is carried out at a temperature of from 40°C to 300°C, preferably at a temperature of from 60°C to 200°C. The compound (I) thus obtained can be isolated and purified not only by chromatography, but also by other conventional isolation techniques such as recrystallization.

The ketocarboxylic acid derivative (II) and the amidrazone derivative (III) may be either commercially available ones, or prepared by a conventional method. For example, the ketocarboxylic acid derivative (II) can be prepared by the method disclosed in Collection of Czechoslovak Chemical Communication, vol. 29, p. 97, 1964, or Journal of Organic Chemistry, vol. 52, p. 5026, 1987, or Medicinal Chemistry Research, vol. 4, p. 385, 1994, or Kenkyu Houkoku-Sasebo Kogyo Koto Senmon Gakko, vol. 25, p. 119, 1988, etc. The amidrazone derivative (III) can be prepared by the method disclosed in Chemical Reviews, vol. 70, p.

151, 1970, etc.

Among the compounds of the formula (I), the compound (I) wherein R¹ is a phenyl or heterocyclic group substituted by an unsubstituted amino group can be prepared by subjecting the corresponding compound (I) wherein R¹ is a phenyl or heterocyclic group substituted by a nitro group, to reduction. That is, the corresponding compound (I) is subjected to catalytic hydrogenation with using a catalyst such as palladium-carbon, etc., under hydrogen atmosphere so that the nitro group thereof is reduced into an amino group. The solvent used in this reaction is, for example, alcohols (e.g., methanol, ethanol, etc.), ethers (e.g., tetrahydrofuran, dioxane, etc.), halogenated hydrocarbons (e.g., carbon tetrachloride, methylene chloride, etc.), esters (e.g., ethyl acetate, etc.), or a mixture of these solvents. The pressure of the hydrogen gas in the reaction system is usually in the range of from 1 to 4 atms, and the temperature is in the range of from 0 to 60°C. The compound of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof is used in the form of a pharmaceutical composition being suitable for either oral administration or parenteral administration, for example, tablets, capsules, syrups, suspensions, etc. for oral administration, or solutions, emulsions, suspensions, etc. for injection. The administration route of these preparations may be selected according to the preparation form thereof. The compound of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof may also be administered by a rectal route in the form of a suppository. These preparations can be prepared by formulating the active compound (I) by a conventional method with using a conventional carrier, diluent, binder, stabilizer, etc. In the preparation of injection form, the injection form can contain a pharmaceutically acceptable buffering agent, solubilizer, isotonic agent, etc.

The dosage and the administration frequency of the active compound (I) of the present invention vary according to the conditions, ages, weights of the patients, or the administration forms, but the active compound (I) of the present invention is administered in a dose of about 1 to 2000 mg/day /adult, preferably in a dose of 5 to 1000 mg/day/adult, for the oral administration, and 0.1 to 500 mg/day/adult for the injection, one to several times per day. BEST MODE FOR CARRYING OUT THE INVENTION The present invention is illustrated in more detail by the following Examples, but should not be construed to be limited to the Examples. Example 1

Preparation of 6-(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one: To a solution of 2-pyridylamidrazone (1.00 g, 7.34 mmol) in ethanol (10 ml) was added 4-nitrophenylpyruvic acid (1.54 g, 7.34 mmol, 1.0 eq.), and the mixture was heated under reflux for four hours. The mixture was allowed to cool to room temperature, and the precipitated solids were collected by filtration, purified by a flash silica gel column chromatography (hexane-ethyl acetate and methylene chloride-methanol) to give the title compound (506 mg, 22 %) as white solids. m.p. 238-239°C

The compounds of Examples 2-8 were obtained in the same manner as in Example 1.

Table 1

Example 9

Preparation of 6-(4-hydroxybenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one hydrochloride:

2-Pyridylamidrazone (770 mg, 5.66 mmol) and 4-hydroxyphenylpyruvic acid (1.02 g, 5.66 mmol, 1.0 eq.) were treated in the same manner as in Example 1 to give the title compound as basefree, which was further treated with hydrochloric acid-ethanol to give the hydrochloride thereof (644 mg, 30 %) as pale yellow solids. m.p. 238-240°C Example 10

Preparation of 6-(4-aminobenzyl)-3-(2-pyridyl)-l ,2,4-triazin-5-one:

To a solution of 6-(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one (50 mg, 0.162 mmol) in methylene chloride-methanol (1:1, 10 ml) was added 10 % Pd-C (50 % wet, 60 mg) under hydrogen atmosphere, and the mixture was stirred for two hours. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by a flash silica gel column chromatography (hexane-ethyl acetate and methylene chloride-methanol) to give the title compound (16 mg, 35 %) as yellow solids. m.p. 217-220°C Preparation 1

Preparation of 6-(4-trifluoromethylphenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5- one:

To a solution of 2-pyridylamidrazone (78.0 g, 573 mmol) in ethanol (800 ml) was added a solution of 4-trifluoromethylphenylpyruvic acid (125 g, 573 mmol, 1.0 eq.) in ethanol (350 ml), and the mixture was stirred at room temperature for one hour, and heated under reflux for 9 hours. The reaction solution was allowed to cool to room temperature, and the precipitated solids were collected by filtration. The solids thus obtained were dispersed into hot chloroform (300 ml), and then allowed to cool to room temperature. The precipitated yellow prisms were collected by filtration to give the title compound (119 g, 69 %). m.p. 230-233°C Experiment Promoting activity of LDL receptor

LDL receptors were detected from HepG2 cells which were cultured in a medium containing a lipoprotein-free serum, 25-hydroxy cholesterol and a test compound, by the method disclosed in Journal of Biological Chemistry, vol. 266, p. 16764, 1991, except that Western-Blotting was carried out with using Anti- mouse Ig, Horseradish peroxidase (Amersham) instead of ¹²⁵I-anti-mouse IgG, and LDL receptor was detected by ECL western-blotting detection system (Amersham).

The compound of Preparation 1 showed more excellent increasing activity of protein amount of LDL receptor compared with the control group, at 10 μM. EFFECTS OF INVENTION

The compound (I) of the present invention increases the LDL receptor amount by increasing the expression of LDL receptor gene (amount of mRNA), by which can reduce the cholesterol level in the blood. Therefore, the compound (I) of the present invention is useful in the prophylaxis or treatment of hyperlipidemia, especially hypercholesterolemia.

Claims

1. An agent for promoting expression of LDL receptor gene which comprises as an active ingredient a pharmaceutically effective amount of a 1,2,4- triazin-5-one derivative of the formula (I):

or substituted heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted 5- to 6-membered aromatic heteromonocyclic group, an unsubstituted or substituted C1-C₅ alkyl

group, or an unsubstituted or substituted C₂-C₆ alkenyl group, or a

pharmaceutically acceptable salt thereof.

2. The agent according to claim 1, wherein R¹ in the formula (I) is an

unsubstituted or substituted nitrogen-containing aromatic heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted benzyl group, an unsubstituted or substituted furyl group, an unsubstituted or substituted thienyl group, an unsubstituted or substituted pyrrolyl group, an unsubstituted or substituted C_j-

C₆ alkyl group, or an unsubstituted or substituted C -C₆ alkenyl group, or a

pharmaceutically acceptable salt thereof.

3. The agent according to claim 1, wherein R¹ in the formula (I) is an unsubstituted or substituted pyridyl group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted benzyl group, an unsubstituted or substituted furyl group, an unsubstituted or substituted thienyl group, an unsubstituted or substituted pyrrolyl group, an unsubstituted or substituted Cj-Cg alkyl group, or an

unsubstituted or substituted C₂-Cg alkenyl group, or a pharmaceutically

acceptable salt thereof.

4. The agent according to claim 1, wherein the l,2,4-triazin-5-one derivative is selected from the following compounds, or a pharmaceutically acceptable salt thereof:

6-(l-naphthyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(2-naphthyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(3-phenoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(4-phenoxyphenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, 6-((l,l'-biphenyl)-4-yl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-phenyl-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(4-aminophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(4-hydroxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(3,4-dimethoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(2,4-dimethoxyphenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one,

6-(4-(dimethylamino)phenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, 6-(4-cyanophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 3-(2-pyridyl)-6-(4-(trifluoromethyl)phenyl)-l,2,4-triazin-5-one, 3-(2-pyridyl)-6-(3-(trifluoromethyl)phenyl)-l,2,4-triazin-5-one, 6-(4-nitrophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-nitrophenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(4-(methylsulfonyl)phenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-(methylthio)phenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(4-methoxyphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-methylphenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-chloro-6-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-difluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3,4-dichlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2,4-dichlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-iodophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-bromophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(2-fluorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, -(3-chlorophenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(2-chlorophenyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one, -(4-chlorophenyl)-3-(3-pyridyl)-l,2,4-triazin-5-one, -(4-chlorophenyl)-3-(4-pyridyl)- 1 ,2,4-triazin-5-one, -(4-chlorophenyl)-3-(2-pyrazyl)-l,2,4-triazin-5-one, -phenyl-3-(4-pyridyl)-l,2,4-triazin-5-one, -phenyl-3 - (2-py rimidyl) - 1 , 2,4-triazin-5 -one, -phenyl-3-(4-pyrimidyl)-l,2,4-triazin-5-one, -phenyl-3-(5-pyrimidyl)-l,2,4-triazin-5-one, -phenyl-3-(4-pyridazyl)-l,2,4-triazin-5-one, 6-benzyl-3-(2-pyridyl)- 1 ,2,4-triazin-5-one,

6-(4-hydroxybenzyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one,

6-(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(2,4-dichlorobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(4-aminobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(4-nitrobenzyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(2-furyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(2-thienyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(2-(6-chloro)mienyl)-3-(2-pyridyl)-l,2,4-triazin-5-one, 6-(2-(3-bromo)thienyl)-3-(2-pyridyl)-l ,2,4-triazin-5-one,

6-(2-(l-methyl)pyrrolyl)-3-(2-pyridyl)- 1 ,2,4-triazin-5-one,

6-isobutyl-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-(sec-butyl)-3-(2-pyridyl)-l,2,4-triazin-5-one,

6-isopropyl-3-(2-pyridyl)-l,2,4-triazin-5-one, and 6-(N,N-dimethylaminophenyl)-3-phenyl- 1 ,2,4-triazin-5-one.

5. An agent for treating or preventing hyperlipidemia, which comprises as an active ingredient a pharmaceutically effective amount of a l,2,4-triazin-5- one derivative of the formula (I):

or substituted heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted 5- to 6-membered aromatic heteromonocyclic group, an unsubstituted or substituted C_rC₆ alkyl

group, or an unsubstituted or substituted C₂-CG alkenyl group, or a

pharmaceutically acceptable salt thereof.

6. A method of promoting expression of LDL receptor gene which comprises administering to a mammalian a pharmaceutically effective amount of a l,2,4-triazin-5-one derivative of the formula (I):

or substituted heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted 5- to 6-membered aromatic heteromonocyclic group, an unsubstituted or substituted Cj-Cg alkyl

group, or an unsubstituted or substituted C₂-Cg alkenyl group, or a

pharmaceutically acceptable salt thereof.

7. A method of treating or preventing hyperlipidemia, which comprises administering to a mammalian a pharmaceutically effective amount of a 1,2,4- triazin-5-one derivative of the formula (I):

or substituted heterocyclic group; R² is an unsubstituted or substituted phenyl group, an unsubstituted or substituted naphthyl group, an unsubstituted or substituted aralkyl group, an unsubstituted or substituted 5- to 6-membered aromatic heteromonocyclic group, an unsubstituted or substituted Cι-C₆ alkyl

group, or an unsubstituted or substituted C₂-Cg alkenyl group, or a

pharmaceutically acceptable salt thereof.

8. A use of a 1 ,2,4-triazin-5-one derivative of the formula (I):

group, or an unsubstituted or substituted C₂-C₆ alkenyl group, or a

pharmaceutically acceptable salt thereof, in the preparation of an agent for promoting expression of LDL receptor gene.

9. A use of a 1 ,2,4-triazin-5-one derivative of the formula (I):

group, or an unsubstituted or substituted C₂-Cg alkenyl group, or a

pharmaceutically acceptable salt thereof, in the preparation of an agent for treating or preventing hyperlipidemia.

10. A 1 ,2,4-triazin-5-one derivative of the formula (I):

wherein R¹ is an unsubstituted or substituted nitrogen-containing aromatic

heterocyclic group containing 2 nitrogen atoms, and R² is an unsubstituted or

substituted phenyl group, or R¹ is an unsubstituted or substituted pyridyl group,

and R² is an unsubstituted or substituted benzyl group or an unsubstituted or substituted furyl group, or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition which comprises a pharmaceutically effective amount of the compound as set forth in claim 10 in admixture with a pharmaceutically acceptable carrier or diluent.