JP2003335680A - ACAT-1 inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an ACAT-1 inhibitor containing a phosphonic acid diester derivative. <P>SOLUTION: This ACAT-1 inhibitor comprises the phosphonic acid diester derivative represented by the general formula, e.g. diisopropyl-4-[(4-(4- fluorophenyl)-5-methylthiazol-2-yl)carbamoyl]benzyl phosphonate as an active ingredient. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an ACAT-1 inhibitor containing a phosphonic acid diester derivative (acyl-coenzyme A:
cholesterol acyltransferase-1 inhibitor).

[0002]

2. Description of the Related Art ACAT is an intracellular enzyme that catalyzes the reaction of transferring long-chain fatty acids from acyl coenzyme A to the hydroxyl group at the 3-position of cholesterol to produce cholesterol ester (Chang, TY, et al., Annu. . Rev. Biochem.,
66, 613-638 (1997)). The general role of this enzyme is to esterify excess intracellular free cholesterol and keep the free cholesterol level constant, and it has different roles depending on organs. For example, in the small intestine, cholesterol is absorbed from the intestinal tract into the small intestinal epithelium, converted into cholesterol ester by ACAT, and then incorporated as a constituent lipid of kynamicron. In the liver, AC
The cholesterol ester synthesized by AT is incorporated as a constituent lipid present in the core of VLDL and released into the blood. In steroid hormone-producing cells such as the adrenal cortex and macrophages with atherosclerotic lesions, cholesterol ester is significantly accumulated by the action of ACAT.

Therefore, administration of a drug having an inhibitory activity on ACAT suppresses esterification of cholesterol in the small intestinal epithelium in the small intestine and increases the free cholesterol level in the small intestine epithelium, so that the intestinal lumen and The cholesterol gradient is lost and the absorption of cholesterol is inhibited, thus lowering blood cholesterol levels can be expected. In the liver, inhibition of cholesterol ester synthesis by ACAT inhibition promotes hepatic intracellular degradation of VLDL and suppresses extracellular secretion of VLDL, and thus a decrease in blood LDL level can be expected. In addition, at the lesion site of arteriosclerosis, accumulation of cholesterol ester at the lesion site is suppressed by ACAT inhibition, and a direct anti-atherosclerotic effect can be expected.

As a drug having the above ACAT inhibitory activity (ACAT inhibitor), FR145237 (NipponRinsho, 2001 Mar;
59 Suppl.3: 675-680), F-1394 (Nippon Yakurigaku Z
asshi, 2001 Dec; 118 (6): 389-395), Dup128 (Nippon
Rinsho, 2001 Mar; 59 Suppl.3: 675-680), E5324 (Jp
n. J. Pharmacol., 1999 Feb; 79 (2): 151-158), CL277.
082 (Metabolism, 1998 Mar; 47 (3): 325-332), NTE-12
2 (Jpn. J. Pharmacol., 2001 May; 86 (1): 120-123) and other urea agents having a structure derived from urea (H ₂ N-CO-NH ₂ ), 58-035 (J. Pharm. Sci., 2001 Nov; 90 (11): 1859
-1867), CI-976 (J. Pharm. Sci., 2001 Nov; 90 (11):
1859-1867), CI-1011 (Biochem. Pharmacol., 2002 Feb
1; 63 (3): 349-360) and other amide (-NH-CO-) structures are known.

However, many ACAT inhibitors to date have been studied and developed as antihyperlipidemic agents with emphasis on the cholesterol absorption inhibitory action.

Recently, ACAT is a type that exists only in the small intestine
It was reported that there are two subtypes (ACAT-2) and a type present in liver, macrophages, adrenal gland and small intestine (ACAT-1). According to this subtype, it was revealed that most of the ACAT inhibitors developed so far were aimed at the inhibition of ACAT-2.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an ACAT-1 inhibitor having a novel structure which has hitherto been unknown.

The applicant of the present invention, in the course of continuing research and development of active ingredient compounds that can be used in the pharmaceutical field, first found that the lipid-lowering action, the cataract prevention and treatment actions,
A new series of phosphonic acid diester derivatives having hypoglycemic action have been developed (Patent 2787407).

As a result of subsequent research, the present applicant has found that in the above phosphonic acid diester derivative, ACAT
It was found that there is a compound having -1 inhibitory activity, and the present invention has been completed here.

[0010]

The present invention has the following general formula:
There is provided an ACAT-1 inhibitor comprising the phosphonic acid diester derivative represented by (1) as an active ingredient. General formula (1):

[0011]

[Chemical 2]

[In the formula, A represents N or CR ¹ . R ¹ is a hydrogen atom, a lower alkyl group, a halogen atom on the phenyl ring,
1-3 of groups selected from the group consisting of lower alkyl groups, lower alkoxy groups, cyano groups, nitro groups and phenyl groups.
A phenyl group which may have a lower alkoxyoxalyl group, a lower alkoxycarbonyl lower alkyl group,
Lower alkoxycarbonyl group, phenylcarbonyl lower alkyl group, phenyl lower alkyl group, naphthyl group, 1,
A 4-benzodioxanyl group or a thienyl group is shown. R ² is a hydrogen atom, a halogen atom, a nitro group, a C _1-12 -alkyl group, a lower alkoxycarbonyl group, a phenyl group which may have a halogen atom or a lower alkyl group as a substituent, a phenylcarbonyl group, a phenylsulfonyl group,
A phenyl lower alkyl group having a halogen atom on the phenyl ring, a halogen-substituted lower alkyl group, a mercapto group or a lower alkylthio group. R ³ represents a hydrogen atom, a lower alkyl group or a phenyl group. R ⁴ and R ⁵ are the same or different and each represents a lower alkyl group or a phenyl lower alkyl group. ]

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The above general formula (1) representing a phosphonate diester derivative which is an active ingredient of the ACAT-1 inhibitor of the present invention.
And the other groups used in the present specification, in each case when they are used as a group shown in each formula and when they are used as a substituent of the group, specifically, Is the street. The term "lower" used for each group containing carbon in the present specification shall mean "having 1 to 6 carbon atoms".

The lower alkyl group includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl and te.
Examples thereof include linear or branched alkyl groups having 1 to 6 carbon atoms such as rt-butyl, pentyl and hexyl groups. Further, the C _1-12 -alkyl group includes heptyl, octyl, nonyl, decyl, undecyl, dodecyl groups and the like in addition to the above lower alkyl group.

No phenyl group which may have 1-3 halogen groups, lower alkyl groups, lower alkoxy groups, cyano groups, nitro groups and phenyl groups on the phenyl ring may be used. In addition to the substituted phenyl group, for example, 4-chlorophenyl, 4-bromophenyl, 2-chlorophenyl, 3-chlorophenyl, 4-fluorophenyl, 4-iodophenyl, 2,3-dichlorophenyl,
3,4-dichlorophenyl, 2,4-dichlorophenyl, 2,4,6-
Trichlorophenyl, 4-methylphenyl, 4-ethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-propylphenyl, 4-butylphenyl, 3,4-dimethylphenyl, 2,4,6-trimethylphenyl, 4 -Hexylphenyl,
4-methoxyphenyl, 4-ethoxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-propoxyphenyl, 4-butoxyphenyl, 3,4-dimethoxyphenyl, 2,
4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,
2-cyanophenyl, 3-cyanophenyl, 4-cyanophenyl, 2-nitrophenyl, 3-nitrophenyl, 4-nitrophenyl, 2-bromo-4-cyanophenyl, 4-bromo-2-cyanophenyl, 4- Halogen atom on the phenyl ring such as chloro-2-nitrophenyl, 2-biphenyl, 3-biphenyl, 4-biphenyl group, linear or branched alkyl group having 1-6 carbon atoms, 1-carbon number Examples thereof include a phenyl group having 1-3 linear or branched alkoxy groups, cyano groups, nitro groups, and groups selected from the group consisting of phenyl groups.

Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

The lower alkoxy group is a linear or branched alkoxy group having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy and hexyloxy groups. Can be illustrated.

The lower alkoxyoxalyl group has 1-6 carbon atoms such as methoxyoxalyl, ethoxyoxalyl, propoxyoxalyl, isopropoxyoxalyl, butoxyoxalyl, isobutoxyoxalyl, tert-butoxyoxalyl, pentyloxyoxalyl and hexyloxyoxalyl groups. The oxalyl group having a linear or branched alkoxy group can be exemplified.

As the lower alkoxycarbonyl lower alkyl group, methoxycarbonylmethyl, 2- (methoxycarbonyl) ethyl, 3- (methoxycarbonyl) propyl, 4
-(Methoxycarbonyl) butyl, 5- (methoxycarbonyl) pentyl, 6- (methoxycarbonyl) hexyl, ethoxycarbonylmethyl, propoxycarbonylmethyl,
Alkoxy moieties such as isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, tert-butoxycarbonylmethyl, pentyloxycarbonylmethyl, and hexyloxycarbonylmethyl groups are straight-chain or branched alkoxy groups with 1-6 carbon atoms. And the alkyl moiety is a linear or branched alkyl group having 1 to 6 carbon atoms.

The lower alkoxycarbonyl group has 1-6 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl groups. The carbonyl group having a linear or branched alkoxy group can be exemplified.

As the phenylcarbonyl lower alkyl group, phenylcarbonylmethyl, 2- (phenylcarbonyl) ethyl, 3- (phenylcarbonyl) propyl, 4- (phenylcarbonyl) butyl, 5- (phenylcarbonyl) pentyl, 6- ( Examples thereof include a phenylcarbonylalkyl group in which the alkyl moiety such as a phenylcarbonyl) hexyl group is a linear or branched alkyl group having 1 to 6 carbon atoms.

The phenyl lower alkyl group is a straight chain having 1-6 carbon atoms such as benzyl, 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl, 5-phenylpentyl and 6-phenylhexyl groups. Examples thereof include a phenylalkyl group which is a branched or branched chain alkyl group.

The naphthyl group includes 1-naphthyl and 2-naphthyl groups.

The 1,4-benzodioxanyl group includes 1,4-benzodioxan-5-yl and 1,4-benzodioxan-6-yl groups.

The phenyl which may have a halogen atom or a lower alkyl group as a substituent includes, in addition to an unsubstituted phenyl group, for example, 4-chlorophenyl, 4-bromophenyl, 2-chlorophenyl, 3-chlorophenyl,
4-fluorophenyl, 4-iodophenyl, 4-methylphenyl, 4-ethylphenyl, 2-methylphenyl, 3-methylphenyl, 4-propylphenyl, 4-isopropylphenyl, 4-butylphenyl, 4-isobutylphenyl, 4-tert
Examples thereof include a phenyl group having a halogen atom or a lower alkyl group such as -butylphenyl, 4-pentylphenyl and 4-hexylphenyl groups.

The phenyl lower alkyl group which may have a halogen atom on the phenyl ring is, for example, 2-chlorobenzyl, 2- (2-chlorophenyl) ethyl, 3- (in addition to the unsubstituted phenyl lower alkyl group. 2-chlorophenyl) propyl, 4- (2-chlorophenyl) butyl, 5- (2-chlorophenyl) pentyl, 6- (2-chlorophenyl) hexyl, 2-fluorobenzyl, 2-bromobenzyl, 2-iodobenzyl, 3- Chlorobenzyl, 2- (3-chlorophenyl) ethyl, 3- (3-chlorophenyl) propyl, 4- (3-chlorophenyl) butyl, 5- (3-chlorophenyl) pentyl, 6- (3-
(Chlorophenyl) hexyl, 4-chlorobenzyl, 2- (4-chlorophenyl) ethyl, 3- (4-chlorophenyl) propyl, 4- (4-chlorophenyl) butyl, 5- (4-chlorophenyl) pentyl, 6- (4- (Chlorophenyl) hexyl, etc.,
Examples thereof include a phenyl lower alkyl group having a halogen atom on the phenyl ring.

Examples of halogen-substituted lower alkyl groups include trifluoromethyl, trichloromethyl, tribromomethyl, pentafluoroethyl, heptafluoropropyl,
Examples thereof include linear or branched alkyl groups having 1 to 6 carbon atoms, which are substituted with a halogen atom, such as nonafluorobutyl, undecafluoropentyl, and tridecafluorohexyl groups.

The lower alkylthio group has a linear or branched alkyl group having 1 to 6 carbon atoms such as methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, tert-butylthio, pentylthio and hexylthio groups. A thio group can be illustrated.

As the derivative represented by the general formula (1), which is particularly suitable as the active ingredient compound of the ACAT-1 inhibitor of the present invention, compounds belonging to the following groups can be mentioned. (a): In the general formula (1), a compound in which R ¹ is a phenyl group having a halogen atom as a substituent on the phenyl ring and R ² is a lower alkyl group (which can exhibit particularly excellent ACAT-1 inhibitory action) (b): A compound in the formula (1), wherein R ¹ is a phenyl group having a halogen atom as a substituent on the phenyl ring and R ² is a hydrogen atom.

The derivative represented by the above-mentioned general formula (1) is prepared according to the method described in the applicant's previous patent (Patent No. 2787407), for example, with a suitable amine such as 2-amino-4-phenylthiazole. , 4-[(diethoxyphosphoryl) methyl] benzoyl chloride and other suitable carboxylic acid halide derivatives can be produced by reacting with a deoxidizing agent in a suitable solvent. The details are as described in the reference example below.

The target compound thus obtained can be easily isolated and purified by a conventional separation and purification means such as adsorption chromatography, preparative thin layer chromatography, recrystallization and solvent extraction.

The ACAT-1 inhibitor of the present invention is prepared in the form of a general pharmaceutical composition by using a pharmaceutically acceptable carrier together with the compound represented by the general formula (1) and put into practical use. It

The pharmaceutically acceptable carrier used in the pharmaceutical composition of the present invention is a diluent or excipient usually used depending on the use form of the preparation, such as a filler, a bulking agent or a binder. , Moisturizers, disintegrants, surfactants, lubricants and the like. These are appropriately selected and used according to the dosage unit form of the pharmaceutical preparation to be adjusted.

As the dosage unit form of the pharmaceutical preparation, various forms can be appropriately selected according to the purpose of treatment. Typical examples thereof include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.), ointments and the like.

In the case of molding in the form of tablets, as a pharmaceutically acceptable carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, phosphoric acid. Excipients such as potassium; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone and other binders; sodium carboxymethylcellulose, carboxymethylcellulose calcium, low Degree of substitution Hydroxypropyl cellulose, dry starch, sodium alginate, agar powder, naminaran powder, sodium hydrogen carbonate, calcium carbonate, and other disintegrants; polyoxyethylene sorbitan fatty acid Surface active agents such as tellers, sodium lauryl sulfate, stearic acid monoglyceride; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oils; absorption promoters such as quaternary ammonium base, sodium lauryl sulfate; glycerin, starch Humectants such as; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; purified talc, stearates, boric acid powders, lubricants such as polyethylene glycol and the like can be used. Further, the tablets may be tablets coated with a usual coating, if necessary, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double-layered tablets, multi-layered tablets.

In the case of molding in the form of pills, as a pharmaceutically acceptable carrier, for example, glucose, lactose,
Excipients such as starch, cocoa butter, hydrogenated vegetable oil, kaolin and talc; binders such as gum arabic powder, tragacanth powder, gelatin and ethanol; disintegrating agents such as laminaran and agar can be used.

In forming a suppository, as a pharmaceutically acceptable carrier, for example, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides, etc. can be used.

Capsules are usually prepared by mixing the compound of the present invention with various pharmaceutically acceptable carriers exemplified above and filling hard gelatin capsules, soft gelatin capsules and the like. .

When prepared as injections such as solutions, emulsions and suspensions, these are preferably sterilized and isotonic with blood. In making these forms, for example, water, ethanol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester and the like can be used as the diluent. In this case, a sufficient amount of salt, glucose or glycerin for preparing an isotonic solution may be contained in the pharmaceutical preparation, and a usual solubilizing agent, buffer, soothing agent, etc. are added. You may.

In the case of preparation in the form of an ointment such as paste, cream or gel, white petrolatum, paraffin, glycerin, cellulose derivative, polyethylene glycol, silicone, or bentonite can be used as a diluent.

Further, the pharmaceutical composition of the present invention may contain colorants, preservatives, perfumes, flavors, sweeteners and other pharmaceuticals, if necessary.

The amount of the compound of the present invention (active ingredient compound) blended in the pharmaceutical composition of the present invention is not particularly limited and is appropriately selected from a wide range. It is usually added to a pharmaceutical composition in an amount of about 0.5-90% by weight, preferably about 1-85% by weight.

The administration method of the pharmaceutical preparation of the present invention is not particularly limited, and it is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are intravenously or intramuscularly alone or mixed with a usual fluid replacement such as glucose and amino acid. ,
It is administered intradermally, subcutaneously or intraperitoneally, and suppositories are administered rectally.

The dose of the pharmaceutical preparation of the present invention is appropriately selected depending on the usage, the age and sex of the patient, other conditions, the degree of disease and the like. The amount of the compound of the present invention which is usually an active ingredient is
The daily dose for an adult is about 0.5-20 mg / kg, preferably about 1-10 mg / kg. The formulation can be administered once a day or in 2-4 divided doses.

[0045]

EXAMPLES In order to explain the present invention in more detail, the production examples of the compounds of the present invention will be given as reference examples, and then the pharmacological test examples carried out on the compounds of the present invention and pharmaceutical preparations containing the compounds of the present invention as active ingredients will be described. Here's an example.

[0046]

[Reference Example 1] Production of diisopropyl 4-[(4- (4-fluorophenyl) -5-methylthiazol-2-yl) carbamoyl] benzylphosphonate (1) 2-amino-4- (4-fluorophenyl) Production of 5-5-methylthiazole 179 g of 4-fluoropropiophenone and aluminum chloride (I
II) 1 g and 1 g of chloroform were dissolved in 1000 mL of chloroform, and 197 g of bromine was slowly added dropwise to this mixture while stirring with ice cooling. After stirring for 1 hour under ice cooling, the reaction mixture was poured into 500 mL of ice water. The chloroform layer was separated, washed successively with 500 mL of saturated aqueous sodium hydrogen carbonate and 500 mL of saturated saline, and then dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain 4'-fluoro-2-bromopropiophenone. Without purification, 600 mL of ethanol and 86 g of thiourea were added, and the mixture was added at 70 ° C for 12
Stir for hours. After the solvent was distilled off under reduced pressure, 1000 mL of ethyl acetate and 800 m of a 2N sodium hydroxide aqueous solution were added to the reaction mixture obtained.
L was added for liquid separation. The ethyl acetate layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the obtained crude crystals were recrystallized from ethyl acetate-n-hexane to give 172 g of the title compound.
Got (2) Preparation of diisopropyl 4-[(4- (4-fluorophenyl) -5-methylthiazol-2-yl) carbamoyl] benzylphosphonate 4-[(diisopropoxyphosphoryl) methyl] benzoyl
A solution of chloride (111.6 g) in methylene chloride (350 mL) was stirred with ice cooling, and to this was added 2-amino-4- (4-fluorophenyl) -5-methylthiazole (72.9 g, pyridine solution 4) obtained in (1) above.
00 mL was slowly added dropwise. The mixture was stirred at room temperature for 12 hours, 500 mL of water was added thereto, and methylene chloride was distilled off under reduced pressure. The obtained crude crystals were recrystallized from ethanol-water to give 146 g of colorless crystals of the title compound. Table 1 shows the structure and physical properties of the obtained compound.

[0047]

[Reference Example 2-12] In the same manner as in Reference Example 1, each compound shown in Table 1 was synthesized. The structure and physical properties of the obtained compound are shown in Table 1.
Will be described in parallel. In the table, the abbreviations of groups indicate the following. Et: ethyl group, i-Pr: isopropyl group, OMe: methoxy group,
OEt: ethoxy group

[0048]

【table 1】

[0049]

[Pharmacological Test Example 1] ACAT-1 Inhibitory Action Test 1 Each compound obtained in Reference Example 1-12 (see Table 1 and referred to as Compound No. 1-12 corresponding to Reference Example) and described in Table 2-6 below. Using each compound as a test substance, their ACAT-1 inhibitory activities were tested as follows.

[0050]

[Table 2]

[0051]

[Table 3]

[0052]

[Table 4]

[0053]

[Table 5]

[0054]

[Table 6]

ACAT-1 enzyme activity was measured by the reconstitution method (recon
Instituted vesicle assay) [J. Lipid Res., 29 , 1683-1
692 (1988), Biochem. Biophys. Acta, 982 , 187-195.
(1989), J. Biol. Chem., 270 , 29532-29540 (1995)].

Preparation of I. Broken Homoginate SW-13 cells (human adrenocortical carcinoma-derived cells) become confluent on a culture plate in a carbon dioxide incubator in L-15 medium containing 10% fetal bovine serum (FBS). Was cultured up to.

Methods described in literature [hypotonic shock and scr
apping method, Anal. Biochem., 116 , 298-302 (198
According to 1)], Broken Homoginate was collected. Protein quantification (B
radford method) and stored at −80 ° C. until use.

II. Cholesterol / Phosphatidylcholine (C
hol / PC) vesicle preparation Chang's method [Chang, TY, et al., Anal. Bioche
m., 157 , 323-330 (1986)], according to Chol / PC vesicle (C
hol / PC = 3.9 mM / 12.8 mM) was prepared.

III. Preparation of 5 × DOC / PC 50 mg of phosphatidylcholine (50 mg)
g / mL sodium deoxycholate-Buffer A (50mM Tris-HCl,
5mM EDTA, 0.05mM PMSF (phenylmethyl sulfonyl fluori
de, Wako Pure Chemical Industries, Ltd., pH 7.8).

IV. Preparation of Enzyme Solution To 2.6 mL of Broken Homoginate with a protein concentration of 2.5 mg / mL, 5 × DO was added.
C / PC (0.65 mL) was added, the mixture was stirred, and then left in ice for 20 minutes. To this, 22 mL of Chol / PC vesicle was added, stirred, and left standing in ice for 20 minutes. After centrifugation, the suspended matter was removed and this was used as the enzyme solution.

V. Assay The test substance was dissolved in DMSO to a concentration of 1 × 10 ⁻² mol / L.

A test substance or DM is placed in a screw cap glass test tube.
SO (as a control) 2.5 μL, enzyme solution 200 μL and substrate solution (150 mM potassium phosphate buffer (pH 7.0), 15 mg / mL
BSA (FFA free), 2mM DTT and 0.1mM [1- ¹⁴ C] oleoyl co
enzyme A (8.0 Ci / mol)] 50 μL was added. The reaction was carried out at 37 ° C for 30 minutes. Hexane 4 mL, 2M NaCl 1 mL and ^[3 H] -chole
The reaction was stopped by adding 1 mL (about 10,000 dpm) of ethanol containing steryl oleate. After shaking for 5 minutes, centrifugation was performed, 2 mL of the upper hexane phase was transferred to a glass test tube, and 1 mL was transferred to a scintillation vial.

For the hexane phase in the glass test tube, the solvent was removed under a nitrogen gas stream, the obtained lipid extract was redissolved in 100 μL of a chloroform / methanol (2: 1) mixed solution, and then spotted on a TLC plate. . Develop the TLC plate with hexane / diethyl ether / acetic acid (73: 25: 2) and use a bioimage analyzer (BAS2000II, Fuji Film Co., Ltd.).
Then, ¹⁴ C of the cholesterol ester fraction was quantified.

For the hexane phase in the scintillation vial, scintillation cocktail was added, ³ H was counted, and the extraction efficiency was calculated from the ³ H amount of the added [ ³ H] -cholesteryl oleate-added ethanol. The amount of total cholesterol ester produced was calculated from the extraction efficiency. Compared with the case of the control, the amount of total cholesterol ester produced that decreases when the test substance is added is displayed as a percentage,
It was defined as the ACAT-1 enzyme inhibition rate.

VI. Results The results are shown in Table 7-9 below.

[0066]

[Table 7]

[0067]

[Table 8]

[0068]

[Table 9]

VII. Discussion From the results shown in Table 7-9, each compound belonging to the general formula (1), which is an active ingredient in the present invention, has excellent ACA.
It is apparent that it has T-1 inhibitory activity.

The fact that such a compound having ACAT-1 inhibitory activity is effective as an arteriosclerosis preventive agent and a cholesterol absorption inhibitor is described in, for example, The Journal of Biologi.
calChemistry, Vol.276, No.28, July 14, pp.21324-21
330, 2000 and The Journal of Biological Chemistr
y, Vol.275, No.36, September 8, pp.28083-28092,200
It is clear from the description of 0.

[0071]

[Pharmacological test example 2] ACAT-1 inhibitory effect test 2 (THP-1 cell foaming inhibitory effect test) The THP-1 cell foaming inhibitory effect (ACAT-1 inhibitory effect) of the test substances shown in Table 1-6 is shown. It tested as follows.

I. Test method To a 24-well plate, 200 nM phorbol 12-myristate 13-acetate (PMA) was added to each well at 7.5 × 10 ⁵ cells per well.
THP-1 cells adjusted with a 10% FBS-RPMI1640 culture medium were seeded and cultured in a carbon dioxide gas incubator for 3 days to differentiate into macrophage-like cells. With RPMI 1640 medium
After washing once, the culture solution was washed with 5% Lipoprotein Deficient Se
rum (LPDS; RJ Mayer, et al., J. Biol. Chem., 26
6 , 20070 (1991): DE Vance, et al., Biochem. Bio
phys. Acta, 792 , 39 (1984))-RPMI1640 1 mL / well, and further cultured for 8 hours. 8 hours later, protein concentration 50μ
g / mL Acetyl LDL (Ac LDL; Hideki Hakama et al., “Arteriosclerosis + Hyperlipidemia Research Strategy”, pp36-41 (1996) Shujunsha), BSA- [ ¹⁴ C] oleate complex (JL Goldstein, et.
al, Method. Enzymol., 98 , 241 (1983)) 2.5 μL and test substance (final concentration: 1 × 10 ^-5 mol / L) 5% LPDS-RP
The culture medium was replaced with 500 μL of MI1640 culture medium. After culturing for 16 hours, the cells were washed once with 0.3% BSA-PBS (-) and twice with PBS (-). In order to extract intracellular lipid components, 0.5 mL of hexane / 2-propanol (3: 2) was added to each well and left standing. After 30 minutes, the extracts were pooled in glass test tubes. Repeat the same extraction operation once more, combine with the previous extract,
The solvent was removed under a stream of nitrogen gas. Redissolve the resulting lipid extract in 100 μL of chloroform / methanol (2: 1) and
Spotted on C plate. TLC plates are hexane /
After development with diethyl ether / acetic acid (73: 25: 2), autoradiography performed ¹⁴ % of the cholesterol ester fraction.
C was quantified. Bio Image Analyzer B for quantification
AS2000II (manufactured by Fuji Film Co., Ltd.) was used. Also,
0.1N NaOH-0.1% SDS 0.3 in each well after lipid extraction
mL was added, and the cells attached to the plate were peeled off with a rubber policeman and collected. The amount of protein in this cell lysate
It was quantified with the BCA Protein Assay kit (PIERCE).

Quantitative amount of cholesterol ester (pmol)
Value was divided by the amount of protein (mg), and the decrease rate (%) was calculated by comparing it with that without addition of the test substance, and this was calculated as the THP-1 cell foaming inhibition rate (%) of the test substance. As the test substance AC
It was used as an index of AT-1 activity.

II. Results The results of the test are shown in Table 10 below.

[0075]

[Table 10]

III. Discussion Similar to the results shown in Table 7-9 as well as the results shown in Table 10, the compound of the present invention represented by the general formula (1) has excellent ACAT-1 inhibitory activity. It is clear.

The fact that such a compound having an ACAT-1 inhibitory activity is effective as an arteriosclerosis preventive agent and a cholesterol absorption inhibitor is described, for example, in The Journal of Biologi.
calChemistry, Vol.276, No.28, July 14, pp.21324-21
330, 2000 and The Journal of Biological Chemistr
y, Vol.275, No.36, September 8, pp.28083-28092,200
It is clear from the description of 0.

[0078]

[Formulation Example 1] As an active ingredient, the compound of the present invention obtained in Reference Example 1 is used, and tablets each containing 300 mg thereof (2000 tablets)
Was prepared according to the following formulation. The compound of the present invention obtained in Reference Example 1 600 g Lactose (Japanese Pharmacopoeia) 67 g Corn starch (Japanese Pharmacopoeia) 33 g Carboxymethylcellulose calcium (Japanese Pharmacopoeia) 25 g Methylcellulose (Japanese Pharmacopoeia) 12 g Magnesium stearate (Japanese Pharmacopoeia) Direction) 3 g, that is, according to the above formulation, the compound of the present invention obtained in Reference Example 1,
Lactose, corn starch and calcium carboxymethylcellulose are thoroughly mixed, and the mixture is granulated using an aqueous methylcellulose solution, passed through a 24 mesh sieve, mixed with magnesium stearate and pressed into tablets to obtain the desired tablets. It was

[0079]

[Formulation Example 2] Using the compound of the present invention obtained in Reference Example 1 as an active ingredient, a hard gelatin capsule (2000 capsules) containing 200 mg per capsule was prepared according to the following formulation. The compound of the present invention obtained in Reference Example 1 400 g Crystalline cellulose (Japanese Pharmacopoeia) 60 g Corn starch (Japanese Pharmacopoeia) 34 g Talc (Japanese Pharmacopoeia) 4 g Magnesium stearate (Japanese Pharmacopoeia) 2 g That is, according to the above prescription Each component was pulverized into fine powders, mixed so as to form a uniform mixture, and then filled into a gelatin capsule for oral administration having a desired size to obtain a target capsule.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07F 9/6558 C07F 9/6558 (72) Inventor Akiko Kuroda 105 Kitahama, Saida, Narato-cho, Tokushima Prefecture ( 72) Inventor Yasuhide Inoue Hama 11-54, Benai Tenji, Naraido-machi, Naruto City, Tokushima Prefecture 72-72 Inventor Akifumi Hagi 2-10-1, Minamikurahoncho, Tokushima City, Tokushima Prefecture 103 (72) Shinya Miki 57-12 Nishikubo, Kitamura, Kitajima-cho, Itano-gun, Tokushima (72) Inventor Toshihiro Yoshinaga 95-2 Miyanonishi, Kitanohama, Senyo-cho, Naruto-shi, Tokushima C-122 Casa Esperar Kitahama (72) Inventor Masako Doi Naruto, Tokushima 64, Sayo, Saitama, Hamatabe Nishi (72) Inventor, Yoshihiko Tsuda 127 Maehama, Maehama, Kureshima, Narai, Naruto, Tokushima Prefecture 4C086 AA01 AA02 DA34 MA01 MA04 NA14 ZA45 ZC33 4H050 AA01 AA03 AB23

Claims (3)

[Claims] [Claim 1] General formula (1): [In the formula, A represents N or CR ¹ . R ¹ has a hydrogen atom, a lower alkyl group, 1-3 on the phenyl ring, a halogen atom, a lower alkyl group, a lower alkoxy group, a cyano group, a nitro group and a group selected from the group consisting of a phenyl group. A phenyl group, a lower alkoxyoxalyl group, a lower alkoxycarbonyl lower alkyl group, a lower alkoxycarbonyl group, a phenylcarbonyl lower alkyl group,
Phenyl lower alkyl group, naphthyl group, 1,4-benzodioxanyl group or thienyl group. R ² is a hydrogen atom, a halogen atom, a nitro group, a C _1-12 -alkyl group, a lower alkoxycarbonyl group, a phenyl group which may have a halogen atom or a lower alkyl group as a substituent, a phenylcarbonyl group, a phenylsulfonyl group, A phenyl lower alkyl group having a halogen atom on the phenyl ring, a halogen-substituted lower alkyl group, a mercapto group or a lower alkylthio group. R ³ represents a hydrogen atom, a lower alkyl group or a phenyl group. R ⁴ and R ⁵ are the same or different and each represents a lower alkyl group or a phenyl lower alkyl group. ]
An ACAT-1 inhibitor comprising a phosphonic acid diester derivative represented by as an active ingredient. 2. The ACAT-1 inhibitor according to claim 1, which comprises a compound of the general formula (1) in which A is CR ¹ as an active ingredient. 3. A is CR ¹ , R ³ is a hydrogen atom, and
2. The ACAT-1 inhibitor according to claim 1, which comprises a compound of the general formula (1) in which R ⁴ and R ⁵ are lower alkyl groups as an active ingredient.