HK1093976B - 2,4-bis(trifluoroethoxy)pyridine compound and medicine containing the same - Google Patents

Description

2, 4-bis (trifluoroethoxy) pyridine compound and medicine containing the same

Technical Field

The present invention relates to a 2, 4-bis (trifluoroethoxy) pyridine compound which has a potent inhibitory effect on acyl CoA cholesterol acyltransferase (ACAT) and is useful for the prevention and treatment of hyperlipidemia, various arteriosclerosis and the like by oral administration, a pharmaceutical composition containing the compound, and an intermediate for the production thereof.

Background

acyl-CoA cholesterol acyltransferase (ACAT) is an enzyme that promotes the synthesis of cholesterol esters from cholesterol, and is an enzyme that plays an important role in the metabolism and absorption of cholesterol in the digestive tract. Although most ACAT inhibitors, which are currently used as anti-hyperlipidemia agents, anti-arteriosclerosis agents, promote the lowering of blood cholesterol by acting on ACAT enzymes of the small intestine and liver, they have problems of causing side effects of intestinal bleeding, intestinal tract disorders, diarrhea and liver injury.

Recent studies have shown that by suppressing the foaming of macrophages, which play an important role in the formation of lesions of arteriosclerosis, atrophy of the lesions of arteriosclerosis themselves can be expected. That is, foamed macrophages (cholesterol esters stored intracellularly as fat droplets) are observed in the focus of atherosclerosis, and the foaming of the macrophages is closely related to the progression of the lesion. In addition, ACAT activity present in the vascular wall is enhanced in the lesion site of arteriosclerosis, and since cholesterol ester is stored in the vascular wall, ACAT activity in the vascular wall and arteriosclerosis are closely related (non-patent document 1).

Therefore, inhibition of cholesterol esterification by ACAT inhibitors in the vascular wall is associated with storage of free cholesterol into cells of the vascular wall, and the stored free cholesterol is released from the cells by High Density Lipoprotein (HDL) and transferred to the liver (reverse transfer by HDL) to be metabolized, and thus storage of cholesterol esters in arteriosclerotic lesion sites is expected to be inhibited (non-patent document 2). The ACAT inhibitors thus inhibiting ACAT present in the vascular wall are considered to have a direct anti-arteriosclerotic effect.

First, the present inventors considered that selective inhibition of ACAT present in the vascular wall could be a preventive/therapeutic agent for arteriosclerosis with less side effects than those of drugs inhibiting macrophage foaming, and as a result of earnest studies, they found that the following general formula (A)

(wherein Ar is an aromatic group which may have a substituent,

is a 2-valent residue of optionally substituted benzene, pyridine, cyclohexane or naphthalene, X represents NH, oxygen atom or sulfur atom, Y represents sulfur atom, Z represents single bond, l represents an integer of 0 to 15, m represents an integer of 2 or 3, n represents an integer of 1 to 3)

The compound represented by (i) or a salt or solvate thereof is useful as a prophylactic/therapeutic agent for hyperlipidemia and arteriosclerosis, and is a compound that selectively inhibits ACAT present in blood vessels (patent document 1).

Non-patent document 1: exp. mol. Pathol.44, 329-Alkand 339(1986)

Non-patent document 1: biochim. biophysis. acta.200115, 1530 (1): 111-122

Patent document 1: international publication No. 98/54153 pamphlet

Disclosure of The Invention

Problems to be solved by the invention

Among the compounds described in patent document 1, it has been confirmed that the compound represented by the following formula (B) and a salt thereof have high water solubility and strong ACAT inhibitory activity, and have a significant drug effect on various animal models. However, although the compounds described in patent document 1, as represented by compound (B), have excellent drug effects based on ACAT inhibitory action in animal experiments, in vitro experiments using human liver microsomes have revealed rapid metabolism and low survival rate of the prototype. These facts indicate that the blood concentration of these compounds in humans is not yet known. In recent years, from the viewpoint of drug interaction, compounds having higher antimetabolite activity are expected to be present in human liver microsomes because they can be drugs having higher safety.

However, since the compound (B) has a plurality of functional groups which are generally metabolized in the organism and these functional groups are considered to have an important effect on the expression of pharmacological activity, it is very difficult to maintain the ACAT inhibitory effect and improve the metabolic stability.

Means for solving the problems

Accordingly, the present inventors have conducted various studies to find a compound which improves the anti-metabolic property of human liver microsomes, is well absorbed orally, and can obtain a high blood concentration, and as a result, have unexpectedly found that a 2, 4-bis (trifluoroethoxy) pyridine compound represented by the following formula (1) shows a higher blood concentration (Cmax), a larger area under the blood concentration time curve (AUC), and a good oral absorbability, although the water solubility is lower than that of the compound (B). Further, they have also shown excellent ACAT inhibitory activity and are therefore useful compounds as preventive and therapeutic agents for hyperlipidemia and arteriosclerosis, and have completed the present invention.

Namely, the present invention provides the following general formula (1)

(in the formula, X¹Represents a fluorine atom or a hydrogen atom)

Disclosed are a 2, 4-bis (trifluoroethoxy) pyridine compound or a salt thereof, and a method for producing the same.

Further, the present invention provides the following general formula (2)

(in the formula, X¹Represents a fluorine atom or a hydrogen atom)

A piperazine compound shown below or a salt thereof.

Further, the present invention provides the following general formula (4)

(in the formula, R¹Represents a hydrogen atom, a chloroacetyl group, a bromoacetyl group or an iodoacetyl group)

A pyridine compound shown below or a salt thereof.

In addition, the present invention provides 2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-nitropyridine.

In addition, the present invention provides N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- (2-hydroxyethyl) piperazin-1-yl ] acetamide.

The present invention also provides a pharmaceutical composition comprising the compound represented by the above general formula (1) or a salt thereof as an active ingredient.

In addition, the invention provides the application of the compound shown in the general formula (1) or the salt thereof in the preparation of medicaments.

The present invention also provides a method for treating arteriosclerosis, which is characterized by administering an effective amount of a compound or salt represented by the above general formula (1).

ADVANTAGEOUS EFFECTS OF INVENTION

The compound (1) of the present invention selectively inhibits ACAT present in the vascular wall, has excellent metabolic stability of human liver microsomes, shows good oral absorbability, and is useful as a prophylactic or therapeutic agent for hyperlipidemia and arteriosclerosis.

Brief description of the drawings

FIG. 1 is a graph showing the stability of compounds (1a) and (1B) and compound (B) hydrochloride to human liver microsomal metabolism.

Best Mode for Carrying Out The Invention

The compound (1) of the present invention is structurally characterized by having 1 or 2 fluorine atoms on the benzimidazole ring and 2, 2, 2-trifluoroethoxy groups on the pyridine ring. Such a specific chemical structure is not described at all in patent document 1.

The present invention comprises the following two compounds and their salts.

Examples of the salt of the compound (1) of the present invention include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, and organic acid salts such as methanesulfonate, maleate, fumarate, citrate, butyrate, lactate, tartrate, ascorbate, malate, mandelate, salicylate, pantothenate, tannate, ethanedisulfonate, benzenesulfonate, p-toluenesulfonate, glutamate, aspartate, trifluoroacetate, pamoate and gluconate.

The compound (1) or a salt thereof of the present invention may be in the form of a solvate. The solvate is a compound to which a solvent used in production, purification, or the like, for example, water, alcohol, or the like is added, and is not particularly limited as long as it does not adversely affect the ACAT inhibitory activity or the like. The solvate is preferably a hydrate.

The compound (1) of the present invention can be produced, for example, by the following production method.

(wherein R represents a protecting group, Y represents an alkyl group or an arylsulfonyl group, and X represents¹Represents a hydrogen atom or a fluorine atom, X²Represents a chlorine atom, a bromine atom or an iodine atom)

That is, 2, 4-dichloro-6-methyl-3-nitropyridine (5) is reacted with 2, 2, 2-trifluoroethanol to obtain compound (6), the nitro group is reduced to obtain compound (7), and the compound is reacted with haloacetic acid or a reactive derivative thereof to obtain compound (3).

On the other hand, the piperazine ethanols (8) having protected amino groups are sulfonylated to give compounds (9), and the compounds (9) are reacted with thiol derivatives (10a) to give compounds (11). Compound (11) can also be obtained by reacting compound (8) with a thiol derivative (10a) or (10b) in the presence of a phosphide. Compound (2) can be obtained by removing the protecting group (R) of compound (11).

The compound (1) of the present invention can be obtained by reacting the compound (2) obtained above with the compound (3).

Therefore, the above-mentioned compound (2), the following compound (4) and the above-mentioned compound (2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-nitropyridine (6)) are useful as production intermediates of the compound (1) of the present invention.

(in the formula, R¹Represents a hydrogen atom, a chloroacetyl group, a bromoacetyl group or an iodoacetyl group)

The above-mentioned reaction steps will be described one by one.

The reaction of 2, 4-dichloro-6-methyl-3-nitropyridine (5) with 2, 2, 2-trifluoroethanol is carried out in a mixed solvent of 2, 2, 2-trifluoroethanol as a solvent or 2, 2, 2-trifluoroethanol and Dimethylformamide (DMF), Tetrahydrofuran (THF), Dimethylsulfoxide (DMSO), or the like, in the presence of an alkali carbonate such as potassium carbonate or sodium carbonate, an alkali hydroxide such as potassium hydroxide or sodium hydroxide, or an alkali hydride such as sodium hydride, potassium hydride, or lithium hydride, at room temperature to reflux temperature for 5 to 24 hours, preferably at reflux temperature for 15 to 20 hours.

The reduction of the compound (6) is preferably 1) a reaction with a sulfur-containing reducing agent such as sodium dithionite, sodium sulfide, sodium hydrogen sulfide or hydrogen sulfide, 2) a reduction reaction with a metal reducing agent such as zinc, iron or stannous chloride, or 3) a catalytic reduction reaction in a hydrogen atmosphere. The reaction with the sulfur-containing reducing agent can be carried out, for example, by dissolving the compound (6) in a solvent such as isopropyl alcohol, ethanol, or THF, and adding an aqueous solution of the sulfur-containing reducing agent at 80 ℃ to react for 10 minutes to 2 hours. The reaction with the metal reducing agent can be carried out, for example, by dissolving the compound (6) in an alcohol solvent such as ethanol or isopropanol, acetic acid, or a water-containing solvent thereof, and reacting at 0 to 100 ℃ for 30 minutes to 24 hours. Acids such as hydrochloric acid and sulfuric acid may be added as necessary for the reaction. The catalytic reduction reaction is carried out by dissolving the compound (6) in a single or mixed solvent such as dioxane, acetic acid, methanol, ethanol, or isopropanol, and reacting the resulting solution at 0 to 50 ℃ for 30 minutes to 12 hours, preferably at room temperature for 30 minutes to 3 hours, in a hydrogen atmosphere in the presence of a catalyst such as raney nickel, palladium on carbon, palladium hydroxide, or palladium black.

Examples of the haloacetic acid to be reacted with the compound (7) include chloroacetic acid, bromoacetic acid and iodoacetic acid. Examples of the reactive derivative of a haloacetic acid include haloacetyl halide and haloacetic anhydride. It is preferable to react the compound (7) with a halogen-substituted acetyl halide. The reaction of the compound (7) with the haloacetyl halide can be carried out, for example, by reacting in a solvent such as methylene chloride, chloroform, ethyl acetate, acetonitrile, toluene or the like in the presence of a base such as N, N-dimethylaniline, triethylamine, pyridine, 4-dimethylaminopyridine, 4-pyrrolidinopyridine or the like at 0 to 50 ℃ for 10 to 5 hours, preferably at 0 ℃ for 10 to 60 minutes.

The synthesis of the piperazineethanols (8) to the compounds (11) can employ the route a using an alkyl or arylsulfonylation reaction and the route b using a phosphide-induced reaction.

In the scheme a, the alkyl or arylsulfonylation reaction of the piperazineethanol (8) is carried out by reacting sulfonyl chloride such as methanesulfonyl chloride, benzenesulfonyl chloride or p-toluenesulfonyl chloride as an alkyl or arylsulfonylation agent in a solvent such as DMF, THF, ethyl acetate or acetonitrile in the presence of a base such as triethylamine, pyridine, N-diisopropylethylamine, N-dimethylaniline or 4-dimethylaminopyridine at 0 to 50 ℃ for 30 minutes to 3 hours.

As the protecting group (R) of the amino group of piperazineethanol (8), various protecting groups used in peptide synthesis can be used. Preferred protecting groups include alkoxycarbonyl groups such as benzyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl and tert-butoxycarbonyl, and formyl groups.

The reaction of the compound (9) with the compound (10a) is carried out by reacting in a solvent such as DMF, DMSO, acetonitrile or the like in the presence of a base such as potassium carbonate, sodium carbonate or the like and a catalyst such as 18-crown-6 or the like at room temperature to 100 ℃ for 1 to 5 hours, preferably at 50 to 80 ℃ for 1 to 2 hours.

Route b is carried out by reacting piperazineethanols (8) with thiol derivatives (10a) or (10b) in the presence of a phosphide.

Examples of the phosphorus compound include a phosphine reagent used in the mitsunobu reaction, a phosphorus reagent comprising the phosphine reagent and an azo reagent or a succinic acid reagent such as dimethyl maleate, N, N, N ', N' -tetramethylfumaramide, and a phosphonium ylide reagent.

Preferable examples of the route b include 1) a method (method A) of reacting a thiol derivative (10a) in the presence of a phosphine reagent and an azo reagent or a succinic acid reagent such as dimethyl maleate, N, N, N ', N' -tetramethylfumaramide; 2) a method (method B) in which the thiol derivative (10a) is reacted in the presence of a phosphonium ylide reagent; 3) a method (method C) in which the thiol derivative (10b) is reacted in the presence of a phosphine reagent.

< method A >

The method A can be carried out by dissolving the compound (8), the thiol derivative (10a) and the phosphine reagent in a reaction solvent, adding an azo reagent or a succinic acid reagent thereto, and reacting at 0 to 100 ℃ and preferably at room temperature to 80 ℃ for 2 to 24 hours under an argon gas or nitrogen atmosphere.

The phosphine reagent used in the reaction may, for example, be a trialkylphosphine such as trimethylphosphine, triethylphosphine, tripropylphosphine, triisopropylphosphine, tributylphosphine, triisobutylphosphine or tricyclohexylphosphine, or a triarylphosphine such as triphenylphosphine or diphenylphosphino polystyrene, and among them, trimethylphosphine, tributylphosphine or triphenylphosphine is preferred.

Examples of the azo reagent include diethyl azodicarboxylate (DEAD), 1 ' -azobis (N, N-dimethylformamide) (TMAD), 1 ' - (azodicarbonyl) dipiperidine (ADDP), 1 ' -azobis (N, N-diisopropylformamide) (TIPA), 1, 6-dimethyl-1, 5, 7-hexahydro-1, 4, 6, 7-tetrad-2, 5-dione (1, 6-dimethyl-1, 5, 7-hexahydro-1, 4, 6, 7-tetrahydrozo-2, 5-dione, DHTD), and diethyl azodicarboxylate is particularly preferable.

The reaction solvent may be DMF, THF, dioxane, acetonitrile, nitromethane, acetone, ethyl acetate, benzene, chlorobenzene, toluene, chloroform, dichloromethane, or the like, and DMF, THF, dioxane, acetonitrile are preferred. Particularly preferred are DMF and THF.

< method B >

The method B can be carried out by dissolving the compound (8), the thiol derivative (10a) and the phosphonium ylide reagent in a reaction solvent and reacting them at room temperature to 120 ℃ and preferably 80 ℃ to 100 ℃ for 2 hours to 12 hours under an argon gas or nitrogen atmosphere.

Examples of the phosphonium ylide reagent used in the present reaction include alkanoylmethylene trialkylphosphine, alkanoylmethylene triaryl phosphorane, alkoxycarbonylmethylene trialkylphosphine, alkoxycarbonylmethylene triaryl phosphorane, cyanomethylene trialkylphosphine, and cyanomethylene triaryl phosphorane. The trialkyl group may, for example, be trimethyl, triethyl, tripropyl, triisopropyl, tributyl, triisobutyl or tricyclohexyl, and the triaryl group may, for example, be triphenyl or diphenyl polyethylene.

In addition, in the present reaction, a method of producing a phosphonium ylide reagent in a reaction system by adding the compound (8), the thiol derivative (10a), a base and a phosphonium halide reagent to a reaction solvent may be used.

Examples of the phosphonium halide used in this case include (cyanomethyl) trialkylphosphonium halide, (cyanomethyl) triarylphosphonium halide, (alkylcarbonylmethyl) trialkylphosphonium halide, (alkylcarbonylmethyl) triarylphosphonium halide, (alkoxycarbonylmethyl) trialkylphosphonium halide and (alkoxycarbonylmethyl) triarylphosphonium halide.

Of the above phosphonium halide reagents, (cyanomethyl) trialkylphosphonium halide and (cyanomethyl) triarylphosphonium halide can be prepared by reacting the corresponding haloacetonitrile with the corresponding trialkylphosphine or triarylphosphine (テトラヘドロン, Vol. 57, 5451-5454 , 2001), and the other phosphonium halide reagents can be prepared by reacting the corresponding alkanoyl halomethyl and alkoxycarbonyl halomethyl with the corresponding trialkylphosphine or triarylphosphine in the same manner.

The trialkyl phosphine and triaryl phosphine used herein may be the same ones as those shown in the description of method A, and among them, trimethyl phosphine, tributyl phosphine, and triphenyl phosphine are preferred, and trimethyl phosphine is particularly preferred.

The alkanoyl group of the above-mentioned alkanoyl halomethyl group is preferably a formyl group, an acetyl group, a propionyl group, a butyryl group or the like, among which the acetyl group and the propionyl group are preferred, and the alkoxy group of the alkoxycarbonyl halomethyl group is exemplified by a methoxy group, an ethoxy group, a propoxy group, a butoxy group or the like, among which the methoxy group, the ethoxy group and the butoxy group are preferred.

The halogen atom is preferably chlorine, bromine or iodine.

Examples of the base include organic bases such as triethylamine, N-diisopropylethylamine, 1, 4-diazabicyclo [2, 2, 2] octane (DABCO), 1, 8-diazabicyclo [5, 4, 0] undec-7-ene (DBU), and 1, 5-diazabicyclo [4, 3, 0] nona-5-ene (DBU), and inorganic bases such as potassium carbonate, sodium carbonate, cesium carbonate, lithium diisopropylamide, and potassium hexamethylsilazide.

The reaction solvent is preferably dioxane, THF, toluene, benzene, DMF, DMSO, acetonitrile, propionitrile, or the like, and particularly preferably propionitrile.

< method C >

The method C is carried out by dissolving the compound (8), the thiol derivative (10b) and the phosphine reagent in the same reaction solvent as in the method A and reacting them at room temperature to 100 ℃ preferably 60 ℃ to 100 ℃ for 2 hours to 48 hours under an argon gas or nitrogen atmosphere.

The phosphine reagent used in the reaction is the same trialkyl phosphine and triaryl phosphine as shown in the method A, and specifically, trimethyl phosphine, triethyl phosphine, tripropyl phosphine, triisopropyl phosphine, tributyl phosphine, triisobutyl phosphine, tricyclohexyl phosphine, triphenyl phosphine, diphenyl phosphine polystyrene and the like are exemplified, and among them, trimethyl phosphine, tributyl phosphine, and triphenyl phosphine are preferable, and trimethyl phosphine and triphenyl phosphine are particularly preferable.

The thiol derivative (10a) can be produced by the method described in patent document 1 or a method based on the method. The thiol derivative (10b) can be easily produced from the thiol derivative (10 a).

The deprotection reaction of the compound (11) can be carried out by a known method corresponding to the protecting group, for example, hydrolysis, reduction reaction, etc.

The reaction of the compound (2) with the compound (3) obtained as described above is carried out in the presence of a base such as potassium carbonate, sodium carbonate, potassium hydrogencarbonate, sodium hydrogencarbonate and the like, in DMF, THF, acetonitrile or a single aqueous solvent thereof at room temperature to 50 ℃ for 5 to 30 hours, preferably at room temperature for 10 to 20 hours.

Further, the compound (1) of the present invention can be also obtained by a method represented by the following reaction formula, in which 1- (2-hydroxyethyl) piperazine (12) and a haloacetamide compound (3) are reacted to obtain a compound (13), and the compound (13) is reacted with a thiol derivative (10a) or (10b) in the presence of a phosphorus compound.

(in the formula, X¹Same as above)

The reaction of the compound (12) and the compound (3) is carried out based on the method of synthesizing the compound (1) from the compound (2).

The reaction of the compound (13) with the thiol derivative (10a) or (10b) is carried out based on the reaction of the compound (8) with the thiol derivative (10a) or (10 b).

Therefore, the above-mentioned compound (N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- (2-hydroxyethyl) piperazin-1-yl ] acetamide (13)) is useful as an intermediate for producing the compound (1) of the present invention.

The method for separating and purifying the compound (1) of the present invention thus obtained can be carried out by appropriately combining washing, extraction, recrystallization, various chromatographies, and the like. In addition, the conversion into the aforementioned acid addition salts can be carried out by a usual method.

When the metabolism of human liver microsomes in vitro was examined, as shown in fig. 1, the survival rate of the prototype after 30 minutes was 27% for compound (1a) and 62% for compound (1B) relative to 16% for the hydrochloride salt of compound (B). It is understood from this that the anti-metabolic property of the compound (1) of the present invention to human liver microsomes is greatly improved.

In addition, when the solubility in water was investigated, as shown in table 3, the solubility of the compound (1) of the present invention was greatly reduced relative to the hydrochloride salt of the compound (B). This indicates that the compound (1) of the present invention has a problem in oral absorption.

However, when oral experiments were carried out using male and female rats, the compound (1) of the present invention unexpectedly showed higher values relative to the hydrochloride of the compound (B), with a blood concentration (Cmax) 2 to 3 times higher than the hydrochloride of the compound (B), and a total area under the drug administration curve (AUC) 2 to 4 times higher than the hydrochloride of the compound (B). From this, it was confirmed that the compound (1) of the present invention showed higher oral absorbability as compared with the hydrochloride salt of the compound (B).

In addition, in vitro ACAT inhibitory activity as shown in table 1, the compound (1) of the present invention showed the same potent inhibitory activity as compound (B) hydrochloride.

From the above results, it is understood that the compound (1) of the present invention exhibits ACAT inhibitory activity of the same level as that of the compound (B), and exhibits higher human liver microsome resistance and higher oral absorbability than that of the compound (B), and therefore, the compound (1) of the present invention is useful as a prophylactic/therapeutic agent for hyperlipidemia and arteriosclerosis.

The compound (1) of the present invention is useful as a prophylactic and therapeutic agent for hyperlipidemia, arteriosclerosis, carotid and cerebral arteriosclerosis, cerebrovascular disorders, ischemic heart diseases, ischemic bowel diseases, coronary arteriosclerosis, nephrosclerosis, arteriosclerotic nephrosclerosis, malignant nephrosclerosis, acute mesangial vascular occlusion, chronic intestinal colic, ischemic colitis, aortic aneurysm, occlusive Arteriosclerosis (ASO), etc., due to its excellent ACAT inhibitory activity.

When the compound (1) of the present invention is used as a pharmaceutical, the compound (1) or a salt thereof of the present invention may be used alone or in the form of tablets, capsules, granules, powders, injections, suppositories, and the like, using a pharmaceutically acceptable carrier such as an excipient, a binder, a diluent, or the like. These preparations can be produced by a known method. For example, when the compound (1) is prepared into an oral preparation, it can be prepared by formulating the compound with an appropriate combination of an excipient such as starch, mannitol or lactose, a binder such as sodium carboxymethylcellulose or hydroxypropylcellulose, a disintegrant such as crystalline cellulose or calcium carboxymethylcellulose, a lubricant such as talc or magnesium stearate, a fluidity improver such as light silicic anhydride, or the like.

The medicament of the present invention may be administered orally or parenterally, preferably orally.

The dose of the drug of the present invention varies depending on the weight, age, sex, symptom, etc. of a patient, and the compound (1) of the present invention is usually administered in 1 to 500mg, preferably 5 to 200mg, preferably 1 to 3 times per day for an adult.

Examples

The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the examples.

Production example 1

Production of 5, 6-difluoro-2-mercaptobenzimidazole:

4, 5-difluoro-2-nitroaniline (5.75g, 33.03mmol) was dissolved in acetic acid (100mL) and concentrated hydrochloric acid (2.3mL), and zinc powder (6.91g, 105.6mmol) was added, typically over 10 minutes, with vigorous stirring in an ice bath. After stirring at the same temperature for 20 minutes, further stirring at room temperature for 130 minutes. Zinc powder (1.20g, 18.35mmol) was added at the same temperature over a further 5 minutes and stirred at the same temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the resulting residue was neutralized with saturated sodium bicarbonate water and filtered with celite. The filtrate was extracted with chloroform, and the organic layer was washed with saturated brine. After drying over anhydrous sodium sulfate, concentration was performed under reduced pressure to obtain a brown oily substance (4.73 g).

The brown oily substance was dissolved in ethanol (200mL), and potassium O-ethylxanthate (15.75g, 98.25mmol) was added thereto, followed by heating and refluxing for 14 hours. The reaction mixture was concentrated under reduced pressure, the resulting residue was extracted with ethyl acetate-1 mol/L hydrochloric acid, and the organic layer was washed with saturated brine. After drying over anhydrous sodium sulfate, concentration was performed under reduced pressure, and the obtained residue was crystallized from chloroform-hexane to obtain 5, 6-difluoro-2-mercaptobenzimidazole (5.58g, calculated yield 91%) as pale brown powder.

Production example 2

Production of 1-tert-butoxycarbonyl-4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazine:

to a solution of 1-tert-butoxycarbonyl-4- (2-hydroxyethyl) piperazine (7.40g, 32.13mmol) in THF (100mL) were added triethylamine (4.36g, 43.09mmol), 4-dimethylaminopyridine (200mg, 1.64mmol) and methanesulfonyl chloride (7.40g, 38.76mmol) in this order with stirring under ice-cooling. After warming to room temperature, the mixture was stirred for 50 minutes. The reaction mixture was filtered, the filtrate was concentrated under reduced pressure, the resulting residue was dissolved in DMF (200mL), and 5, 6-difluoro-2-mercaptobenzimidazole (5.00g, 26.86mmol), potassium carbonate (8.64g, 62.51mmol) and 18-crown-6 (500mg, 1.92mmol) were added in this order at room temperature, followed by stirring at 80 ℃ for 90 minutes. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (silica gel 200g, hexane: acetone 8: 1 to 1: 1). Crystallization from acetone-diethyl ether-hexane gave 1-tert-butoxycarbonyl-4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazine (7.26g, yield 68%) as colorless crystals.

mp192.3-193.0℃

IR(KBr)：3061，2976，2836，1672，1475，1427(cm^-1).

¹H-NMR(400MHz，CDCl₃)δ：1.50(9H，s)，2.51-2.68(4H，m)，2.94(2H，t，J＝5.4Hz)，3.28(2H，t，J＝5.4Hz)，3.45-3.65(4H，m)，6.85-7.62(2H，m).

Example 1

Production of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazinea3 trifluoroacetate:

1-tert-Butoxycarbonyl-4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazine (7.26g, 18.22mmol) was added to trifluoroacetic acid (50mL) and dissolved therein over 15 minutes while stirring under ice-cooling. After stirring for 10 minutes under ice-cooling, diethyl ether (100mL) and hexane (100mL) were added to the reaction mixture, and the resulting crystals were separated by filtration. The resulting crystals were recrystallized from ethanol-ether to give 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazinedi3 trifluoroacetate (9.58g, yield 82%) as pale yellow powder.

mp141.2-142.9℃

IR(KBr)：3417，3026，2749，2483，1671，1484(cm^-1).

¹H-NMR(400MHz，DMSO-d₆)δ：2.78-3.26(10H，m)，3.49(2H，t，J＝7.2Hz)，7.51(2H，t，J＝9.0Hz)，8.76(2H，m).

Production example 3

Production of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] -4-formylpiperazine:

1-formyl-4- (2-hydroxyethyl) piperazine (1.11g, 7.0mmol), 5, 6-difluoro-2-mercaptobenzimidazole (1.30g, 7.0mmol), and diisopropylethylamine (3.62g, 28.0mmol) were dissolved in propionitrile (50mL), cyanomethyltrimethylphosphonium iodide (6.80g, 28.0mmol) was added, and the mixture was stirred at 92 ℃ for 1 hour under argon atmosphere. After cooling, water (100mL) was poured and extracted with chloroform (100 mL. times.3). The organic layer was washed with saturated brine and dried over sodium sulfate anhydrate, followed by concentration under reduced pressure. The crude product thus obtained was crystallized from acetone-diethyl ether to obtain 1.78g (yield 78%) of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] -4-formylpiperazine as a yellow crystalline powder.

mp197.0-198.0℃

IR(KBr)cm^-1：3441，2825，1648，1476，1431，1363.

¹H-NMR(DMSO-d₆)：δ2.38(2H，t，J＝5.1Hz)，2.44(2H，t，J＝5.0Hz)，2.69(2H，t，J＝7.0Hz)，3.23-3.38(4H，m)，3.41(2H，t，J＝7.0Hz)，7，38-7.58(2H，m)，7.97(1H，s)，12.8(1H s).

MS(m/z)：326(M⁺)，140(100).

Example 2

Production of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazine:

1- [2- (5, 6-Difluorobenzimidazol-2-ylthio) ethyl ] -4-formylpiperazine (1.70g, 5.2mmol) was dissolved in methanol (20mL), 12N hydrochloric acid (2mL) was added thereto, the mixture was stirred at room temperature for 18 hours, the reaction mixture was concentrated under reduced pressure, and a saturated ammonia-methanol solution was added thereto, and the mixture was stirred at room temperature for 5 minutes. The residue obtained by concentrating the solvent under reduced pressure was purified by silica gel column chromatography (chloroform: saturated ammonia-methanol: 100: 3), whereby 1.40g (yield 90%) of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazine was obtained as a brown oil.

IR(KBr)cm^-1：2925，2853，1664，1602，1478，1435，1364.

¹H-NMR(CDCl₃)：δ2.61-2.82(4H，m)，3.00(2H，t，J＝4.8Hz)，3.10(4H，t，J＝4.8Hz)，3.16(2H，t，J＝4.8Hz)，7.16-7.42(2H，m).

MS(m/z)：298(M⁺)，70(100).

Production example 4

Production of 2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-nitropyridine:

2, 4-dichloro-6-methyl-3-nitropyridine (30g, 144.9mmol) was dissolved in 2, 2, 2-trifluoroethanol (250mL), and potassium carbonate (50g, 361.8mmol) was added thereto, followed by heating and refluxing for 21 hours. The reaction solution was diluted with water and extracted with chloroform. The organic layer was washed with saturated brine, dried over sodium sulfate anhydrate, and concentrated under reduced pressure to give 2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-nitropyridine (45.40g, yield 94%) as a pale yellow solid.

mp72.8-73.2℃

IR(KBr)：3432，3111，2975，1610，1585，1535(cm^-1).

¹H-NMR(400MHz，CDCl₃)δ：2.50(3H，s)，4.49(2H，q，J＝7.7Hz)，4.85(2H，q，J＝8.3Hz)，6.53(1H，s).

Elemental analysis C₁₀H₈F₆N₂O₄

Calculated values: c, 35.94; h, 2.41; the content of N, 8.38,

measured value: c, 35.94; h, 2.45; and N, 8.49.

Example 3

Production of 3-amino-2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridine:

to a solution of 2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-nitropyridine (45.00g, 134.7mmol) in isopropanol (300mL) was added a solution of sodium dithionite (78.00g, 448.0mmol) in water (300mL) with stirring at 80 ℃, after 15 minutes from the start of the reaction, a solution of sodium dithionite (16.50g, 94.8mmol) in water (51mL) was added, after 25 minutes from the start of the reaction, a solution of sodium dithionite (11.10g, 63.8mmol) in water (51mL) was added, and the mixture was stirred for 10 minutes. After completion of the reaction, a 4mol/L aqueous sulfuric acid solution (201mL) was added to the reaction mixture, and the mixture was stirred at 90 ℃ for 30 minutes. After cooling, 28% aqueous ammonia (360mL) was added under ice cooling, and the mixture was stirred for 30 minutes. The reaction solution was diluted with water and extracted with chloroform. The organic layer was washed with saturated brine, dried over sodium sulfate anhydrous, and concentrated under reduced pressure. The obtained crystals were recrystallized from hexane to obtain 3-amino-2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridine as yellow needle crystals (32.91g, yield 80%).

mp53.5-53.8℃

IR(KBr)：3453，3314，2968，1603，1505，1456(cm^-1).

¹H-NMR(400MHz，CDCl₃)δ：2.34(3H，s)，3.66(2H，br.s)，4.39(2H，q，J＝8.0Hz)，4.79(2H，q，J＝8.6Hz)，6.35(1H，s).

Elemental analysis C₁₀H₁₀F₆N₂O₂·0.55H₂O

Calculated values: c, 38.24; h, 3.56; the content of N, 8.92,

measured value: c, 37.96; h, 3.19; and N, 8.94.

Example 4

Production of 2-bromo-N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide:

to a solution of 3-amino-2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridine (42.29g, 139.0mmol) in dichloromethane (600mL) was added N, N-dimethylaniline (20.46g, 168.8mmol), and a solution of bromoacetyl bromide (28.73g, 142.3mmol) in dichloromethane (100mL) was added under ice-cooling stirring, followed by stirring for 10 minutes. The reaction solution was diluted with water and extracted with chloroform. The organic layer was washed with saturated brine, dried over sodium sulfate anhydrate, and concentrated under reduced pressure. The resulting crystals were recrystallized from chloroform-hexane to give colorless needle crystals of 2-bromo-N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (50.25g, yield 85%).

mp152.8-154.0℃

IR(KBr)：3250，3053，1677，1597，1541，1456(cm^-1).

¹H-NMR(400MHz，CDCl₃)δ：2.43(3H，s)，4.02(2H，s)，4.42(2H，q，J＝7.9Hz)，4.78(2H，q，J＝8.5Hz)，6.47(1H，s)，7.49(1H，br s).

Elemental analysis C₁₂H₁₁BrF₆N₂O₃

Calculated values: c, 33.90; h, 2.61; the content of N, 6.59,

measured value: c, 34.13; h, 2.66; and N, 6.65.

Example 5

Production of 2- [4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (compound 1 b):

to a mixed solution of 1- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazinedi3 trifluoroacetate (4.00g, 6.25mmol) and potassium carbonate (31.26mmol) in acetonitrile (100mL) and water (30mL) was added 2-bromo-N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (2.20g, 5.22mmol) over 15 minutes under ice-cooling and stirring. After stirring at room temperature for 15 hours, the reaction mixture was diluted with water and extracted with chloroform. The organic layer was washed with saturated brine, dried over sodium sulfate anhydrate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel 150g, hexane: acetone 4: 1 to 2: 1 to 1: 1). The resulting crystals were recrystallized from chloroform-hexane to give 2- [4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide as colorless crystals (3.04g, yield 91%).

mp191-192℃

IR(KBr)：3275，1686，1604，1591，1509(cm^-1).

¹H-NMR(400MHz，DMSO-d₆)δ：2.38(3H，s)，2.42-2.62(8H，m)，2.67(2H，t，J＝6.7Hz)，3.30(2H，s)，3.40(2H，t，J＝6.7Hz)，4.82(2H，q，J＝8.8Hz)，4.90(2H，q，J＝8.8Hz)，6.91(1H，s)，7.47(2H，m)，8.77(1H，s)，12.82(1H，br.s).

Elemental analysis C₂₅H₂₆F₈N₆O₃S

Calculated values: c, 46.73; h, 4.08; n, 13.08

Measured value: c, 46.55; h, 4.12; n, 12.94

Production example 5

Production of 5-fluoro-2-mercaptobenzimidazole:

4-fluoro-2-nitroaniline (8.00g, 51.22mmol) was dissolved in methanol (100mL), and 10% palladium-carbon powder (0.80g) was added and stirred at room temperature for 4 hours under a hydrogen atmosphere. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to purify the resulting residue by column chromatography (silica gel 150g, hexane: ethyl acetate: 1: 4) to obtain a brown oily substance (5.67g, yield 88%).

The brown oily substance (5.64g, 44.72mmol) was dissolved in ethanol (150mL), and potassium O-ethylxanthate (8.60g, 53.65mmol) was added and the mixture was refluxed for 3 hours. Further, potassium O-ethylxanthate (1.43g, 8.92mmol) was added, and the mixture was refluxed for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by column chromatography (silica gel 150g, hexane: ethyl acetate: 2: 1) to give 5-fluoro-2-mercaptobenzimidazole (5.93g, yield 79%) as brown powder.

Production example 6

Production of 1-tert-butoxycarbonyl-4- [2- (5-fluorophenylimidazol-2-ylthio) ethyl ] piperazine:

1-tert-butoxycarbonyl-4- (2-hydroxyethyl) piperazine (6.00g, 26.05mmol) was dissolved in THF (36mL), triethylamine (3.43g, 33.90mmol) and 4-dimethylaminopyridine (159mg, 1.30mmol) were added, and a solution of methanesulfonyl chloride (3.58g, 31.25mmol) in THF (9mL) was added dropwise under ice cooling. After stirring for 1 hour, the reaction mixture was filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was dissolved in DMF (90 mL). While stirring at room temperature, 5-fluoro-2-mercaptobenzimidazole (4.82g, 28.66mmol), potassium carbonate (5.40g, 39.07mmol) and 18-crown-6 (688mg, 2.60mmol) were added in this order, and the mixture was stirred at 80 ℃ for 2 hours. The reaction mixture was concentrated under reduced pressure, and water was added to the obtained residue, followed by extraction with ethyl acetate and washing of the organic layer with water and saturated brine. After drying over anhydrous sodium sulfate, the resulting residue was purified by silica gel column chromatography (silica gel 150g, hexane: ethyl acetate 2: 1 to 1: 2) and concentrated under reduced pressure to give 1-tert-butoxycarbonyl-4- [2- (5-fluorobenzimidazolyl-2-ylthio) ethyl ] piperazine (7.28g, yield 73%).

¹H-NMR(400MHz，CDCl₃)δ：1.49(9H，s)，2.63(4H，t，J＝4.9Hz)，2.94(2H，t，J＝5.9Hz)，3.29(2H，t，J＝5.9Hz)，3.58(4H，t，J＝4.9Hz)，6.93(1H，td，J＝9.2，2.5Hz)，7.19(1H，dd，J＝9.2，2.5Hz)，7.40(1H，dd，J＝9.2，4.9Hz).

Example 6

Production of 1- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazinea3 trifluoroacetate salt:

1-tert-Butoxycarbonyl-4- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazine (6.50g, 17.08mmol) was added and dissolved in trifluoroacetic acid (17mL) under ice-cooling and stirring over 30 minutes. After returning to room temperature and stirring for 30 minutes, diethyl ether and hexane were added, and the resulting solid was separated by filtration. The resulting residue was washed with diethyl ether to give 1- [2- (5-fluorophenylimidazol-2-ylthio) ethyl ] piperazinecarbonyl 3 trifluoroacetate as brown powder (10.50g, yield 99%).

mp127.7-129.3℃

IR(KBr)：3143，3032，2731，1789，1747，1660(cm^-1).

¹H-NMR(400MHz，DMSO-d₆)δ：3.29-3.47(8H，m)，3.48(2H，t，J＝6.6Hz)，3.62(2H，t，J＝6.6Hz)，7.03(1H，t，J＝9.0Hz)，7.32(1H，d，J＝9.0Hz)，7.48(1H，dd，J＝9.0，4.4Hz)，9.36(2H，br)，13.76(3H，br).

Production example 7

Production of 1- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] -4-formylpiperazine:

1-formyl-4- (2-hydroxyethyl) piperazine (1.20g, 7.6mmol), 5-fluoro-2-mercaptobenzimidazole (1.28g, 7.6mmol), and diisopropylethylamine (3.93g, 30.4mmol) were dissolved in propionitrile (50mL), cyanomethyltrimethylphosphonium iodide (7.39g, 30.4mmol) was added, and the mixture was stirred at 92 ℃ for 1 hour under an argon atmosphere. After cooling, water (100mL) was added and the mixture was extracted with chloroform (100 mL. times.3). The organic layer was washed with saturated brine, dried over sodium sulfate anhydrate, and concentrated under reduced pressure. The crude product thus obtained was crystallized from acetone-ether to give 1.87g (yield 80%) of 1- [2- (5-fluorobenzimidazolyl-2-ylthio) ethyl ] -4-formylpiperazine as brown crystalline powder.

mp173.0-175.0℃

IR(KBr)cm^-1：3435，3051，2953，2825，1648，1503，1446.

¹H-NMR(DMSO-d₆)：δ2.38(2H，t，J＝5.2Hz)，2.44(2H，t，J＝5.0Hz)，2.70(2H，t，J＝7.0Hz)，3.22-3.38(4H，m)，3.42(2H，t，J＝7.0Hz)，6.87-6.98(1H，m)，7.23(1H，br s)，7.39(1H，br s)，7.97(1H，s)，12.6(1H s).

MS(m/z)：308(M⁺)，140(100).

Example 7

Production of 1- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazine:

1- [2- (5-Fluorobenzimidazol-2-ylthio) ethyl ] -4-formylpiperazine (1.80g, 5.8mmol) was dissolved in methanol (20mL), and 12N hydrochloric acid (2mL) was added thereto, followed by stirring at room temperature for 18 hours. The reaction was concentrated under reduced pressure and saturated ammonia-methanol solution was added under stirring at room temperature within 5 minutes. The solvent was removed under reduced pressure, and the obtained residue was purified by silica gel column chromatography (chloroform: saturated ammonia-methanol: 100: 3), whereby 1.33g (yield 81%) of 1- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazine was obtained as a brown oil.

IR(KBr)cm^-1：3059，2947，2815，1626，1602，1482，1444，1408.

¹H-NMR(DMSO-d₆)：δ2.30-2.45(4H，m)，2.62(2H，t，J＝6.8Hz)，2.67(4H，t，J＝4.8Hz)，3.39(2H，t，J＝6.8Hz)，6.90-6.98(1H，m)，7.23(1H，dd，J＝9.5，2.5Hz)，7.39(1H，dd，J＝8.8，4.9Hz).

MS(m/z)：280(M⁺)，70(100).

Example 8

Production of 2- [4- [2- (5-fluorophenylimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (compound 1 a):

to a suspension of 1- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazinea3 trifluoroacetate (6.92g, 11.12mmol) and 2-bromo-N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (4.50g, 10.59mmol) in acetonitrile (90mL) was slowly added potassium carbonate (5.85g, 42.33 mmol). After stirring at room temperature for 5 hours, water (100mL) was added to the reaction mixture, followed by extraction with ethyl acetate, and the organic layer was washed with saturated brine. After drying over anhydrous sodium sulfate, the residue obtained by concentration under reduced pressure was purified by silica gel column chromatography (chloroform: methanol: 50: 1). The resulting crystals were recrystallized from acetone-ether to give 2- [4- [2- (5-fluorobenzoimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide in the form of pale brown prisms (4.72g, yield 71%).

mp182.0-182.7℃

IR(KBr)：3282，2824，1509，1413，1272，1166(cm^-1).

¹H-NMR(400MHz，CDCl₃)δ：2.41(3H，s)，2.66-2.91(8H，m)，2.97(2H，t，J＝5.1Hz)，3.25(2H，t，J＝5.1Hz)，3.29(2H，s)，4.41(2H，q，J＝8.0Hz)，4.75(2H，q，J＝8.5Hz)，6.45(1H，s)，6.93(1H，td，J＝9.0，2.3Hz)，7.10-7.56(2H，m)，8.28(1H，s)，13.14(1H，br.s)

Elemental analysis C₂₅H₂₇F₇N₆O₃S

Calculated values: c, 48.08; h, 4.36; n, 13.46

Measured value: c, 47.98; h, 4.38; n, 13.31

Example 9

Preparation of N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- (2-hydroxyethyl) piperazin-1-yl ] acetamide

To a solution of 1- (2-hydroxyethyl) piperazine (1.95g, 15.0mmol) and 2-bromo-N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methyl-3-pyridin-3-yl ] acetamide (5.00g, 12.5mmol) in acetonitrile (30ml) was added potassium carbonate (2.25g, 16.3mmol), and the mixture was stirred at room temperature for 5 hours. The reaction solution was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over sodium sulfate anhydrate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (developing solvent: ammonia-saturated methanol/chloroform-1/20), whereby 5.40g (yield 91%) of N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- (2-hydroxyethyl) piperazin-1-yl ] acetamide was obtained as colorless crystals.

¹H-NMR(CDCl₃)δ：2.42(3H，s)，2.48-2.82(8H，m)，2.57(2H，t，J＝5.3Hz)，3.17(2H，s)，3.63(2H，t，J＝5.3Hz)，4.41(2H，q，J＝8.0Hz)，4.75(2H，q，J＝8.5Hz)，6.47(1H，s)，8.38(1H，br s).

Example 10

Production of 2- [4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide (compound 1 b):

diethyl azodicarboxylate (40% w/v toluene solution, 11.0mL, 25.3mmol) was added dropwise to a solution of N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- [ 2-hydroxyethyl ] piperazin-1-yl ] acetamide (4.0g, 8.43mmol), 5, 6-difluoro-2-mercaptobenzimidazole (5.8g, 31.2mmol), and triphenylphosphine (7.8g, 29.7mmol) in N, N-dimethylformamide (170mL) under argon atmosphere under ice-cooling, and stirred at the same temperature for 1.5 hours. Ethyl acetate and 1mol/L hydrochloric acid were added to the reaction mixture to separate an aqueous layer, and an organic layer was extracted with 1mol/L hydrochloric acid. The aqueous layers were combined, made alkaline with 1mol/L aqueous sodium hydroxide solution and extracted with ethyl acetate. The organic layer was washed with water and saturated brine in this order, dried over sodium sulfate anhydrate, and then removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (developing solvent; chloroform: ammonia saturated methanol: 100: 3) to give 2- [4- [2- (5, 6-difluorobenzimidazol-2-ylthio) ethyl ] piperazin-1-yl ] -N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] acetamide in the form of colorless crystals (4.9g, yield 90.1%).

Test example 1 test of ACAT inhibitory Activity in J774A cells

J774 cells (2X 10) were seeded in 24-well plates⁵Cells/well), cultured with 500 μ L DMEM (10% FBS) for 24 hours. After changing the medium, 25-hydroxycholesterol (10. mu.g/mL) and ACAT inhibitor (final concentration 0, 10) were added^-9～10^-5mol/L) for 18 hours. After washing with 0.9% sodium chloride, 250. mu.L of lipid was extracted with hexane-isopropanol (3: 2). The lipid was extracted again with 250. mu.L of hexane-isopropanol (3: 2), and after removing the extraction solvent, Cholesterol Ester (CE) was quantified by the fluorometric method. In addition, the cells after lipid extraction were subjected to protein quantification (micro BCA method) to determine the amount of CE corresponding to 1mg of protein. Calculating IC from CE production ratio of test substance to blank₅₀(concentration of drug that inhibits 50% CE production). In the test, N is 4.

As a result, as shown in table 1, it was confirmed that the compounds (1a) and (1b) had strong ACAT inhibitory activity.

[ TABLE 1]

	ACAT inhibitory activity: J774A cells: IC (integrated circuit)(nM)
		Compound (1a)	87
Compound (1b)	75
		Compound (B). HCl	59

Test example 2 human liver microsome metabolic stability test

According to the following Table 2, NRS (NADPH-producing system) sample solution and 16% human serum albumin were added to 0.1mol/L phosphate buffer solution (pH7.4), and then a test compound (100. mu.M) in acetonitrile (0.01mL) was added thereto. After pre-incubation at 37 ℃ for 5 minutes in a warm bath, HUMAN LIVER MICROSOMES (POOLED HUMAN LIVER MICROMES, Lot. No. 20GENTEST) were added thereto, and the reaction was carried out at 37 ℃ for 30 minutes in a warm bath. 0.25mL of the reaction mixture was separated 0 and 30 minutes after the start of the reaction, and extraction was performed^＊The amount of the detection reagent was measured by HPLC. The remaining rate of the sample after 30 minutes was calculated as (peak area after 30 minutes/peak area at 0 minutes) × 100.

As a result, as shown in fig. 1, it was confirmed that the stability of the compound (1a) and the compound (1B) against the metabolism of human liver microsomes was significantly improved as compared with the hydrochloride of the compound (B).

[ TABLE 2]

Composition of human liver microsome reaction solution (1mL)

Human liver microsomes (POOLED): 0.05mL of the composition contains 1mg of protein	0.05mL
		NRS (NADPH producing system) sample solution: 0.25mL of the composition contains beta-nicotinamide adenine dinucleotide phosphate oxidized form 2 mgD-glucose 6-phosphate 2 sodium 2mg of glucose 6-phosphate dehydrogenase at position 0.8	0.25mL
16% human serum albumin	0.25mL
		0.1mol/L phosphate buffer (pH7.4)	0.44mL
Acetonitrile solution of sample (100. mu.M)	0.01mL
		Total up to	1mL

Extraction operation

To each sample were added 1.0mL of glycine buffer (pH10), 0.1mL of internal standard substance, and 5.0mL of t-butyl methyl ether, followed by shaking for 10 minutes, centrifugation at 2500 rpm for 10 minutes, and separation of the organic layer.

Test example 3 solubility test (Japanese pharmacopoeia I liquid)

Each test compound was dissolved in acetonitrile to prepare a 100. mu.M solution, and the solution was added to the Japanese pharmacopoeia I solution to prepare a 1000ng/mL solution. After stirring the solution for 10 minutes, 1mL of the solution was aspirated by a syringe and passed through a filter having a pore size of 0.2 μm (HLC-DISK 13 water system/solvent system Kanto chemical Co., Ltd.). 0.5mL of the filtrate was used to conduct extraction^＊The amount of the detection reagent was measured by HPLC.

As a result, as shown in table 3, the solubility of compound (1a) and compound (1B) was lower than that of the hydrochloride salt of compound (B). Oral absorbability of the compound of the present invention is expected to be low from solubility.

[ TABLE 3 ]

Extraction operation

The method is the same as the metabolic stability test of human liver microsome.

Test example 4 oral test in rats

Test compounds were dissolved in 0.01N hydrochloric acid solution and administered orally to male or female SD rats at 10mg/5 mL/kg. Blood was collected at 0.25mL 30, 60, 120, 180, 240, 360 min post-dose. The collected blood was centrifuged at 9,000g and 4 ℃ for 5 minutes to obtain plasma. The obtained plasma is kept at-30 deg.C until the measurement is carried out, and extraction is carried out^＊The plasma concentrations of the compounds were determined by LC/MS/MS. The results are shown in table 4, and compound (1a) and compound (1B) showed higher Cmax and higher AUC values under the drug time curve than compound (B) hydrochloride, and thus confirmed to have a higher Cmax and a higher AUC than compound (B) hydrochlorideThe acid salt has better oral absorbability.

[ TABLE 4 ]

(10mg/kg·p.o.)

Extraction operation

The method is the same as the metabolic stability test of human liver microsome.

Compared with the compound (B) described in patent document 1, the compound (1) of the present invention exhibits excellent metabolic stability of human liver microsomes and retains a potent ACAT inhibitory activity. Although compound (1) of the present invention has poor solubility in water as compared with compound (B) hydrochloride, it shows good oral absorbability in oral tests in male and female rats. This is expected to result in a good bioavailability to humans.

Claims (4)

1. General formula (1)

2, 4-bis (trifluoroethoxy) pyridine compound or pharmaceutically acceptable salt thereof, wherein X is¹Represents a fluorine atom or a hydrogen atom.

2. A process for producing the compound of claim 1 or a pharmaceutically acceptable salt thereof, which comprises reacting a compound of the formula (2)

Piperazine compounds of the formula (3)

A pyridine compound shown in the formula, wherein X¹Represents a fluorine atom or a hydrogen atom, X²Represents a chlorine atom, a bromine atom or an iodine atom.

N- [2, 4-bis (2, 2, 2-trifluoroethoxy) -6-methylpyridin-3-yl ] -2- [4- (2-hydroxyethyl) piperazin-1-yl ] acetamide.

4. A pharmaceutical composition as a prophylactic or therapeutic agent for hyperlipidemia and/or arteriosclerosis, which comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.