JP2003267969A - Fluorescent labeled ligand - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and comprehensive method for testing an LXR- binding ability. <P>SOLUTION: A compound represented by the general formula (I) (R<SP>1</SP>and R<SP>2</SP>are each independently H, a halogen, a 1 to 20C alkyl, or the like) or its salt. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a compound having both LXR binding ability and fluorescent ability or a salt thereof, and an LXR binding ability test method using the compound.

[0002]

BACKGROUND OF THE INVENTION Liver X Receptor (LXR) is one of the important factors that regulate lipids, and its ligand is expected as a therapeutic drug for hyperlipidemia, arteriosclerosis and the like. LXR
To find a better ligand, finding an efficient method helps to find a better ligand at an early stage,
This will lead to more efficient creation of LXR ligands.

By the way, as a method for investigating the affinity (LXR binding ability) of a test compound for LXR, a method using a radioactive label and a method using a fluorescent label can be mentioned. Among these, as a method using a fluorescent label, for example, LX described in International Patent Publication WO 01/82917.
A method is known in which the R coactivator fragment is labeled with rhodamine. The method is LXR
Since a specific coactivator is used, only the ligand that recruits that coactivator can be detected, and in order to detect the ligand that recruits other coactivators, it is necessary to construct a corresponding assay system for a while. It had drawbacks. Further, an exhaustive and comprehensive search for LXR modulators containing agonists, partial agonists, partial antagonists, and antagonists is not a simple method because this method requires a lot of labor. Further, the method using a radiolabeled body of a known ligand does not have the above-mentioned drawbacks, but it is difficult to synthesize and handle and it cannot be said to be a simple method.

[0004]

DISCLOSURE OF THE INVENTION As a result of various investigations on the method for detecting an LXR ligand, the present inventors have found that a compound having the general formula (I) of the present invention or a salt thereof has good LXR binding ability and fluorescence. The present inventors have completed the present invention by discovering that they can be used in a simple and more comprehensive method for testing LXR binding ability, which has the ability to combine.

[0005]

The present invention has the general formula (I)

[0006]

[Chemical 3]

(Where R¹ And R² Independently, hydrogen source
Child; halogen atom; C₁-C₂₀Alkyl group; halogen source
Child, hydroxyl group, mercapto group, C₁-C₆ Alkoxy group, C₁-
C₆Alkylthio group, cycloalkyl group, and hetero
The same or different selected from the substituent group consisting of aryl groups
C substituted with 1 to 6 substituents₁-C₂₀Alkyl group;
Aryl group; halogen atom, hydroxyl group, mercapto group, di
Toro group, cyano group, amino group, C₁-C₆Alkoxy group, C₁-
C₆Alkylthio group and C₁-C₆Consisting of an alkyl group
The same or different substituents selected from the group of substituents from 1 to
4-substituted aryl group; aralkyl group; halogen source
Child, hydroxyl group, mercapto group, nitro group, cyano group, amine
No group, C₁-C₆Alkoxy group, C₁-C₆Alkylthio group, and
C₁-C₆Selected from the group of substituents consisting of alkyl groups
Aralkyl substituted by 1 to 4 by the same or different substituents
A group; or; General formula (II) -A¹-D-G¹-A²-G²-FL (II) [In the formula, A¹ And A² Is independently C₁-C₆Alkylene group;
Is a halogen atom, hydroxyl group, mercapto group, C₁-C₆ Al
Coxy group, C₁-C₆An alkylthio group, an aryl group, and
The same selected from the group of substituents consisting of heteroaryl groups
Or C substituted by 1 to 3 with different substituents₁-C₆Arche
Group, D is a single bond; phenylene group; or; halo
A group of substituents consisting of a gen atom, a hydroxyl group, and a mercapto group.
1 to 3 substituted with the same or different substituents selected from
Phenylene group, G¹ And G² Independent and single
Compound; -O-;-S-;-OC (= O)-;-OC (= S)-;-NHC (= O)-;-NHSO
₂-;-NHC (= O) NH-; or; -NHC (= S) NH-, FL is fluorescent
Indicates a group. ] The group represented by is shown. ) Compound represented by
Or a salt thereof. [However, (i) R¹ Is represented by the general formula (II).
R if not² Is a group represented by the general formula (II)
And (ii) R² Is not a group represented by the general formula (II)
Is R¹ Is a group represented by the general formula (II). ] General above
In the compound represented by the formula (I) or a salt thereof,
Suitably, 1) R¹ Is a group represented by the general formula (II), or
Is its salt, 2) In 1), R²Is C₁-C₂₀An alkyl group; or
Halogen atom, hydroxyl group, mercapto group, C₁-C₆ Arcoki
Shi group, C₁-C₆Alkylthio group, cycloalkyl group, and
And selected from the group of substituents consisting of heteroaryl groups
C 1 to 6 substituted with the same or different substituents₁-C₂₀A
A compound that is a rualkyl group, or a salt thereof, 3) In 2), R²Is C₁-C₆An alkyl group; or
Rogen atom, hydroxyl group, mercapto group, C₁-C₆ Alkoxy
Base, C₁-C₆Alkylthio group, cycloalkyl group, and
And selected from the group of substituents consisting of heteroaryl groups
C 1 to 6 substituted with the same or different substituents₁-C₆Al
A compound that is a kill group, or a salt thereof, 4) In 3), R²Is not a halogen atom or hydroxyl group
The same or different substituents selected from the
Up to 6 substituted C₁-C₆A compound that is an alkyl group, or
That salt, 5) In 4), R²Are the same or different halogen atoms
1 to 6 substituted C₁-C ₆A compound that is an alkyl group, or
Is its salt, 6) In 5), R²Is 2,2,2-trifluoroethyl
A compound that is a group, or a salt thereof, 7) R² Is a group represented by the general formula (II), or
Is its salt, 8) In 7), R¹Is a hydrogen atom, or
That salt, 9) The compound in 7) in which D is a single bond, or
Is the salt of 10) A¹Is C₂-C_FiveA compound that is an alkylene group, or
Is the salt of 11) In 10), A¹A compound in which is a propylene group
Thing or a salt thereof, 12) G¹Is -NH-C (= O)-, or a salt thereof.
, 13) A²Is C₁-C₆A compound that is an alkylene group, or
Is the salt of 14) A²Is a pentamethylene group, or
Is salt, 15) G²Is -NH-C (= O)-, or a salt thereof.
, 16) A compound in which FL is a fluorescein group, or a compound thereof
It is salt. Further, the present invention is represented by the above general formula (I).
Liver X Receptor (LXR) binding using compound or its salt
Noh test method.

The above-mentioned test method is preferably a test method including the step of 17) measuring the fluorescence polarization degree.

Examples of the above-mentioned "alkyl group" include methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, s-butyl, tert-butyl,
n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2
-Methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl , 2-ethylbutyl,
Mention may be made of straight-chain or branched-chain alkyl groups having 1 to 20 carbon atoms such as heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl and icosyl. It is preferably a straight chain or branched chain alkyl group having 1 to 6 carbon atoms, and more preferably a straight chain or branched chain alkyl group having 1 to 4 carbon atoms.

The "halogen atom" in the above is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and preferably a fluorine atom or a chlorine atom.

Examples of the "C ₁ -C ₆ alkoxy group" in the above include methoxy, ethoxy, n-propoxy,
Isopropoxy, n-butoxy, isobutoxy, s-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, 2-methylbutoxy, neopentyloxy, n-hexyloxy, 4-methylpentyloxy,
3-methylpentyloxy, 2-methylpentyloxy, 3,3-dimethylbutoxy, 2,2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,3 -A linear or branched alkoxy group having 1 to 6 carbon atoms such as dimethylbutoxy is preferable, and a linear or branched alkoxy group having 1 to 4 carbon atoms is preferable.

Examples of the above-mentioned "alkylthio group" include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, s-butylthio, tert-butylthio, n-pentylthio, isopentylthio, 2- Methylbutylthio, neopentylthio, 1-ethylpropylthio, n-hexylthio, isohexylthio, 4-methylpentylthio, 3
-Methylpentylthio, 2-methylpentylthio, 1-
Methylpentylthio, 3,3-dimethylbutylthio,
Carbon number such as 2,2-dimethylbutylthio, 1,1-dimethylbutylthio, 1,2-dimethylbutylthio, 1,3-dimethylbutylthio, 2,3-dimethylbutylthio, 2-ethylbutylthio It represents a linear or branched alkylthio group having 1 to 6 carbon atoms, preferably a linear or branched alkylthio group having 1 to 4 carbon atoms.

The "cycloalkyl group" in the above may be a condensed ring such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl or adamantyl 3 to 1
A 0-membered saturated cyclic hydrocarbon group can be mentioned, and a 5- to 10-membered saturated cyclic hydrocarbon group is preferable.

Examples of the above-mentioned "heteroaryl group" include furyl, thienyl, pyrrolyl, azepinyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-.
Examples thereof include a 5- to 7-membered aromatic heterocyclic group containing 1 to 3 sulfur atoms, oxygen atoms and / or nitrogen atoms such as oxadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl and pyrazinyl. , And preferably pyridyl.

Examples of the above-mentioned "aryl group" include aromatic hydrocarbon groups having 5 to 14 carbon atoms such as phenyl, indenyl, naphthyl, phenanthrenyl, anthracenyl, and preferably phenyl group. is there.

As the above-mentioned "aralkyl group",
"Aryl group" refers to a group bonded to an "alkyl group",
For example, benzyl, α-naphthylmethyl, β-naphthylmethyl, indenylmethyl, phenanthrenylmethyl,
Anthracenylmethyl, diphenylmethyl, triphenylmethyl, 1-phenethyl, 2-phenethyl, 1-naphthylethyl, 2-naphthylethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1
-Naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl, 2-phenylbutyl, 3-phenylbutyl, 4-phenylbutyl, 1-naphthylbutyl, 2-naphthylbutyl, 3-naphthylbutyl, 4 -Naphthylbutyl, 1-phenylpentyl, 2-
Phenylpentyl, 3-phenylpentyl, 4-phenylpentyl, 5-phenylpentyl, 1-naphthylpentyl, 2-naphthylpentyl, 3-naphthylpentyl,
4-naphthylpentyl, 5-naphthylpentyl, 1-phenylhexyl, 2-phenylhexyl, 3-phenylhexyl, 4-phenylhexyl, 5-phenylhexyl, 6-phenylhexyl, 1-naphthylhexyl, 2
-Naphthylhexyl, 3-naphthylhexyl, 4-naphthylhexyl, 5-naphthylhexyl, 6-naphthylhexyl can be mentioned, and preferably "alkyl group"
Is an "aralkyl group" having 1 to 4 carbon atoms, and more preferably benzyl.

Examples of the "C ₁ -C ₆ alkylene group" in the above include methylene, methylmethylene, ethylene,
Mention may be made of a linear or branched alkylene group having 1 to 6 carbon atoms such as propylene, trimethylene, tetramethylene, 1-methyltrimethylene, 2-methyltrimethylene, 3-methyltrimethylene, pentamethylene and hexamethylene. It is preferably a straight chain or branched chain alkylene group having 1 to 4 carbon atoms, and more preferably methylene, ethylene, trimethylene or tetramethylene.

The "fluorescent group" in the above means a group having a property of emitting light by transition between electronic states having the same spin multiplicity, and examples of such a group include dansyl group, azulenyl group and fluorescein. Examples thereof include a fluorescein group.

"The salt" means the compound (I) of the present invention.
Means a salt thereof, and therefore refers to the salt, and such salt is preferably sodium salt, potassium salt,
Alkali metal salts such as lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; ammonium salts such as ammonium salts Inorganic salt, t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N , N′-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt,
Amine salts such as organic salts such as piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; halogen such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide Atomic hydrogenates, nitrates,
Inorganic acid salts such as perchlorates, sulfates and phosphates; lower alkane sulfonates such as methane sulfonate, trifluoromethane sulfonate and ethane sulfonate, benzene sulfonate, p-toluene sulfonate Organic acid salts such as salts of aryl sulfonates, acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates; and glycine salts , Lysine salt,
Mention may be made of amino acid salts such as arginine salts, ornithine salts, glutamate and aspartate.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The compound (I) of the present invention can be obtained by Method A or Method B described below.

[0021]

[Chemical 4]

In the above, R¹, R², A², G², And FL
Has the same meaning as above, R^1aIs the above R¹Or, the formula-
A¹-D-G¹-H (where A¹, D and G¹Means the same as above
You ) Represents a group represented by^2aIs the above R²Or some
Is the expression-A¹-D-G¹-H (where A¹, D and G¹Is the same as above
Show significance. ) Represents a group represented by^1bIs the above R
¹Or, the formula-A¹-D-G¹-X (where A¹, D and G¹Is
It has the same meaning as above and X represents a leaving group. )
Represents a group, R^2aIs the above R²Or, the formula-A¹-D-G¹-H
(In the formula, A¹, D and G¹Means the same as above, and X means
Indicates a leaving group. ) Is shown, and X is a leaving group.
You However, i) R^1aIs R¹If, then R^2aIs the expression-A¹-D-G ¹-H
Ii) R^2aIs R²If, then R^1aIs
Expression-A¹-D-G¹A group represented by -H, iii) R^1bIs R¹Is
If R^2bIs the expression-A¹-D-G¹A group represented by -X, iv) R
^2bIs R²If, then R^1bIs the expression-A¹-D-G¹Group represented by -X
Is.

The leaving group in the above is not particularly limited as long as it is a functional group which reacts with a nucleophile to cause a substitution reaction. Examples of such a group include a hydroxyl group; a cyano group; and chlorine. “Halogen atom” such as atom, bromine atom, iodine atom; “Lower alkylsulfonyloxy group” such as methanesulfonyloxy, ethanesulfonyloxy;
A “halogen-substituted lower alkylsulfonyloxy group” such as trifluoromethanesulfonyloxy; an arylsulfonyloxy group such as benzenesulfonyloxy, a lower alkylated arylsulfonyloxy group such as p-toluenesulfonyloxy, a parachlorobenzenesulfonyloxy group An "aromatic sulfonyloxy group" such as a halogen-substituted arylsulfonyloxy group can be mentioned.

Each step of Method A to Method B will be described in detail below. (Method A) This step is a known method or a known method for Compound 1 which is obtained according to the methods C to E described below, is known, or is easily obtained according to the known method. Is a step of obtaining a compound (I) by carrying out a substitution reaction using the compound 2 easily obtained according to.

Examples of the solvent used in this step include halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; ethyl formate, ethyl acetate,
Esters such as propyl acetate, butyl acetate, diethyl carbonate; diethyl ether, diisopropyl ether,
Tetrahydrofuran, dioxane, dimethoxyethane,
Ethers such as diethylene glycol dimethyl ether; methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t
Alcohols such as butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, methyl cellosolve; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2 -Pyrrolidone, N-methylpyrrolidinone, amides such as hexamethylphosphorotriamide; and sulfoxides such as dimethyl sulfoxide and sulfolane, and preferably,
N, N-dimethylformamide or dimethyl sulfoxide.

In this reaction, a buffer solution can be added if necessary, and there is no particular limitation as long as it is a buffer solution usually used in organic synthesis.
A pH 6-8 buffer can be mentioned, and a pH 7 phosphate buffer is preferable.

The reaction temperature varies depending on the raw material compound and the reaction reagent, but it is -20 ° C to 80 ° C, and preferably 0 ° C.
℃ to 50 ℃.

The reaction time varies depending on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 1 hour to 48 hours, preferably 1 hour to 24 hours.
It's time.

After completion of the reaction, for example, the target compound of this reaction is prepared by concentrating the reaction mixture, adding water and an immiscible organic solvent such as ethyl acetate, washing with water, and separating the organic layer containing the target compound. It is obtained by drying over magnesium sulfate or the like and then distilling off the solvent.

If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

This step can also be carried out according to the following method using a condensing agent when the bond produced by the substitution is an ester bond.

That is, as the "condensing agent" used, (i) a combination of a phosphoric ester such as diethylphosphoryl cyanide, diphenylphosphoryl azide, diethyl cyanophosphate and the following bases: (ii) 1,3-dicyclohexyl Carbodiimide, 1,
3-diisopropylcarbodiimide, 1-ethyl-3-
Carbodiimides such as (3-dimethylaminopropyl) carbodiimide; combinations of the carbodiimides and the following bases; the carbodiimides and N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-di A combination of N-hydroxys such as carboximides; (iii) 2,2'-dipyridyl disulfide, 2,
A combination of disulfides such as 2'-dibenzothiazolyl disulfide and phosphines such as triphenylphosphine and tributylphosphine; (iv) N, N'-disuccinimidyl carbonate, di-2-pyridyl Carbonate, S, S'-bis (1-
Carbonates such as phenyl-1H-tetrazol-5-yl) dithiocarbonate; (v) phosphinic chlorides such as N, N'-bis (2-oxo-3-oxazolidinyl) phosphinic chloride; vi) N, N'-disuccinimidyl oxalate,
N, N'-diphthalimido oxalate, N, N'-bis (5-norbornene-2,3-dicarboxyimidyl) oxalate, 1,1'-bis (benzotriazolyl) oxalate, 1,1 Oxalates such as'-bis (6-chlorobenzotriazolyl) oxalate and 1,1'-bis (6-trifluoromethylbenzotriazolyl) oxalate; (vii) the phosphines and diethyl azodicarboxylate, A combination of an azodicarboxylic acid ester such as 1,1 ′-(azodicarbonyl) dipiperidine or an azodicarboxamide; a combination of the phosphines and the following bases: (viii) N-ethyl-5-phenylisoxazolium N-lower alkyl-5 such as -3'-sulfonate
Arylisoxazolium-3'-sulfonates; (ix) Diheteroaryl diselenides such as di-2-pyridyl diselenide; (x) Arylsulfonyl tria such as p-nitrobenzenesulfonyl triazolide Zodides; (xi) 2-chloro-1-methylpyridinium 2-halo-1-lower alkylpyridinium such as iodide
Halides; (xii) 1,1′-oxalyldiimidazole, N,
Imidazoles such as N'-carbonyldiimidazole; (xiii) 3-lower alkyl-2-halogen-benzothiazolium fluoroborates such as 3-ethyl-2-chloro-benzothiazolium fluoroborate (Xiv) 3-lower alkyl-benzothiazole-2-cerone such as 3-methyl-benzothiazole-2-theron; (xv) phenyldichlorophosphate, phosphate such as polyphosphate ester; (xvi) chloro Halogenosulfonyl isocyanates such as sulfonyl isocyanates; (xvii) trimethylsilyl chloride, halogenosilanes such as triethylsilyl chloride; (xviii) combination of lower alkanesulfonyl halides such as methanesulfonyl chloride with the following bases: (xix) N, N, N ', N'-TE Mention may be made of N, N, N ', N'-tetra-lower alkylhalogenoformamidium chlorides such as lamethylchloroformamidium chloride, preferably carbodiimides and phosphines and azodicarbonates. It is a combination of acid esters or azodicarboxamides. The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but preferably, aliphatic hydrocarbons such as hexane and heptane; benzene, toluene, Aromatic hydrocarbons such as xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate, etc. Esters; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile;
Formamide, N, N-dimethylformamide, N, N
Examples include amides such as -dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide.

Examples of the base used include N-methylmorpholine, triethylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4- (N, N-). Dimethylamino) pyridine, 2,6-di (tert-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline,
Mention may be made of organic bases such as N, N-diethylaniline.

The reaction temperature is −20 ° C. to 80 ° C., preferably 0 ° C. to room temperature.

The reaction time mainly depends on the reaction temperature, the starting compound,
Depending on the type of reaction reagent or solvent used,
Usually, it is 10 minutes to 3 days, preferably 30 minutes to 1 day.

After completion of the reaction, the target compound is prepared according to a conventional method.
Taken from the reaction mixture.

For example, the reaction mixture is appropriately neutralized, and if insolubles are present, they are removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water,
The organic layer containing the target compound is separated, dried over anhydrous magnesium sulfate or the like, and then the solvent is distilled off.

If necessary, the obtained compound can be further purified by a conventional method such as recrystallization or silica gel column chromatography.

The compound obtained in this step can be a mixture of positional isomers on the fluorescent group, but even a mixture can be used in the LXR binding capacity test method without purification. (Method B) This step is a known method or a known method for Compound 3 which is obtained according to Method C to Method E described below, is publicly known, or is easily obtained according to known methods. Is a step of obtaining a compound (I) by carrying out a substitution reaction using a compound 4 easily obtained according to.

This step is performed in the same manner as Method A.

Compounds 1 and 3 used in the above methods A to B can be obtained by the following methods C to E.

[0042]

[Chemical 5]

In the above, R ¹ , R ² , R ^1a , R ^2a , R ^2b , A
¹ , D and G ¹ have the same meanings as described above, X ¹ and X ^{2 each} independently represent a leaving group, and Alk represents a C ₁ -C ₆ alkyl group.

The leaving group for X ¹ and X ² is as described above.
The same as the leaving group in X can be mentioned, and a halogen atom is preferable.

The C ₁ -C ₆ alkyl group in Alk has the same meaning as the above-mentioned alkyl group, and a methyl group or an ethyl group is preferred.

Each step of methods C to E will be described in detail below. (Method C) (Step C-1) In this step, compound 6 which is known or can be easily obtained from a known compound is reacted with compound 6 which is known or easily obtained from a known compound. Let me
In this step, compound 7 is obtained.

Examples of the solvent used in this step include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated carbonization such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene. Hydrogens; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate; diethyl ether, diisopropyl ether
Ethers such as ter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2.
Examples include amides such as -pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane, and ethers or amides are preferable.

Examples of the base used in this step include alkali metal carbonates such as sodium carbonate, potassium carbonate and lithium carbonate; alkali metal hydrogen carbonates such as sodium hydrogen carbonate, potassium hydrogen carbonate and lithium hydrogen carbonate. Alkali metal hydrides such as lithium hydride, sodium hydride and potassium hydride; alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, barium hydroxide and lithium hydroxide; sodium fluoride, Inorganic bases such as alkali metal fluorides such as potassium fluoride can be mentioned, and sodium hydride and sodium carbonate are preferred.

The reaction temperature varies depending on the raw material compound and the reaction reagent, but it is -20 ° C to 120 ° C, and preferably 0 ° C.
℃ to 100 ℃.

The reaction time varies depending on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 1 hour to 48 hours, and preferably 12 hours to 2 hours.
4 hours.

After the completion of the reaction, the target compound of this reaction is obtained by, for example, concentrating the reaction mixture, adding water and an immiscible organic solvent such as ethyl acetate, washing with water, separating the organic layer containing the target compound, It is obtained by drying over magnesium sulfate or the like and then distilling off the solvent.

If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography and the like. (Step C-2) In this step, compound 7 obtained in step C-1 is subjected to an azidation reaction and then a reduction reaction to obtain compound 1a. <Azidation reaction> Examples of the solvent used in this reaction include esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether
Tell, diisopropyl ether, tetrahydrofuran,
Ethers such as dioxane, dimethoxyethane, diethylene glycol dimethyl ether; methanol, ethanol, n-propanol, isopropanol, n-
Butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin,
Alcohols such as octanol, cyclohexanol, methyl cellosolve; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-
Examples thereof include amides such as methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide; sulfoxides such as dimethylsulfoxide and sulfolane, and ethers or amides are preferable.

The reagent used in this reaction is not particularly limited as long as it is usually used for azidation, but sodium azide is preferred.

The reaction temperature varies depending on the starting compound and the reaction reagent, but it is -20 ° C to 120 ° C, and preferably 0 ° C.
℃ to 100 ℃.

The reaction time varies depending on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 1 hour to 48 hours, and preferably 1 hour to 10 hours.

After the completion of the reaction, the target compound of this reaction is prepared by, for example, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, It is obtained by drying over magnesium sulfate or the like and then distilling off the solvent.

If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography and the like. <Reduction reaction> This reaction is carried out in a hydrogen atmosphere in the presence of a catalyst.

Examples of the solvent used in this reaction include esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane. Ethers such as diethylene glycol dimethyl ether; methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, Alcohols such as diethylene glycol, glycerin, octanol, cyclohexanol, methyl cellosolve; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone, hexamethyl Phosphoro Amides such as amides; dimethyl sulfoxide, sulfoxides such as sulfolane;
N-methylmorpholine, triethylamine, tripropylamine and tributy can be mentioned, and alcohols are preferable.

The catalyst used is not particularly limited as long as it is a catalyst containing a 0-valent or 2-valent palladium metal and is used in organic synthesis, and examples thereof include metal palladium, palladium-carbon, and hydroxide. Palladium, palladium chloride, palladium (II) acetate, tris (dibenzylideneacetone) dipalladium-chloroform, allyl palladium chloride, [1,2-bis (diphenylphosphino) ethane] palladium dichloride, bis (tri-o-toluylphosphine ) Palladium dichloride, bis (triphenylphosphine) palladium dichloride, tetrakis (triphenylphosphine) palladium, dichloro [1,1'-bis (diphenylphosphino) ferrocene] palladium, and a reaction liquid with a ligand for these The catalyst produced in the solution when added to the inside can be mentioned. Examples of the ligand added to the reaction solution include 1,1'-bis (diphenylphosphino) ferrocene, bis (2-diphenylphosphinophenyl) ether, 2,2'-bis (diphenylphosphino)
-1,1'-Binaphthol, 1,3-bis (diphenylphosphino) propane, 1,4-bis (diphenylphosphino) butane, tri-o-toluylphosphine, 2-diphenylphosphino-2'-methoxy Mention may be made of phosphorus ligands such as -1,1'-binaphthyl and 2,2-bis (diphenylphosphino) -1,1'-binaphthyl. Of the above palladium catalysts, palladium acetate, tris (dibenzylideneacetone) dipalladium-chloroform, palladium acetate and the ligand bis (2-diphenylphosphinophenyl) are preferred.
Combination of ethers and tris (dibenzylideneacetone) dipalladium-chloroform with ligand 1,1'-bis
A combination of (diphenylphosphino) ferrocene, and more preferably a combination of palladium acetate and a ligand bis (2-diphenylphosphinophenyl) ether, and
A combination of tris (dibenzylideneacetone) dipalladium-chloroform and the ligand 1,1′-bis (diphenylphosphino) ferrocene.

The reaction temperature varies depending on the starting compound and the reaction reagent, but it is -20 ° C to 120 ° C, and preferably 0 ° C.
℃ to 100 ℃.

The reaction time varies depending on the reaction temperature, the starting compound, the reaction reagent or the type of solvent used, but is usually 1 hour to 48 hours, and preferably 1 hour to 10 hours.

After completion of the reaction, for example, the target compound of this reaction is prepared by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, and separating the organic layer containing the target compound. It is obtained by drying over magnesium sulfate or the like and then distilling off the solvent.

If necessary, the obtained compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography and the like.

When X ² in compound 7 is a hydroxyl group, compound 3a can be obtained by directly performing the following step C-3 without performing the step C-2. (Step C-3) This step is a reaction for adding a leaving group to the compound 1a obtained in the step C-1 or the step C-2.

In contrast to this step, the reaction between the (α) hydroxyl group and the sulfonyl halide or the halogenation of the (β) hydroxyl group can be selected depending on the desired leaving group. The solvent used for the reaction of the (α) hydroxyl group and the sulfonyl halide is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but is preferably an aromatic hydrocarbon such as benzene. Examples thereof include halogenated hydrocarbons such as dichloromethane and chloroform; ethers such as diethyl ether, tetrahydrofuran, dioxane and dimethoxyethane, and ethers (particularly tetrahydrofuran) are preferable.

Examples of the base used include triethylamine, diisopropylamine, isoprobeylamine, N-methylmorpholine, pyridine and 4- (N, N-
Examples thereof include organic bases such as dimethylamino) pyridine, N, N-dimethylaniline and N, N-diethylaniline, and triethylamine is preferable.

The reaction temperature will differ depending on the solvent, raw materials, reagents, etc., but is usually -20 to 50 ° C., and preferably 0 to 25 ° C.

The reaction time varies depending on the solvent, raw materials, reagents, reaction temperature and the like, but is usually 5 minutes to 10 hours, and preferably 10 minutes to 3 hours.

After completion of the reaction, the target compound of this step is collected from the reaction mixture according to a conventional method. For example, after the reaction is completed, water is added to the reaction solution, a solvent immiscible with water (eg, benzene, ether, ethyl acetate, etc.) is added to extract the target compound, and then the extracted organic layer is washed with water and anhydrous magnesium sulfate, etc. After drying with, the target compound is obtained by distilling off the solvent. The desired compound thus obtained can be further purified, if necessary, by a conventional method, for example, recrystallization, reprecipitation or chromatography. The target compound of this step can be used in the next step without purification. (Β) Halogenation of hydroxyl group The phosphines used are, for example, triC1-C6 alkylphosphine such as trimethylphosphine, triethylphosphine, tripropylphosphine, tributylphosphine, tripentylphosphine or trihexylphosphine; Tri-C6-, such as indenylphosphine or trinaphthylphosphine
C10 arylphosphine; or triC6 optionally having C1-C4 alkyl as a substituent, such as tolyldiphenylphosphine, tritolylphosphine, trimesitylphosphine, tributylphenylphosphine or tri-6-ethyl-2-naphthylphosphine. -C10 arylphosphines, preferably tri-C1-C6 alkylphosphines (especially trimethylphosphine, triethylphosphine, tripropylphosphine or tributylphosphine) or tri-C6-C10 arylphosphines (especially triphenylphosphine, triindenyl). Phosphine or trinaphthylphosphine), and more preferably triC6-C10 arylphosphine (particularly triphenylphosphine).

The halogenating agent used is a carbon tetrahalide, such as carbon tetrabromide.

The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting material to some extent, but for example, aliphatic hydrocarbons such as hexane, heptane, ligroin or petroleum ether; Aromatic hydrocarbons such as benzene, toluene or xylene; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene or dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane or Ethers such as diethylene glycol dimethyl ether; nitriles such as acetonitrile; formamide, dimethylformamide, dimethylacetamide, hexamethylphosphoramide (HMPA) or hexamethylphospholast Amides such as amide (HMPT); or to give a dimethyl sulfoxide or sulfoxides such as sulfolane, preferably a halogenated hydrocarbon.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but is usually −10 ° C. to 100 ° C., and is preferably 0.
C. to 50.degree.

The reaction time varies depending on the starting compounds, reagents and reaction temperature, but is usually 5 minutes to 10 hours, preferably 10 minutes to 3 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture by a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (Method D) (Step D-1) In this step, compound 8 which is known or easily obtained from a known compound is reacted with compound 8 which is known or easily obtained from a known compound. Let me
In this step, compound 1a is obtained.

This step is performed in the same manner as Step C-1. (Step D-2) This step is a step of obtaining a compound 1b by introducing a leaving group into the compound 1a obtained in the step D-1.

This step is performed in the same manner as the step C-3. (Method E) In this step, compound 11 which is known or can be easily obtained from a known compound is reacted with compound 10 which is known or is easily obtained from a known compound to obtain compound 11. It is a process.

Examples of the solvent used in this step include aromatic hydrocarbons such as benzene, toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene. Kinds; Esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate; diethyl ether, diisopropyl ether
Ethers such as ter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2.
Examples include amides such as -pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane, and ethers or amides are preferable.
Further, this reaction can be carried out without a solvent, if necessary.

The reagent used in this reaction is not particularly limited as long as it is a substance usually used for sulfonamidation, and examples thereof include sulfonyl halides, preferably sulfonyl chloride.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but will usually be -20 ° C. to 120 ° C., and is preferably 0.
C. to 100.degree.

The reaction time varies depending on the starting compounds, reagents and reaction temperature, but is usually 1 hour to 48 hours, preferably 12 hours to 24 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (Step E-2) In this step, the compound 11 obtained in the step E-1 is subjected to 1) reduction reaction of ester to alcohol, and a) halogenation and (b) nitration, if desired. , (C) The nitrile reduction reaction is continuously performed, or 2) The ester is reduced to an aldehyde (α) and then the Wittig reaction is performed (β), the catalytic reduction (γ) is performed, and then the Curtius transition (δ) is performed. This is the step of obtaining compound 1b. 1) Reduction reaction of ester to alcohol Examples of the solvent used in this reaction include, for example,
Aromatic hydrocarbons such as benzene, toluene, xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane ,
Examples thereof include ethers such as dimethoxyethane and diethylene glycol dimethyl ether, and ethers are preferable.

As the reducing agent used in this step, diisobutylaluminum hydride, di (methoxyethoxy)
Examples thereof include organoaluminum hydride-based reducing agents such as aluminum sodium dihydride, and preferably,
It is diisobutylaluminum hydride.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but is usually -78 ° C. to 100 ° C., and preferably −
The temperature is 78 ° C to 20 ° C.

The reaction time varies depending on the starting compounds, reagents and reaction temperature, but is usually 1 hour to 48 hours, and preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.

The target compound of this reaction is, if desired,
An amino group can be introduced by sequentially performing (a) halogenation, (b) nitration, and (c) reduction reaction. (a) Halogenation As the solvent used in this reaction, for example, benzene,
Aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate Esters such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N- Methyl-2-pyrrolidone, N
Examples include amides such as -methylpyrrolidinone and hexamethylphosphorotriamide, and amides are preferable.

The halogenating agent used in this reaction includes thionyl halides such as thionyl chloride, thionyl bromide and thionyl iodide; sulfuryl halides such as sulfuryl chloride, sulfuryl bromide and sulfuryl iodide; phosphorus trichloride, Phosphorus tribromide,
Phosphorus trihalides such as phosphorus triiodide; phosphorus pentachlorides such as phosphorus pentachloride, phosphorus pentabromide, phosphorus pentaiodide or phosphorus oxychloride, phosphorus oxybromide, phosphorus oxyiodide Examples thereof include phosphorus oxyhalogenides; halogenated phosphines such as triphenylphosphine dibromide, and triphenylphosphine dibromide is preferable.

Examples of the base used in this reaction include alkali metal carbonates such as sodium carbonate, potassium carbonate and lithium carbonate; alkali metal hydrogen carbonates such as sodium hydrogen carbonate, potassium hydrogen carbonate and lithium hydrogen carbonate; Alkali metal hydrides such as lithium hydride, sodium hydride, potassium hydride; Alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, barium hydroxide, lithium hydroxide; sodium fluoride, fluorine Inorganic bases such as alkali metal fluorides such as potassium iodide; alkali metal alkoxides such as sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium t-butoxide, lithium methoxide; methyl mercaptan Sodium, ethyl mercap Mercaptans alkali metals such as emissions sodium; N- methylmorpholine, triethylamine, tripropylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N
-Methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4- (N, N-dimethylamino) pyridine, 2,6-di (t-butyl) -4-methylpyridine,
Quinoline, N, N-dimethylaniline, N, N-diethylaniline, 1,5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.4.0] undec-7-ene (DB
U) such as organic bases may be mentioned, and preferably,
Pyridine, sodium carbonate and sodium hydroxide.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but will usually be -20 ° C. to 120 ° C., and is preferably 0.
C. to 50.degree.

The reaction time will differ depending on the starting compounds, reagents and reaction temperature, but will usually be 1 hour to 48 hours, and is preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (b) Nitrilation Examples of the solvent used in this reaction include esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate and diethyl carbonate; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxy. Ethers such as ethane and diethylene glycol dimethyl ether; methanol, ethanol, n-
Propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol
Alcohols such as alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2.
-Pyrrolidone, N-methylpyrrolidinone, amides such as hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane; water; and a mixed solvent thereof, and preferably water-
It is a mixed solvent of alcohols.

The reagent used in this reaction is not particularly limited as long as it is a cyanate salt and is usually used, but examples thereof include potassium cyanide.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but will usually be -20 ° C. to 120 ° C., and is preferably 0.
C. to 50.degree.

The reaction time varies depending on the starting compound, the reagent and the reaction temperature, but is usually 1 hour to 48 hours, preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (C) Reduction reaction As the solvent used in this reaction, for example, benzene,
Aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxy Ethers such as ethane and diethylene glycol dimethyl ether can be mentioned, and ethers are preferable.

Examples of the reducing agent used in this reaction include alkali metal borohydrides such as sodium borohydride and lithium borohydride, aluminum hydride compounds such as lithium aluminum hydride and lithium triethoxide aluminum hydride. , A hydride reagent such as sodium tellurium hydride, a borane-tetrahydrofuran complex, a chloroborane-dimethyl sulfide complex, a diciamylborane, and a boron complex such as 9-borabicyclo [3.3.1] nonane (9-BBN) can be mentioned, and preferred. Is a borane-tetrahydrofuran complex.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but will usually be -20 ° C. to 120 ° C., and is preferably 0.
C. to 50.degree.

The reaction time will differ depending on the starting compounds, reagents and reaction temperature, but will usually be 1 hour to 48 hours, and is preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. 2) Reduction of (α) ester to aldehyde Examples of the solvent used in this reaction include:
Aromatic hydrocarbons such as benzene, toluene, xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, dichlorobenzene; diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane ,
Examples thereof include ethers such as dimethoxyethane and diethylene glycol dimethyl ether, and ethers are preferable.

As the reducing agent used in this step, diisobutylaluminum hydride, di (methoxyethoxy)
Examples thereof include organoaluminum hydride-based reducing agents such as aluminum sodium dihydride, and preferably,
It is diisobutylaluminum hydride.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but is usually −78 ° C. to 100 ° C., and preferably −
The temperature is 78 ° C to 20 ° C.

The reaction time will differ depending on the starting compounds, reagents and reaction temperature, but will usually be 1 hour to 48 hours, and is preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (Β) Wittig reaction Examples of the solvent used in this reaction include:
Ethers formamides such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N
-Methylpyrrolidinone, amides such as hexamethylphosphorotriamide; sulphoxides such as dimethylsulfoxide and sulfolane, and preferably tetrahydrofuran, N, N-dimethylformamide or dimethylsulfoxide. .

The Wittig reagent used in this reaction is not particularly limited as long as it is a reagent capable of obtaining the desired compound, but is a reagent having a carboxy group.

Examples of the base used in this reaction include alkali metal hydrides such as lithium hydride, sodium hydride and potassium hydride; sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, Examples thereof include alkali metal alkoxides such as potassium t-butoxide and lithium methoxide; organometallic bases such as butyllithium, lithium diisopropylamide and lithium bis (trimethylsilyl) amide, and lithium bis (trimethylsilyl) is preferable. ) Amide, lithium diisopropylamide, sodium hydride or potassium t-butoxide.

The reaction temperature will differ depending on the raw material compounds, reagents, etc., but is usually -78 ° C. to 100 ° C., and preferably −
The temperature is 78 ° C to 25 ° C.

The reaction time will differ depending on the starting compounds, reagents and reaction temperature, but will usually be 1 hour to 48 hours, and is preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (Γ) Catalytic reduction This reaction is carried out in the same manner as in <reduction reaction> in step C-2. (Δ) Curtius transition As the solvent used in this reaction, for example, benzene,
Aromatic hydrocarbons such as toluene and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene; ethyl formate, ethyl acetate, propyl acetate, butyl acetate, diethyl carbonate Esters such as; t
-Butanol; diethyl ether, diisopropyl ether
Ethers such as ter, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2.
Examples thereof include amides such as -pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide, and aromatic hydrocarbons or t-butanol are preferable.

Examples of the base used include N-methylmorpholine, triethylamine, tripropylamine, tributylamine, diisopropylethylamine,
Dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4- (N,
N-dimethylamino) pyridine, 2,6-di (t-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline, N, N-diethylaniline, 1,5-diazabicyclo [4.3.0]. ] Nona-5-ene (DBN),
1,4-diazabicyclo [2.2.2] octane (DA
BCO), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), and organic bases such as triethylamine or diisopropylamine.

The reagent used is t-butanol,
It is diphenylphosphoric acid azide.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but is usually 0 ° C. to 120 ° C., and preferably 25 ° C.
To 100 ° C.

The reaction time varies depending on the raw material compounds, reagents and reaction temperature, but is usually 1 hour to 48 hours, and preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography.

After the completion of this reaction, if necessary, a de-Boc reaction is carried out.

Examples of the solvent used in the de-Boc reaction include aromatic hydrocarbons such as benzene, toluene and xylene; halogens such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene and dichlorobenzene. Hydrocarbons; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; methanol;
, Ethanol, n-propanol, isopropanol
, N-butanol, isobutanol, t-butanol
Alcohols such as alcohol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol and methyl cellosolve; formamide, N,
Examples thereof include amides such as N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphorotriamide, and preferably dioxane or dichloroethane. . When acetic acid is used as the acid in this reaction, acetic acid can also be used as the solvent.

The acid used in the de-Boc reaction is not particularly limited as long as it is used as an acid catalyst in a normal reaction, but hydrochloric acid, hydrobromic acid, sulfuric acid, perchlorine are preferable. Acids, inorganic acids such as phosphoric acid, or organic acids such as acetic acid, formic acid, oxalic acid, methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid can be mentioned, and preferably Is an organic acid, more preferably a strong organic acid.

The reaction temperature will differ depending on the starting compounds, reagents, etc., but will usually be -20 ° C. to 120 ° C., and is preferably 0.
C. to 100.degree.

The reaction time will differ depending on the starting compounds, reagents and reaction temperature, but will usually be 1 hour to 48 hours, and is preferably 1 hour to 12 hours.

After completion of the reaction, the target compound of this reaction is collected from the reaction mixture according to a conventional method. For example, the reaction mixture is appropriately neutralized, and if insoluble matter is removed by filtration, water and an immiscible organic solvent such as ethyl acetate are added, and after washing with water, an organic layer containing the target compound is obtained. It is obtained by separating, drying over anhydrous magnesium sulfate and the like, and distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation or chromatography. (Step E-3) This step is a step of producing a compound 3b by introducing a leaving group into the compound 1b obtained in the step E-2.

This step is performed in the same manner as Step C-3.

The compound (I) of the present invention or a salt thereof has both LXR binding activity and fluorescence. Therefore, the compound (I) of the present invention is useful because it can be used in a test method for LXR binding ability of a test compound.

[0122]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is described in more detail below by showing Examples, Reference Examples and Test Examples, but the scope of the present invention is not limited to these.

[0123]

EXAMPLES Example 1 2- (6-Hydroxy-3-oxo-3H-xanthen-9-yl) -5-({[6-
Oxo-6-({2- [4-({(2,2,2-trifluoroethyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino } Sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid and 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl)-
6-({[6-oxo-6-({2- [4-({(2
2,2-Trifluoroethyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid Acid mixture

[0124]

[Chemical 6]

(1a) 4-Chlorosulfonylbenzoic acid methyl ester potassium hydroxide (12.8 g, 228 mmol) was dissolved in water (30 mL), and diethyl ether (120 m) was added.
L) was added. N-methyl-N-nitroso-p-
Toluenesulfonamide (purity 78%, 12.0 g, 9
0.8 mmol) was added little by little, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was separated, the aqueous layer was removed, and this organic layer was used as a diazomethane-diethyl ether solution.

4-Chlorosulfonylbenzoic acid (5.00
g, 22.7 mmol) was dissolved in a mixed solvent of ethyl acetate-tetrahydrofuran (4: 1, V / V, 100 mL), the diazomethane-diethyl ether solution prepared above was added, and the mixture was stirred at room temperature for 15 minutes. The reaction solution is
Concentrate to give the title compound as a white solid (5.31 g,
Yield 100%) was obtained.

¹ H NMR (CDCl ₃ , 500 MHz): δ 4.00 (3H, s),
8.13 (2H, d, J = 9 Hz), 8.28 (2H, d, J = 9 Hz). (1b) 4-({4- [2,2,2-trifluoro-1-
Hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester 4- [2,2,2-trifluoro-1-hydroxy-1-
(Trifluoromethyl) ethyl] aniline (4.49 g,
17.3 mmol) in pyridine (30 mL),
4-Chlorosulfonylbenzoic acid methyl ester prepared in Example 1 (1a) (4.89 g, 20.8 mmol)
And 4-N, N-dimethylaminopyridine (20 mg,
0.164 mmol) was added and the mixture was stirred at room temperature for 12 hours.

Water (200 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The obtained organic layers were combined, 1N hydrochloric acid (100 mL × 1), saturated aqueous sodium hydrogencarbonate (100 mL × 1) and saturated saline (100 mL).
It was washed successively with mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude title compound.

The crude target compound thus obtained was subjected to silica gel column chromatography (hexane: ethyl acetate,
4: 1-7: 3, V / V) to give the title object compound (7.92 g, yield 100%) as a white solid.

¹ H NMR (DMSO-d ₆ , 400 MHz): δ 3.88 (3H,
s), 7.23 (2H, d, J = 9 Hz), 7.56 (2H, d, J = 9 Hz), 7.
97 (2H, d, J = 8 Hz), 8.13 (2H, d, J = 8 Hz), 8.64 (1
H, s), 10.86 (1H, br.). (1c) 4-({(2,2,2-trifluoroethyl)-
4- [2,2,2-trifluoro-1-hydroxy-1-
(Trifluoromethyl) ethyl] anilino} sulfonyl)
Methyl benzoate 4-({4- [2,2,2] prepared in Example 1 (1b))
-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester (7.92 g, 17.3 mmol) was dissolved in dry N, N-dimethylformamide (80 mL),
Potassium tert-butoxide (2.14 g, 19.1)
mmol) and trifluoromethanesulfonic acid (2, 2,
2-Trifluoroethyl) ester (4.70 g, 20.
2 mmol) in that order, and the mixture was stirred at 60 ° C. for 3 hours under a nitrogen atmosphere.

Water (250 mL) was added to the reaction mixture under ice cooling, and the mixture was extracted with ethyl acetate (150 mL × 3). The obtained organic layers were combined, washed successively with water (200 mL × 3) and saturated saline (200 mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to give the crude title compound. Got

The crude target compound thus obtained was subjected to silica gel column chromatography (hexane: ethyl acetate, 1
7: 3, V / V) to give the title object compound (5.70 g, yield 61%) as a pale yellow solid.

Mp 153-156 ° C .; ¹ H NMR (CDCl ₃ , 500 MHz): δ 3.97 (3H, s), 4.26 (2H,
q, J = 8 Hz), 7.16 (2H, d, J = 9 Hz), 7.66 (2H, d, J = 9
Hz), 7.71 (2H, d, J = 9 Hz), 8.13 (2H, d, J = 9 Hz); IR (KBr) ν _max 3447, 1730 cm ^-1 ; MS (FAB) m / z: 540 (M + H) ⁺ . (1d) 4-Hydroxymethyl-N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-({(2,2,2-trifluoroethyl) prepared in Example 1 (1c). ) -4- [2,2,2-trifluoro-1
-Hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester (5.40)
g, 10.0 mmol) was dissolved in dry tetrahydrofuran (125 mL), and diisobutylaluminum hydride-1M-toluene solution (5
5.0 mL, 55.0 mmol) was added, and the mixture was stirred at room temperature for 1 hour.

Methanol (10 mL) and sodium sulfate decahydrate (45 g) were added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes. The solid in the reaction solution was filtered through Celite, washed with tetrahydrofuran (50 mL), and the filtrate was concentrated under reduced pressure to give a crude title compound.

The crude target compound thus obtained was subjected to silica gel column chromatography (hexane: ethyl acetate,
7: 3-1: 1, V / V) and recrystallized from methylene chloride to give the title compound (5.1 as a white powder).
1 g, yield 100%) was obtained.

Mp 127-130 ° C .; ¹ H NMR (DMSO-d ₆ , 400 MHz): δ 5.47 (2H, t, J = 6 Hz),
7.30 (2H, d, J = 9 Hz), 7.50 (2H, d, J = 8 Hz), 7.58 (2
H, d, J = 8 Hz), 7.66 (2H, d, J = 9 Hz); IR (KBr) ν _max 3465, 3275, 1730 cm ^-1 ; MS (FAB) m / z: 512 (M + H) ⁺ . (1e) 4-bromomethyl-N- (2,2,2-trifluoroethyl) -N- {4 -[2,2,2-trifluoro-
1-Hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-hydroxymethyl-N prepared in Example 1 (1d)
-(2,2,2-trifluoroethyl) -N- {4-
Dissolve [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (5.11 g, 10.0 mmol) in dry N, N-dimethylformamide (40 mL). Then
Under a nitrogen atmosphere, dibromotriphenylphosphorane (6.
34 g, 15.0 mmol) was added and the mixture was stirred at room temperature for 1 hour.

Saturated aqueous sodium hydrogen carbonate solution (10
0 mL) was added, and the mixture was extracted with ethyl acetate (100 mL × 3). The obtained organic layers were combined, and water (150 mL × 3) was added.
And successively washed with saturated saline (150 mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude title compound.

The crude target compound thus obtained was subjected to silica gel column chromatography (hexane: ethyl acetate,
9: 1-4: 1, V / V) to give the title object compound (5.30 g, yield 92%) as a colorless oil.

[0139]¹H NMR (CDCl₃, 500MHz): δ 4.24 (2H, q,
J = 8 Hz), 4.48 (2H, s), 7.18 (2H, d, J = 9 Hz), 7.49
(2H, d, J = 8 Hz), 7.56 (2H, d, J = 8 Hz), 7.70 (2H,
d, J = 9 Hz); IR (KBr) ν_max 3465, 3275, 1730 cm^-1; MS (FAB) m / z: 512 (M + H)⁺. (1f) 4-cyanomethyl-N- (2,2,2-trif
Loloethyl) -N- {4- [2,2,2-trifluoro-
1-hydroxy-1- (trifluoromethyl) ethyl] fu
Phenyl} benzenesulfonamide 4-Bromomethyl-N- produced in Example 1 (1e)
(2,2,2-Trifluoroethyl) -N- {4- [2,
2,2-trifluoro-1-hydroxy-1- (trifluoro
(Methyl) ethyl] phenyl} benzenesulfonamide
(5.30 g, 9.23 mmol) was mixed with ethanol-water.
Dissolve in a mixed solvent (4: 1, V / V, 75 mL) and cyan
Potassium iodide (1.98 g, 30.4 mmol) was added,
The mixture was stirred at 60 ° C for 2 hours.

Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The obtained organic layers were combined, washed with saturated brine (200 mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude title compound.

The obtained crude target compound was subjected to silica gel column chromatography (hexane: ethyl acetate,
4: 1-7: 3, V / V) to give the title object compound (4.32 g, yield 90%) as a white powder.

Mp 145-148 ° C .; ¹ H NMR (CDCl ₃ , 500 MHz): δ 3.85 (2H, s), 4.12 (2H,
q, J = 7 Hz), 7.18 (2H, d, J = 9 Hz), 7.46 (2H, d, J = 8
Hz), 7.62 (2H, d, J = 8 Hz), 7.71 (2H, d, J = 9 Hz); IR (KBr) ν _max 3391 cm ^-1 ; MS (FAB) m / z: 521 (M + H) ⁺ . (1g) 4- (2-aminoethyl) -N- (2,2,2
-Trifluoroethyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-cyanomethyl prepared in Example 1 (1f) -N-
(2,2,2-Trifluoroethyl) -N- {4- [2,
2,2-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (100 mg, 0.19 mmol) was dissolved in dry tetrahydrofuran (1 mL) to prepare a borane-tetrahydrofuran complex-1M tetrahydrofuran solution. (3.0m
L, 3.0 mmol) was added, and the mixture was heated under reflux for 7 hours under a nitrogen atmosphere. Methanol (1 mL) was added to the reaction solution,
It was concentrated under reduced pressure.

The residue is a mixed solvent of methanol-water (4: 1,
V / V, 5 mL), add concentrated hydrochloric acid (1 mL),
The mixture was heated under reflux for 2 hours. The reaction mixture was made basic with 1N aqueous sodium hydroxide solution (10 mL) and washed with ethyl acetate (20 mL).
Extracted with mL × 3). The obtained organic layers were combined, washed with saturated brine (30 mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude title compound.

The crude target compound thus obtained was purified by preparative thin layer chromatography (methylene chloride: methanol: isopropylamine, 87: 10: 3, V / V) to give the title target compound as a white foam. (95 mg, yield 95
%) Was obtained.

¹ H NMR (CDCl ₃ , 400 MHz): δ 2.85 (2H, t,
J = 7 Hz), 2.98 (2H, t, J = 7 Hz), 4.24 (2H, q, J = 8 H
z), 7.13 (2H, d, J = 9 Hz), 7.27 (2H, d, J = 8 Hz), 7.
50 (2H, d, J = 8 Hz), 7.71 (2H, d, J = 9 Hz); MS (FAB) m / z: 525 (M + H) ⁺ . (1h) 2- (6-hydroxy- 3-oxo-3H-xanthen-9-yl) -5-({[6-oxo-6-
({2- [4-({(2,2,2-trifluoroethyl)
-4- [2,2,2-trifluoro-1-hydroxy-1
-(Trifluoromethyl) ethyl] anilino} sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid and 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl) -6- ( {[6-
Oxo-6-({2- [4-({(2,2,2-trifluoroethyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino } Sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid mixture 4- (2-aminoethyl) prepared in Example 1 (1g)
-N- (2,2,2-trifluoroethyl) -N- {4-
[2,2,2-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (19.0 mg, 0.0172 mmol) was dissolved in dimethyl sulfoxide (0.5 mL), and Fluor was added.
escein-5 (6) -carboxamidoca
proic acid N-succinimidyl
ester (Fluka) (6.0 mg, 0.010)
2 mmol) and neutral phosphoric acid pH standard solution (pH 6.8)
6) (0.05 mL) was added, and the mixture was stirred at 50 ° C. for 10 hours.

Water (10 mL) and 1N hydrochloric acid (1
mL) was added, and the mixture was extracted with ethyl acetate (10 mL × 3). The obtained organic layers were combined, washed successively with water (10 mL × 1) and saturated saline (10 mL × 1), dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to give the crude title compound. Got

The crude target compound thus obtained was subjected to preparative thin layer chromatography (ethyl acetate: methanol, 19:
1, V / V) to give the title compound (13 mg, yield 76%) as a yellow glass.

IR (KBr) ν _max 3399, 2926, 1744, 1642,
1613, 1542, 1508 cm ^-1 ; MS (FAB) m / z: 996 (M + H) ⁺ ; HRMS (FAB) m / z: calcd for C46H39F9N3O10S (M + H) ⁺ :
996.2212; found: 996.2205; (Example 2) 2- (6-hydroxy-3-oxo-3H)
-Xanthen-9-yl) -5-({[6-oxo-6
-({2- [3-({(2,2,2-trifluoroethyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl)) Phenyl] ethyl} amino) hexyl] amino}
Carbonyl) benzoic acid and 2- (6-hydroxy-3-)
Oxo-3H-xanthen-9-yl) -6-({[6
-Oxo-6-({2- [3-({(2,2,2-trifluoroethyl) -4- [2,2,2-trifluoro-1-
Hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid mixture

[0149]

[Chemical 7]

(2a) 3-Chlorosulfonylbenzoic acid methyl ester Using 3-chlorosulfonylbenzoic acid (5.20 g, 22.6 mmol) instead of 4-chlorosulfonylbenzoic acid, Example 1- (1a) ) According to the method described in
The title object compound (5.60 g, yield 100%) was obtained.

¹ H NMR (CDCl ₃ , 500 MHz): δ 4.00 (3H, s),
7.74 (1H, t, J = 8 Hz), 8.23 (1H, m), 8.41 (1H, dt,
J = 1,8 Hz), 8.70 (1H, t, J = 2 Hz). (2b) 3-({4- [2,2,2-trifluoro-1-
Hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester 3-chlorosulfonylbenzoic acid methyl ester (2.14 g, 9.12 mmol) was used in place of 4-chlorosulfonylbenzoic acid methyl ester. Then, the title object compound (3.79 g, yield 91%) was obtained according to the method described in Example 1- (1b).

¹ H NMR (DMSO-d ₆ , 400 MHz): δ 3.88 (3H,
s), 7.23 (2H, d, J = 9 Hz), 7.56 (2H, d, J = 8 Hz), 7.
74 (1H, t, J = 8 Hz), 8.08 (1H, d, J = 8 Hz), 8.17 (1
H, s), 8.34 (1H, d, J = 8 Hz), 8.61 (1H, s), 10.79 (1
H, br). (2c) 3-({(2,2,2-trifluoroethyl)-
4- [2,2,2-trifluoro-1-hydroxy-1-
(Trifluoromethyl) ethyl] anilino} sulfonyl)
Benzoic acid methyl ester 4-({4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) Benzoic acid methyl ester instead of 3-({4
-[2,2,2-trifluoro-1-hydroxy-1-
(Trifluoromethyl) ethyl] anilino} sulfonyl)
Benzoic acid methyl ester (3.79 g, 8.29 mmo
Using l), and according to the method described in Example 1- (1c), the title object compound (2.03 g, yield 45%) was obtained.

¹ H NMR (CDCl ₃ , 400 MHz): δ 3.92 (3H, s),
4.13 (2H, q, J = 8 Hz), 7.16 (2H, d, J = 9 Hz), 7.57
(1H, t, J = 8 Hz), 7.69? 7.76 (3H, m), 8.18 (1H,
s), 8.28 (1H, d, J = 8 Hz); IR (KBr) νmax, 3437, 1730 cm-1; MS (FAB) m / z: 540 (M + H) +. (2d) 3-hydroxy Methyl-N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4- ({(2,2,2-trifluoroethyl) -4-
Instead of [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester, 3-({(2,2,2-
Trifluoroethyl) -4- [2,2,2-trifluoro-
1-Hydroxy-1- (trifluoromethyl) ethyl] anilino} sulfonyl) benzoic acid methyl ester (1.9
0 g, 3.52 mmol) was used in Example 1- (1
According to the method described in d), the title object compound (1.67
g, yield 93%) was obtained.

¹ H NMR (CDCl ₃ , 400 MHz): δ 4.19 (2H, q,
J = 8 Hz), 4.66 (2H, s), 5.64 (1H, br), 7.14 (2H, d,
J = 8 Hz), 7.44? 7.52 (3H, m), 7.59 (1H, d J = 8 H
z), 7.68 (2H, d, J = 8 Hz); IR (KBr) νmax, 3298, 1711 cm-1; MS (FAB) m / z: 540 (M + H) +. (2e) 3-bromomethyl -N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro-
1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-hydroxymethyl-N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro -1-
Instead of hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide, 3-hydroxymethyl-N- (2,2,2-trifluoroethyl) -N-
{4- [2,2,2-trifluoro-1-hydroxy-1
Using-(trifluoromethyl) ethyl] phenyl} benzenesulfonamide (1.48 g, 2.89 mmol) and following the method described in Example 1- (1e), the title compound (1.60 g, yield) was obtained. 96%).

¹ H NMR (CDCl ₃ , 400 MHz): δ 4.12 (2H, q,
J = 8 Hz), 4.39 (2H, s), 7.15 (2H, d, J = 8 Hz), 7.47
(1H, t, J = 9 Hz), 7.50 (1H, s), 7.56 (1H, d, J = 8 H
z), 7.63 (1H, d, J = 8 Hz), 7.71 (2H, d, J = 8 Hz); IR (KBr) νmax, 3442, 1709 cm-1; MS (FAB) m / z: 512 ( M + H) +. (2f) 3-Cyanomethyl-N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro-
1-Hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-bromomethyl-N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro- 1-hydroxy-1- (trifluoromethyl) ethyl] phenyl}
Instead of benzenesulfonamide, 3-bromomethyl-
N- (2,2,2-trifluoroethyl) -N- {4-
Using [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (1.01 g, 1.76 mmol),
The title object compound (781 mg, yield 85%) was obtained according to the method described in Example 1- (1f).

¹ H NMR (CDCl ₃ , 500 MHz): δ 3.86 (2H, s),
4.25 (2H, q, J = 8 Hz), 7.13 (2H, d, J = 8 Hz), 7.16
(1H, t, J = 8 Hz), 7.21 (1H, s), 7.54 (1H, d, J = 8 H
z), 7.57 (1H, d, J = 8 Hz), 7.72 (2H, d, J = 8 Hz) IR (KBr) νmax 3491 cm-1; MS (FAB) m / z: 521 (M + H) +. (2g) 3- (2-aminoethyl) -N- (2,2,2
-Trifluoroethyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide 4-cyanomethyl-N- (2,2,2 -Trifluoroethyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl}
Instead of benzenesulfonamide, 3-cyanomethyl-
N- (2,2,2-trifluoroethyl) -N- {4-
Described in Example 1- (1g) using [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (51.7 mg, 0.0994 mmol). According to the method described above, the title object compound (33.5 mg, yield 64%) was obtained.

¹ H NMR (CDCl ₃ , 400 MHz): δ 2.58? 2.64
(4H, m), 3.41 (2H, br), 4.32 (2H, q, J = 8 Hz), 7.07
(2H, d, J = 8 Hz), 7.26 (1H, s), 7.34 (1H, d, J = 8 H)
z), 7.50 (1H, t, J = 8 Hz), 7.66? 7.70 (3H, m); MS (FAB) m / z: 525 (M + H) +. (2h) 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl) -5-({ [6-oxo-6-
({2- [3-({(2,2,2-trifluoroethyl)
-4- [2,2,2-trifluoro-1-hydroxy-1
-(Trifluoromethyl) ethyl] anilino} sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid and 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl) -6- ( {[6-
Oxo-6-({2- [3-({(2,2,2-trifluoroethyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino } Sulfonyl) phenyl] ethyl} amino) hexyl] amino} carbonyl) benzoic acid mixture 4- (2-aminoethyl) -N- (2,2,2-trifluoroethyl) -N- {4- [2,2 2,2-trifluoro
Instead of 1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide, 3- (2-
Aminoethyl) -N- (2,2,2-trifluoroethyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl}
Benzenesulfonamide (4mg, 0.007628m
mol), according to the method described in Example 1- (1h), the title compound (2.7 mg, yield 80%).
Got

IR (KBr) νmax 3390, 2932, 1748, 1642,
1612, 1541, 1508, 1450, 1358 cm-1; MS (FAB) m / z: 996 (M + H) +; HR-MS (FAB) m / z: calc for C46H39F9N3O10S (M + H) +:
996.2212; found: 996.2219; (Example 3) 2- (6-hydroxy-3-oxo-3H
-Xanthen-9-yl) -5-[({6-oxo-6
-[(3-{(phenylsulfonyl) -4- [2,2,
2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} propyl) amino] hexyl} amino) carbonyl] benzoic acid and 2- (6-hydroxy-3-oxo-3H-xanthene-9- Ill)
-6-[({6-oxo-6-[(3-{(phenylsulfonyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino}}
A mixture of propyl) amino] hexyl} amino) carbonyl] benzoic acid

[0159]

[Chemical 8]

(3a) N- (3-aminopropyl) -N
-{4- [2,2,2-trifluoro-1-hydroxy-
1- (Trifluoromethyl) ethyl] phenyl} benzenesulfonamide N- {4- [2,2,2-trifluoro-1-] produced by the method described in WO200054759, Example 2.
Hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide (400 mg, 1.0 mm
ol) was dissolved in dimethylformamide (5 ml), and potassium carbonate (168 mg, 1.2 mmol) and 1-bromo-3-chloropropane (0.15 ml, 1.
5 mmol) was added and the mixture was stirred at 50 ° C. for 7 hours.

The reaction solution was diluted with water (100 ml) and then extracted with ethyl acetate. The obtained organic phase was washed with saturated saline and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure to obtain a colorless oily residue.

This was purified by silica gel column chromatography (hexane: ethyl acetate, 7: 3, V / V), and the target compound, N- (3-chloropropyl) -N- {4- [2]. , 2,2-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl}
Benzenesulfonamide and by-product N-allyl-
370 mg of an inseparable mixture with N- {4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] phenyl} benzenesulfonamide were obtained.

This mixture was mixed with dimethylformamide (5 m
l) dissolved in sodium azide (131 mg,
2.0 mmol) was added and the mixture was stirred at 60 ° C. for 7 hours.

The reaction solution was diluted with water (100 ml) and then extracted with ethyl acetate. The obtained organic phase was washed with saturated saline and dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure to obtain 373 mg of a colorless oily residue.

This crude product was dissolved in ethanol (10 ml) and added with 10% palladium on carbon catalyst (113 m).
g) was added, and the mixture was vigorously stirred at room temperature for 1 hour under a hydrogen gas atmosphere. The solvent was distilled off from the filtrate obtained by filtering the reaction solution under reduced pressure to obtain an oily residue. This product was diluted with ethyl acetate, and the generated crystals were collected by filtration to give the title object product (124 mg, yield 27%) as colorless crystals.

Mp 187? 188 ℃; IR (neat) ν _max 1346, 1263, 1215, 1168 cm ^-1 ; ¹ H NMR (CDCl ₃ + DMSO-d ₆ , 400MHz): δ 1.46 (2H, quinte
t, J = 7Hz), 2.69 (2H, t, J = 7Hz), 3.54 (2H, t, J = 7 H
z), 7.00-7.03 (2H, m), 7.34-7.61 (7H, m); MS (FAB) m / z: 457 (M + H) ⁺ ; HRMS (ESI) m / z: calcd for C ₁₈ H ₁₉ F ₆ N ₂ O ₃ S (M + H) ⁺ :
457.1021 Found 457.1027. (3b) 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl) -5-[({6-oxo-6-
[(3-{(phenylsulfonyl) -4- [2,2,2
-Trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} propyl) amino] hexyl}
Amino) carbonyl] benzoic acid and 2- (6-hydroxy-3-oxo-3H-xanthen-9-yl) -6
-[({6-oxo-6-[(3-{(phenylsulfonyl) -4- [2,2,2-trifluoro-1-hydroxy-1- (trifluoromethyl) ethyl] anilino} propyl) amino] Hexyl} amino) carbonyl] benzoic acid mixture N- (3-aminopropyl) -N- {4- [2,2,2-trifluoro-1-hydroxy-1- (prepared in Example 3- (3a). Trifluoromethyl) ethyl] phenyl}
Benzenesulfonamide (82mg, 0.18mmo
1) was dissolved in dimethylsulfoxide (3 ml), and Fluorescein-5 (6) -carbox was added thereto.
amidocaproic acid N-succi
nimidyl ester (Fluka) (100m
g, 0.17 mmol) and pH 6.9 phosphate buffer (0.50 ml) were added, and the mixture was stirred at room temperature for 2 hours.

The reaction solution was diluted with ethyl acetate, and this was diluted with water,
Then, it was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude title compound.

The crude target compound thus obtained was treated with silica gel.
Repeat column chromatography (ethyl acetate) twice
And purified to give the title object compound (132 mg, yield 79
%) Was obtained. RT 4.95 (Inertsil ODS-3 (GL science) 4.6mmFx250mmL,
 Acetonitryle: Buffer (2% acetic acid-2% triethylamine water
Solution v / v) 6: 4, v / v, 1 ml / min); IR (neat) ν_max 3384, 2938, 1742, 1643, 1613, 1544,
 1508, 1176, 1111 cm ^-1; MS (FAB) m / z: 928 (M + H)⁺; HRMS (ESI) m / z: calcd for C₄₅H₄₀F₆N₃O_TenS (M + H)⁺ :
 928.2339 Found 928.2342. (Test example) LXR binding assay 1) Preparation of human LXRα Plymerase chain reaction:
It is called "PCR" below. ) By the method, glutathione-S-tran
Glutathione-S-transferase: "GS
"T". : PGEX-4T-3 (Amersham Pharmacia biotec
derived from h). ) And human LXRα ligand bindin
Ligand binding domain: Below, "LB
"D". ) Fusion protein (hereinafter “GST-LXRα LB
"D". ) Obtained recombinant expression vector
And culturing E. coli transformed with the vector,
GST-LXRα LBD was recovered from the culture.

First, the coding sequence for human LXRα (Gen
BANK accession No. U22662 (PJWilly et al, Genes
Dev. 9 (9), 1033-1045 (1995)) nucleotides 597 to 1379), and a recognition sequence for restriction enzyme SalI or NotI was added to both ends of the gene to express expression vector pGEX-4T-3. A set of oligonucleotides having the following nucleotide sequences were synthesized for incorporation in (Amersham Pharmacia biotech). 5'-CACGACGTCGACCATGCCCATCCTTGCCC-3 ': SEQ ID NO: 1 (α1) in the sequence listing 5'-CACGACGCGGCCGCTCATTCGTGCACATCCCAGAT-3': SEQ ID NO: 2 (α2) in the sequence listing, then human liver cDNA library (PJ Willy
et al, Genes Dev. 9 (9), 1033-1045 (1995)) as a template, and the above α1 and α2 as primers,
45 seconds at 94 ° C, then 1 minute at 58 ° C, then 72
After repeating the heat retention cycle consisting of 1 minute at ℃ 6 times,
45 seconds at 94 ° C, then 1 minute at 62 ° C, then 72
PCR was carried out by repeating a heat retention cycle consisting of 1 minute at ℃ 23 times. The amplified polynucleotide fragment was cloned into TOPO Vector (manufactured by Invitrogen), and the nucleotide sequence of the insert portion (insert) was determined by a conventional method (SEQ ID NO: 5 in the sequence listing).

Then, the obtained recombinant vector was treated with restriction enzyme SalI and NotI sites, and then inserted (inse
rt) to recover pGEX-4T-3 (Amersham Pharmacia biotec
subcloned into h).

Next, E. coli strain TOP10F 'strain (manufactured by Invitrogen) was transformed with the obtained recombinant vector. The transformed strain was treated with L-broth containing ampicillin at a concentration of 100 μg / ml.
th medium (tryptone (Difco) 10 g, yeast extract (Difco) 5 g and sodium chloride 5 g
Dissolved in 1 L of water. ) Inoculate 5 ml, 3
After shaking culture at 7 ° C for 4 hours, 5.0% (volume / volume) was added to 1 L of L-broth medium containing 100 μg / ml of ampicillin.
(Volume) and inoculated, and shaking culture was carried out at 37 ° C. for 4 hours.
Then 0.1 mM isopropyl-β-D-thiogalactoside (Iso
Propyl β-D-Thiogalactoside: "IPTG": Amersha
m Pharmacia biotech) was added and shaking culture was performed at 25 ° C. for 17 hours.

After completion of the culture, centrifugation was carried out at 8,000 × g for 10 minutes to collect the precipitated fraction, which was suspended in 50 ml of Dulbecco's phosphate buffer (pH 7.1: manufactured by GIBCO: hereinafter referred to as “PBS”). did. The suspension is sonicated and 1% (volume /
(Volume) Triton X-100 was added and gently shaken at room temperature for 30 minutes. The supernatant was recovered by centrifugation at 11000 xg for 15 minutes, and the glutathione sepharose 4B gel (Amersham P
harmacia biotech) 0.5 ml was added, and the mixture was gently shaken for 30 minutes. The gel was recovered by centrifugation, washed with PBS and 50 mM Tris-HCl buffer (pH 8.0) three times each, and then 10 mM reduced glutathione solution (50 mM Tris-HCl, p
H 8.0) 2 ml was added. Collect the supernatant by centrifugation,
12.5% polyacrylamide sodium dodecyl sulfate
As a result of performing gel electrophoresis (hereinafter referred to as "SDS-PAGE"), the molecular weight of the obtained fusion protein under reducing conditions was about 58,000. In addition, the protein concentration of the fusion protein was determined by protein assay staining solution (BIO RAD
It was measured by the Bradford method using a standard protein (bovine serum albumin) and a standard protein. 2) Similar to the preparation 1) of human LXRβ, a recombinant expression vector encoding a fusion protein of LBD of GST and human LXRβ (hereinafter referred to as “GST-LXRβ LBD”) was obtained by the PCR method, and the vector was obtained. Escherichia coli transformed with Escherichia coli was cultured, and GST-LX was extracted from the culture.
Rβ LBD was recovered.

First, the sequence encoding LBD of human LXRβ (GenBANK accession No. U07132; DM Shinar et al,
Nucleotide number 836 in Gene 147 (2), 273-276 (1994)
To 1630) encoding restriction enzymes at both ends of the gene
Expression vector pGEX-4T-3 (Amersham Pharmacia biotech) with EcoRI or XhoI recognition sequence added.
A set of oligonucleotides having the following nucleotide sequences were synthesized for integration into 5'-TCAGCCGAATTCGCCTGGGGCTTCCCCTGGTGG-3 ': SEQ ID NO: 3 (β1) in the sequence listing 5'-CCTAGCCTCGAGTCACTCGTGGACGTCCCAGA-3': SEQ ID NO: 4 (β2) in the sequence listing Next, a polynucleotide encoding human LXRβ (J.
Willy et al, Genes Dev. 9 (9), 1033-1045 (1995)) as a template, and using β1 and β2 as primers described above, PCR was performed according to the method described in 1) to amplify the amplified poly. Nucleotide fragments are converted into TOPO Vector
Cloned into an inserter (inser
The nucleotide sequence of t) was determined (SEQ ID NO: 6 in the sequence listing).

Then, the resulting recombinant vector was treated with restriction enzymes EcoRI and XhoI, and
Was recovered and subcloned into pGEX-4T-3.

Next, E. coli strain TOP10F 'strain (manufactured by Invitrogen) was transformed with the obtained recombinant vector. The transformant was cultured according to the method described in 1), and the desired fusion protein was recovered from the culture according to the method described in 1). The molecular weight under reducing conditions by SDS-PAGE was about 58,000. Further, the protein concentration of the fusion protein was measured according to the method described in 1). 3) LXR binding assay buffer using fluorescence polarization (10 mM HEPES, 0.1 mM EDTA 3Na, 10 mM (±) -dithiothreitol (DTT), 20% (volume / volume) Ultrapure
Bovine Gamma Globulin 5mg / ml (PanVera):
1 nM of the compound of Example 1 or Example 3 or 5 nM of the compound of Example 2 was added to the "binding buffer)" (hereinafter referred to as "FITC-ligand addition binding buffer"). To a 96-well flat bottom black plate (Corning Costar), 90 μl of FITC-ligand addition binding buffer was added, and 10 μl of GST-LXRα LBD obtained in 1) or GST-LXRβ LBD obtained in 2) was added, respectively. As a control, FITC-ligand added binding buffer 90μl, 50mM Tris-HCl buffer (pH 8.0) 10μ
l were added respectively. As a blank, binding buffer 90
μl and 10 μl of 50 mM Tris-HCl (pH 8.0) were added respectively. Then, after incubating the plate at 4 ° C for 12 hours,
Incubate at room temperature for 1 hour and measure fluorescence polarization analyzer Analyst (L
The fluorescence polarization degree was measured by using a jL company. The measurement conditions are shown below: Kinetic read cycle: 1 of 1 Read start delay time: 0 s Time delay between kinetic reads: <n / a> Microplate format: Corning Costar 96 PS solid Shake time: 0 s Temperature: room temperature Serial number: AN0107 Read sequence: row Mode sequence: well Detection mode: FPS Light sensor: HC-120 Excitation side: Top Emission side: Top Lamp: Continuous Readings per well: 3 Time between readings: 100 ms Integration time: 100000 us Attenuator mode : o Motion settling time: 150 ms Z Height: 2 mm Numeric Excitation filter: 1 Fluorescein 485 nm Emission filter: 1 Fluorescein 530 nm Beamsplitter: Top Fluorescein (505 nm) Excitation polarizer filter: s Emission polarizer filter: s Detector counting: Digital Flash lamp voltage: <n / a> Delay after flash: <n / a> Sensitivity setting: <n / a> A / D converter gain: <n / a> Integrating gain: <n / a> Detection mode: FPP Light sensor: HC-120 Excitation side: Top Emission side: Top Lamp: Continuous Readings per well: 3 Time between reading s: 100 ms Integration time: 100000 us Attenuator mode: out Motion settling time: 150 ms Z Height: 2 mm Numeric Excitation filter: 1 Fluorescein 485 nm Emission filter: 1 Fluorescein 530 nm Beamsplitter: Top Fluorescein (505 nm) Excitation polarizer filter : s Emission polarizer filter: p Detector counting: Digital Flash lamp voltage: <n / a> Delay after flash: <n / a> Sensitivity setting: <n / a> A / D converter gain: <n / a> Integrating gain The value obtained by subtracting the average value of the fluorescence polarization degree (mP) of the control from the fluorescence polarization degree (P) obtained in the presence of <n / a> protein was defined as the fluorescence polarization degree (ΔmP). A Klotz Plot was prepared by plotting the fluorescence polarization degree (ΔmP) on the vertical axis and the protein concentration on the horizontal axis. The result

1 to

FIG. 3 shows. As shown in the figure, an increase in the degree of fluorescence polarization was observed depending on the protein concentration, and the compounds of the present invention (Example 1, Example 2 and Example 3) were GST-LXRα LBD and GST-LXRβ LBD, respectively. It was confirmed to be combined. The Kd value of the compound of Example 1 was 90.4 nM for GST-LXRα LBD and 316 nM for GST-LXRβ LBD.
Kd value of the compound is 143nM for GST-LXRα LBD, GST-LX
1.17 μM against Rβ LBD, Kd of the compound of Example 2
Values are 76.3 nM for GST-LXRα LBD and for GST-LXRβ LBD
It was 499 nM.

The compounds of the present invention are useful as compounds used in the LXR binding ability test method. (Measurement of Ki value) Using the compound of Example 1, T0901317 (Proc. Natl. Acad. Sci., 9 known as an LXR agonist)
8, 1477-1482 (2001)) was used to test the LXR binding ability of the ligand. An LXR-added binding buffer was prepared with the following composition.

LXR-added binding buffer HEPES 10 mM EDTA 3Na 0.1 mM (±) -dithiothreitol (DTT) 10 mM Ultrapure Bovine Gamma Globulin 5 mg / ml (PanVera) 20% (v / v) GST-LXRα LBD or GST-LXRβ LBD 40 μg / ml The compound of Example 1 1 nM T0901317 was adjusted to 1 mM with DMSO, and then serially diluted with DMSO from 1 mM to 1/10 in 9 steps.

95 μl of LXR-added binding buffer was added to a 96-well flat bottom black plate (Corning Costar), and 5 μl of each concentration of T0901317 was added thereto. At this time, what added 5 microliters of DMSO instead of T0901317 was used as a negative control. Furthermore, as a positive control, 5 μl of DMSO was added to 95 μl of FITC-ligand-added binding buffer. As a blank DMSO 5 in 95 μl binding buffer
μl was added. After standing overnight at 4 ° C., the temperature was returned to room temperature, and the fluorescence polarization degree was measured under the conditions described above using a fluorescence polarization measurement apparatus Analyst (LjL).

The binding activity was represented by the inhibition rate calculated by the following formula. Inhibition rate (%) = 100− (X−A) / (B−A) × 100 X: Fluorescence polarization degree (mP) when T0901317 was added A: Fluorescence polarization degree of positive control (mP) B: Fluorescence of negative control The polarization degree (mP) measurement result is shown in FIG. In Figure 4, the horizontal axis is T09013
The logarithm (logM) of 17 concentrations is shown, and the vertical axis shows the inhibition rate (%). As is clear from FIG. 4, T0901317 showed a higher concentration-dependent inhibition rate, and T0901317 showed higher GST-LXRα LBD.
Alternatively, it was confirmed that it was bound to GST-LXRβ LBD. Ki of T0901317 is 52.7nM for GST-LXRα LBD, GST-LX
It was 49.2 nM for Rβ LBD.

[0180]

The compound having the general formula (I) of the present invention or a salt thereof has good LXR binding ability and fluorescence ability,
It can be used as a simple and more comprehensive method for testing LXR binding ability. Further, the LXR binding ability test method using the compound of the present invention is useful as a simple and comprehensive method for testing LXR binding ability.

[0181]

[Sequence Listing Free Text] SEQ ID NO: 1: sense primer for amplifying DNA encoding ligand binding domain of human LXRα SEQ ID NO: 2: antisense for amplifying DNA encoding ligand binding domain of human LXRα Primer SEQ ID NO: 3: Sense primer for amplifying DNA encoding ligand binding domain of human LXRβ SEQ ID NO: 4: Antisense primer for amplifying DNA encoding ligand binding domain of human LXRβ

[0182]

[Sequence list]                                SEQUENCE LISTING <110> Sankyo, Co., Ltd. <110> Kenji Wakabayashi; Kozo Oda <120> Fluorescent Labeled Ligand <130> 2002007SL <140> <141> <160> 6 <170> PatentIn Ver. 2.1 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> 1 <223> Description of Artificial Sequence: A sense primer for amplifying  a DNA coding a ligand biding domain of human LXR alpha. <400> 1 cacgacgtcg accatgccca tccttgccc 29 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> 2 <223> Description of Artificial Sequence: An antisense primer for ampli fying a DNA coding a ligand biding domain of human LXR alpha. <400> 2 cacgacgcgg ccgctcattc gtgcacatcc cagat 35 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> 3 <223> Description of Artificial Sequence: A sense primer for amplifying  a DNA coding a ligand biding domain of human LXR beta. <400> 3 tcagccgaat tcgcctgggg cttcccctgg tgg 33 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> 4 <223> Description of Artificial Sequence: An antisense primer for ampli fying a DNA coding a ligand biding domain of human LXR beta. <400> 4 cctagcctcg agtcactcgt ggacgtccca ga 32 <210> 5 <211> 783 <212> DNA <213> Homo sapiens <400> 5 catgccacat ccttgccccc cagggcttcc tcaccccccc aaatcctgcc ccagctcagc 60 ccggaacaac tgggcatgat cgagaagctc gtcgctgccc agcaacagtg taaccggcgc 120 tccttttctg accggcttcg agtcacgcct tggcccatgg caccagatcc ccatagccgg 180 gaggcccgtc agcagcgctt tgcccacttc actgagctgg ccatcgtctc tgtgcaggag 240 atagttgact ttgctaaaca gctacccggc ttcctgcagc tcagccggga ggaccagatt 300 gccctgctga agacctctgc gatcgaggtg atgcttctgg agacatctcg gaggtacaac 360 cctgggagtg agagtatcac cttcctcaag gatttcagtt ataaccggga agactttgcc 420 aaagcagggc tgcaagtgga attcatcaac cccatcttcg agttctccag ggccatgaat 480 gagctgcaac tcaatgatgc cgagtttgcc ttgctcattg ctatcagcat cttctctgca 540 gaccggccca acgtgcagga ccagctccag gtagagaggc tgcagcacac atatgtggaa 600 gccctgcatg cctacgtctc catccaccat ccccatgacc gactgatgtt cccacggatg 660 ctaatgaaac tggtgagcct ccggaccctg agcagcgtcc actcagagca agtgtttgca 720 ctgcgtctgc aggacaaaaa gctcccaccg ctgctctctg agatctggga tgtgcacgaa 780 tga 783 <210> 6 <211> 795 <212> DNA <213> Homo sapiens <400> 6 cctggggctt cccctggtgg atctgaggca ggcagccagg gctccgggga aggcgagggt 60 gtccagctaa cagcggctca agaactaatg atccagcagt tggtggcggc ccaactgcag 120 tgcaacaaac gctccttctc cgaccagccc aaagtcacgc cctggcccct gggcgcagac 180 ccccagtccc gagatgcccg ccagcaacgc tttgcccact tcacggagct ggccatcatc 240 tcagtccagg agatcgtgga cttcgctaag caagtgcctg gtttcctgca gctgggccgg 300 gaggaccaga tcgccctcct gaaggcatcc actatcgaga tcatgctgct agagacagcc 360 aggcgctaca accacgagac agagtgtatc accttcttga aggacttcac ctacagcaag 420 gacgacttcc accgtgcagg cctgcaggtg gagttcatca accccatctt cgagttctcg 480 cgggccatgc ggcggctggg cctggacgac gctgagtacg ccctgctcat cgccatcaac 540 atcttctcgg ccgaccggcc caacgtgcag gagccgggcc gcgtggaggc gttgcagcag 600 ccctacgtgg aggcgctgct gtcctacacg cgcatcaaga ggccgcagga ccagctgcgc 660 ttcccgcgca tgctcatgaa gctggtgagc ctgcgcacgc tgagctctgt gcactcggag 720 caggtcttcg ccttgcggct ccaggacaag aagctgccgc ctctgctgtc ggagatctgg 780 gacgtccacg agtga 795

[Brief description of drawings]

FIG. 1 shows the results of an LXR binding ability test method using the compound of Example 1.

FIG. 2 shows the results of the LXR binding ability test method using the compound of Example 3.

FIG. 3 shows the results of the LXR binding ability test method using the compound of Example 2.

FIG. 4 shows the results of testing the LXR binding ability of T0901317 using the compound of Example 1.

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 4B024 AA01 AA11 BA63 CA04 DA06                       EA04 FA02 GA11 HA01 HA03                 4C062 HH21

Claims (2)

[Claims] 1. A general formula (I) [Chemical 1] (In the formula, R¹ And R² Independently, hydrogen atom; halogen
Atom; C₁-C₂₀Alkyl group; halogen atom, hydroxyl group,
Lucapto group, C₁-C₆ Alkoxy group, C₁-C₆Alkylchi
O group, cycloalkyl group, and heteroaryl group
The same or different substituents selected from the group consisting of
To 6 substituted C₁-C₂₀Alkyl group; Aryl group; Ha
Rogen atom, hydroxyl group, mercapto group, nitro group, cyano
Group, amino group, C₁-C₆Alkoxy group, C₁-C₆Alkylchi
O group and C₁-C₆Select from the substituent group consisting of alkyl groups
1 to 4 substituted with the same or different substituents selected.
Reel group; aralkyl group; halogen atom, hydroxyl group, mel
Capto group, nitro group, cyano group, amino group, C₁-C₆Al
Coxy group, C₁-C₆Alkylthio group and C₁-C₆Archi
Group selected from the group of substituents consisting of
An aralkyl group substituted with 1 to 4 substituents; or; General formula (II) -A¹-D-G¹-A²-G²-FL (II) [In the formula, A¹ And A² Is independently C₁-C₆Alkylene group;
Is a halogen atom, hydroxyl group, mercapto group, C₁-C₆ Al
Coxy group, C₁-C₆An alkylthio group, an aryl group, and
The same selected from the group of substituents consisting of heteroaryl groups
Or C substituted by 1 to 3 with different substituents₁-C₆Arche
Group, D is a single bond; phenylene group; or; halo
A group of substituents consisting of a gen atom, a hydroxyl group, and a mercapto group.
1 to 3 substituted with the same or different substituents selected from
Phenylene group, G¹ And G² Independent and single
Compound; -O-;-S-;-OC (= O)-;-OC (= S)-;-NHC (= O)-;-NHSO
₂-;-NHC (= O) NH-; or; -NHC (= S) NH-, FL is fluorescent
Indicates a group. ] The group represented by is shown. ) Compound represented by
Or its salt. However, (i) R¹ Is a group represented by the general formula (II)
R if not² Is a group represented by the general formula (II),
(ii) R² Is not a group represented by the general formula (II), R¹ 
Is a group represented by the general formula (II). 2. General formula (I) [Chemical 2] (In the formula, R¹ And R² Independently, hydrogen atom; halogen
Atom; C₁-C₂₀Alkyl group; halogen atom, hydroxyl group,
Lucapto group, C₁-C₆ Alkoxy group, C₁-C₆Alkylchi
O group, cycloalkyl group, and heteroaryl group
The same or different substituents selected from the group consisting of
To 6 substituted C₁-C₂₀Alkyl group; Aryl group; Ha
Rogen atom, hydroxyl group, mercapto group, nitro group, cyano
Group, amino group, C₁-C₆Alkoxy group, C₁-C₆Alkylchi
O group and C₁-C₆Select from the substituent group consisting of alkyl groups
1 to 4 substituted with the same or different substituents selected.
Reel group; aralkyl group; halogen atom, hydroxyl group, mel
Capto group, nitro group, cyano group, amino group, C₁-C₆Al
Coxy group, C₁-C₆Alkylthio group and C₁-C₆Archi
Group selected from the group of substituents consisting of
An aralkyl group substituted with 1 to 4 substituents; or; General formula (II) -A¹-D-G¹-A²-G²-FL (II) [In the formula, A¹ And A² Is independently C₁-C₆Alkylene group;
Is a halogen atom, hydroxyl group, mercapto group, C₁-C₆ Al
Coxy group, C₁-C₆An alkylthio group, an aryl group, and
The same selected from the group of substituents consisting of heteroaryl groups
Or C substituted by 1 to 3 with different substituents₁-C₆Arche
Group, D is a single bond; phenylene group; or; halo
A group of substituents consisting of a gen atom, a hydroxyl group, and a mercapto group.
1 to 3 substituted with the same or different substituents selected from
Phenylene group, G¹ And G² Independent and single
Compound; -O-;-S-;-OC (= O)-;-OC (= S)-;-NHC (= O)-;-NHSO
₂-;-NHC (= O) NH-; or; -NHC (= S) NH-, FL is fluorescent
Indicates a group. ] The group represented by is shown. ) Compound represented by
Or Liver X Receptor (LXR) binding capacity test using its salt
Law. However, (i) R¹ Is not a group represented by general formula (II)
If R² Is a group represented by the general formula (II), and (ii) R² 
Is not a group represented by the general formula (II), R¹ Is general
It is a group represented by the formula (II).