JP2009013126A - Prion protein structure transformation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a compound efficiently inhibiting the formation of an infectious prion protein via binding to a normal prion protein, to provide a prion protein structure transformation inhibitor comprising the composition, and to provide a prophylactic or curative agent for prion disease. <P>SOLUTION: The compound is represented by formula (1) [wherein, R<SP>1</SP>to R<SP>4</SP>are each H, a halogen atom, (substituted) hydrocarbon group or the like; R<SP>5</SP>to R<SP>12</SP>are each H, a halogen atom, (substituted) hydrocarbon group or the like; X is a single bond or linkage group; ring Z<SP>1</SP>and ring Z<SP>2</SP>are each a (substituted) nitrogen-containing ring; wherein, at least one of R<SP>1</SP>to R<SP>4</SP>is a group other than H]. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel compound capable of suppressing the production of infectious prion through binding to a binding pocket of normal prion protein, a prion protein structure conversion inhibitor containing the compound as an active ingredient, and prevention of prion disease -It is related with the therapeutic agent and the method of screening the compound which has the structure conversion inhibitory activity of prion protein using the said compound.

  Prion disease is a general term for diseases caused by prion protein abnormalities. For example, sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kool (SBPrusiner, Science, 216, 136 -144 (1982), SBPrusiner, Science, 252, 1515-1522 (1991), SBPrusiner, Prions. Proc. Natl. Acad. Sci. USA, 95, 13363-13383 (1998)), mutant Jacob disease ( Neurodegenerative diseases such as G. Chazot et al., Lancet, 347, 1181-1181 (1996) and RGWill et al., Lancet, 347, 921-925 (1996)) are known. As methods for treating such prion diseases, methods such as inactivation of prions using radiation, boiling, chemicals, etc., and methods of attenuating infectivity by various means have been tried. The tolerance of was not necessarily effective.

Here, prion disease is a disease caused by abnormality of prion protein. Specifically, normal prion (PrP ^C ) undergoes structural transformation and is infected via amyloid precursor special structure (PrP *). It is thought that the type prion (PrP ^Sc ) is generated, and the infectious type prion binds with the normal type prion in the body, and the normal type prion is successively converted into the infectious type prion and the symptoms progress. (SBPrusiner, Science, 216, 136-144 (1982), SBPrusiner, Science, 252, 1515-1522 (1991), K. Kuuwata et al., Biochemistry 41, 12277-12283 (2002)). In V. Perrier et al., Proc. Natl. Acad. Sci. USA 97, 6073-6078 (2000), an anti-prion drug is a compound that binds to a prion protein having a normal type structure and inhibits structural conversion. However, its pharmacological effect has not been proven.

  To date, mice (R. Riek et al., Nature, 382, 180-182 (1996)), hamsters (TLJames et al., Proc. Natl. Acad. Sci. USA 94, 10086-10091 (1997)) Cattle (F. Lopez Garcia, R. Zahn, R. Riek, K. Wuthrick, Proc. Natl. Acad. Sci. USA 97, 8334-8339 (2000)), humans (L. Calzolai et al., Proc. Natl. Acad. Sci. USA 97, 8340-8345 (2000)), three-dimensional structure determination by NMR is performed. These structures are quite similar, and amino acid residues No. 128 to No. 231 are composed of a structure composed of an α helix and an incomplete soft β sheet. No structure is formed from the N end to the 113th, and a hydrophobic cluster is formed from the 113th to the 128th, forming a plurality of structures (H. Liu et al., Biochemistry 38, 5362- 5377 (1999)). Until now, prion intermediates have been observed in relation to the structural transformation of prion protein (H. Zhang et al., Biochemistry 36, 3543-53 (1997), S. Hornemann, RAGlockshuber, Proc. Natl. Acad. Sci. USA 95, 6010-6014 (1998), ACApetri, WKSurewicz, Biol. Chem. 277, 44589-44592 (2002)). Recently, the prion folding intermediate PrP * has been identified and characterized by high-pressure NMR (K. Kuuwata et al., Biochemistry 41, 12277-12283 (2002)). In PrP *, helices B and C are denatured, but helix A is retained. PrP * is found to be present at 1% under physiological conditions (K. Kuuwata et al., Biochemistry 41, 12277-12283 (2002)).

  Based on the above findings, the present inventors conducted a docking simulation focusing on the binding pocket between the AS 2 loop in the normal prion structure and the C-terminal side of helix B, and the normal prion protein. We screened for compounds that suppress the structural transformation through binding and strongly suppress the formation of the folding intermediate PrP *. As such compounds, 2-pyrrolidin-1-yl-N- [4- [4- (2 -pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl] -acetamide has been obtained (Japanese Patent Laid-Open No. 2005-120002). However, this compound can bind to normal prions to suppress structural conversion, but there has been a demand for a compound that can exhibit superior effects by reducing the amount of infectious prions produced.

V.Perrier et al., Proc.Natl.Acad.Sci.U.S.A. 97, 6073-6078 (2000) R. Riek et al., Nature, 382, 180-182 (1996) T.L.James et al., Proc.Natl.Acad.Sci.U.S.A. 94, 10086-10091 (1997) F.LopezGarcia, R.Zahn, R.Riek, K.Wuthrick, Proc.Natl.Acad.Sci.U.S.A. 97, 8334-8339 (2000) L. Calzolai et al., Proc. Natl. Acad. Sci. U.S.A. 97, 8340-8345 (2000) H. Liu et al., Biochemistry 38, 5362-5377 (1999) H. Zhang et al., Biochemistry 36, 3543-53 (1997) S. Hornemann, R.A. Glockshuber, Proc. Natl. Acad. Sci. U.S.A. 95, 6010-6014 (1998) A.C.Apetri, W.K.Surewicz, Biol.Chem.277, 44589-44592 (2002) K. Kuuwata et al., Biochemistry 41, 12277-12283 (2002) JP 2005-120002 A

An object of the present invention is to provide a compound capable of efficiently suppressing the production of infectious prion protein through binding to normal prion protein, a prion protein structure conversion inhibitor containing the compound, and prevention / treatment of prion disease It is to provide an agent.
Another object of the present invention is to provide a method for efficiently screening for a compound excellent in the effect of suppressing the structural transformation of prion protein.

  As a result of intensive studies to achieve the above object, the present inventors have found that, according to a compound having a specific structure, the prion protein is structurally converted from a normal form to an infectious form by firmly binding to the prion protein. The present invention has been completed by finding that it effectively suppresses the above and exerts an excellent effect in preventing the onset and progression of prion diseases.

（式中、Ｒ^１〜Ｒ^４は、同一又は異なって、水素原子、ハロゲン原子、置換基を有していてもよい炭化水素基、置換基を有していてもよい複素環式基、カルボキシル基、置換オキシカルボニル基、置換若しくは無置換カルバモイル基、シアノ基、アシル基、ニトロ基、硫黄酸基、硫黄酸エステル基、ヒドロキシル基、置換オキシ基、メルカプト基、置換チオ基、又は置換若しくは無置換アミノ基を示す。Ｒ^５〜Ｒ^１２は、同一又は異なって、水素原子、ハロゲン原子、置換基を有していてもよい炭化水素基、置換基を有していてもよい複素環式基、カルボキシル基、置換オキシカルボニル基、置換若しくは無置換カルバモイル基、シアノ基、アシル基、ニトロ基、硫黄酸基、硫黄酸エステル基、ヒドロキシル基、置換オキシ基、メルカプト基、置換チオ基、又は置換若しくは無置換アミノ基を示す。Ｘは単結合又は連結基を示す。環Ｚ^１及び環Ｚ^２は、それぞれ置換基を有していてもよい窒素原子含有環を示す。但し、Ｒ^１〜Ｒ^４の少なくとも一つは水素原子以外の基を示す）
で表される化合物を提供する。 That is, the present invention provides the following formula (1):

(Wherein R ^{1 to} R ⁴ are the same or different and are a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group, carboxyl Group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercapto group, substituted thio group, or substituted or unsubstituted R ^{5 to} R ¹² are the same or different and each represents a hydrogen atom, a halogen atom, a hydrocarbon group which may have a substituent, or a heterocyclic group which may have a substituent. , Carboxyl group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercap Group, a substituted thio group, or .X showing a substituted or unsubstituted amino group is a single bond or a linking group. Ring Z ¹ and the ring Z ² is a good nitrogen atom-containing ring which may have a substituent Provided that at least one of R ^{1 to} R ⁴ represents a group other than a hydrogen atom)
The compound represented by these is provided.

で表される化合物が用いられる。 In the formula (1), preferably, R ^{1 to} R ⁴ are hydrogen atoms or optionally substituted hydrocarbon groups, and R ^{5 to} R ¹² are the same or different and are hydrogen atoms. , halogen atom, or an optionally substituted hydrocarbon group, a hydroxyl group, a substituted oxy group, a mercapto group, or a substituted thio group, X is C ₁ -C ₄ alkylene group, an oxygen atom (ether bond; -O-), or a sulfur atom (thioether bond; -S-), and ring Z ¹ and ring Z ² are the same or different and each may be a 5-membered or 6-membered ring optionally having a substituent. A nitrogen atom-containing ring, two of R ^{1 to} R ⁴ are hydrogen atoms and two are groups other than hydrogen atoms, or three of R ^{1 to} R ⁴ are hydrogen atoms and one is a group other than hydrogen atoms . Particularly preferably, the following formula (2)

The compound represented by these is used.

  The present invention also provides a prion protein structure conversion inhibitor containing the compound of the present invention as an active ingredient.

  Furthermore, the present invention provides a preventive / therapeutic agent for prion diseases comprising the compound of the present invention as an active ingredient. Examples of the prion disease include sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kool, and mutant Jacob disease.

  The present invention further provides a kit comprising the compound of the present invention and / or a derivative thereof, a prion-infected cell, and a means for detecting an infectious prion protein.

  The present invention also uses the above-described compound of the present invention and / or a derivative thereof to screen for a compound having prion protein structure conversion inhibitory activity based on the binding strength predicted by docking simulation with a normal prion protein. Provide a method.

  In the present specification, “normal prion protein” means a prion protein that is expressed in normal cells and has no infectivity, and “infectious prion protein” means normal prion protein and amino acid. It means a prion protein having the same sequence but different steric structure and having infectivity. Further, “infecting prion” means a state of being infected with infectious prion protein.

  Since the compound of the present invention binds firmly to the prion protein, it is extremely excellent in the effect of suppressing the structural conversion of the prion protein. A compound that exhibits such an effect is useful as a prion protein structural conversion inhibitor that has an effect of suppressing the production of infectious prions resulting from the structural conversion of normal prions. Since the compound of the present invention exerts the above-described action, it can provide a preventive / therapeutic agent for prion disease that is excellent in preventing the onset of prion disease and the progression of symptoms. According to the screening method of the present invention, since the above compound is used, a compound excellent in the effect of suppressing the structural transformation of the prion protein can be obtained efficiently.

The compound of the present invention is represented by the formula (1). In formula (1), R ^{1 to} R ⁴ are the same or different and are a hydrogen atom, a halogen atom, a hydrocarbon group which may have a substituent, or a heterocyclic group which may have a substituent. , Carboxyl group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercapto group, substituted thio group, or substituted Or an unsubstituted amino group is shown. R ¹ and R ² , R ³ and R ⁴ may be bonded to each other to form a ring.

Examples of the halogen atom include fluorine, chlorine, bromine and iodine atoms. Examples of the hydrocarbon group include an aliphatic hydrocarbon group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group, and a group in which a plurality of these are bonded. Examples of the aliphatic hydrocarbon group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, s-butyl, t-butyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, vinyl, allyl, ethynyl, 1-propynyl group, and the like. And a linear or branched aliphatic hydrocarbon group (alkyl group, alkenyl group, alkynyl group) having about 1 to 20 carbon atoms (preferably 1 to 10, more preferably 1 to 8). Examples of the alicyclic hydrocarbon group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclooctyl, cyclodecyl, cyclododecyl, norbornyl, adamantyl, and tricyclo [5.2.1.0 ^2,6 ] decyl group. And an alicyclic hydrocarbon group having about 3 to 20 carbon atoms (preferably 3 to 15 carbon atoms) such as a cycloalkyl group, a cycloalkenyl group, and a bridged carbocyclic group. Examples of the aromatic hydrocarbon group include aromatic hydrocarbon groups having about 6 to 14 carbon atoms such as phenyl and naphthyl groups.

  Examples of the group in which an aliphatic hydrocarbon group and an alicyclic hydrocarbon group are bonded include cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, 4-methylcyclohexyl group, and the like. Examples of the group in which an aliphatic hydrocarbon group and an aromatic hydrocarbon group are bonded include, for example, an aralkyl group such as benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl; 2-methylphenyl, 3-phenyl Examples include methylphenyl and 4-methylphenyl groups.

These hydrocarbon groups may have one or two or more substituents. Examples of the substituent include a halogen atom (fluorine, chlorine, bromine, iodine atom), a heterocyclic group (pyrrolidino group, pyrrolidino group, morpholino group, imidazolyl group, indole group, nitrogen atom, oxygen atom and sulfur atom). 3 to 20-membered heterocyclic group containing at least one selected hetero atom), carboxyl group, substituted oxycarbonyl group (alkoxy-carbonyl group, cycloalkyloxycarbonyl group, aryloxycarbonyl group, aralkyloxy) Carbonyl group etc.), substituted or unsubstituted carbamoyl group (carbamoyl group; mono- or di-hydrocarbon group substituted carbamoyl group such as methylcarbamoyl, dimethylcarbamoyl group etc.), cyano group, acyl group (formyl, acetyl, propionyl group etc.) Aliphatic acyl group; cyclohexa Alicyclic acyl groups such as carbonyl groups; aromatic acyl groups such as benzoyl groups; acetoacetyl groups, etc., nitro groups, sulfur acid groups (sulfonic acid groups, sulfinic acid groups), sulfur acid ester groups (sulfonic acid ester groups) , Sulfinic acid ester groups), hydroxyl groups, substituted oxy groups (alkoxy groups, cycloalkyloxy groups, aryloxy groups, acyloxy groups, etc.), mercapto groups, substituted thio groups (methylthio, ethylthio groups, etc.) C ₁ -C ₆ alkylthio A cycloalkylthio group such as a cyclohexylthio group; an arylthio group such as a phenylthio group; an acylthio group such as an acetylthio group), a substituted or unsubstituted amino group (amino group, mono- or di-C ₁ -C ₆ alkylamino group, etc.) , An oxo group or a group in which a plurality of these are bonded. The carboxyl group, sulfur acid group, hydroxyl group, and mercapto group may be protected with a protecting group. As the protecting group, a protecting group conventionally used in the field of organic synthesis can be used.

The heterocyclic ring constituting the heterocyclic group in R ^{1 to} R ⁴ includes an aromatic heterocyclic ring and a non-aromatic heterocyclic ring. As such a heterocyclic ring, for example, a heterocyclic ring containing an oxygen atom as a hetero atom (for example, 5-membered ring such as furan, tetrahydrofuran, oxazole, isoxazole, 4-oxo-4H-pyran, tetrahydropyran, morpholine, etc. 6-membered ring, condensed ring such as benzofuran, isobenzofuran, 4-oxo-4H-chromene, chromane, isochroman, etc.), heterocycle containing a sulfur atom as a hetero atom (for example, 5 such as thiophene, thiazole, isothiazole, thiadiazole) 5-membered rings, 6-membered rings such as 4-oxo-4H-thiopyran, condensed rings such as benzothiophene, etc.), heterocycles containing nitrogen atoms as heteroatoms (eg, pyrrole, pyrrolidine, pyrazole, imidazole, triazole, etc.) Ring, pyridine, pyridazine, pyrimi Emissions, pyrazine, piperidine, 6-membered ring such as piperazine, indole, indoline, quinoline, acridine, naphthyridine, quinazoline, other condensed rings purine) and the like. These heterocyclic groups may have a substituent (for example, the same group as the substituent that the hydrocarbon group may have, and the hydrocarbon group exemplified above).

Examples of the substituted oxycarbonyl group in R ^{1 to} R ⁴ include C _{1 to} C ₆ alkoxy-carbonyl groups such as methoxycarbonyl and ethoxycarbonyl; cycloalkyloxycarbonyl groups such as cyclohexyloxycarbonyl group; and aryls such as phenoxycarbonyl group An oxycarbonyl group; and an aralkyloxycarbonyl group such as a benzyloxycarbonyl group. Examples of the substituted or unsubstituted carbamoyl group include a carbamoyl group; a mono- or di-hydrocarbon group-substituted carbamoyl group such as a methylcarbamoyl group and a dimethylcarbamoyl group. Examples of the acyl group include aliphatic acyl groups such as formyl, acetyl, propionyl groups (C _{1 to} C ₇ aliphatic acyl groups); cycloaliphatic acyl groups such as cyclohexanecarbonyl groups; aromatic acyls such as benzoyl groups Group; acetoacetyl group and the like. Examples of the sulfur acid group include a sulfonic acid group and a sulfinic acid group. Examples of the sulfur acid ester group include a sulfonic acid ester group (such as a sulfonic acid C _{1 to} C ₄ alkyl ester group) and a sulfinic acid ester group (such as a sulfinic acid C _{1 to} C ₄ alkyl ester group). The substituted oxy group, for example, methoxy, ethoxy, isopropyloxy, C ₁ -C ₆ alkoxy group or a butoxy group; such as phenoxy group an aryloxy group; a cycloalkyl group such as cyclohexyl group, acetyloxy, propionyloxy And acyloxy groups such as C ₁ -C ₇ acyloxy groups. The substituted thio group, for example, methylthio, C ₁ -C ₆ alkylthio groups such as ethylthio group; arylthio group phenylthio group; cycloalkylthio groups such as cyclohexyl thio group acetylthio acylthio group such as (C ₁ -C ₇ Acylthio group, etc.). Examples of the substituted or unsubstituted amino group include amino groups; mono- or di-C ₁ -C ₆ alkylamino groups such as methylamino, dimethylamino, ethylamino and diethylamino groups; amidino groups; guanidino groups and the like. The

In the formula (1), R ¹ and R ² , R ³ and R ⁴ may be bonded to each other. Examples of the ring include 3 to 15 members (preferably 4 to 12 members, more preferably a cyclopropane ring, cyclobutane ring, cyclopentane ring, cyclopentene ring, cyclohexane ring, cyclohexene ring, norbornane ring, norbornene ring, and adamantane ring. Is a 5- to 8-membered) non-aromatic carbocycle (cycloalkane ring, cycloalkene ring, bridged carbocycle) and the like. These rings may have a substituent (for example, the same group as the substituent which the hydrocarbon group may have), and other rings (non-aromatic ring or aromaticity). Ring) may be condensed.

In the present invention, at least one of R ^{1 to} R ⁴ is a group other than a hydrogen atom. Specifically, two of R ^{1 to} R ⁴ are hydrogen atoms and two are groups other than hydrogen atoms, or three of R ^{1 to} R ⁴ are hydrogen atoms and one is a group other than hydrogen atoms, R ^{1 to} R All ⁴ are groups other than a hydrogen atom. In particular, a compound in which three of R ^{1 to} R ⁴ are hydrogen atoms and one is a group other than a hydrogen atom is preferably used. As a group other than a hydrogen atom, a side chain site of an amino acid other than glycine and derivatives thereof are preferably used. Here, the side chain site of an amino acid means a functional group other than hydrogen, amino group, and carboxyl group that binds to the α-carbon of the amino acid, such as methyl group in alanine, isopropyl group in valine, carboxy group in aspartic acid Examples include a methyl group, a carbamoylethyl group in glutamine, a benzyl group in phenylalanine, and the like. Of these, a C _{1 to} C ₁₂ aliphatic hydrocarbon group, a group in which an aliphatic hydrocarbon group and an aromatic hydrocarbon group are bonded, and derivatives thereof, particularly a methyl group and a benzyl group are preferable.

R ^{5 to} R ¹² in Formula (1) are the same or different and are a hydrogen atom, a halogen atom, a hydrocarbon group which may have a substituent, a heterocyclic group which may have a substituent, Carboxyl group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercapto group, substituted thio group, and substituted or An unsubstituted amino group is shown, and those exemplified above can be used. Of these, a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group, a hydroxyl group, a substituted oxy group, a mercapto group, or a substituted thio group is preferable, and in particular, a hydrogen atom, C _{1 to} C _4. alkyl group, a hydroxyl _group, C 1 -C ₄ alkyl group are preferably used.

Examples of the linking group in X include divalent groups such as linear or branched alkylene groups such as methylene, methylmethylene, dimethylmethylene, ethylene, propylene, and trimethylene groups (such as C _{1 to} C ₆ alkylene groups). Aliphatic hydrocarbon group; 1,2-cyclopentylene, 1,3-cyclopentylene, 1,2-cyclohexylene, 1,3-cyclohexylene, 1,4-cyclohexylene, cyclopentylidene, cyclohexylidene Divalent alicyclic hydrocarbon group such as a group; carbonyl group; oxygen atom (ether bond; -O-); sulfur atom (thioether bond; -S-); oxycarbonyl group (ester bond; -COO-, ring Z ¹ , ring Z ³ , ring Z 5, and ring Z 7 may be bonded to the right or left side; aminocarbonyl group (amide bond; —CONH -The side bonded to the adjacent group may be the right side or the left side); and a group in which a plurality of these groups are bonded. Preferred examples of the linking group include a C _{1 to} C ₄ alkylene group, an oxygen atom (ether bond; —O—), a sulfur atom (thioether bond; —S—), and a group in which two or more of these are bonded.

The monocyclic or polycyclic nitrogen atom-containing ring in ring Z ¹ and ring Z ² is not particularly limited as long as it contains at least one nitrogen atom as an atom constituting the ring, and is an aromatic heterocyclic ring or non-aromatic ring. Any of the family heterocycles may be used. Such rings include, for example, nitrogen atom-containing 5-membered rings such as pyrrole, pyrrolidine, pyrazole, imidazole, and triazole, nitrogen-containing 6-membered rings such as pyridine, pyridazine, pyrimidine, pyrazine, piperidine, piperazine, and indole. And condensed rings such as indoline, quinoline, acridine, naphthyridine, quinazoline, and purine; and rings that contain a nitrogen atom and an oxygen atom in the atoms constituting the ring such as oxazolidine and morpholine. These rings may have a substituent. As the substituent, the same groups as described above can be used. Among them, a hydrocarbon group such as an alkyl group (C _{1 to} C ₁₀ hydrocarbon group), a hydroxyl group, a halogen atom, a nitro group, a carboxy group, and an alkoxycarbonyl group. An alkyl group substituted with a group, an oxo group, or an aromatic heterocyclic group. Typical examples of the ring having such a substituent include nitrogen atom-containing monocyclic or polycyclic rings such as 2-pyrrolidone, succinimide, maleimide, and proglutamic acid. Preferably, a nitrogen atom-containing 5-membered ring or 6-membered ring which may have a substituent is used, more preferably pyrrolidine, piperidine, morpholine, maleimide, succinimide, etc., particularly pyrrolidine, piperidine and the like are used.

As a preferable compound of the present invention, for example, in the formula (1), R ^{1 to} R ⁴ are a hydrogen atom or a hydrocarbon group which may have a substituent, and R ^{5 to} R ¹² are same or different, a hydrogen atom, a halogen atom, or an optionally substituted hydrocarbon group, a hydroxyl group, a substituted oxy group, a mercapto group, or a substituted thio group, X is C ₁ -C ₄ alkylene group , An oxygen atom (ether bond; —O—), or a sulfur atom (thioether bond; —S—), and ring Z ¹ and ring Z ² may be the same or different and may have a substituent. Examples of the compound include a member ring or a six-membered nitrogen atom-containing ring, and at least one of R ^{1 to} R ⁴ is a hydrocarbon group which may have a substituent. As typical examples of the compound of the present invention, structural formulas of compounds containing the compound represented by the formula (2) are shown in FIGS.

（式中、Ｒ^５〜Ｒ^１０及びＸは前記に同じ）
で表される化合物を、当該式（３）に含まれる一方のアミノ基を保護基（例えばtert−ブトキシカルボニル基：Ｂｏｃ等）で保護した後、下記式（３ａ）

（式中、Ｚ^１、Ｒ^１、Ｒ^２及びＸは前記に同じ。但し、Ｒ^１とＲ^２の少なくとも一方が水素原子以外の基を示す）
で表される化合物と反応させ、次いで保護基で保護されたアミノ基を脱保護し、下記式（３ｂ）

（式中、Ｚ^２、Ｒ^３、Ｒ^４は前記に同じ。但し、Ｒ^３とＲ^４の少なくとも一方が水素原子以外の基を示す）
で表される化合物と反応させる工程（Ａ）により得ることができる。 The compound of this invention can be manufactured using a well-known method. Specifically, for example, a compound in which at least one of R ¹ and R ³ and at least one of R ³ and R ⁴ in formula (1) is a group other than a hydrogen atom can be obtained according to the following step. That is, the following formula (3)

(Wherein R ^{5 to} R ¹⁰ and X are the same as above)
After protecting one amino group contained in the formula (3) with a protecting group (for example, tert-butoxycarbonyl group: Boc etc.), the compound represented by the following formula (3a)

(Wherein Z ¹ , R ¹ , R ² and X are the same as above, provided that at least one of R ¹ and R ² represents a group other than a hydrogen atom)
And then deprotecting the amino group protected with a protecting group, and the following formula (3b)

(In the formula, Z ² , R ³ and R ⁴ are the same as above, provided that at least one of R ³ and R ⁴ represents a group other than a hydrogen atom.)
It can obtain by the process (A) made to react with the compound represented by these.

で表される化合物と反応させ、次いで下記式（４ａ）

（式中、環Ｚ^１は前記に同じ）
で表される化合物と反応させ、次いで保護基で保護されたアミノ基を脱保護した後、前記式（３ｂ）で表される化合物を導入する工程（Ｂ）により得ることができる。 Moreover, the compound whose R < ¹ > and R < ² > in Formula (1) are both hydrogen atoms can be obtained according to the following process, for example. That is, after protecting one amino group contained in the formula (3) with a protecting group in the compound represented by the formula (3), the following formula (3c)

And then reacting with the compound represented by formula (4a)

(Wherein ring Z ¹ is the same as above)
And then deprotecting the amino group protected by the protecting group, and then introducing the compound represented by the formula (3b) (B).

（式中、環Ｚ^２は前記に同じ）
で表される化合物を反応させる工程（Ｃ）により得ることができる。 The compound in which R ³ and R ⁴ in the formula (1) are both hydrogen atoms is, for example, a compound represented by the formula (3c) instead of the compound represented by the formula (3c) in the step (A). And then the following formula (4b)

(Wherein ring Z ² is the same as above)
It can obtain by the process (C) with which the compound represented by this is made to react.

  In said process (A)-(C), the usage-amount of the compound represented by Formula (3a)-(3c) and Formula (4a), (4b) is 1 equivalent of the compound represented by Formula (2). On the other hand, it is about 0.05 to 50 equivalents, preferably 0.1 to 10 equivalents, and more preferably about 0.3 to 5 equivalents. The compounds represented by the formulas (3a) to (3c) and the formulas (4a) and (4b) can be used in the same equivalent amount as the compound represented by the formula (3).

Here, the compounds represented by the formulas (3a) and (3b) use, for example, an amino acid as a raw material, and after protecting the carboxyl group of the amino acid with a protecting group, introduce the ring Z ¹ into the α-amino group Then, the protective group can be deprotected to give a carboxyl group (D).

  Each reaction in the steps (A) to (D) is performed in the presence or absence of a solvent. Examples of the solvent include aliphatic hydrocarbons such as hexane, heptane, and octane; alicyclic hydrocarbons such as cyclohexane; aromatic hydrocarbons such as benzene, toluene, xylene, and ethylbenzene; chloroform, dichloromethane, 1,2- Halogenated hydrocarbons such as dichloroethane; ethers such as diethyl ether, dimethoxyethane, tetrahydrofuran and dioxane; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate, ethyl acetate, isopropyl acetate and butyl acetate; N, N-dimethylformamide; Amides such as N, N-dimethylacetamide; nitriles such as acetonitrile, propionitrile, and benzonitrile. These solvents are used alone or in admixture of two or more.

  The temperature of each reaction can be selected from the range of, for example, 0 to 150 ° C., preferably 20 to 120 ° C., more preferably 40 to 80 ° C., depending on the type of substrate, the type of reaction, etc. It is also possible to carry out at room temperature. Each reaction can be performed at normal pressure or under pressure. The reaction time can be appropriately selected from the range of about 30 minutes to 48 hours, for example, depending on the reaction temperature and pressure. After completion of the reaction, the reaction product can be separated and purified by general separation and purification means such as liquid property adjustment, filtration, concentration, crystallization, washing, recrystallization, extraction, distillation, sublimation purification, column chromatography and the like.

  The compound of the present invention has an action of binding to a normal prion protein and preventing structural transformation. Therefore, the compound of the present invention is useful as an active ingredient of a structure conversion inhibitor of normal prion protein and a prophylactic / therapeutic agent for prion disease. The compound of the present invention can also be used by adding to foods and beverages. Here, the prion disease in the present invention is a disease caused by an infectious prion protein produced by structural conversion of a normal prion protein, such as sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob. Diseases, GSS, FFI, Kuru, mutant Jacob disease and the like.

  The prion protein structure conversion inhibitor and the prion disease preventive / therapeutic agent of the present invention contain the compound of the present invention as an active ingredient, and may further contain other pharmaceutically acceptable ingredients. These drugs can be used for animals such as mammals such as humans, cows, sheep and horses. Examples of the pharmaceutically acceptable ingredients include excipients, lubricants, binders, disintegrants mainly used for solid preparations, solvents, dissolution aids, and suspending agents mainly used for liquid preparations. , Tonicity agents, buffering agents, soothing agents, and the like, and antiseptics, antioxidants, coloring agents, sweetening agents, adsorbents, wetting agents, and the like can also be used.

  Examples of the dosage form include injections, creams, ointments, beverages, aerosols, skin gels, eye drops, in which an effective amount of a compound is dissolved, dispersed, and emulsified in a diluent or dispersion medium such as water or physiological saline. Liquid preparations such as nasal drops; solid preparations such as tablets, capsules, powders, granules, tablets, remedies, suppositories, etc. can be used, and these active ingredients are encapsulated in liposomes, sustained-release materials, etc. It may be in the form of an enclosure or a carrier carried on a carrier.

  The drug can be administered either orally or parenterally, and it can be administered into the whole body by intraarterial, intravenous, intramuscular, subcutaneous, intraperitoneal, intrarectal or by respiration, percutaneous, nasal, ophthalmic, etc. Or it can carry out by methods, such as local administration. Moreover, the chemical | medical agent of this invention can also be directly administered to a brain with an intraventricular pump.

  The doses of the prion protein structure conversion inhibitor and the prion disease preventive / therapeutic agent of the present invention are the types of the active compound, the administration method, the species to be administered, age, weight, symptoms, drug characteristics, medical history, respectively. For example, the dose is in the range of several μg to several tens of mg per kg of body weight per time, and can be administered several times a week to several times a day.

  The pharmacological activity test is not particularly limited as long as it is a method capable of evaluating the structure conversion inhibitory activity of normal prion protein. For example, the detection of infectious prion protein produced by prion-infected cells in the presence of a test substance is detected. Means can be used. The prion-infected cell can be generated by infecting a prion-infected cell with a prion by a known method. For detection of infectious prion protein, a known method capable of detecting a specific protein can be used, and a quantitative detection method is preferably used. As the quantitative detection method, for example, a means for recognizing a specific protein such as an antibody, a nucleic acid, or an analog thereof (peptide, PNA, etc.), and an image analysis means (ELISA) for a protein labeled with a phosphor or radiation. , ECL-plus Western blotting, etc.) are often combined with a means for quantifying a recognized protein.

  As a specific example of a pharmacological activity test, a mouse neuron cell line is infected with prion, and then cultured for a certain period in a medium to which a test substance (drug) is added at various concentrations. And the like can be used for the detection of antibodies with antibodies and the like and quantification by image analysis or the like. In the above test, it can be evaluated that the smaller the amount of infectious prion protein produced, the better the prion protein structural change inhibitory activity and the higher the effect of treating and preventing prion diseases.

  In the above test method, the compound of the present invention has a relative value of, for example, 20 or less, preferably 15 or less, and more preferably 15 or less, when the production amount of infectious prion protein when the compound is not added is 100. Has an effect of reducing it to 13.5 or less, particularly about 12 or less. The compounds of the present invention are also represented by 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl]-which has already been reported by the inventors. Compared with phenyl] -acetamide (see Japanese Patent Application Laid-Open No. 2005-120002), it has an excellent effect of suppressing the production of infectious prion protein, and in particular, a high effect is obtained with the compound represented by the formula (2). Can do. Thus, since the compound of the present invention is extremely excellent in the effect of suppressing the structural conversion of normal prion protein, it can be used as an active ingredient of a prion protein structural conversion inhibitor or a preventive / therapeutic agent for prion disease. Can be effectively prevented, and after the onset of symptoms, an excellent therapeutic effect can be exhibited by preventing or delaying the progression of symptoms.

  The kit of the present invention includes a compound and / or a derivative thereof, a cell infected with a prion, and a means for detecting an infectious prion protein. As a means for detecting cells infected with prions and infectious prion protein, those exemplified in the pharmacological evaluation test can be used. The kit of the present invention can be used for screening for a compound that suppresses the structural conversion of prion protein, testing, evaluation, research, and the like using the compound.

  The compound of the present invention can also be used in a method for efficiently screening a compound optimized for the above pharmaceutical use based on the compound. For screening, a known technique such as in vivo, in vitro, in silico or the like can be used. In particular, in the present invention, it is preferable to use a method of performing in vitro and / or in vivo screening for a candidate compound obtained by in silico screening (virtual screening), because it is possible to efficiently select a compound that can exert an excellent pharmacological effect. . For the in silico screening, for example, a screening method using docking simulation described later is preferably used.

  The method of the present invention uses the compound of the present invention and / or a derivative thereof to screen for a compound having a prion protein structure conversion inhibitory activity based on the binding strength predicted by docking simulation with a normal prion protein. Is the method. For screening, a library is usually used. The library may be composed of the derivative of the present invention, and may be a known or unknown compound created on a computer using software or the like. Commercially available products can also be used

  The derivative of the compound of the present invention includes, for example, a compound partially modified by introducing a known substituent into the compound of the present invention. As such substituents, for example, those exemplified above can be used. However, derivatives of the compounds of the present invention include 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl] -acetamide. Absent. A library can be constructed with the above-described derivatives of the compound of the present invention and used for optimizing lead compounds using the structure of the compound of the present invention. Hereinafter, the compounds of the present invention, derivatives thereof, and libraries used for docking simulation may be collectively referred to as “test substances”.

  The docking simulation is generally performed on a computer by a docking simulation software program based on the three-dimensional structure information of the test substance and the normal prion protein. The three-dimensional structure information can be obtained as registered information in a known database such as Protein Data Bank (PDB) for known proteins, and individually created by a method such as modeling using a general-purpose program for new substances. be able to.

  The three-dimensional structure information of the test substance of the present invention can be created using modeling software such as ChemOffice manufactured by CambridgeSoft. For example, after modeling individually by manual operation on the GUI using ChemOffice, create 3D structure information by minimizing energy using the AM1 method with the semi-empirical molecular modeling application CSMOPAC attached to ChemOffice. Can do. By further processing the information obtained in this manner, such as addition of electric charge or deletion of hydrogen atoms, the interaction with the protein can be analyzed with higher accuracy. Specifically, charges were added using the MQEq method (modified charge equilibrium method: Chem-Bio Info. J. 1, 35-40 (2001), etc.). The three-dimensional structure information of the compound of the present invention and the library compound can be obtained by a known method, and is not limited to the above method.

  The three-dimensional structure information of prion protein can be obtained from Protein Data Bank (PDB). For example, the three-dimensional structure information of normal mouse prion protein can use registration information PDB code 1AG2 whose structure is determined by NMR. Furthermore, processes such as addition of charges and deletion of hydrogen atoms can be performed according to the Amber United-atom model using AutoDockTools (manufactured by Scripps Research Institute).

  Prediction of binding strength by docking simulation, for example, focuses on the binding pockets present in normal prion protein, predicts the binding free energy between the test substance and normal prion protein, It can be carried out by a method for evaluating that the bond is strong. For example, a method disclosed in JP 2005-120002 A can be used.

  As the binding pocket, for example, a binding pocket between the A-S2 loop and the C-end side of helix B can be used, and “focusing on the binding pocket” means that the binding site is sufficiently centered. This means setting a grid box of a large size. The size of the grid box is not particularly limited, but can be set to, for example, a size of 45 mm × 45 mm × 30 mm.

  As the software program for docking simulation, one or a plurality of software programs for docking simulation can be used in combination, and for example, known programs such as DOCK, AutoDock, and GOLD can be used. For example, according to the AutoDock, it is possible to perform an automatic docking simulation by the flexible docking method in which the position of the normal prion protein is fixed and only the structural change of the compound is considered.

  More specifically, the automatic docking simulation using AutoDock searches the optimal binding state of the library and normal prion protein by a Lamarckian genetic algorithm, and tens of millions per compound for each compound. The method for evaluating the docking mode is repeated several times, and the lowest free energy is predicted as the binding free energy between the compound and the prion protein. On the other hand, for the compound of the present invention and normal prion protein, the binding free energy is predicted by the same method as described above. It can be evaluated that the lower the value of the binding free energy thus obtained, the stronger the binding, that is, the better the effect of suppressing the structural change of the prion protein.

  Specifically, the compound represented by the formula (2) has a binding free energy predicted based on the above method as low as −1.43 kcal / mol, and its effectiveness by a pharmacological activity test has been confirmed. Based on this finding, compounds having a binding free energy of less than −11.43 kcal / mol, for example, all the compounds shown in FIG. 2 to FIG. 9 are as useful as or more than the compounds represented by formula (2). It is estimated that there is.

  Among the compounds represented by the formula (1), a compound group that is estimated to be useful as a therapeutic / preventive agent for prion diseases using binding free energy as an index includes, for example, compounds having binding free energy of less than −14.00 kcal / mol ( For example, 6LQ-C-5G, 5LR-C-5G, MLR-C-5G, 6LK-C-5G, 5LA-S-6DH, 6LN-C-5G, 5G-C-6DH in FIGS. , 6LR-C-5G, 5LH-C-5G, etc.), followed by a compound group of −14.00 kcal / mol or more and less than −13.00 kcal / mol, −13.00 kcal / mol or more, − It can be estimated that it is effective in the order of the compound group of less than 12.00 kcal / mol. Furthermore, since the compound group having a bond free energy of -12.00 kcal / mol or more and less than -11.00 kcal / mol has the same bond free energy as the compound represented by formula (2), the formula (2) It can be presumed that the possibility of exhibiting the same degree of pharmacological activity as that of the compound is high.

  Furthermore, these compounds or their derivatives are used in a method for screening a compound having a prion protein structure conversion inhibitory activity, and based on the binding strength predicted by docking simulation with a normal prion protein, treatment of prion disease -It can also be used to search for active ingredients of prophylactic agents.

  Compared with known compounds that bind to prion protein, the compound of the present invention has stronger binding to normal prion protein by docking simulation and can exhibit superior pharmacological activity by the above test. Therefore, the compound of the present invention can be used not only as an active ingredient of a prion protein structure conversion inhibitor or a prion disease prophylactic / therapeutic agent itself, but also by creating a library comprising derivatives of the compound to further increase the pharmacological effect. It can be preferably used for the development of a drug excellent in biocompatibility and the like.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

で表される２−ピロリジン−１−イル−Ｎ−［４−｛４−（２−ピロリジン−１−イル−アセチルアミノ）−ベンジル｝−フェニル］−プロピオンアミドの合成
４，４’−メチレンジアニリン１２ｇに対し、１／２当量のジカルボン酸ジ−ｔｅｒｔ−ブチル（Ｂｏｃ無水物）を反応させ、アミンの片方のみがBoc基で保護された反応生成物について、ニンヒドリン試薬等を用いて、薄相クロマトグラフィー（TLC）で確認しながら順相カラムクロマトグラフィにより精製を行い、ほぼ当量の４−（４−ｔｅｒｔ−ブトキシカルボニルアミノベンジル）アニリン１８ｇを得た。 Example 1
Following formula (2)

Synthesis of 2-pyrrolidin-1-yl-N- [4- {4- (2-pyrrolidin-1-yl-acetylamino) -benzyl} -phenyl] -propionamide represented by the formula 4,4′-methylenedi About 12 g of aniline, 1/2 equivalent of di-tert-butyl dicarboxylate (Boc anhydride) was reacted, and the reaction product in which only one of the amines was protected with a Boc group was diluted with a ninhydrin reagent or the like. Purification was performed by normal phase column chromatography while confirming by phase chromatography (TLC) to obtain 18 g of approximately equivalent 4- (4-tert-butoxycarbonylaminobenzyl) aniline.

で表される化合物２．８ｇを得た。 Fmoc-L-alanine was reacted with 4 g of 4- (4-tert-butoxycarbonylaminobenzyl) aniline obtained using a solution obtained by dissolving 20.4 g of HBTU and 8.2 g of HOBt in 100 mL of DMF as a condensing agent. . After the reaction mixture was stirred at room temperature for 1 hour, the condensing agent was removed by partitioning with dichloromethane, and the Fmoc group was deprotected with a DMF solution containing 20% piperidine in the residue. The mixture is purified by normal phase column chromatography, and the following formula (X)

2.8 g of a compound represented by the formula:

で表される化合物を１．８ｇ得た。得られた前記式（Ｙ）で表される化合物１ｇに対し、ジクロロエタン中、ＴＦＡを反応させてＢｏｃ基を脱保護し、次いで以下に示す方法で１−ピロリジン酢酸との縮合反応を行った。ＤＭＦ中、４当量の１−ピロリジン酢酸、ＨＢＴＵ、及びＨＯＢｔの共存下、室温で２時間撹拌後、溶媒を洗浄、留去し、次いで順相カラムクロマトグラフィ及び逆相HPLCにより精製を行い、目的の２−ピロリジン−１−イル−Ｎ−［４−｛４−（２−ピロリジン−１−イル−アセチルアミノ）−ベンジル｝−フェニル］−プロピオンアミドを収率12.3％で得た。
以下、２−ピロリジン−１−イル−Ｎ−［４−｛４−（２−ピロリジン−１−イル−アセチルアミノ）−ベンジル｝−フェニル］−プロピオンアミドを、「5G-C-5LA」と称する。
［目的化合物 のスペクトルデータ］
¹³C-NMR（CDCl₃） δ：168.9, 137.0, 136.9, 136.1, 135.9, 129.4, 119.7, 119.7, 64.2, 59.8, 54.6, 51.2, 40.8, 38.6, 29.7, 24.1, 23.6, 16.4
HR-FAB-MS: m/z 434.2680 ([M+H]⁺）C₂₆H₃₄N₄O₂ By adding 2.8 g of 1,4-dibromobutane to 1 g of NaHCO _{3 in} 100 mL of ethanol and heating and stirring at 50 ° C. with respect to 2 g of the compound represented by the formula (X) thus obtained, the following formula (Y)

As a result, 1.8 g of a compound represented by the formula: 1 g of the obtained compound represented by the formula (Y) was reacted with TFA in dichloroethane to deprotect the Boc group, and then subjected to a condensation reaction with 1-pyrrolidineacetic acid by the method shown below. After stirring for 2 hours at room temperature in the presence of 4 equivalents of 1-pyrrolidineacetic acid, HBTU and HOBt in DMF, the solvent was washed and distilled off, and then purified by normal phase column chromatography and reverse phase HPLC. 2-Pyrrolidin-1-yl-N- [4- {4- (2-pyrrolidin-1-yl-acetylamino) -benzyl} -phenyl] -propionamide was obtained in a yield of 12.3%.
Hereinafter, 2-pyrrolidin-1-yl-N- [4- {4- (2-pyrrolidin-1-yl-acetylamino) -benzyl} -phenyl] -propionamide is referred to as “5G-C-5LA”. .
[Spectral data of target compound]
¹³ C-NMR (CDCl ₃ ) δ: 168.9, 137.0, 136.9, 136.1, 135.9, 129.4, 119.7, 119.7, 64.2, 59.8, 54.6, 51.2, 40.8, 38.6, 29.7, 24.1, 23.6, 16.4
HR-FAB-MS: m / z 434.2680 ([M + H] ⁺ ) C ₂₆ H ₃₄ N ₄ O ₂

Example 2
In silico screening using compound 5G-C-5LA obtained in Example 1 [Library creation]
A library composed of derivatives obtained by introducing various substituents into the compound obtained in Example 1 is prepared.
[Obtaining three-dimensional structure information]
The three-dimensional structure information of the compound obtained in Example 1 was individually modeled manually by GUI operation using ChemOffice (CambridgeSoft), and then energy minimization was performed using AM1 method with CS MOPAC attached to ChemOffice. Created. The charge was applied using the MQEq method (modified charge balance method) developed by Nakano et al.
For the three-dimensional structure information of the normal prion protein, a normal mouse prion protein (PDB code: 1AG2) registered in the Protein Data Bank (PDB) and whose structure was determined by NMR was used. Addition of charge information to each atom of the prion protein and subsequent hydrogen deletion were performed by the Amber United-Atom model method using AutoDockTools developed and published at the Script Institute of the United States.

[Coupling site prediction]
The binding site of the normal prion protein with the compound obtained in Example 1 was predicted based on information on the slow fluctuation of NMR. A grid box with a size of 45 mm x 45 mm x 30 mm was set around the vicinity predicted to be a binding site in this way and used for in silico screening.
[In silico screening]
For the library compound and normal mouse prion protein, an automatic docking simulation using AutoDock was performed to predict the binding free energy of both. Specifically, the optimal binding state is searched by a Lamarckian genetic algorithm, and 25 million docking modes are evaluated once for one compound, and this is repeated 10 times, and the lowest among them. The free energy was predicted as the binding free energy of the compound and mouse prion protein (J. Computational Chemistry, 19: 1639-1662 (1998)). The binding free energy of the compound obtained in Example 1 obtained by the above simulation was −11.43 kcal / mol.

  Table 1 shows compounds (less than −11.43 kcal / mol) whose binding free energy values were lower than those obtained in Example 1 among the library compounds as a result of the simulation. In Table 1, “name” indicates a compound corresponding to the structural formula shown in FIGS.

  From these results, the compounds shown in Table 1 can bind to the normal prion protein more firmly than the compound 5G-C-5LA obtained in Example 1, and normality is achieved through the binding. It can be easily predicted that the compound is excellent in activity to inhibit the structural conversion of the type prion protein. Here, 5G-C-5LA has been confirmed to be a compound that exhibits an excellent pharmacological activity of suppressing the production of infectious prion protein, as shown in an evaluation test described later. Therefore, it can be predicted that the compounds shown in Table 1 can exhibit pharmacological activity equivalent to or higher than that of 5G-C-5LA. Therefore, these compounds are extremely useful as an active ingredient constituting a preventive / therapeutic agent for prion diseases or as a lead compound for searching for such compounds. Further, by obtaining a compound having a structure conversion inhibitory activity of prion protein by in silico screening using these compounds themselves or derivatives thereof, compounds useful for the prevention / treatment of prion disease can be easily searched. .

Evaluation test The compound obtained in Example 1 was subjected to a pharmacological test by the following method.
[cell]
Mouse hypothalamic neuronal lineage GT1 can infect mouse prions. In order to evaluate the anti-prion action of the compounds, we used the GT ^FK-1 cell line (N. Nishida et al., J. Virol, 74, 320-325 (2000)). These are Fukuoka-1 strains stably infected with mouse-compatible prions derived from GSS (O. Milhavet et al., Proc. Natl. Acad. Sci. USA, 97, 13937-13942 (2000)).
[Prion infection treatment]
GT1-7 cells were cultured in DMEM medium containing penicillin (GIBCO / BRL). Every 3-4 days, cells were trypsinized, diluted 1: 5 and passaged. GT1-7 cells stably infected with Fukuoka-1 or 22L strain are described in O. Milhavet et al., Proc. Natl. Acad. Sci. USA 97, 13937-13942 (2000). Compound stock solutions (19 of 59 selected compounds) were prepared in 100 mM DMSO and stored at 4 ° C. Prior to use, the compounds were diluted with media. Control cells were treated with medium containing only solvent (0.1%). Approximately 2 × 10 ⁵ cells were placed in each well of a 6-well plate and drug treatment was initiated 15 hours after compound addition. Cells were harvested after 72 hours of incubation.

[Quantification of infectious prion protein]
The collected cells were lysed in 150 μl of 1 × Triton / DOC lysis buffer (0.5% tritonX-100, 0.5% deoxycholic acid, 150 mM NaCl, 25 mM Tris HCl pH 7.5, 2 mM EDTA and pepstatin, leupepsin). Protein concentration was measured by BCS assay (Pierce) and each sample was normalized to 2 mg protein. To analyze PrPsc production, the protein was digested with proteinase K at a concentration of 10 μg / mg for 30 minutes at 37 ° C. Digestion was inhibited with PMSF (2 mM) and samples were subjected to SDS / PAGE using a 15% SDS / PAGE gel. The protein was then transferred to a PVDF membrane (Immobilon-P, Amersham, USA). Anti-mouse PrP antibody (SS28) was used for PrPres. For detection of PrPC, SAF32 antibody (Spi-Bio, France) was used as the primary antibody. The signal was visualized by ECL-plus Western blotting (Amersham) and exposed to X-ray film (Konica). Signal quantification was performed by scanning the membrane with Fluorochem (Alpha Innotech, US).

These results are shown in the graph of FIG. In FIG. 1, “DMSO 0.1%” indicates that the compound was not added, “5G-C-5LA” indicates that the compound synthesized in Example 1 was added, and “GN8” Infection with the addition of 2-pyrrolidin-1-yl-N- [4- [4- (2-pyrrolidin-1-yl-acetylamino) -benzyl] -phenyl] -acetamide disclosed in 120002 The production amount of the type prion protein is shown as a relative value. As a result, when “DMSO 0.1%” was taken as 100, GN8 was 15.97, whereas “5G-C-5LA” as the compound of the present invention was 13.16. Cells treated with 5G-C-5LA for 72 hours had significantly reduced infectious prion protein production compared to DMSO0.1%, which was not treated with the same compound, and GT ^FK-1 It was found that the production of scrapie protein was strongly suppressed in the cell lineage. In addition, it was confirmed that 5G-C-5LA suppressed the production of infectious prion protein by 20% or more compared to GN8.

  According to the compound of the present invention, it is possible to efficiently develop a drug useful for the prevention and treatment of prion diseases.

It is a graph which shows the evaluation test result of the pharmacological activity in an Example. 1 is a structural formula showing an example of a compound of the present invention. It is structural formula which shows the other example of the compound of this invention. It is a structural formula showing still another example of the compound of the present invention. It is a structural formula showing still another example of the compound of the present invention. It is a structural formula showing still another example of the compound of the present invention. It is a structural formula showing still another example of the compound of the present invention. It is a structural formula showing still another example of the compound of the present invention. It is a structural formula showing still another example of the compound of the present invention.

Claims (8)

Following formula (1)

(Wherein R ^{1 to} R ⁴ are the same or different and are a hydrogen atom, a halogen atom, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group, carboxyl Group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercapto group, substituted thio group, or substituted or unsubstituted R ^{5 to} R ¹² are the same or different and each represents a hydrogen atom, a halogen atom, a hydrocarbon group which may have a substituent, or a heterocyclic group which may have a substituent. , Carboxyl group, substituted oxycarbonyl group, substituted or unsubstituted carbamoyl group, cyano group, acyl group, nitro group, sulfur acid group, sulfur acid ester group, hydroxyl group, substituted oxy group, mercap Group, a substituted thio group, or .X showing a substituted or unsubstituted amino group is a single bond or a linking group. Ring Z ¹ and the ring Z ² is a good nitrogen atom-containing ring which may have a substituent Provided that at least one of R ^{1 to} R ⁴ represents a group other than a hydrogen atom)
A compound represented by In formula (1), R ^{1 to} R ⁴ are a hydrogen atom or a hydrocarbon group which may have a substituent, and R ^{5 to} R ¹² are the same or different, and a hydrogen atom, a halogen atom, substituent a hydrocarbon group which may have a hydroxyl group, a substituted oxy group, a mercapto group, or a substituted thio group, X is C ₁ -C ₄ alkylene group, an oxygen atom (an ether bond; -O-) Or a sulfur atom (thioether bond; -S-), and ring Z ¹ and ring Z ² are the same or different and may have a substituent, a 5-membered or 6-membered nitrogen atom-containing ring The compound according to claim 1, wherein at least one of R ^{1 to} R ⁴ is a hydrocarbon group which may have a substituent. Following formula (2)

A compound represented by   A prion protein structure conversion inhibitor comprising the compound according to any one of claims 1 to 3 as an active ingredient.   A preventive / therapeutic agent for prion diseases comprising the compound according to any one of claims 1 to 3 as an active ingredient.   6. The prion disease prevention according to claim 5, wherein the prion disease is at least one selected from the group consisting of sheep scrapie, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, GSS, FFI, Kuru, and mutant Jacob disease.・ Therapeutic agent.   A kit comprising the compound and / or derivative thereof according to any one of claims 1 to 3, a prion-infected cell, and a means for detecting an infectious prion protein.   A compound having a structure conversion inhibitory activity of prion protein based on binding strength predicted by docking simulation with normal prion protein using the compound and / or derivative thereof according to any one of claims 1 to 3. How to screen.

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