JPH08311050A - Suppressor against nitrogen monoxide formation and its screening - Google Patents

Abstract

PURPOSE: To obtain a new compound useful as a curing agent for a disease associating the degeneration damage to nervous tissue, such as Alzheimer disease and capable of selectively suppressing NO-generation by cells which generate an induced type of a nitrogen monoxide-synthesizing enzyme (iNOS) in glare cells, macrophages, etc. CONSTITUTION: This is a compound of formula I [R is a heterocyclic group; X is CH2 , CO or CN=CH; m is an integer of 0-3; p is 1, 2] or its salt, e.g. a compound of formula II. A compound of formula I is obtained e.g. by reacting an aldehyde derivative of formula III with a phosphonium salt of formula IV (Q is a halogen). Further, an NO-generation inhibitor is obtained by using a compound of formula I or its pharmacologically allowable salt as an effective component. Furthermore, the evaluation of an inhibiting activity against NO- generation of a compound of formula I is performed by adding a β-amyloid protein or its fractions together with a compound to be tested to intracerebral cells, by which NO is derived by the β-amyloid protein or its fractions, and culturing the intracerebral cells followed by measuring an NO formation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a nitric oxide production inhibitor and a screening method, and more specifically, a compound which selectively suppresses nitric oxide production from inducible nitric oxide synthase (iNOS) producing cells and Regarding its use.

[0002]

2. Description of the Related Art Nitric oxide (NO) is a physiological activity in the body of mammals, for example, as a vasodilator in the vascular system [Pharmacol.
ev.) Vol. 43, pp. 109-142 (1991)],
In the leukocyte system, [C
urr. Opin. Immunol. Volume 3, pp. 65-70 (19
91)], and as a neurotransmitter in the nervous system [Neuron, Vol. 8, pp. 3-11 (1992)], etc.
It is considered to have various roles. Basically, NO is produced from L-arginine by NO synthase (NOS). Currently, at least two types of N
Presence of OS is confirmed. One is the non-inductive type (consti
tutive type), which is present in vascular endothelial cells and nerve cells, is calcium / calmodulin dependent, and is activated by various receptor stimuli under physiological conditions. The other is inducible
ype), which is induced in macrophages and vascular endothelial cells by various cytokines. The latter is calcium calmodulin-independent, with NO synthesis lasting longer than the former once activated. Interestingly, it has been pointed out that NO synthesized by the latter acts cytotoxically in various ways. Sepsis
s), endotoxemia, etc.-When NO is excessively produced in vivo, hypotension and shock are induced by its vasorelaxant action, cerebral ischemic disease and certain inflammations. In sexual encephalopathy (viral encephalitis) and the like, it has been suggested that the chemical reactivity of NO itself as a radical may cause various cell and tissue disorders. So far, compounds such as NOS inhibitors and NO eliminators have been used in order to analyze the mechanism of expression of such pathophysiological activity of NO in vivo. However, on the other hand, regarding the administration of these compounds to a biological system, not only adverse effects on metabolic systems other than NO synthesis system, but also suppression of NO production due to non-inducible NOS, thereby circulatory system of living body, There is also concern that it may impair nerve function.

[0003]

Therefore, iNOS such as glial cells and phagocytic cells can be obtained without affecting NO production from cNOS-retaining cells such as vascular endothelial cells and nerve cells.
If NO production from producing cells can be suppressed, the involvement of NO in the pathological condition can be clarified, and it can be applied to the treatment of diseases. In particular, in the central nervous system, it is considered possible to treat Alzheimer's disease, ischemic diseases, etc. without impairing higher-order nerve functions.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have revealed that NOS inducer containing β-amyloid protein induces iNOS in glial cells and the like to produce NO. Furthermore, by screening the inhibitory substance, a compound that selectively suppresses NO generation due to iNOS induced in glial cells and phagocytic cells was found. By using the compound, it becomes possible to treat diseases associated with degenerative disorders of nerve tissue without affecting the NO production derived from cNOS, which is said to play a physiological role. Further, the screening method using the inducer can be a useful means for discovering a therapeutic drug for a disease associated with a neurodegenerative disorder such as Alzheimer's disease.

That is, the present invention provides (1) general formula (I)

Embedded image

(In the formula, R is an optionally substituted heterocyclic group, and X is -CH _2- , -CO- or -CH = CH-
, M is an integer of 0 to 3, and p is 1 or 2. ) Or a salt thereof, (2) a nitric oxide production inhibitor comprising the compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof, (3) β-
Nitric oxide is induced by amyloid protein or its fragments.Brain cells are cultured by adding β-amyloid protein or its fragments and a test compound to the brain cells, and measuring the amount of nitric oxide produced. A method for determining the nitric oxide production inhibitory activity of a characteristic test compound, and (4)
The present invention provides a nitric oxide synthase inducer containing β-amyloid protein or a fragment thereof.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION In the above general formula (I), the heterocyclic group in the optionally substituted heterocyclic group represented by R is, for example, one sulfur atom, nitrogen atom or oxygen atom. 5 to 7-membered heterocyclic group containing, 5 containing 2 to 4 nitrogen atoms
A 6-membered heterocyclic group, a 5- or 6-membered heterocyclic group containing 1-2 nitrogen atoms and 1 sulfur atom or oxygen atom, and these heterocyclic groups contain 2 or less nitrogen atoms. It may be condensed with a 6-membered ring, a benzene ring or a 5-membered ring containing one sulfur atom. Specific examples of the heterocyclic group include, for example, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-
Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl,
6-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 2-pyrrolyl, 3-pyrrolyl, 2
-Imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-pyrazolyl, 4-pyrazolyl, isothiazolyl, isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 1,2 , 4-triazole-3
-Yl, 1,3,4-triazol-2-yl, 1,2,3
-Triazol-4-yl, tetrazol-5-yl,
Benzimidazol-2-yl, indol-3-yl, benzpyrazol-3-yl, 1H-pyrrolo [2,
3-b] pyrazin-2-yl, 1H-pyrrolo [2,3-
b] Pyridin-6-yl, 1H-imidazo [4,5-b]
Pyridin-2-yl, 1H-imidazo [4,5-c] pyridin-2-yl, 1H-imidazo [4,5-b] pyrazin-2-yl and the like (preferably one of oxazolyl, thiazolyl and the like And a 5- or 6-membered heterocyclic group containing a nitrogen atom and one oxygen atom or a sulfur atom).

In the above general formula (I), the heterocyclic group represented by R may have 1 to 3 substituents at any position on the ring. As such a substituent, an aliphatic chain hydrocarbon group, an alicyclic hydrocarbon group, an aryl group, an aromatic heterocyclic group,
Non-aromatic heterocyclic group, halogen atom, nitro group, optionally substituted amino group, optionally substituted acyl group, optionally substituted hydroxyl group, optionally substituted thiol group, ester And a carboxyl group which may be compounded (preferably an aliphatic chain hydrocarbon group, an aryl group, an aromatic heterocyclic group, etc.) and the like. As such an aliphatic chain hydrocarbon group, a linear or branched aliphatic hydrocarbon group, for example, an alkyl group, preferably an alkyl group having 1 to 10 carbon atoms, an alkenyl group, preferably 2 carbon atoms. An alkenyl group of -10, an alkynyl group,
Preferred is an alkynyl group having 2 to 10 carbon atoms. Preferred examples of the alkyl group include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, heptyl, octyl, nonyl,
Decyl etc. are mentioned. Preferable examples of the alkenyl group include, for example, vinyl, allyl, isopropenyl, 1-
Propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-ethyl-1-butenyl, 3-methyl-2-butenyl, 1-pentenyl,
2-pentenyl, 3-pentenyl, 4-pentenyl, 4
-Methyl-3-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and the like can be mentioned. Preferable examples of the alkynyl group include, for example, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl and 1-.
Pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like can be mentioned. Of these, an alkyl group (particularly methyl) and the like are preferable.

Examples of the alicyclic hydrocarbon group include saturated or unsaturated alicyclic hydrocarbon groups such as cycloalkyl group, cycloalkenyl group and cycloalkadienyl group. The alicyclic hydrocarbon group has 10 carbon atoms.
The following are preferable, and preferable examples of the cycloalkyl group include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo [2.2.1] heptyl, bicyclo [2.2.2]. ] Octyl, bicyclo [3.2.1] octyl, bicyclo [3.2.2] nonyl, bicyclo [3.3.
1] nonyl, bicyclo [4.2.1] nonyl, bicyclo [4.3.1] decyl and the like. Preferable examples of the cycloalkenyl group include 2-cyclopenten-1-yl, 3-cyclopenten-1-yl, 2-cyclohexen-1-yl, 3-cyclohexen-1-yl and the like. Preferable examples of cycloalkadienyl group include, for example, 2,4-cyclopentadiene-1-
Yl, 2,4-cyclohexadiene-1-yl, 2,5-
Examples thereof include cyclohexadiene-1-yl. The aryl group means a monocyclic or condensed polycyclic aromatic hydrocarbon group, and preferable examples thereof include those having 6 carbon atoms.
Phenyl, naphthyl, anthryl, phenanthryl, acenaphthylenyl and the like of about 14 to 14 are mentioned, and among them, phenyl, 1-naphthyl, 2-naphthyl and the like (especially phenyl) are preferable.

Preferred examples of the aromatic heterocyclic group include furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, flazanyl, 1,2,3-thiadiazolyl, 1,2,
4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,
In addition to carbon atoms such as 2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl and triazinyl, 1 to 4 heteroatoms selected from oxygen atom, sulfur atom and nitrogen atom. 5- to 6-membered aromatic monocyclic heterocyclic groups including; for example, benzofuranyl, isobenzofuranyl, benzo [b] thienyl, indolyl, isoindolyl, 1H-
Indazolyl, benzimidazolyl, benzoxazolyl, 1,2-benzisoxazolyl, benzothiazolyl, 1,2-benzisothiazylyl, 1H-benzotriazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, naphthyridinyl , Purinyl, pteridinyl, carbazolyl, α-
Carborinyl, β-carbolinyl, γ-carbolinyl,
Acridinyl, phenoxazinyl, phenothiazinyl,
Phenazinyl, phenoxathinyl, thianthrenyl,
Phenatridinyl, phenatrolinyl, indoridinyl, pyrrolo [1,2-b] pyridazinyl, pyrazolo [1,
5-a] pyridyl, imidazo [1,2-a] pyridyl, imidazo [1,5-a] pyridyl, imidazo [1,2-b] pyridazinyl, imidazo [1,2-a] pyrimidinyl, 1,
2,4-triazolo [4,3-a] pyridyl, 1,2,4-
Bicyclic or tricyclic (preferably, monocyclic heterocyclic group such as triazolo [4,3-b] pyridazinyl) bonded to 1 or 2 of the monocyclic heterocyclic group and / or the aryl group. And a bicyclic) fused aromatic heterocyclic group (preferably a 5- or 6-membered aromatic monocyclic heterocyclic group such as furyl).

Preferable examples of the non-aromatic heterocyclic group include
For example, oxiranyl, azetidinyl, oxetanyl,
5-6 member containing 1 to 4 heteroatoms selected from oxygen atom, sulfur atom and nitrogen atom in addition to carbon atoms such as thietanyl, pyrrolidinyl, tetrahydrofuryl, thiolanyl, piperidyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl and piperazinyl And the non-aromatic monocyclic heterocyclic group. Examples of the halogen include fluorine, chlorine, bromine and iodine, and fluorine and chlorine are particularly preferable. The hydroxyl group which may be substituted has a hydroxyl group and an appropriate substituent for the hydroxyl group, in particular, a hydroxyl group-protecting group, for example, alkoxy, alkenyloxy, aralkyloxy, acyloxy and the like. Aryloxy is mentioned. The alkoxy has 1 to 10 carbon atoms.
Alkoxy (e.g., methoxy, ethoxy, propoxy,
Isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, heptyloxy, nonyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy and the like) are preferable.
Examples of alkenyloxy include those having 1 to 10 carbon atoms such as allyloxy, crotyloxy, 2-pentenyloxy, 3-hexenyloxy, 2-cyclopentenylmethoxy and 2-cyclohexenylmethoxy, and examples of aralkyloxy include: , Phenyl-C _1-4 alkyloxy (eg, benzyloxy, phenethyloxy, etc.)
Is mentioned. As acyloxy, alkanoyloxy having 2 to 4 carbon atoms (eg, acetyloxy, propionyloxy, n-butyryloxy, iso-butyryloxy, etc.) is preferable. Phenoxy as aryloxy,
4-chlorophenoxy etc. are mentioned.

The thiol group which may be substituted includes a thiol group and an appropriate substituent for the thiol group,
Particularly, those having an thiol-protecting group, for example, alkylthio, aralkylthio, acylthio and the like can be mentioned. Alkylthio has 1 carbon atom
An alkylthio of 10 (eg methylthio, ethylthio,
Propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, isopentylthio, neopentylthio, hexylthio, heptylthio, nonylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio and the like) are preferable. Aralkylthio includes, for example, phenyl
And -C _1-4 alkylthio (eg, benzylthio, phenethylthio, etc.). Acylthio has 2 carbon atoms
Alkanoylthio of 4 to 4 (eg, acetylthio, propionylthio, n-butyrylthio, iso-butyrylthio, etc.) are preferable. As the optionally substituted amino group,
1 or 2 amino of alkyl having 1 to 10 carbons, alkenyl having 2 to 10 carbons, aromatic group or acyl group having 2 to 10 carbons (eg, alkanoyl having 2 to 10 carbons, benzoyl, naphthoyl, etc.) Substituted with a group (-NH ₂ group) (eg, methylamino, dimethylamino, ethylamino, diethylamino, dibutylamino, diallylamino, cyclohexylamino, phenylamino, N-methyl)
-N-phenylamino, acetylamino, propionylamino, benzoylamino and the like). As the optionally substituted acyl, formyl or alkyl having 1 to 10 carbons, alkenyl having 2 to 10 carbons,
Cycloalkyl having 3 to 10 carbon atoms, cycloalkenyl having 3 to 10 carbon atoms, or one in which an aromatic group (eg, the aryl group etc.) and a carbonyl group are bonded (eg, acetyl,
Propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, octanoyl, cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl, crotonyl, 2-cyclohexenecarbonyl, benzoyl, nicotinoyl and the like). Examples of the optionally esterified carboxyl group include alkoxycarbonyl (eg, those having 2 to 5 carbon atoms such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and butoxycarbonyl),
Aralkyloxycarbonyl (eg, phenyl-C _1-4 alkyloxy-carbonyl such as benzyloxycarbonyl), aryloxycarbonyl (eg, phenoxycarbonyl, p-tolyloxycarbonyl, etc.) and the like can be mentioned.

In the above general formula (I), the substituent of the heterocyclic group represented by R is an alicyclic hydrocarbon residue, an aryl group, an aromatic heterocyclic group or a non-aromatic heterocyclic group. when,
Further, each has at least one suitable substituent, preferably 1
~ 3 may have, and as the substituent, for example,
Lower alkyl group, lower alkenyl group, lower alkynyl group, lower cycloalkyl group, aryl group (eg, phenyl, naphthyl, etc.), the aromatic heterocyclic group, the non-aromatic heterocyclic group, aralkyl group (eg, phenyl- C _1-4 alkyl etc.), amino group, N-monosubstituted amino group, N, N-disubstituted amino group, amidino group, acyl group (eg C _2-10 alkanoyl, benzoyl, naphthoyl etc.), carbamoyl group, N-monosubstituted carbamoyl group, N, N-disubstituted carbamoyl group, sulfamoyl group, N-monosubstituted sulfamoyl group, N, N-disubstituted sulfamoyl group, carboxyl group, lower alkoxycarbonyl group, hydroxyl group, lower alkoxy group, Lower alkenyloxy group, lower cycloalkyloxy group, aralkyloxy group (eg, phenyl-C
_1-4 alkyloxy etc.), aryloxy group (eg phenoxy, naphthoxy etc.), mercapto group, lower alkylthio group, aralkylthio group (eg phenyl-C _1-4)
Alkylthio, etc.), arylthio groups (eg, phenylthio, naphthylthio, etc.), sulfo groups, cyano groups, azido groups, nitro groups, nitroso groups, halogens and the like. The above-mentioned “substitution” means a substituent on the ring of the heterocyclic group represented by R (preferably an aliphatic chain hydrocarbon group or an alicyclic hydrocarbon group, more preferably an aliphatic chain hydrocarbon group). Group), and the above “lower” means having 6 or less carbon atoms. In the above general formula (I), X is -C.
H ₂ -, - CO- or -CH = CH-, preferably -
CH ₂ - or -CH = CH-, more preferably -C
H = CH-. m is an integer of 0 to 3, preferably 0 or 1, and more preferably 0. p is 1 or 2, preferably 1.

The salt of the compound (I) of the present invention is preferably a pharmacologically acceptable salt, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid. , Salts with basic or acidic amino acids, and the like. Preferable examples of the salt with an inorganic base include, for example, alkali metal salts such as sodium salt and potassium salt; calcium salt,
Alkaline earth metal salts such as magnesium salts; as well as aluminum salts, ammonium salts and the like. Preferable examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N′-dibenzylethylenediamine and the like. Preferable examples of the salt with an inorganic acid include salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like. Suitable examples of salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p. -Salts such as toluene sulfonic acid are included. Suitable examples of salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, and suitable examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, etc. Can be mentioned.

The compound (I) of the present invention or a pharmacologically acceptable salt thereof has a nitric oxide production inhibitory action and low toxicity, and as it is or a known pharmacologically acceptable carrier or excipient. Senile dementia, Alzheimer's disease, against humans or non-human mammals (eg, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.) by mixing with excipients, bulking agents, etc. It can be used as a therapeutic agent for cerebral nerve cell disorder diseases such as cerebral ischemic diseases. The method of administration is usually used orally as tablets, capsules (including soft capsules and microcapsules), powders, granules, etc., but in some cases parenterally as injections, suppositories, pellets, etc. it can.
Although the dose varies depending on the administration route, administration subject, target disease, symptoms, etc., it is used as a therapeutic drug for senile dementia in adults (about 5
When administered orally in an amount of 0 kg), about 10 to 500 mg, preferably about 50 to 100 mg, in terms of compound (I) or a salt thereof, is preferably administered once to three times a day. The object compound (I) of the present invention or a pharmaceutically acceptable salt thereof is mixed with a pharmaceutically acceptable carrier according to a conventional method, and a solid preparation such as tablets, capsules, granules and powders; or syrup. It can be administered orally or parenterally as a liquid preparation such as an agent or an injection.

As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances conventionally used as a formulation material are used. Excipients, lubricants, binders, disintegrants in solid formulations; solvents in liquid formulations, It is added as a solubilizing agent, suspending agent, isotonicity agent, buffer, soothing agent and the like. In addition, formulation additives such as antiseptics, antioxidants, coloring agents and sweeteners can be used, if necessary. Preferable examples of excipients include lactose, sucrose, D-mannitol, starch, crystalline cellulose, light anhydrous silicic acid and the like. Preferable examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Preferable examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone and the like. Preferable examples of the disintegrant include, for example, starch, carboxymethyl cellulose,
Examples include carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium and the like. Preferable examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like. Preferable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Suitable examples of the suspending agent include, for example, stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate, and other surfactants; Polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose,
Hydrophilic polymers such as hydroxyethyl cellulose and hydroxypropyl cellulose are included. Preferable examples of the tonicity agent include sodium chloride, glycerin, D-mannitol and the like. Preferable examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Preferable examples of soothing agents include benzyl alcohol and the like. Preferable examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Preferable examples of the antioxidant include sulfite, ascorbic acid and the like. The method for producing the object compound (I) of the present invention is described below.

[0017]

[Chemical 4]

[In the formula, Q represents a halogen atom, and other symbols have the same meanings as above. Examples of the halogen atom represented by Q include chlorine, bromine and iodine. In this method, an aldehyde derivative (II) is reacted with a phosphonium salt (III) to produce an unsaturated aldehyde derivative (I). The reaction of (II) and (III) is carried out in an appropriate solvent in the presence of a base according to a conventional method. Examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene, dioxane, tetrahydrofuran,
Ethers such as dimethoxyethane, alcohols such as methanol, ethanol and propanol, N, N-dimethylformamide, dimethyl sulfoxide, chloroform, dichloromethane, 1,2-dichloroethane, 1,
Examples include 1,2,2-tetrachloroethane and a mixed solvent thereof. Examples of the base include alkali metal salts such as sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate and sodium hydrogen carbonate, amines such as pyridine, triethylamine and N, N-dimethylaniline,
Examples thereof include metal hydrides such as sodium hydride and potassium hydride, sodium ethoxide, sodium methoxide, potassium t-butoxide, and the like, and the amount of these bases to be used is preferably about 1 to 5 mol relative to the compound (III).
The amount of compound (III) used is 1 to 5 relative to compound (II).
It is about 1 mol, preferably about 1 to 3 mol. This reaction is generally -50 ° C to 150 ° C, preferably about -10 ° C to 100 ° C.
Done in. The reaction time is 0.5 to 30 hours.

[0019]

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[In the formulas (IV) and (V), A'and A "are the same or different and represent a lower alkyl group, and other symbols have the same meanings as above.] A'or A" Examples of the lower alkyl group include those having 1 to 4 carbon atoms such as methyl, ethyl, propyl and butyl. In this method, first, an aldehyde derivative (II) is treated with a phosphonoacetic acid or a γ-phosphonocrotonic acid derivative (I
V) to produce unsaturated ester derivative (V). The reaction of (II) and (IV) is performed by the compound (I
It is carried out in the same manner as the reaction of I) with compound (III). Then, the compound (V) is subjected to a reduction reaction to give an alcohol derivative.
(VI) is produced. This reduction reaction can be performed by a method known per se. For example, reduction with a metal hydride, reduction with a metal-hydrogen complex compound, reduction with diborane and substituted borane, etc. are used. That is, this reaction is a compound
It is carried out by treating (V) with a reducing agent. Examples of the reducing agent include alkali metal borohydrides (eg, sodium borohydride, lithium borohydride, etc.), metal-hydrogen complex compounds such as lithium aluminum hydride and diborane, but use diisobutylaluminum hydride. Is more advantageously performed. This reaction is carried out in an organic solvent that does not affect the reaction. Examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene, halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloromethane, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane. Ethers such as diethyl ether, tetrahydrofuran, dioxane, alcohols such as methanol, ethanol, propanol, isopropanol, and 2-methoxyethanol, amides such as N, N-dimethylformamide, and mixed solvents thereof, or the like, and a reducing agent. It is appropriately selected and used according to the type of. The reaction temperature is -20 ° C to 150 ° C,
Particularly, 0 ° C to 100 ° C is suitable, and the reaction time is about 1 to 2
It takes about 4 hours.

Then, the compound (VI) is subjected to an oxidation reaction to produce an aldehyde derivative (II). This oxidation reaction can be carried out by a method known per se. For example, oxidation with manganese dioxide, oxidation with chromic acid, oxidation with dimethyl sulfoxide, etc. are used. That is, this reaction is carried out by treating compound (VI) with an oxidizing agent.
Although manganese dioxide and chromic anhydride are used as the oxidizing agent, manganese dioxide is advantageously used. This reaction is carried out in an organic solvent that does not affect the reaction. Examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene, halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloromethane, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane. Ethers such as diethyl ether, tetrahydrofuran, dioxane, dimethyl sulfoxide, or a mixed solvent thereof are appropriately selected and used depending on the kind of the oxidizing agent. Reaction temperature is -20 ° C to 150
C., especially 0.degree. C. to 100.degree. C. is suitable, and the reaction time is about 1 to 24 hours.

The aldehyde derivative obtained according to Method A and Method B can be isolated and purified by known separation and purification means such as concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, chromatography and the like. . Method A and B
Among the starting compound (II) of the method, the benzaldehyde derivative represented by the general formula (II-1) can be produced, for example, according to the methods C to E.

[0023]

[Chemical 6]

[Wherein n represents 0 or an integer of 1 to 3,
Other symbols have the same meanings as above. In this method, first, a compound represented by the general formula (VII) is reacted with a reaction-corresponding amount of triphenylphosphine,
A phosphonium salt derivative represented by (VIII) is produced. This reaction is carried out in a solvent, and examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as tetrahydrofuran, dioxane and dimethoxyethane, acetonitrile and a mixed solvent thereof. This reaction is carried out at 10 ° C to 200 ° C, preferably at 30 ° C.
It is carried out at ˜150 ° C. for 0.5 to 50 hours. Then,
The phosphonium salt (VIII) and the aldehyde derivative (IX) are subjected to a condensation reaction to produce a compound of general formula (X). This condensation reaction is performed in the same manner as Method A. The compound (X) is obtained as a mixture of isomers of (E) -form and (Z) -form with respect to the newly formed double bond, and these (E) -form and (Z) -form are known means. In the same manner as in the reduction reaction of the compound (V) in Method B, the compound (XI) is produced after isolation in step (b) or without isolation. Compound (XI)
Produces the benzaldehyde derivative (II-1) in the same manner as in the oxidation reaction from the compound (VI) to the compound (II) in Method B.

[0025]

[Chemical 7]

[In the formula (XII), Q'represents a halogen atom, and other symbols have the same meanings as described above. Examples of the halogen atom represented by Q ′ include chlorine, bromine and iodine. In this method, first, phosphonium salt
Compound (XIII) is produced by subjecting (VIII) and aldehyde derivative (XII) to a condensation reaction. This condensation reaction is performed in the same manner as Method A. The compound (XIII) is obtained as a mixture of isomers of (E) -form and (Z) -form with respect to the newly formed double bond, and these (E) -form and (Z) -form are obtained.
After the isolation of each of the -forms by a known means, or the mixture without isolation, the mixture is treated with butyllithium, sec-butyllithium, tert-butyllithium, methyllithium, phenyllithium or the like to give a lithio compound. , N, N
-Compound by reacting with dimethylformamide (DMF)
(II-1) is produced. Compound from compound (XIII)
The reaction to (II-1) is preferably diethyl ether,
The reaction is carried out at -100 ° C to 50 ° C for about 1 to 24 hours using ethers such as tetrahydrofuran and dioxane as a solvent. The amount of N, N-dimethylformamide (DMF) used is 1 to 5 molar equivalents relative to compound (XIII).

The benzaldehyde derivative (II-1) produced according to the methods C and D is a known separation and purification means,
For example, it can be isolated and purified by concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, chromatography and the like. Compound (II-2) can be produced by subjecting the compound represented by general formula (II-1) to a reduction reaction according to method E.

[0028]

Embedded image

[Each symbol in the formula has the same meaning as described above. The compound (II-2) can be produced by subjecting the compound (II-1) to a reduction reaction. This reduction reaction is performed according to a conventional method in a solvent in the presence of a catalyst, in a hydrogen atmosphere at 1 to 150 atm. Examples of the solvent include alcohols such as methanol, ethanol, propanol, isopropanol and 2-methoxyethanol, aromatic hydrocarbons such as benzene, toluene and xylene, ethyl ether,
Ethers such as isopropyl ether, dioxane, tetrahydrofuran, chloroform, dichloromethane,
Examples thereof include halogenated hydrocarbons such as 1,1,2,2-tetrachloroethane, ethyl acetate, acetic acid, and mixed solvents thereof. As a catalyst, a metal such as a nickel compound, a transition metal catalyst such as palladium, platinum, or rhodium can be advantageously used. Reaction temperature is 0-100
° C, preferably 10 to 80 ° C, reaction time 0.5 to 50
Time. The benzaldehyde derivative (II-2) produced according to the method E can be isolated and purified by known separation and purification means, for example, concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, chromatography and the like. it can. C
The intermediate (X) in the method can also be produced, for example, according to the method F.

[0030]

[Chemical 9]

[In the formula, each symbol has the same meaning as described above. In this method, first, a compound represented by the general formula (XIV) is reacted with a reaction-corresponding amount of triphenylphosphine,
A phosphonium salt derivative represented by (XV) is produced. This reaction is carried out in the same manner as the reaction of compound (VII) of method C and triphenylphosphine. Then, the compound (X) is produced by reacting the phosphonium salt (XV) with the compound (XVI). The reaction between (XV) and (XVI) is a compound of Method C.
It is carried out in the same manner as the reaction of (VIII) and (IX). Compound (X) produced according to Method F can be isolated and purified by a known separation and purification means, for example, concentration, concentration under reduced pressure, solvent extraction, crystallization, recrystallization, phase transfer, chromatography and the like. Among the raw material compounds (II) in Method A and Method B, the compound in which X is CO, m is 2, and R is 5-methyl-2-phenyl-4-oxazolyl is a compound of Journal of Medicinal Chemistry.
al Chemistry), 1992, 35, 1853. As a typical example of the above compound (I), structural formula (I-3) or (I-4)

[0032]

[Chemical 10]

A compound represented by the formula (preferably compound (I
-4)).

To carry out the method for determining the NO production inhibitory activity of the present invention, first, the protein or a fragment thereof and a test compound are added to brain cells in which NO is induced by the β-amyloid protein or a fragment thereof. , Culturing cells in the brain.
β-amyloid protein is a constituent protein of amyloid fibrils deposited in the brain of patients with Alzheimer's disease and the like [Proc. Natl. Acad. Sci. USA, 82, 4245-4249 (1985)]. The fragments include
A part of the protein or precursor protein is included and is not particularly limited, but in the present invention, β-amyloid protein (1
-40) or β-amyloid protein (1-42) is preferable.

The cells to which these proteins or fragments thereof and the test compound are added are iNOS-producing cells, and examples of such cells include glial cells, phagocytic cells, vascular endothelial cells, vascular smooth muscle cells, and mesangial cells. , Hepatocytes, etc. are known, and a large amount of NO is produced by various cytokines such as lipopolysaccharide, interferon, and interleukin-1. On the other hand, cNOS is present in platelets and pancreatic B cells in addition to vascular endothelial cells and nerve cells, and it is activated by various stimulants such as bradykinin and glutamic acid, although it depends on the cell type. Glial cells are roughly divided into microglia and macroglia.
Macroglia are classified into astrocytes and oligodendrocytes. The glial cells referred to here may be any of the above-mentioned glial cells, but microglia or astrocytes are particularly preferable. The vascular cells may be vascular endothelial cells or vascular smooth muscle cells. Furthermore, cells in the brain include blood-derived phagocytes or lymphocytes. The phagocytic cells are classified into macrophages, monocytes, neutrophils, eosinophils, basophils, etc. Among them, macrophages and monocytes are preferable, but not limited thereto. Culturing of cells and measurement of the amount of NO produced can be carried out by a method known per se. iN
In addition to nerve cells, glial cells or vascular cells may be damaged by NO caused by OS. That is, the iNOS selective inhibitor obtained by the above screening method is applicable as a broad therapeutic agent for central degenerative disorders, and the compound (I) of the present invention is
The screening method of the present invention exhibits excellent NO production inhibitory activity.

The NO synthase inducer of the present invention is useful as a reagent used in the method for determining the NO production inhibitory activity of the present invention, and comprises the above β-amyloid protein or a fragment thereof,
Furthermore, as an NO synthase inducer, lipopolysaccharide and interferon-α, together with them,
NO production can be enhanced by containing cytokines such as β, γ, interleukin-1, and tumor necrosis factor alone or in combination. Among them, those containing interferon-γ and / or lipopolysaccharide in coexistence are particularly preferable. The inducer can be made into various dosage forms such as liquids and freeze-dried products by a method known per se, and can also be made into a kit form by combining with known diluents and the like.

Hereinafter, reference examples and examples will be described in order to explain the present invention in more detail, but the present invention is not limited to these reference examples and examples. Reference Example 1 4-chloromethyl-5-methyl-2- (2-naphthyl)
A mixture of oxazole (10.0 g), triphenylphosphine (11.1 g) and acetonitrile (100 ml) was heated to reflux for 18 hours. Crystals of [5-methyl-2- (2-naphthyl) -4-oxazolylmethyl] triphenylphosphonium chloride precipitated after cooling (19.3 g, 8
(8%) was collected by filtration and washed with acetonitrile and diethyl ether. Melting point 285-286 [deg.] C. Elemental analysis Calculated as C ₃₃ H ₂₇ NOPCl: C, 76.22; H, 5.23; N, 2.69 Analytical value: C, 76.14; H, 5.50; N, 2.63

Reference Example 2 In the same manner as in Reference Example 1, (5-methyl-2-phenyl-4-oxazolyl) was obtained by reacting 4-chloromethyl-5-methyl-2-phenyloxazole with triphenylphosphine. Methyl) triphenylphosphonium chloride was obtained. Melting point 277-278 [deg.] C. Elemental analysis calculated as C ₂₉ H ₂₅ NOPCl: C, 74.12; H, 5.36; N, 2.98 Analytical value: C, 73.79; H, 5.32; N, 2.97

Reference Example 3 In the same manner as in Reference Example 1, by reacting 2-chloromethyl-5-methyl-4-phenylthiazole with triphenylphosphine, (5-methyl-4-phenyl-2-thiazolylmethyl) tri Phenylphosphonium chloride was obtained. Melting point 256-257 [deg.] C. Elemental analysis calculated as C ₂₉ H ₂₅ NPSCl: C, 71.67; H, 5.18; N, 2.88 Analytical value: C, 71.53; H, 5.15; N, 2.91

Reference Example 4 In the same manner as in Reference Example 1, 4-chloromethyl-2- (2-
The reaction of furyl) -5-methyloxazole with triphenylphosphine gave [2- (2-furyl) -5-methyl-4-oxazolylmethyl] triphenylphosphonium chloride. Melting point 284-285 [deg.] C. Elemental analysis Calculated as C ₂₇ H ₂₃ NO ₂ PCl: C, 70.51; H, 5.04; N, 3.05 Analytical value: C, 70.25; H, 4.97; N, 3.09

Reference Example 5 [5-Methyl-2- (2-naphthyl) -4-oxazolylmethyl] triphenylphosphonium chloride (18.4 g)
Was suspended in DMF (200 ml), and 60% sodium hydride (oily, 1.42 g) was gradually added at 0 ° C. After stirring for 1 hour at room temperature, methyl 4-formylbenzoate (5.8
0 g) was added and the mixture was stirred for another 3 hours. The reaction mixture was poured into ice water, and the precipitated crystals were collected by filtration. Purify by silica gel column chromatography, and elute with ethyl acetate-chloroform (5:95, v / v) to obtain 4-[(E) -2.
Methyl-[5-methyl-2- (2-naphthyl) -4-oxazolyl] vinyl] benzoate (10.3 g, 79%) was obtained. Recrystallized from dichloromethane-methanol. Pale yellow prism crystal. Melting point 216-217 [deg.] C.

Reference Example 6 In the same manner as in Reference Example 5, (5-methyl-4-phenyl-
2-Thiazolylmethyl) triphenylphosphonium chloride and methyl 4-formylbenzoate give 4-
Methyl [(E) -2- (5-methyl-4-phenyl-2-thiazolyl) vinyl] benzoate was obtained. Recrystallized from ethyl acetate. Colorless plate crystals. Melting point 156-157 [deg.] C.

Reference Example 7 In the same manner as in Reference Example 5, by reacting [2- (2-furyl) -5-methyl-4-oxazolylmethyl] triphenylphosphonium chloride with methyl 4-formylbenzoate, 4 -[(E) -2- [2- (2-furyl) -5-methyl-
Methyl 4-oxazolyl] vinyl] benzoate was obtained. Recrystallized from ethyl acetate. Colorless prism crystals. Melting point 142-
143 ° C.

Reference Example 8 In the same manner as in Reference Example 5, (5-methyl-2-phenyl-
The reaction of 4-oxazolylmethyl) triphenylphosphonium chloride with methyl 4-formylbenzoate gives 4
Methyl-[(E) -2- (5-methyl-2-phenyl-4-oxazolyl) vinyl] benzoate was obtained. Recrystallized from ethyl acetate. Colorless prism crystals. Melting point 164-165 [deg.] C.

Reference Example 9 4-[(E) -2- [5-methyl-2- (2-naphthyl) -4]
Methyl -oxazolyl] vinyl] benzoate (9.30 g) was suspended in THF (250 ml), and lithium aluminum hydride (955 mg) was gradually added at 0 ° C. After stirring at room temperature for 1 hour, water (5 ml) was added and the insoluble material was filtered off. The crystals obtained by concentrating the filtrate under reduced pressure were recrystallized from dichloromethane-methanol to give 4-[(E) -2- [5-methyl-2].
-(2-Naphtyl) -4-oxazolyl] vinyl] benzyl alcohol (8.00 g, 93%) was obtained. Colorless prism crystals. Melting point 173-174 [deg.] C.

Reference Example 10 Methyl 4-[(E) -2- (5-methyl-4-phenyl-2-thiazolyl) vinyl] benzoate (9.5 g), palladium-carbon (5%, 1.0 g) And a mixture of dioxane (70 ml) -methanol (60 ml) was subjected to catalytic reduction reaction at 1 atm and room temperature. The catalyst was filtered off, the filtrate was concentrated under reduced pressure, 4-
Methyl [2- (5-methyl-4-phenyl-2-thiazolyl) ethyl] benzoate (9.0 g, 94%) was obtained. Recrystallized from hexane. Colorless plate crystals. Melting point 52-53 [deg.] C.

Reference Example 11 In the same manner as in Reference Example 10, methyl 4-[(E) -2- (5-methyl-2-phenyl-4-oxazolyl) vinyl] benzoate was subjected to a reduction reaction to give 4- Methyl [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl] benzoate was obtained. Recrystallized from hexane. Colorless needles. Melting point 59-
60 ° C.

Reference Example 12 In the same manner as in Reference Example 9, methyl 4- [2- (5-methyl-4-phenyl-2-thiazolyl) ethyl] benzoate was subjected to a reduction reaction with lithium aluminum hydride to give 4 −
[2- (5-Methyl-4-phenyl-2-thiazolyl) ethyl] benzyl alcohol was obtained. Recrystallized from ethyl acetate-hexane. Colorless prism crystals. Melting point 62-63
° C.

Reference Example 13 Methyl 4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl] benzoate was subjected to a reduction reaction with lithium aluminum hydride in the same manner as in Reference Example 9 to obtain 4 −
[2- (5-methyl-2-phenyl-4-oxazolyl)
Ethyl] benzyl alcohol was obtained. Recrystallized from ethyl acetate-hexane. Colorless plate crystals. Melting point 103-104
° C.

Reference Example 14 In the same manner as in Reference Example 9, methyl 4-[(E) -2- [2- (2-furyl) -5-methyl-4-oxazolyl] vinyl] benzoate was added to lithium aluminum hydride. Was subjected to a reduction reaction with to obtain 4-[(E) -2- [2- (2-furyl) -5-methyl-4-oxazolyl] vinyl] benzyl alcohol. Recrystallized from ethyl acetate-hexane. Colorless prism crystals. Melting point 150-151 [deg.] C.

Reference Example 15 4-[(E) -2- [5-methyl-2- (2-naphthyl) -4]
-Oxazolyl] vinyl] benzyl alcohol (7.80
g), active manganese dioxide (15.6 g) and chloroform (300 ml) were stirred at room temperature for 1 day. After removing manganese dioxide by filtration, the filtrate was concentrated under reduced pressure, and the obtained crystal was recrystallized from dichloromethane-methanol.
[(E) -2- [5-Methyl-2- (2-naphthyl) -4-oxazolyl] vinyl] benzaldehyde (6.56 g, 85
%). Pale yellow needles. Melting point 162-163 [deg.] C.

Reference Example 16 In the same manner as in Reference Example 15, 4- [2- (5-methyl-4-)
Phenyl-2-thiazolyl) ethyl] benzyl alcohol was subjected to an oxidation reaction with manganese dioxide to give 4- [2- (5
-Methyl-4-phenyl-2-thiazolyl) ethyl] benzaldehyde was obtained. Recrystallized from hexane. Colorless prism crystals. Mp 66-67 ° C.

Reference Example 17 In the same manner as in Reference Example 15, 4-[(E) -2- [2- (2-
Furyl) -5-methyl-4-oxazolyl] vinyl] benzyl alcohol was subjected to oxidation reaction with manganese dioxide to give 4-[(E) -2- [2- (2-furyl) -5-methyl-
4-Oxazolyl] vinyl] benzaldehyde was obtained. Recrystallized from ethyl acetate. Colorless prism crystals. Melting point 209
~ 210 ° C.

Reference Example 18 (5-Methyl-2-phenyl-4-oxazolylmethyl)
Triphenylphosphonium chloride (25.4 g) in ethanol solution of sodium ethoxide [sodium (1.4
g) and ethanol (300 ml)] under ice cooling. After stirring the mixture at room temperature for 5 minutes, 4-bromobenzaldehyde (10.0 g) was added. After stirring at room temperature for 2 hours, the reaction mixture was poured into water and extracted with ethyl acetate. After ethyl acetate layer was washed with water, dried (MgSO _4),
It was concentrated under reduced pressure and the residue was subjected to silica gel chromatography. From the portion eluted with ether-hexane (1:20, v / v), 4-[(E) -2- (4-bromophenyl) vinyl] -5-methyl-2-phenyloxazole was obtained.
(13.1 g, 71%) was obtained. Recrystallized from ethyl acetate-hexane. Colorless prism crystals. Melting point 138-139
° C.

Reference Example 19 4-[(E) -2- (4-bromophenyl) vinyl] -5-methyl-2-phenyloxazole (13.0 g) in tetrahydrofuran (140 ml) solution at -70 ° C. -Butyl lithium in hexane solution (1.6 M, 28.7 ml) was added dropwise. After stirring this mixture at -70 ° C for 15 minutes,
A solution of N, N-dimethylformamide (4.2 g) in tetrahydrofuran (10 ml) was added dropwise at the same temperature. The reaction mixture was stirred at -70 ° C for 30 minutes, warmed to room temperature, 1N HCl (150 ml) was added dropwise, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried (MgSO _4), and concentrated under reduced pressure, the residue was subjected to silica gel chromatography. From the portion eluted with ethyl acetate-hexane (1: 2, v / v), 4-[(E) -2- (5-methyl-2-phenyl-
4-oxazolyl) vinyl] benzaldehyde (5.9
g, 54%). Recrystallized from ethyl acetate-hexane. Light brown prismatic crystals. Melting point 158-159 [deg.] C.

Reference Example 20 4-[(E) -2- [5-methyl-2- (2-naphthyl) -4]
-Oxazolyl] vinyl] benzaldehyde (4.00
g), triethylphosphonoacetate (2.64 g) and DMF
To a mixture of (70 ml) at 0 ° C. was slowly added 60% sodium hydride (oily, 475 mg). After stirring at room temperature for 2 hours, the reaction mixture was poured into ice water, and the precipitated crystals were collected by filtration. Recrystallize from dichloromethane-ethanol to give 4-
Ethyl [(E) -2- [5-methyl-2- (2-naphthyl) -4-oxazolyl] vinyl] cinnamate (4.33 g, 90
%). Pale yellow prism crystal. Melting point 186-187
° C.

Reference Example 21 In the same manner as in Reference Example 20, 4-[(E) -2- (5-methyl-2-phenyl-4-oxazolyl] vinyl] benzaldehyde was reacted with triethylphosphonoacetate to give 4-
Ethyl [(E) -2- (5-methyl-2-phenyl-4-oxazolyl] vinyl] cinnamate was obtained and recrystallized from ethyl acetate.Light brown plate crystals, melting point 161-162 ° C.

Reference Example 22 In the same manner as in Reference Example 20, 4- [2- (5-methyl-4-)
By the reaction of phenyl-2-thiazolyl) ethyl] benzaldehyde with triethylphosphonoacetate, 4- [2- (5
-Methyl-4-phenyl-2-thiazolyl) ethyl] ethyl cinnamate was obtained. Recrystallized from hexane. Colorless prism crystals. Melting point 69-70 [deg.] C.

Reference Example 23 In the same manner as in Reference Example 20, 4-[(E) -2- [2- (2-
Furyl) -5-methyl-4-oxazolyl] vinyl] benzaldehyde was reacted with 4-ethylphosphonoacetate to give 4-[(E) -2- [2- (2-furyl) -5-methyl-
Obtained ethyl 4-oxazolyl] vinyl] cinnamate. Recrystallized from ethanol. Pale yellow prism crystal. Melting point 123
~ 124 ° C.

Reference Example 24 4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl] benzyl alcohol (11.3 g), active manganese dioxide (23.0 g) and dichloromethane (200 m)
The mixture of l) was stirred at room temperature for 3 hours. After manganese dioxide was filtered off, the filtrate was concentrated under reduced pressure. Remaining 4- [2
Crystals of-(5-methyl-2-phenyl-4-oxazolyl) ethyl] benzaldehyde were mixed with tetrahydrofuran (15
0 ml) and triethylphosphonoacetate (7.5 g) was added. Then add 60% sodium hydride to this mixture.
(Oiliness, 1.6 g) was gradually added under ice cooling. After stirring at room temperature for 1 hour, the reaction mixture was poured into ice water and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried (MgSO ₄₎
Then, it was concentrated under reduced pressure and ethyl 4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl] cinnamate (7.
9 g, 57%) was obtained. Recrystallized from ethyl acetate-hexane. Colorless prism crystals. Melting point 73-74 [deg.] C.

Reference Example 25 A toluene solution of diisobutylaluminum hydride (1.
5M, 17 ml) was added to 4-[(E) -2- [5-methyl-2-
A suspension of ethyl (2-naphthyl) -4-oxazolyl] vinyl] cinnamate (4.20 g) in dichloromethane (100 ml) was added dropwise at 0 ° C. After stirring at room temperature for 4 hours, methanol (2 ml) and then water (6 ml) were added at 0 ° C. The insoluble material was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, and eluted with ethyl acetate-chloroform (5:95, v / v) to (E) -3- [4
-[(E) -2- [5-methyl-2- (2-naphthyl) -4-
Crystals of oxazolyl] vinyl] phenyl] -2-propenol were obtained. Recrystallized from chloroform-ethanol,
Pale yellow prism crystals (3.04 g, 81%) were obtained. Melting point 1
84-185 ° C.

Reference Example 26 In the same manner as in Reference Example 25, reduction reaction of ethyl 4-[(E) -2- (5-methyl-2-phenyl-4-oxazolyl) vinyl] cinnamate with diisobutylaluminum hydride. And (E) -3- [4-[(E) -2- (5-methyl-2)
-Phenyl-4-oxazolyl) vinyl] phenyl] -2
-Propenol was obtained. Recrystallized from ethyl acetate. Pale yellow prism crystal. Melting point 165-166 [deg.] C.

Reference Example 27 In the same manner as in Reference Example 25, 4- [2- (5-methyl-4-)
Phenyl-2-thiazolyl) ethyl] ethyl cinnamate is subjected to a reduction reaction with diisobutylaluminum hydride,
(E) -3- [4- [2- (5-methyl-4-phenyl-2
-Thiazolyl) ethyl] phenyl] -2-propenol was obtained. Recrystallized from hexane. Colorless plate crystals. Melting point 93
~ 94 ° C.

Reference Example 28 In the same manner as in Reference Example 25, 4- [2- (5-methyl-2-
Phenyl-4-oxazolyl) ethyl] ethyl cinnamate was subjected to a reduction reaction with diisobutylaluminum hydride to give (E) -3- [4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl. ] Phenyl] -2-propenol was obtained. Recrystallized from ethyl acetate-hexane. Colorless needles. Melting point 102-103 [deg.] C.

Reference Example 29 In the same manner as in Reference Example 25, 4-[(E) -2- [2- (2-
Furyl) -5-methyl-4-oxazolyl] vinyl] ethyl cinnamate was subjected to a reduction reaction with diisobutylaluminum hydride to give (E) -3- [4-[(E) -2- [2- (2 −
Furyl) -5-methyl-4-oxazolyl] vinyl] phenyl] -2-propenol was obtained. Recrystallized from ethyl acetate. Colorless prism crystals. Melting point 148-149 [deg.] C.

Example 1 (E) -3- [4-[(E) -2- [5-methyl-2- (2-naphthyl) -4-oxazolyl] vinyl] phenyl] -2-propenol (2. 80 g), activated manganese dioxide (8.40
g) and chloroform (150 ml) at room temperature for 16
Stir the time. After manganese dioxide was filtered off, the filtrate was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography, and from the portion eluted with chloroform, 4-
[(E) -2- [5-Methyl-2- (2-naphthyl) -4-oxazolyl] vinyl] cinnamaldehyde (2.50 g, 9
0%). Recrystallized from dichloromethane-methanol. Pale yellow prism crystal. Melting point 213-214 [deg.] C.

Example 2 In the same manner as in Example 1, (E) -3- [4-[(E) -2-
(5-Methyl-2-phenyl-4-oxazolyl] vinyl] phenyl] -2-propenol was subjected to an oxidation reaction with active manganese dioxide to give (E) -4-[(E) -2- (5-methyl- 2-Phenyl-4-oxazolyl) vinyl] cinnamaldehyde was obtained and recrystallized from ethyl acetate, pale yellow prism crystals, melting point 191-192 ° C.

Example 3 In the same manner as in Example 1, (E) -3- [4- [2- (5-methyl-4-phenyl-2-thiazolyl) ethyl] phenyl] -2-propenol was treated with active dioxide. Subjected to an oxidation reaction with manganese, 4- [2- (5-methyl-4-phenyl-
2-thiazolyl) ethyl] cinnamaldehyde was obtained. Recrystallized from ethyl acetate-hexane. Colorless prism crystals.
Melting point 94-95 [deg.] C.

Example 4 In the same manner as in Example 1, (E) -3- [4- [2- (5-methyl-2-phenyl-4-oxazolyl) ethyl] phenyl] -2-propenol was treated with active dioxide. Subjected to an oxidation reaction with manganese, 4- [2- (5-methyl-2-phenyl-
4-oxazolyl) ethyl] cinnamaldehyde was obtained.
Recrystallized from ethyl acetate-hexane. Colorless prism crystals. Melting point 99-100 [deg.] C.

Example 5 In the same manner as in Example 1, (E) -3- [4-[(E) -2-
[2- (2-furyl) -5-methyl-4-oxazolyl]
Vinyl [phenyl] -2-propenol was subjected to an oxidation reaction with active manganese dioxide to give 4-[(E) -2- [2- (2
-Furyl) -5-methyl-4-oxazolyl) vinyl] cinnamaldehyde was obtained. Recrystallized from ethyl acetate-hexane. Pale yellow prism crystal. Melting point 199-200 ° C.

Example 6 Preparation of Microglial Cells Two days old Sprague-Dawle
y) After collecting the cerebral cortex of the rat and segmenting it,
HBSS (Hanks' Balaced Salt) containing 25% trypsin (Flow) and 0.01% DNase I (Sigma)
Solution, Mg ⁺⁺ , Ca ^++- free, Sigma) at 37 ℃
And incubated for 10 minutes. Fetal calf serum (FCS) was added to 10% and centrifuged at 1000 rpm for 2 minutes to remove the supernatant, and then 10% FCS,
DMEM (Dulbecco's mod containing 20 mM HEPES
ified. The cells were suspended in Eagle's medium, Flow). After filtering the cell suspension with sterile lens paper, T1
Cells were seeded in 75 flasks (Nunc) at a concentration of 2 × 10 ⁷ cells / flask, and the medium was exchanged after 2 days, and the medium was exchanged every 3 days thereafter to obtain primary glial cells. Culture 12
Rotating shaker (TEITE)
C, BR-300 L), shaken 200 times and shaken overnight. The culture medium containing free cells was seeded on a 9 cm dish (Terumo) and allowed to stand at 37 ° C. for 1 hour, non-adherent cells were removed by suction, and the cells were washed twice more with the culture medium. The remaining adherent cells were collected with a rubber policeman to obtain microglia. The assay of microglia was performed by the incorporation of Ac-LDL.
8% or more microglia [The Journal of Ne
uroscience, 6 , 2163-2178 (1986)].

Example 7 NO production induction test by β-amyloid protein fragment DMEM (Dulbecco's modified. Eagle's me) containing 10% FCS and 20 mM HEPES so that the number of isolated microglia was 8 × 10 ⁴ per well.
medium, 96-well microplate
(Nunc Inc.). β-amyloid protein fragment 1-
40 [β-AP (1-40)] or 1-42 [β-AP
(1-42)] was added at the same time as seeding. After incubating at 37 ° C. under 5% carbon dioxide gas (CO ₂ ) for 48 hours, the nitrite concentration in the culture supernatant was measured by Flow Injection As.
measured by say The Flow Injection Assay is the method of Yui et al. [J. Biol. Chem. Volume 266, 3369-3
371 (1991)]. Figure 1 is 10μ
Β-AP (1-40) of M induces nitrite ion production in microglia, which is a nonselective inhibitor of NO synthase ^NG -monomethyl-L-arginine (NMM).
It is shown to be inhibited by A). This means that β-A
It means that P (1-40) induces NO production of microglia. In addition, 30 μM β-AP (1-
42) also obtained similar results (Fig. 2).

Example 8 Neurotoxicity Test with NO Isolation of isolated microglia in 10% FCS, 20 mM HE
It was suspended in DMEM containing PES and 100 μl of the medium was added in advance to Transwell (Coaster,
2.5 × 10 ⁵ pieces were added to a diameter of 6.5 mm). 30 μM β-AP (1-40) was added, and seeded in advance on a 24-well cluster plate (Coaster), and transferred to each well of cultured nerve cells. 37
After incubation for 24 hours at 5 ° C and 5% CO ₂ , the life and death of nerve cells immediately under the transwell was determined by Fluorescein-diac.
Staining by etate (FDA) [J. Histochemistry and
Cytochemistry 32, 1084-1090
(1984)]. Nerve cells were collected from Sprague-Dawley rat cerebral cortex on the 17th day of embryonic development by the method of Hatanaka et al. [Develop. Brain Re
s. Vol. 30, p. 47-56 (1986)]. After suspending in DMEM / F12 / N12 medium (Sigma), the cells were seeded on a culture plate, and cells on the 7th day of culture were used. FIG. 3 shows that nerve cells are damaged by the action of β-AP (1-40) on microglia and suppressed by NMMA. This is β-AP
By the action of (1-40), nerve cells are damaged by NO produced by microglia, which means that nerve cell injury is inhibited by suppressing NO production from microglia. Similar results were obtained using β-AP (1-42).

Example 9 (1) Preparation of vascular endothelial cells and NO production test Vascular endothelial cells (BAE) were isolated from bovine aorta by the method of Rone et al.
[Proc. Soc. Exp. Biol. Med. Volume 184, Fourth
95 to 503 (1987)] and used for subculturing 6 to 9 generations. BAE is treated with 0.1% trypsin, then 100 U / ml penicillin G (Meiji Seika),
100 μg / ml streptomycin (Meiji Seika), 10%
MEM Earl (Flow) and Ham F containing FCS
-12 (Nissui Pharmaceutical Co., Ltd.) was suspended in an equal volume of mixed medium, seeded on a 48-well culture plate (Coaster), and cultured at 37 ° C. in a 5% CO ₂ incubator until confluent. After removing the supernatant by suction, Ca ²⁺ , Mg 2 ⁺ -containing Hanks'
Wash twice with Balanced Salts Solution (HBS),
HBS containing 2 μM calcium ionophore A23187 (Sigma), 0.1% bovine serum albumin (Seikagaku) was added. The compound was added simultaneously with the addition of A23187. After incubating at 37 ° C for 4 hours,
The concentration of nitrite ion in the supernatant was measured by Flow Injection Assa.
Measured at y.

(2) Various compounds were screened in accordance with the methods shown in Examples 7 and 9 (1) to obtain a representative compound [Structural Formula (I-4)] obtained in Example 2, i.
FIG. 4 shows the result of examining the suppressing effect on NO caused by NOS and cNOS. This is because the present compound efficiently suppresses NO production due to iNOS induced by β-AP (1-40) in microglia.
This means that NO generation due to cNOS is not affected at all. From the rat brain, the method of Bredt et al. [Pr
oc. Natl. Acad. Sci. USA Vol. 87, 682
.About.685 (1990)], the compound of structural formula (I-4) showed almost no inhibitory activity at 8.5 μM with respect to the activity of cNOS (neuronal NOS) purified according to the method described in (1). Figure 5
Is a result of investigating NO generation by a similar method with respect to NMMA which is an existing NOS inhibitor. The compound of structural formula (I-4) is superior in both iNOS selectivity and activity, although it shows a slight selectivity for iNOS. In addition,
This compound also efficiently suppressed NO production from microglia by β-AP (1-42) stimulation.

Example 10 Effect of Interferon-γ (IFN-γ) on NO Production by β-Amyloid Protein Fragment IFN on β-AP (1-40) -stimulated NO production from microglia according to the method described in Example 7. −
The effect of γ was investigated. FIG. 6 shows that IFN-γ is 1 depending on the concentration.
It shows that NO production stimulated by 0 μM β-AP (1-40) is significantly enhanced. That is, β-AP
(1-40) and IFN-γ are NO by microglia
It is meant to have a strong synergistic effect on production. In addition, FIG. 7 shows that the compound of the structural formula (I-4) is 10 μm.
It is shown that NO production from microglia stimulated by M β-AP (1-40) + 1U / ml IFN-γ stimulation is strongly suppressed. Similarly, when β-AP (1-42) was used, NO production was strongly enhanced by IFN-γ, and NO production was suppressed by the compound of structural formula (I-4).

[0077]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to suppress NO production from iNOS producing cells such as glial cells and phagocytic cells without affecting NO production from cNOS-bearing cells such as vascular endothelial cells and nerve cells. Provided are a compound, a nitric oxide production inhibitor using the same, and a novel method for determining the nitric oxide production inhibitory activity of a test compound, which is useful for screening such a compound. The disease can be treated.

[Brief description of drawings]

FIG. 1 is a graph showing the influence of NMMA on nitrite production by β-AP (1-40).

FIG. 2 is a graph showing the effect of NMMA on nitrite production by β-AP (1-42).

FIG. 3 is a graph showing the effect of NMMA on neuronal damage caused by β-AP (1-40).

FIG. 4 iNO for compounds of structural formula (I-4)
The graph which shows the inhibitory effect with respect to NO resulting from S and cNOS.

FIG. 5: iNOS and cNOS for NMMA
The graph which shows the suppression effect with respect to NO resulting from.

FIG. 6 is a graph showing the effect of IFN-γ on NO production by β-AP (1-40) stimulation.

FIG. 7 is a compound of structural formula (I-4), β-AP (1
-40) and a graph showing the effect of NO production due to IFN-γ stimulation.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C07D 413/04 307 G01N 31/00 H G01N 31/00 33/50 Z 33/50 A61K 37/02 ABR

Claims (4)

[Claims] 1. A compound of the general formula (I) (In the formula, R is an optionally substituted heterocyclic group, and X is-
CH ₂ -, - CO- or -CH = CH-, m is an integer of 0 to 3, p is 1 or 2. ) The compound or its salt represented by this. 2. A compound represented by the general formula (I): (In the formula, R is an optionally substituted heterocyclic group, and X is-
CH ₂ -, - CO- or -CH = CH-, m is an integer of 0 to 3, p is 1 or 2. ) A nitric oxide production inhibitor comprising a compound represented by the formula (4) or a pharmaceutically acceptable salt thereof. 3. A β-amyloid protein or its fragment and a test compound are added to a brain cell in which nitric oxide is induced by the β-amyloid protein or its fragment, and the brain cell is cultured to obtain nitric oxide. A method for determining the nitric oxide production inhibitory activity of a test compound, which comprises measuring the production amount. 4. A nitric oxide synthase inducer containing a β-amyloid protein or a fragment thereof.