EP1087950A1

EP1087950A1 - New 2-aminocarbonyl-5(2h)-isoxazolones as ligands of a dfp-binding site for treatment of cns-diseases

Info

Publication number: EP1087950A1
Application number: EP99929179A
Authority: EP
Inventors: Stephan Lensky; Bernd Riedl; Chantal FÜRSTNER; Jens ERGÜDEN; Frank BÖSS; Bernard Schmidt; Franz-Josef Van Der Staay; Werner Schröder; Joachim Schuhmacher; Delf Schmidt
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 1998-06-19
Filing date: 1999-06-11
Publication date: 2001-04-04
Also published as: JP2002518490A; DE19827387A1; CA2335190A1; US6344471B1; WO1999067229A1; AU4607999A

Abstract

The invention relates to new 2-aminocarbonyl-5(2H)-isoxazolones used as selective ligands of a high-affinity binding site of diisopropyl-fluorophosphate (DFP) on brain membranes, for prophylaxis and treatment of diseases of the central nervous system, especially cognitive disorders, depression, schizophrenia and anxiety.

Description

New 2-aminocarbonyl-5 (2H) -isoxazolones as ligands of a DFP binding site for the treatment of CNS diseases

The present invention relates to new 2-aminocarbonyl-5 (2H) isoxazolones as selective ligands of a high affinity binding site of diisopropyl fluorophosphate (DFP) on brain membranes for the prophylaxis and treatment of diseases of the central nervous system, in particular cognitive disorders, depression, schizophrenia and Anxiety.

Studies in the so-called Morris test, an animal model for learning and memory, showed that diisopropyl fluorophosphate (DFP) has a beneficial effect on learning and memory processes (J Pharmacol. Exp. Ther. 1996, 278, 697-708). The optimal dosage of DFP for the procognitive effect in young adult rats is 0.03 mg / kg, given orally. Increasing the dose above this value does not lead to a further increase in learning performance, but to a return to the control level or further to a deterioration in cognitive performance compared to control animals. Although DFP inhibits cholinesterase, significant inhibition of cholinesterase in the brain of rats is achieved ex vivo only at doses of> 3 mg / kg orally, ie at

Dosages that are a factor of about 100 higher than the active dose in the learning test. Cholinesterase inhibitors that differ structurally from phosphoric acid esters, e.g. Tacrine, or physostigmine, show no improvement in cognitive performance in this animal model.

The discrepancy between cognitively stimulating dosages on the one hand and acetylcholinesterase-inhibiting dosages on the other hand indicates the involvement of a second mechanism of action, which is more sensitive to DFP than cholinesterase. A high affinity binding site in the chicken spinal cord was identified for DFP (Biochem. Pharmacol. 1994, 48, 2073-2079), which differs from the catalytically active centers of acetylcholinesterase and butyrylcholinesterase. The function of the new, high-affinity binding site for DFP is not yet known.

It has now been found for the first time that high-affinity, specific binding sites for DFP occur in membranes of the mammalian brain. It has been detected in membranes of the cerebral cortex of rats, calves and humans.

Surprisingly, the high affinity binding site for DFP is not a previously known molecular target of DFP. In particular, the competition profile of the binding site does not match that of acetylcholinesterase, butyrylcholinesterase, neuropathy target esterase, prolylendopeptidase and dipeptidyl peptidase II. In addition, selective ligands for 84 known neurotransmitter receptors, enzymes and ion channels of the mammalian brain were tested for competition of [ ³ H] DFP in vitro. None of the compounds showed affinity, even at very high, already physiologically irrelevant concentrations.

In addition to DFP itself, only the well-known acetylcholinesterase inhibitor dichlorvos binds to the high-affinity DFP binding site.

Cerebral tissue preparations from mammals, preferably from rats, calves or humans, are therefore suitable for locating ligands of the high-affinity DFP binding site.

The high-affinity DFP binding site is obtained from conventional mammalian cerebral tissue, preferably by homogenizing and centrifuging the tissue and resuspending the precipitate.

After that, the ligand usually becomes this in different concentrations

Mix given and pre-incubated. To measure binding, DFP is added and the mixture is incubated. The DFP is usually labeled, preferably radioactively labeled. DFP is added in concentrations of less than 50 nM, preferably in a concentration of 0.1-20 nM, very particularly preferably 5-15 nM.

Ligands are usually understood to mean modulators, i.e. Substances that affect the activity of the binding site. The modulators can act agonistically or antagonistically on the binding site.

Binding sites in the sense of the invention are endogenous proteins, preferably enzymes or receptors.

The properties of the new, high-affinity binding site for DFP explain the above-mentioned learning and memory-enhancing effects of DFP. Therefore, substances that influence this binding site, such as DFP, should also have the cognitive-enhancing effect of DFP. Such substances are therefore suitable both for the therapeutic and for the preventive treatment of cognitive disorders in general, in particular dementias of the Alzheimer type.

In addition, the antimicrobial and antileukaemic effect of l- (l-aminocarbonyl) -2, l-benzisoxazol-3 (lH) -one is known (J Med. Chem. 1984, 27, 1212-1215).

The synthesis of 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone has also been described (Can. J. Chem. 1981, 59, 175-179).

The present invention relates to compounds of the general formula (I) in which

R ¹ and R ^{2 are the} same or different and

represent (C, -C ₈ ) alkyl, which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ⁵ and which is optionally selected one or more times by radicals from the group (C, -C ₄ ) alkoxy , (C, -C ₃ ) perfluoroalkoxy, halogen or NR R ^{7 is} substituted.

stand for (C ₃ -C ₈ ) cycloalkyl, which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ⁸ and which is optionally selected one or more times by radicals from the group (C, -C ₄ ) alkoxy , (C _r C ₃ ) -perfluoroalkoxy, halogen or NR ⁹ R '° is substituted,

represent aryl- (CH ₂ ) _m ,

wherein

R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ^{10 are the} same or different and are independently hydrogen, (C, -C) alkyl, (C, -C) acyl, (C, -C ₄ ) - Alkoxy-carbonyl, carbamoyl, mono- or di- (C, -C ₄ ) -alkylamino-carbonyl, and

m represents 0, 1, 2 or 3,

or R ^{1 represents} hydrogen, aryl-S (O) _n , (C, -C ₄ ) alkoxy, aryl-O or halogen and R ^{2 has} the meaning given above, and

wherein

n represents 0, 1 or 2,

or

R 'and R ² together with the adjacent carbon atoms form a 5-10-membered, monounsaturated carbocycle which is optionally interrupted by an oxygen or sulfur atom or by a radical NR "and which is optionally selected one or more times by radicals from the Group (C, -C ₄ ) alkoxy, (C, -C ₃ ) perfluoroalkoxy, halogen or NR ¹² R ^{13 is} substituted,

wherein

R ", R ¹² and R ^{13 are} identical or different and independently of one another have the meaning given for R ⁵ ,

R ³ and R ^{4 are the} same or different and independent of each other

stand for (C, -C ₈ ) alkyl, which may be replaced by an oxygen or

Sulfur atom or is interrupted by a radical NR ¹⁴ and which is optionally substituted one or more times by radicals selected from the group (C ₃ -C ₆ ) cycloalkyl, (C, -C ₄ ) alkoxy, halogen or NR ¹⁵ R ¹⁶ ,

stand for (C ₃ -C ₈ ) cycloalkyl, which may be replaced by an oxygen or

Sulfur atom or is interrupted by a residue NR ¹⁷ and that optionally substituted one or more times by radicals selected from the group (C, -C ₄ ) alkoxy, halogen or NR ¹⁸ R ¹⁹ ,

represent aryl- (CH ₂ ),

wherein

R ' ⁴ , R' \ R ' ⁶ , R ¹⁷ , R' ^S and R ' ^{9 are} identical or different and independently of one another have the meaning given for R ³ , and

p represents 0 or 1,

or

R ³ and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 3-10-membered, mono- or bicyclic heterocycle, which optionally contains up to two further heteroatoms selected from the group consisting of nitrogen, oxygen or sulfur, and optionally by residues selected from the group (C, -C ₄ ) -alkyl, (C, -C) -alkoxy, hydroxy, halogen, COOR ²⁰ , aryl-Y, or NR ^I R ^{22 is} substituted,

wherein

R ²⁰ denotes hydrogen or (C, -C ₄ ) alkyl,

Y represents a bond, CH ₂ , CO or CHOH, and

R ²¹ and R ^{22 are the} same or different and independently of one another have the meaning given for R ³ ,

and X represents oxygen or sulfur,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

Physiologically acceptable salts of the compounds according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulfonic acids. For example, particular preference is given to Salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.

Depending on the substitution pattern, the compounds according to the invention can exist in stereoisomeric forms which either behave like image and mirror image (enantiomers) or do not behave like image and mirror image (diastereomers). The invention relates to both the enantiomers or diastereomers or their respective mixtures. The racemic shapes can be just like that

Separate diastereomers in a known manner into the stereoisomerically uniform constituents.

In the context of the invention, (C, -C ₈ ) -alkyl, (C | -C ₆ ) -alkyl or (C _r C ₄ ) -alkyl stand for a straight-chain or branched alkyl radical with 1 to 8 or 1 to 6 or 1 to 4

Carbon atoms. Examples include: methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-pentyl and n-hexyl. A straight-chain or branched alkyl radical having 1 to 4 carbon atoms (C, -C) is preferred. A straight-chain or branched alkyl radical having 1 to 3 carbon atoms (C _] -C ₃ ) is particularly preferred. In the context of the invention, (C, -C _ft ) alkoxy stands for a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms. Examples include: methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy. A straight-chain or branched alkoxy radical having 1 to 4 carbon atoms (C, -C ₄ ) is preferred. A straight-chain or branched alkoxy radical having 1 to 3 carbon atoms (C _r C ₃ ) is particularly preferred.

In the context of the invention, aryl stands for an aromatic or heteroaromatic radical having 5 to 6 ring atoms. Examples include phenyl, fur-2-yl, fur-3-yl, thien-2-yl, thien-3-yl, pyrid-2-yl, pyrid-3-yl, pyrid-4-yl, pyrimidin-2 -yl, pyrimidine-4-yl. Phenyl is preferred. The radicals can in turn be substituted one or more times with halogen atoms, preferably chlorine or fluorine.

In the context of the invention, (C, -C ₈ ) cycloalkyl, (C ₃ -C ₆ ) cycloalkyl etc. stand for cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.

The following may preferably be mentioned: cyclopropyl, cyclopentyl and cyclohexyl.

Halogen in the context of the invention generally represents fluorine, chlorine, bromine and iodine. Fluorine, chlorine and bromine are preferred. Fluorine and chlorine are particularly preferred.

In the context of the invention, (C, -C ₄ ) -acyl represents a straight-chain or branched acyl radical having 1 to 4 carbon atoms. Examples include: formyl, acetyl, propionyl, n-butyryl, iso-butyryl.

In the context of the invention, (C _! -C ₆ ) alkoxycarbonyl represents a straight-chain or branched alkoxycarbonyl radical having 1 to 6 carbon atoms. Examples include: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxycarbonyl and tert-butoxycarbonyl. A straight-chain or branched alkoxycarbonyl radical having 1 to 4 carbon atoms (C, -C ₄ ) is preferred. A straight-chain or branched alkoxycarbonyl radical having 1 to 3 carbon atoms (C, -C ₃ ) is particularly preferred. In the context of the invention, the 5-10-membered, monounsaturated carbocycle stands for cyclopentene-1,2-diyl, cyclohexene-1,2-diyl, cycloheptene-1,2-diyl, cyclooctene-1,2-diyl, cyclonone 1,2-diyl or cyclodecene-1,2-diyl. Cyclohexene-1,2-diyl, cycloheptene-1,2-diyl and cyclooctene-1,2-diyl are preferred. Individual ring atoms can be replaced by an oxygen, sulfur or nitrogen atom. Examples include: 3-pyrroline-3,4-diyl, 1,2,5,6-tetrahydropyridine-3,4-diyl, 5,6-dihydro- (2H) -pyran-3,4-diyl.

The saturated or partially unsaturated 3-12-membered, mono- or bicyclic

Within the scope of the invention, heterocycle stands for a monocyclic or bicyclic ring with 3 to 10 ring atoms, which is bonded to the adjacent carbonyl / thiocarbonyl group via a nitrogen atom and contains up to two further heteroatoms and optionally one or more double or triple bonds. In the case of the bicyclus, the two rings can be linked spirocyclically, or the bridgehead atoms of the two rings are directly adjacent or separated by one or more ring atoms. Examples include aziridin-1-yl, azetidin-1-yl, pyrrolidin-1-yl, pyrrolin-1-yl, piperidin-1-yl, 1,2,5,6-tetrahydropyridin-1-yl, morpholin-4 -yl, thiomorpholin-4-yl, hexahydroazepin-1-yl, 2,3-dihydro- (lH) -indol-l-yl, octahydroindol-1-yl, 8-azabicyclo [3.2.1] octan-8-yl ,

3-aza-bicyclo [3.2.1] octan-3-yl, 3,8-diaza-l-oxa-bicyclo [4.3.0] nonan-8-yl, azacyclodecen-1-yl. Azetidin-1-yl, pyrrolidin-1-yl and pyrrolin-1-yl are preferred.

Compounds of the general formula (I) are preferred

in which

R ¹ and R ^{2 are the} same or different and

stand for (CC ₈ ) -alkyl, which may be replaced by an oxygen or

Sulfur atom is interrupted and if necessary one or more times is substituted by radicals selected from the group consisting of methoxy, ethoxy or fluorine,

represent (C ₃ -C ₆ ) cycloalkyl which is optionally substituted one or more times by radicals selected from the group consisting of methoxy, ethoxy or fluorine,

represents aryl- (CH ₂ ) _m ,

wherein

m represents 0, 1 or 2,

or

R ^{1 represents} hydrogen, aryl-S, aryl-O, fluorine or chlorine and R ^{2 has} the meaning given above,

or

R ¹ and R ² together with the adjacent carbon atoms form a 5-8-membered, monounsaturated carbocycle which is optionally interrupted by an oxygen or sulfur atom and which is optionally selected one or more times by radicals selected from the group consisting of methoxy, ethoxy or fluorine is substituted,

R ³ and R ^{4 are the} same or different and independent of each other

stand for (C, -C ₆ ) alkyl, which may be replaced by an oxygen or

Sulfur atom or is interrupted by a residue NR ¹⁴ and, if appropriate, one or more times by residues selected from the group Cyclopropyl, cyclobutyl, methoxy, ethoxy, fluorine or NR ^{, 3} R ^{16 is} substituted,

represent (C ₃ -C ₆ ) -cycloalkyl which is optionally substituted one or more times by radicals selected from the group consisting of methoxy, ethoxy, fluorine or NR ¹⁸ R ¹⁹ ,

represent aryl- (CH ₂ ), and

wherein

R ¹⁴ , R ' ⁵ , R' ⁶ , R ¹⁷ , R ^1S and R ^{19 are} identical or different and are hydrogen, (C, -C ₃ ) -alkyl, acetyl, propionyl, methoxycarbonyl or ethoxycarbonyl, and

p represents 0 or 1,

or

R ³ and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 4-9-membered, mono- or bicyclic heterocycle which optionally contains up to two further heteroatoms selected from the group consisting of nitrogen, oxygen or sulfur, and if appropriate by radicals selected from the group methyl, ethyl, methoxy, ethoxy, hydroxy, fluorine, COOR ²⁰ , aryl-Y, or NR ²¹ R ²² ,

wherein

R ^{20 represents} hydrogen, methyl or ethyl,

Y represents a bond, CH ₂ or CHOH, and R ² 'and R ^{22 are} identical or different and independently of one another are hydrogen, (C, -C ₃ ) -alkyl, acetyl, propionyl, methoxycarbonyl or ethoxycarbonyl,

and

X represents oxygen or sulfur,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

Compounds of the general formula (I) are particularly preferred

in which

R ¹ and R ^{2 are the} same or different and

stand for (C, -C ₆ ) -alkyl, which is optionally substituted one or more times by fluorine,

represent (C ₃ -C ₆ ) cycloalkyl which is optionally substituted by fluorine,

stands for benzyl,

or

R ^{1 represents} hydrogen, phenyl-S or phenyl-O and R ^{2 has} the meaning given above, or

R ¹ and R ² together with the adjacent carbon atoms form a 5-9-membered, monounsaturated carbocycle which is optionally substituted one or more times by fluorine,

R ³ and R ^{4 are the} same or different and independent of each other

represent (C, -) alkyl which is optionally interrupted by an oxygen or sulfur atom and which is optionally substituted one or more times by radicals selected from the group (C ₃ -C ₆ ) -cycloalkyl or fluorine,

represent phenyl- (CH ₂ ) _P ,

wherein

p represents 0 or 1,

or

R ³ and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 4-9-membered, mono- or bicyclic heterocycle which optionally contains up to two further heteroatoms selected from the group consisting of nitrogen, oxygen or sulfur, and if appropriate by radicals selected from the group methyl, hydroxy, fluorine, COOR ²⁰ , phenyl-Y, or NR ²¹ R ^{22 is} substituted,

wherein

R ^2U means methyl, Y represents a bond or CHOH, and

R ²¹ and R ^{22 are the} same or different and are hydrogen or methyl, and

X represents oxygen,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

Compounds according to claim 1 are very particularly preferred,

in which

R ¹ and R ^{2 are the} same or different and

represent (C ₂ -C ₆ ) alkyl, or

represent cyclopentyl or cyclohexyl,

R ³ and R ^{4 are the} same or different and independently of one another represent methyl or ethyl,

or

R ³ and R ⁴ together with the nitrogen atom form an azetidin-1-yl, pyrrolidin-1-yl or pyrrolin-1 -yl radical, and

X represents oxygen,

and their salts.

In addition, processes for the preparation of compounds of the general formula (I) were found, characterized in that compounds of the general formula (II),

in which

R ¹ and R ^{2 have} the meaning given in claim 1, and R ^{23 represents} (C, -C ₄ ) -alkyl which is optionally substituted by fluorine,

with hydroxylamine as the free base or salt, optionally in a solvent, to give a compound of the general formula (III),

in which

R ¹ , R ² and R ^{23 have} the meaning given above,

implements and then in the presence of a base to give a compound of the general formula (IV),

in which

R ¹ and R ^{2 have} the meaning given above,

cyclized,

thereafter with a compound of the general formula (V),

(Cl ₂ C = X) _q (V)

in which

X represents oxygen or sulfur, and

q means 1, 2 or 3,

optionally in the presence of an inert solvent and a base to give a compound of the general formula (VI),

in which, R, R "and X have the meaning given above,

implements

finally with a compound of the general formula (VII),

HNR ³ R ⁴ (VII)

in which

R " ¹ and R ^{4 have} the meaning given in claim 1,

optionally condensed in the presence of an auxiliary base to the compound of general formula (I).

Starting from compounds of the formula (II), it is also possible to prepare compounds of the general formula (IV) without isolating the compounds of the formula (III) in the meantime.

Customary organic solvents which do not change under the reaction conditions are suitable as solvents for the processes. These include ethers such as diethyl ether, dioxane, tetrahydrofuran, glycol dimethyl ether, or hydrocarbons such as benzene, toluene, xylene, hexane, cyclohexane or petroleum fractions, or halogenated hydrocarbons such as dichloromethane, trichloromethane, tetrachloromethane, dichlorethylene, tricholethylene or tricholethylene, or ethyl acetate, pyridine, dimethyl sulfoxide, N, N-dimethylformamide, hexamethylphosphoric triamide, acetonitrile, acetone or nitromethane. It is also possible to use mixtures of the solvents mentioned. For the processes (II) -> (III) and (III) -> (IV), water or a mixture of water with one of the organic solvents mentioned is also suitable. Water is preferred for these processes. For the processes (IV) -> (VI) and - lo -

(VI) -> (I) are preferably dichloromethane and trichloromethane. Dichloromethane is particularly preferred.

In general, inorganic or organic bases can be used as bases for the processes according to the invention. This preferably includes

Alkali metal hydroxides such as sodium hydroxide or potassium hydroxide, alkaline earth metal hydroxides such as barium hydroxide, alkali metal carbonates such as sodium carbonate, potassium carbonate or cesium carbonate, alkaline earth metal carbonates such as calcium carbonate, or alkali metal or alkaline earth metal alcoholates such as sodium or potassium methanolate, sodium - or potassium ethanolate or potassium tert-butoxide, or organic

Amines (trialkyl (C _r C ₆ ) amines) such as triethylamine, or heterocycles such as 1,4-di-azabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), pyridine, N, N-dimethylaminopyridine, methylpiperidine or morpholine. Sodium hydroxide is preferred for process (II) -> (III) -> (IV) and triethylamine for process (VI) -> (I).

In general, the base is used in an amount of 0.05 mol to 10 mol, preferably 1 mol to 3 mol, based on 1 mol of the compound of the formula (III) or (VI).

The processes according to the invention are generally carried out in a temperature range from -20 ° C. to + 100 ° C., preferably from 0 ° C. to + 60 ° C.

The processes according to the invention are generally carried out at normal pressure. However, it is also possible to use overpressure or light pressure

Vacuum (e.g. in a range of 0.5 to 5 bar).

The process according to the invention can be illustrated by the following formula scheme:

The compounds of the general formulas (II), (V) and (VII) are known or can be prepared by customary methods.

However, cognitive disorders are not the only medical indication of such ligands for the newly found binding site. It was surprisingly found that DFP also has potent effects in an in vivo animal model for the discovery of new antidepressants. This is the "Rat Forced Swimming Test" according to Porsolt (Nature 1977, 266, 730-732). DFP induces in this

A dose-dependent test has a behavior-activating effect, as is known from clinically effective antidepressants. As in the cognition test, the optimal dose is 0.03 mg / kg orally, ie far below the concentrations that would be required for choline esterase inhibition (as described above). Based on these test results, ligands from the high-affinity DFP binding site are also suitable for use in psychiatric indications such as depression, schizophrenia or anxiety. The compounds according to the invention also inhibit ACPH (N-acyl peptide hydrolase).

The new active compounds can be converted in a known manner into the customary formulations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents. Here, the therapeutically active compound should in each case be present in a concentration of about 0.0001 to 90% by weight, preferably 0.0001 to 1.0% by weight, of the total mixture, i.e. in amounts sufficient to achieve the dosage range indicated.

The formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, optionally using emulsifiers and / or dispersants, e.g. in the case of the use of water as a diluent, organic solvents can optionally be used as auxiliary solvents.

The application is carried out in the usual way, preferably orally, transdermally or parenterally, in particular perlingually or intravenously.

In general, it has proven to be advantageous to administer amounts of approximately 0.00001 to 10 mg / kg, preferably approximately 0.0001 to 1 mg / kg, of body weight in the case of intravenous administration in order to achieve effective results.

Nevertheless, it may be necessary to deviate from the amounts mentioned, depending on the body weight or the type of application route, on the individual behavior towards the medication, the type of its formulation and the time or interval at which the administration takes place . So in some cases it may be sufficient to use less than that to make do with the aforementioned minimum quantity, while in other cases the upper limit mentioned must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day.

The ligands of the high affinity DFP binding site are generally suitable for use in the prophylaxis and treatment of diseases of the central nervous system.

However, compounds of the general formula (IX) are excluded,

in which

R ²⁴ and R ^{25 are the} same or different and are independently halogen, (C, -C ₈ ) alkoxy. (C, -C ₈ ) alkenoxy, aryl-S or aryl-O, where (C, -C ₈ ) -

Alkoxy or (C, -C ₈ ) alkenoxy are optionally substituted one or more times by halogen, and

R ^{26 has} the meaning of R ²⁴ and is the same or different from it, or represents optionally substituted (C, -C ₈ ) -alkyl or substituted aryl, and

X has the meaning given above.

In the context of the invention, (C, -C ₈ ) -alkenoxy stands for a straight-chain or branched alkenyloxy residue consisting of 1 to 8 carbon atoms. Examples include: ethenyl, prop-2-en-1-yl, prop-2-en-2-yl, prop-3-enyl or but-1-en-1-yl. Such new drugs are first identified by an in vitro competition test at the high affinity DFP binding site. In principle, Zeil membranes from mammalian species other than rats, including humans, can also be used.

Ligands of the high-affinity DFP binding site in the sense of the invention are those substances which, when applied with [ ³ H] DFP, displace at a concentration of less than 10 nM with a dissociation constant K _d of less than 100,000 nM.

Ligands are preferred whose K _d at this binding site is less than 1000 nM.

Also particularly suitable are those ligands which bind weaker to the acetylcholinesterase by a factor of at least 500, very particularly preferably weaker by a factor of at least 1000.

The selectivity of ligands related to the high affinity DFP binding site

Cholinesterase inhibition can be demonstrated with suitable in vitro tests. One such test is the photometric determination of the cholinesterase activity according to Ellman (Biochem. Pharmacol. 1961, 7, 88-95).

The ligands of the high-affinity DFP binding site are particularly suitable for the

Use for the prophylaxis and treatment of cognitive disorders, anxiety, schizophrenia or depression, especially of dementias of the Alzheimer type.

The efficacy of the substances identified in this way in the treatment and prevention of cognitive disorders is proven with the help of known standard animal models for learning and memory (see, for example, 'Alzheimer's Disease: Biology, Diagnosis and Therapeutics Iqbal et al., Ed .; 1997 , John Wiley, pp. 781-786). Suitable animal models for this are, for example, passive or active avoidance behavior, classic or operant conditioning, spatial orientation tests, or object or subject recognition tests. As a particularly suitable model the so-called Morris test is recommended, which is based on spatial memory (J. Neurosci. Methods 1984, 11, 47-60).

1. High affinity DFP binding site

a) Characterization

Rat brains were homogenized, cooled in 50 mM Tris-HCl buffer, pH 7.4 and centrifuged twice for 30 min at 48,000 × g, the supernatants being discarded and the precipitate being resuspended in 50 mM Tris buffer. The test batch in the binding test initially contained about 0.4 mg of membrane protein, DFP in various concentrations, and 50 mM Tris buffer. This mixture was preincubated for 30 min at 25 ° C. in a water bath before commercially available [ ³ H] DFP (0.1 to 20 nM, 5 nM in standard tests) was added. The samples were then mixed well again and incubated for 120 min at 25 ° C. At the end of the reaction time, 3 ml of ice-cold Tris buffer was added to each sample and the membrane-bound fraction of the ligand was separated from the unbound, free fraction by rapid filtration through Whatman GF / C filters. The filters were washed three times with cold buffer and the bound amount of the radio ligand was determined by ß-scintillation measurement. To determine the specific binding, the non-specific binding determined in the presence of 100 μM non-radioactive DFP was subtracted from the total binding.

The binding measured in this way was 90% specific and saturable. The apparent binding constant K _d was in the whole brain as well as in some

Brain regions such as the cerebral cortex 1.8 + 0.2 nM (mean ± standard error from a total of 21 experiments up to 10 nM). The binding site density B _max was in the range of 347 ± 14.9 pmol / mg membrane protein in these experiments. In the range up to 10 nM [ ³ H] DFP, the Scatchard transformation of the binding data was linear. This implies interaction with a single, non-interacting, high affinity class of Close binding sites for the radioligand in this concentration range. At radioligand concentrations> 10 nM, the total binding increased again disproportionately, which indicates an interaction with other, less affine binding sites at higher concentrations.

b) Differentiation of the high-affinity DFP binding site from cholinesterases: The DFP binding site is not cholinesterases. A comparison of the interaction of unlabelled DFP and physostigmine with both biological targets under largely the same conditions showed that DFP inhibits the binding of [3H] DFP to the high-affinity binding site in rat brain membranes about 200 times more potent than the acetylcholinesterase activity _. (IC50 11 nM vs 2.6 μM). However, physostigmine, which like DFP binds to the acetylcholine binding region of acetylcholinesterase, was only able to inhibit acetylcholinesterase (IC50 33 nM), not to displace DFP from the high-affinity DFP binding site (IC50> 1 mM).

c) Identification of ligands of the high affinity DFP binding site

The displacement of [ ³ H] DFP from the high-affinity DFP binding site is determined analogously to the method described under 1.a) for DFP. Example 1 showed an IC ₅₀ of 4.8 nM.

2. Determination of the selectivity towards acetylcholinesterase

The selectivity of the substances as DFP ligands with regard to cholinesterase inhibition can be demonstrated with suitable in vitro tests. One such test is the photometric determination of the cholinesterase activity according to Ellman (Biochem. Pharmacol. 1961, 7, 88-95). As a rule, the experiment is carried out with raw whole brain rat homogenates. However, brain homogenates from other warm-blooded animals, including humans, are suitable as enzyme preparations, as are commercially available, purified enzyme preparations. In the standard experiment, rat brains are homogenized in 20-fold volume (w / v) 100 mM K ₂ HPO ₄ buffer, pH 8.0. The protein concentration of the homogenate is adjusted to approximately 3 mg / ml by dilution in the buffer. The incubation batch (1500 μl) contains 100 mM phosphate buffer pH 8.0, 330 μM dithiobisnitrobenzoate, 50 μl brain homogenate corresponding to 150 μg protein, various test concentrations of the test substances to be tested and 40 μM acetylthiocholine as the enzyme substrate. In order to enable better comparison of the incubation conditions with those of the [ ³ H] DFP binding test, the reaction mixture is preincubated for 30 min at room temperature before adding the substrate. The enzyme reaction is started by adding substrate. The reaction time is 6 min. The samples are then mixed with 25 ml of 1 mM tacrine solution (final concentration) in order to terminate the reaction, and the yellow color complex in the spectrophotometer at 412 nm is quantified against the corresponding blank value (tacrine addition before substrate addition). The IC _{50 is} calculated from the enzyme activity in the presence of increasing concentrations of test substance in the test and with that for the [ ³ H] DFP-

Competition compared.

Example 1 inhibited acetylcholinesterase with a K _j of> 10 μM. This results in a selectivity of> 2,000 for this substance.

Morris test:

The Morris Test measures spatial orientation learning in rodents. The test is ideal for evaluating the learning and memory-enhancing effects of substances. In this test, rats or mice are trained to locate an invisible platform as the only way out of a water-filled swimming pool. A proven method is to train the animals four times a day over a period of 5 days. The test substances are tested on a daily basis at a defined time, e.g. 30 min before the first swimming attempt per day. Controls receive the corresponding

Vehicle. The learning performance of the animals is expressed in a training-related Shortening the swim distance between the starting position and the platform, as well as reducing the swimming time until reaching the platform, i.e. the better the animal remembers the location of the platform, the shorter the distance covered and the faster the platform is reached. The test is performed on cognitively impaired animals, such as old animals or animals with experimentally induced brain damage.

Rats with an entorhinal cortex lesion are an animal model for Alzheimer's disease. The bilateral lesion of the entorhinal cortex is created by intracerebral injection of the excitotoxin ibotenic acid. It leads to a severe impairment of learning performance in the Morris test.

The learning and memory-improving effect of Example 1 was tested using this animal model. In rats with a bilateral entorhinal cortex lesion, learning of the platform position in the Morris test was greatly slowed, and that

Performance after five days of training was significantly worse than that of control animals that underwent surgery like the lesioned animals but did not receive intracerebral injections of the excitotoxin ibotenic acid.

The intraperitoneal injection of Example 1 in a dose of 0.1 mg per kg

Body weight, 30 minutes before each of the five daily training days, significantly accelerated learning the platform position. The lesionized animals treated with Example 1 already achieved the same level of performance on the second training day as the vehicle-treated animals after 5 training days. Example 1, however, could not completely antagonize the learning disability induced by the entorhinal cortex lesion; considering the extent of the experimentally induced brain damage, this was also not to be expected. 4. Rat Forced Swimming Test:

The effectiveness of the substances in the treatment and prevention of affective disorders is demonstrated with the help of the "Rat Forced Swimming Test" according to Porsolt (Nature 1977, 266, 730-732). The test is based on the observation that rats remain in an immobile position in a hopeless situation ("behavioral despair"). 24 hours before the test, young adult rats (3-4 months old) are placed individually in glass cylinders (height 40 cm, diameter 20 cm) for 20 min, which are filled with water up to a height of 15 cm. In the test, the animals are again transferred to the cylinder and the duration of immobility over a period of

Measured 5 min. The test substances are applied in the period between the two swimming tests. Controls are given the appropriate vehicle.

Table 1:

Behavior-activating effect of DFP in the "Rat Forced Swimming Test". The behavioral effect is indicated as mean change in immobility compared to vehicle-treated control animals ± standard error. Physiological saline was used as vehicle. The significance of the effect was assessed with ANOVA and subsequent Fischer post -hoc analysis checked, ns = not significant.

Output connections:

Example I: 2- (l-methylpropyl) hexan-3-one carboxylic acid ethyl ester

A solution of 10.1 g (62.5 mmol) of hexan-3-one-carboxylic acid ethyl ester in 60 ml of DMF was added dropwise at 0 ° C. to a solution of 8.84 g (68.8 mmol) of potassium tert-butoxide in 25 ml of DMF and then 30 min at this temperature stirs. A solution of 7.59 ml (9.53 g, 68.1 mmol) of 2-bromobutane in 25 ml of DMF was added dropwise over the course of 30 min and the reaction mixture was stirred at room temperature overnight. For working up, the mixture was added to 250 ml of water and extracted with ethyl acetate (3 x 100 ml). The united org. Phases were dried (sodium sulfate), concentrated, and the crude product was purified by chromatography on silica gel (0.04 to 0.63 mm) with 40: 1 petroleum ether. 'H NMR (DMSO, TMS, 400 MHz, δ): 4.1 1 (m, 2H), 3.48 (dd, 1H), 2.49 (m, 2H), 2.08 (m, 1H), 1.48 (m, 2H) , 1.30 (m, 1H), 1.15 (dt, 3H), 1.10 (m, 1H), 0.84 (m, 9H); MS (DCI, NH ₃ ): 215 [M + Hf.

Example II: 2-Cyclopentylhexan-3-oncarboxylic acid ethyl ester

The preparation was carried out analogously to Example I from 4.04 g (25.0 mmol) of ethyl hexan-3-one carboxylate and 2.95 ml (4.10 g, 27.3 mmol) of bromocyclopentane. The

Purification was carried out chromatographically (silica gel, petroleum ether-ethyl acetate 20: 1). MS (EI): 226 [M] ⁺ .

Example III

2-Hexylhexan-3-onecarboxylic acid ethyl ester

1.59 g (69.3 mmol) of sodium were added in portions to 50 ml of ethanol under an argon atmosphere. The mixture was then stirred under reflux until homogeneous

Solution had arisen. 1 1.0 g (69.3 mmol) of hexan-3-oncarboxylic acid ethyl ester was added to the hot alcoholate solution and the mixture was stirred for 45 min. Then 6.51 ml (7.93 g, 52.5 mmol) of 1-iodohexane were slowly added dropwise at the boiling point. After stirring under reflux for 16 h, the mixture was cooled to room temperature, filtered and the solvent was removed in vacuo. The residue was taken up in dichloromethane, filtered again, and concentrated. The crude product was purified chromatographically (silica gel, petroletheπEssigester 30: 1). MS (DCI, NH ₃ ): 309 [M + H] ⁺ .

Preparation Examples:

Example 1: 4-ethyl-3-propyl-2- (l-pyrrolidinylcarbonyl) -5 (2H) -isoxazolone

a) 4-Ethy 1-3-propyl-5 (2H) -isoxazolone

11.53 g (165.9 mmol) of hydroxylamine hydrochloride are dissolved in 25 ml of water and at a temperature of max. 0 ° C with a solution of 13.27 g (331.8 mmol) of sodium hydroxide in 60 ml of water. Then you give without further

Cooling 30.9 g (165.9 mmol) of 2-ethylhexan-3-oncarboxylic acid ethyl ester are added all at once and the mixture is stirred at 50 ° C. for 3 h. For working up, 15.5 ml of conc. Hydrochloric acid added at 0 ° C, diluted with water and extracted three times with dichloromethane. The organic phase is dried over sodium sulfate, concentrated and fractionated in a high vacuum. Kp ^2mbar . 105-1 15 ° C.

b) 4-Ethyl-3-propyl-2- (chlorocarbonyl) -5 (2H) -isoxazolone

15 g (96.65 mmol) of 4-ethyl-3-propyl-5 (2H) -isoxazolone in 150 ml of dichloromethane are mixed at 0 ° C with 27.81 g (144.98 mmol) of diphosgene and the mixture is then 6 hours stirred at 0 ° C. To remove excess phosgene, argon was passed through the reaction mixture at RT for 1 h, the exhaust air being neutralized by sodium hydroxide. The solution was concentrated and the product distilled in vacuo. Kp ^3mbar 125-30 ° C. c) 4-Ethyl-3-propyl-2- (l-pyrrolidinylcarbonyl) -5 (2H) -isoxazolone

At 0 ° C, a solution of 0.43 g (6 mmol) of pyrrolidine in 35 ml of dichloromethane was added dropwise and the mixture was stirred at room temperature overnight. For working up, the residue was chromatographed on silica gel using petroleum ether: ethyl acetate (4: 1). MS (DCI): 253 (M + H).

Analogously to Example 1, the compounds from Table 2 below were prepared from the corresponding starting materials:

Claims

claims

1. Compounds of the general formula (I),

in which

R ¹ and R ^{2 are the} same or different and

represent (C ₁ -C ₈ ) -alkyl which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ⁵ and which is optionally selected one or more times by radicals from the group (C, -C ₄ ) -alkoxy , (C _r C ₃ ) -perfioralkoxy, halogen or NR ⁶ R ^{7 is} substituted,

represent (C ₃ -C ₈ ) cycloalkyl, which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ⁸ and which is optionally selected one or more times by radicals from the group (C | -C ₄ ) alkoxy , (C _r C ₃ ) perfluoroalkoxy, halogen or NR ⁹ R ^{10 is} substituted,

represent aryl- (CH ₂ ) _m ,

wherein

R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ^{10 are} identical or different and are independently of one another hydrogen, (C, -C ₄ ) -alkyl, (C, -C ₄ ) - Acyl, (C _r C ₄ ) -alkoxy-carbonyl, carbamoyl, mono- or di- (C, -C ₄ ) -alkyl-amino-carbonyl, and

m represents 0, 1, 2 or 3,

or

R ^{1 represents} hydrogen, aryl-S (O) _n , (C, -C ₄ ) alkoxy, aryl-O or halogen and R ^{2 has} the meaning given above, and

wherein

n represents 0, 1 or 2,

or

R ¹ and R ² together with the adjacent carbon atoms form a 5-10-membered, monounsaturated carbocycle which is optionally interrupted by an oxygen or sulfur atom or by a radical NR "and which is optionally selected one or more times by radicals from the Group (C, -C ₄ ) alkoxy, (C _r C ₃ ) perfluoroalkoxy, halogen or NR ' ² R ^{13 is} substituted,

wherein

R ³ and R ^{4 are the} same or different and independent of each other represent (C, -C ₈ ) -alkyl which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ¹⁴ and which is optionally selected one or more times by radicals from the group (C ₃ -C ₆ ) -cycloalkyl, ( C, -C) alkoxy, halogen or NR ¹⁵ R ^{16 is} substituted,

represent (C ₃ -C ₈ ) cycloalkyl, which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ¹ 'and which is optionally selected one or more times by radicals selected from the group (C, -C ₄ ) - Alkoxy, halogen or NR ^{I 8} R ^{19 is} substituted,

represent aryl- (CH ₂ ),

wherein

R ¹⁴ , R ¹⁵ , R ^{, δ} , R ' ⁷ , R ¹⁸ and R ^{19 are} identical or different and independently of one another have the meaning given for R ⁵ , and

p represents 0 or 1,

or

R ³ and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 3-10-membered, mono- or bicyclic heterocycle which optionally contains up to two further heteroatoms selected from the group nitrogen, oxygen or sulfur, and if appropriate is substituted by radicals selected from the group (C, -C ₄ ) -alkyl, (C, -C ₄ ) -alkoxy, hydroxy, halogen, COOR ²⁰ , aryl-Y, or NR ²¹ R ²² ,

wherein R ²⁰ denotes hydrogen or (C, -C ₄ ) alkyl,

Y represents a bond, CH ₂ , CO or CHOH, and

R ²¹ and R ^{22 are the} same or different and independently of one another have the meaning given for R ⁵ ,

and

X represents oxygen or sulfur,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

2. Compounds according to claim 1,

in which

R ¹ and R ^{2 are the} same or different and

represent (C, -C ₈ ) -alkyl which is optionally interrupted by an oxygen or sulfur atom and which is optionally substituted one or more times by radicals selected from the group consisting of methoxy, ethoxy or fluorine,

represent (C ₃ -C ₆ ) cycloalkyl which is optionally substituted one or more times by radicals selected from the group consisting of methoxy, ethoxy or fluorine, represents aryl- (CH ₂ ) _m ,

wherein

m represents 0, 1 or 2,

or

R ³ and R ^{4 are the} same or different and independent of each other

stand for (C, -C ₆ ) -alkyl, which is optionally interrupted by an oxygen or sulfur atom or by a radical NR ¹⁴ and which is optionally selected one or more times by radicals from the group cyclopropyl, cyclobutyl, methoxy, ethoxy, fluorine or

NR ¹⁵ R ^{16 is} substituted,

represent (C ₃ -C ₆ ) cycloalkyl which is optionally substituted one or more times by radicals selected from the group consisting of methoxy, ethoxy, fluorine or NR ¹⁸ R ¹⁹ , represent aryl- (CH ₂ ), and

wherein

R ' ⁴ , R ¹⁵ , R' ⁶ , R ¹⁷ , R ' ⁸ and R ^{19 are the} same or different and are hydrogen, (C, -C ₃ ) alkyl, acetyl, propionyl, methoxycarbonyl or ethoxycarbonyl, and

p represents 0 or 1,

or

R ^"1 and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 4-9-membered, mono- or bicyclic heterocycle which optionally contains up to two further heteroatoms selected from the group consisting of nitrogen, oxygen or sulfur, and the optionally substituted by radicals selected from the group consisting of methyl, ethyl, methoxy, ethoxy, hydroxy, fluorine, COOR ²⁰ , aryl-Y, or NR ²¹ R ²² ,

wherein

R ^{20 represents} hydrogen, methyl or ethyl,

Y represents a bond, CH ₂ or CHOH, and

R ²¹ and R ^{22 are} identical or different and independently of one another are hydrogen, (C, -C ₃ ) -alkyl, acetyl, propionyl, methoxycarbonyl or ethoxycarbonyl,

and X represents oxygen or sulfur,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

Compounds according to claim 1,

in which

R ¹ and R ^{2 are the} same or different and

stands for benzyl,

or

R ^{1 represents} hydrogen, phenyl-S or phenyl-O and R ^{2 has} the meaning given above,

or

R ¹ and R ² together with the adjacent carbon atoms form a 5-9-membered, monounsaturated carbocycle which is optionally substituted one or more times by fluorine, R ³ and R ^{4 are the} same or different and independent of each other

represent (C, -C ₆ ) -alkyl which is optionally interrupted by an oxygen or sulfur atom and which is optionally substituted one or more times by radicals selected from the group (C ₃ -C ₆ ) -cycloalkyl or fluorine,

represent phenyl- (CH ₂ ) _,

wherein

p represents 0 or 1,

or

R ³ and R ⁴ together with the nitrogen atom form a saturated or partially unsaturated 4-9-membered, mono- or bicyclic heterocycle, which optionally contains up to two further heteroatoms selected from the group consisting of nitrogen, oxygen or sulfur, and optionally by residues selected from the group methyl, hydroxy, fluorine, COOR ²⁰ , phenyl-Y, or NR ^2I R ^{22 is} substituted,

wherein

R ²⁰ means methyl,

Y represents a bond or CHOH, and R ²¹ and R ^{22 are the} same or different and are hydrogen or methyl,

and

X represents oxygen,

and their salts,

except 2-aminocarbonyl-3-methyl-5 (2H) -isoxazolone.

4. Compounds according to claim 1,

in which

R ¹ and R ^{2 are the} same or different and

represent (C ₂ -C ₆ ) alkyl, or

represent cyclopentyl or cyclohexyl,

R ³ and R ^{4 are the} same or different and independent of each other

represent methyl or ethyl,

or

R ³ and R ⁴ together with the nitrogen atom form an azetidin-1-yl, pyrrolidin-1-yl or pyrrolin-1-yl radical, and X represents oxygen,

and their salts.

5. 4-ethyl-3-propyl-2- (l-pyrrolidinylcarbonyl) dihydro-5 (2H) isoxazolone

6. A process for the preparation of compounds according to claim 1, characterized in that compounds of the general formula (II),

in which

R ¹ and R ^{2 have} the meaning given in claim 1, and

R ^{23 represents} (C, -C ₄ ) -alkyl, which is optionally substituted by fluorine,

in which R, R and R have the meaning given above,

implements

and then in the presence of a base to give a compound of the general formula (IV),

in which

R 'and R ^{2 have} the meaning given above,

cyclized,

thereafter with a compound of the general formula (V),

(Cl ₂ C = X) _q (V)

in which

X represents oxygen or sulfur, and

q means 1, 2 or 3,

optionally in the presence of an inert solvent and a base to give a compound of the general formula (VI), in which,

R ¹ , R ² and X have the meaning given above,

implements

finally with a compound of the general formula (VII),

HNR ³ R ⁴ (VII) in which

R ³ and R ^{4 have} the meaning given in claim 1,

optionally in the presence of an auxiliary base to connect the general

Formula (I) are condensed.

7. Medicament containing a compound of general formula (I) according to one of claims 1 to 5 in admixture with at least one pharmaceutically acceptable, substantially non-toxic carrier or

Excipients.

8. Compounds according to any one of claims 1 to 5 for use as a medicament in the treatment of humans and animals.

. Use of compounds according to one of claims 1 to 4 for the manufacture of medicaments for the treatment and / or prophylaxis of diseases of the central nervous system.

10. Use of compounds according to one of claims 1 to 4 for the manufacture of medicaments for the treatment and / or prophylaxis of cognitive disorders, depression, schizophrenia and anxiety.

1 1. Use of ligands of the high affinity binding site of diisopropyl fluorophosphate, with the exception of compounds of the general formula (IX),

in which

R ²⁴ and R ^{23 are} identical or different and independently of one another are halogen, (C, -C ₈ ) alkoxy, (C, -C ₃ ) alkenoxy, aryl-S or aryl-O, where (C, -C ₈ ) alkoxy or (C _r C ₈ ) alkenoxy optionally substituted one or more times by halogen, and

R ^{26 has} the meaning of R ²⁴ and is the same or different, or for optionally substituted (C, -C ₈ ) alkyl or substituted

Aryl stands, and

X represents oxygen or sulfur,

on mammalian brain membranes for the manufacture of medicinal products

Prophylaxis and treatment of diseases of the central nervous system.

12. Use according to claim 1 1, wherein the dissociation constant K _{d of} the ligands at this binding site is less than 1000 nM.

13. Use according to claim 11 or 12, wherein the dissociation constant K _{d of} the ligands to this binding site is at least 500 times greater than their affinity for acetylcholinesterase.

14. Use of ligands according to claim 12, wherein the dissociation constant K _{d of} the ligands to this binding site is at least a factor of 1000 greater than their affinity for acetylcholinesterase.

15. Use according to any one of claims 1 1 to 14, wherein the central nervous system disease is a cognitive disorder.

16. Use according to claim 15, wherein the cognitive disorder is dementia of the Alzheimer type.

17. Use according to any one of claims 11 to 14, wherein the central nervous system disease is anxiety, schizophrenia or depression.

18. Use of mammalian cerebral tissue preparations to find ligands of the high affinity DFP binding site.

19. Use according to claim 18, wherein the mammals are rat, calf or human.

20. A method for finding ligands of the high-affinity DFP binding site on cerebral tissue, characterized in that

(A) obtains the binding sites from the mammalian brain,

(B) pre-incubated with the ligand,

(C) then incubated with DFP and (D) measures binding.