WO1998037064A1

WO1998037064A1 - Benzamides having dopamine d4 receptor affinity

Info

Publication number: WO1998037064A1
Application number: PCT/US1998/001745
Authority: WO
Inventors: Ashok Tehim; Xin Wang; Jalaj Arora; Adi Treasurywala
Original assignee: Hoechst Marion Roussel, Inc.
Priority date: 1997-02-19
Filing date: 1998-01-30
Publication date: 1998-08-27
Also published as: AU6256198A

Abstract

Described herein are D4 receptor-selective compounds of general formula (I), wherein R1 is a 5- or 6-membered aromatic or heteroaromatic ring, attached at the meta- or para-position of the benzamide phenyl group, wherein said ring is optionally substituted with 1 or 2 groups independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO¿2?-, (loweralkyl)2NSO2-, H(O)C-, HO2C-, trifluoromethyl, trifluoromethoxy, H2N-, loweralkyl-NH- and (loweralkyl)2N-, loweralkylcarbonyl; R?2¿ is selected from an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, and an optionally substituted 6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle; n is 1 or 2; and salts, solvates, stereoisomers or hydrates thereof. Also described is the use of these compounds as pharmaceuticals to treat indications for which a dopamine D4 receptor antagonist is implicated, such as schizophrenia and anxiety.

Description

BENZAMIDES HAVING DOPAMINE D4 RECEPTOR AFFINITY

This invention relates to compounds that bind to the dopamine D4 receptor, to their preparation and their use for therapeutic and drug screening purposes.

Background to the Invention:

Neuronal cell receptors that bind the neurotransmitter dopamine constitute a group of at least five structurally distinct proteins that can now be produced using recombinant DNA techniques. These techniques have been applied to construct cell lines that incorporate the dopamine receptor in their membranes, to provide regenerable and homogeneous substrates with which chemical libraries can be screened to identify potential CNS-active drugs.

Recent evidence strongly implicates the dopamine receptor classified as D4 in the etiology of schizophrenia. It has been suggested that compounds capable of interfering with the function of this receptor, which is present in schizophrenics at levels that are six times normal, would be useful in the treatment of this disease (Seeman et al, Nature, 1993, 365:441). Some drugs currently on the market in fact exhibit the desired antagonism of D4 receptor activity, and bind with relative strong affinity to the receptor. Yet because of their structure, these drugs interact also with related dopamine receptors, particularly the D2 receptor type, which results in significant side effects that include altered motor function and tachycardia. It would be desirable to provide compounds that exhibit not only a high degree of affinity for the D4 receptor, but also a relatively low degree of affinity for the D2 receptor. In this specification, this desired combination of receptor binding properties is referred to as D4 selectivity. Products currently marketed to treat indications in which the D4 receptor function is implicated include the dibenzodiazepine, clozapine, and the dibenzoxazepine, isoloxapine. Analysis of their dopamine receptor binding properties has shown that the preference for binding to the D4 receptor relative to the D2 receptor is about 10 fold, for both products. Similarly, both bind to the D4 receptor with about the same affinity (Ki value approximately 20 nM). More recently, selective D4 receptor antagonists have been identified among other classes of compounds (see US 5,576,336).

It is an object of the present invention to provide a compound that binds to the D4 receptor.

It is another object of the present invention to provide compounds which bind selectively to the D4 receptor, relative particularly to the D2 receptor.

It is a further object of the present invention to provide a pharmaceutical composition comprising a compound of the present invention, as active ingredient.

It is another object of the present invention to provide a method effective to treat medical conditions for which administration of a D4 receptor antagonist is indicated, such as to treat schizophrenia and anxiety.

Summary of the Invention:

According to one aspect of the present invention, there is provided a compound of Formula I:

wherein

R¹ is a 5- or 6-membered aromatic or heteroaromatic ring, attached at the meta- or para- position of the benzamide phenyl group, wherein said ring is optionally substituted with 1 or 2 groups independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl; R is selected from an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, and an optionally substituted 6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle; n is 1 or 2; and salts, solvates, stereoisomers, or hydrates thereof; with the proviso that R¹ is not imidazolo when n is 2 and R² is phenyl.

According to another aspect of the invention, there is provided a pharmaceutical composition comprising a compound of Formula I in an amount effective to antagonize D4 receptor stimulation and a pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise a compound in the context of Formula I wherein R¹ is imidazole, n is 2 and R² is phenyl.

In another of its aspects, the invention provides the use of compounds of Formula I as D4 receptor antagonists for the treatment of medical conditions mediated by D4 receptor stimulation.

In a further aspect of the invention, there is provided an analytical method in which a compound of the invention is used to distinguish, in a receptor population, the D4 receptor from other receptor types, and particularly from the D2 receptor. These and other aspects of the present invention are now described in greater detail hereinbelow. Detailed Description and Preferred Embodiments:

The term "loweralkyl" as used herein means straight or branched chain alkyl radicals containing from one to four carbon atoms and includes methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl and the like. 1-Methylethyl and 1-methylpropyl are also known as isopropyl and sec-butyl respectively.

The term "loweralkoxy" as used herein means straight or branched chain alkoxy radicals containing from one to four carbon atoms and includes methoxy, ethoxy, 1-methylethoxy, propyloxy, butoxy and the like.

The term "halo" as used herein means halide and includes fluoro, chloro, bromo and iodo.

The term "5- or 6-membered aromatic or "heteroaromatic ring" means 5- or 6- membered unsaturated ring systems which contain 1, 2 or 3 heteroatoms selected from O, S and N, and preferably, one or two nitrogen atoms, one or two oxygen atoms, one nitrogen and one oxygen atom, one nitrogen and one sulfur atom, or one sulfur atom, and includes, but is not limited to, thiophene, furan, pyrrole, pyran, pyridine, pyrazine, pyrimidine, pyridazine, isothiazole, isoxazole, pyrazoline, and pyrazine. These ring systems may be "optionally substituted" which means there is either hydrogren or one or two other moieties attached thereto as later described.

The term "6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle" as used herein means a fused 5,6- or 6,6 ring system containing 0 to 4 heteroatoms selected independently from O, S and N, and includes naphthyl, indolo, benzofuranyl, 1,3-benzodioxolyl and the like.

The term "pharmaceutically acceptable salts" means either an acid addition salt or a basic addition salt which is compatible with the treatment of patients for the intended use.

"Pharmaceutically acceptable acid addition salt" is any non-toxic organic or inorganic acid addition salt of the base compounds represented by Formula I or any of its intermediates. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono-, di- and tri-carboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic, p-toluenesulfonic acid and sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Either the mono- or di-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of these compounds are more soluble in water and various hydrophilic organic solvents and which in comparison to their free base forms, generally demonstrate higher melting points.

"Pharmaceutically acceptable basic addition salts" means non-toxic organic or inorganic basic addition salts of the compounds of Formula (I) or any of its intermediates. Examples are alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calcium, magnesium or barium hydroxides; ammonia, and aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline. The selection of the appropriate salt may be important so that the ester is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art.

"Solvate" means a compound of Formula I or the pharmaceutically acceptable salt of Formula I wherein molecules of a suitable solvent are incorporated in a crystal lattice. A suitable solvent is not substantially toxic at the dosage administered as the solvate to achieve the desired effect. Examples of suitable solvents are ethanol and the like.

The term "stereoisomers" is a general term for all isomers of the individual molecules that differ only in the orientation of their atoms in space. It includes mirror image isomers (enantiomers), geometric (cis/trans) isomers, and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers).

The term "patient" means a warm blooded animal, such as for example rat, mice, dogs, cats, guinea pigs, and primates such as humans. The term "treat" or "treating" means to alleviate symptoms, eliminate the causation of the symptoms either on a temporary or permanent basis, or to prevent or slow the appearance of symptoms of the named disorder or condition.

The term "therapeutically effective amount" means an amount of the compound which is effective in treating the named disorder or condition.

The term "pharmaceutically acceptable carrier" is a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to the patient One example of such a carrier is a pharmaceutically acceptable oil typically used for parenteral administration.

The term "schizophrenia" means schizophrenia, schizophreniform disorder, schizoaffective disorder and psychotic disorder wherein the term "psychotic" refers to delusions, prominent hallucinations, disorganized speech or disorganized or catatonic behavior. See Diagnostic and Statistical Manual of Mental Disorder, fourth edition, American Psychiatric Association, Washington D.C.

The invention relates to compounds that bind to the dopamine D4 receptor in a selective manner, relative to the dopamine D2 receptor.

In embodiments of the invention, compounds of Formula I include those in which R¹ is an optionally substituted 5-membered heteroaromatic ring containing 1, 2 or 3 heteroatoms selected from O, S and N, wherein at least one heteroatom is O or S. In preferred embodiments of the invention, R¹ is selected from optionally substituted thienyl and f iranyl. In more preferred embodiments R¹ is selected from thien-2yl and thien-3-yl.

In another embodiment of the invention, R¹ is selected from an optionally substituted phenyl ring and an optionally substituted 6-membered heteroaromatic ring containing 1 or 2 nitrogen atoms. In preferred embodiments, R¹ is optionally substituted phenyl.

In further embodiments of the invention, R¹ is optionally substituted with 1 or 2 groups independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H₂N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl. Preferably, R¹ is selected from phenyl and phenyl substituted with one group selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H₂N-, loweralkyl- NH, (loweralkyl)₂N- and loweralkylcarbonyl. In more preferred embodiments, R¹ is selected from phenyl, 4-trifluoromethylphenyl, 3-aminophenyl, 2-formylphenyl, 4-formylphenyl, 3- thiomethylphenyl, 4-fluorophenyl, 3 -fluorophenyl and 4-chlorophenyl. In the most preferred embodiments of the invention, R¹ is selected from phenyl, 3-fluorophenyl, 4-chlorophenyl and 4-trifluoromethylphenyl. It will be appreciated that, in expressions such as (loweralkyl) N-, the loweralkyl groups may be the same or different, to yield substituents such as CH₃NH- and (CH₃)(CH₃CH₂)N-.

In compounds of the invention, R¹ is attached at the meta- or para-position of the benzamide phenyl group. In preferred embodiments, R¹ is attached at the para-position of the benzamide phenyl group.

In other embodiments of the invention, R² is selected from a 5- or 6-membered aromatic or heteroaromatic ring and a 6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle, both optionally substituted with a group independently selected from H, halo, loweralkyl, trifluoromethyl, trifluoromethoxy, nitro, amino, H₂N-, loweralkyl-NH-, (loweralkyl)₂N-, cyano, and loweralkoxy. Preferably, R² is phenyl.

In compounds of the invention, n is 1 or 2. In preferred embodiments, n is 1.

In specific embodiments of the invention, the compounds of Formula I include: (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-trifluoromethylphenyl)benzamide; (3-R,S)-4-(3-Aminophenyl)-N-( 1 -benzyl-3-pyrrolidinyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(2-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(2-formylphenyl)benzamide; (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-methylthiophenyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(3-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(4-fluorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)l-4-(3-fluorophenyl)benzamide;

(3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-chlorophenyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(4-formylphenyl)benzamide;

(3S)- N-( 1 -Benzyl-3-pyrrolidinyl)-4-phenylbenzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-3-phenylbenzamide;

(3R)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-phenylbenzamide;

(4-R,S)-N-( 1 -Benzyl-4-piperidinyl)-4-phenylbenzamide; (3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-phenylbenzamide;

(4-R,S)-N-( 1 -Benzyl-4-piperidinyl)-3-phenylbenzamide;

(3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-ethylphenyl)benzamide; and

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(4-hydroxyphenyl)benzamide.

Preferred compounds of Formula I include:

(3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-trifluoromethylphenyl)benzamide;

(3-R,S)-4-(3- Aminophenyl)-N-( 1 -benzyl-3-pyrrolidinyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(2-thienyl)benzamide; (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(3-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)l-4-(3-fluorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(4-chlorophenyl)benzamide;

(3S)- N-( 1 -Benzyl-3-pyrrolidinyl)-4-phenylbenzamide;

(3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-phenylbenzamide; and (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-ethylphenyl)benzamide.

More preferred compounds of Formula I include: (3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)-4-(2-thienyl)benzamide; (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(3-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-pyrrolidinyl)l-4-(3-fluorophenyl)benzamide; (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(4-chlorophenyl)benzamide; (3S)- N-( 1 -Benzyl-3-pyπolidinyl)-4-phenylbenzamide; (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-phenylbenzamide; and (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(4-ethylphenyl)benzamide.

The most preferred compounds of Formula I include: (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(2-thienyl)benzamide; (3-R,S)-N-(l-Benzyl-3-pyrrolidinyl)-4-(3-thienyl)benzamide; and (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(4-chlorophenyl)benzamide.

Acid addition salts of the compound of Formula I are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, sulphuric or phosphoric acids and organic acids e.g. succinic, maleic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates may be used for example in the isolation of compounds of Formula I for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.

The compounds of the present invention have chiral centres, e.g. at the 3-position of the pyπolidine ring or 4-position of the piperidine ring. The invention extends to cover all structural and optical isomers of the various compounds, as well as racemic mixtures thereof.

The compounds of the present invention can be prepared by processes analogous to those known in the art. The present invention therefore provides, in a further aspect, a process for the preparation of a compound of Formula I which involves coupling a benzoic acid of Formula A (Scheme 1), wherein R¹ is as defined above, or a reactive derivative thereof, with an amine of Formula B, using standard conditions to form an amide bond which are well documented elsewhere (see for example Takashima, M. et al. US 4,210,660 and "An Introduction to Peptide Chemistry", Bailey, P.D. John Wiley & Sons, 1990).

Scheme 1

B

Alternatively, compounds of Formula I wherein R ,ι is as defined above can be prepared by reacting compounds of Formula C (Scheme 2) wherein R³ is a suitable leaving group, for example halo or triflate (preferably iodo), with a metallo-aryl compound Ri-M, wherein R-_. is as defined above and M is an optionally substituted metal group, using standard palladium catalysed cross coupling techniques. Examples of such M groups include (alkyl)₃Sn-, (alkyl)₂B-, (HO)₂B-, (alkoxy) B-, Li, Cu, chloroZn or haloMg. The most preferred M group is (HO) B-. The reaction takes place in an inert solvent, and optionally in the presence of base, lithium chloride and a suitable catalyst. Suitable catalysts included palladium (II) and palladium (0) species such as palladium (II) acetate, palladium (II) chloride and tetrakis(triphenylphosphine) palladium (0). The prefeπed catalyst is tetrakis(triphenylphosphine) palladium (0). Suitable bases include tertiary amines or sodium carbonate (preferred). Suitable inert solvents include acetonitrile, N,N-dimethylformamide, dimethoxyethane and tetrahydrofuran, with dimethoxyethane being preferred. The reaction takes place at a temperature of from 25-100 °C, preferably 50-100 °C. Compounds of formula RpM can be prepared from reagents of R_t-Y, wherein Y is a suitable leaving group, for example halo or triflate, by standard metalation reactions either independently or in situ. For example, the compound R^M wherein M is (HO)₂B- can be prepared by treating Rj-Br with n-butyl lithium in tetrahydrofuran at -78 °C followed by addition of a trialkylborate, such as trimethylborate, followed by a work-up with IM hydrochloric acid . Scheme 2

Reagents of Formula C may be prepared from a benzoic acid of Formula D (below), wherein R³ is as defined above, or a reactive derivative thereof, and the amine of Formula B using the same conditions described above for the preparation of compounds of Formula I. The benzoic acids of Formulae A and D are either commercially available or can be prepared using standard techniques.

D

The amines of Formula B are commercially available or can be prepared using one of several literature preparations (see for example Ohmori, J. et al. J. Med. Chem. 1996, 39: 2764- 2772 or Rognan, D. et al. EP 539281 ). Alternatively, compounds of Formula I wherein R² is phenyl may be debenzylated under standard conditions, for example catalytic hydrogenation, to provide intermediates of Formula E as shown in Scheme 3. This hydrogenation is normally carried out in an inert solvent in the presence of a catalyst such as palladium on carbon, Raney nickel or platinum oxide in an atmosphere of hydrogen gas and optionally under increased pressure (10-40 psi). Preferred is the hydrogenation in ethyl acetate with palladium on carbon as catalyst at room temperature and atmospheric pressure. Intermediates of Formula E may then be alkylated with reagents of Formula F, wherein Z is a suitable leaving group such as halo, mesylate or tosylate and R² is as defined above, in the presence of a base in an inert solvent. Suitable conditions include sodium hydride in tetrahydrofuran at temperatures in the range of -20 to 30 °C, or potassium carbonate in acetonitrile at refluxing temperatures. Reagents of Formula F are either commercially available or can be prepared using methods analogous to those known in the art.

Scheme 3

The clozapine-like binding profile of the present compounds indicates their utility as pharmaceuticals that may be useful as neuroleptics for the treatment of various conditions in which the D4 receptor is implicated, such as for the treatment of anxiety and schizophrenia. Accordingly, in another of its aspects, the present invention provides pharmaceutical compositions useful to treat D4-related medical conditions, in which a compound of Formula I is present in an amount effective to antagonize D4 receptor stimulation, together with a pharmaceutically acceptable carrier. In a related aspect, the invention provides a method for treating medical conditions for which a D4 antagonist is indicated, which comprises the step of administering to the patient an amount of a compound of Formula I effective to antagonize D4 receptor stimulation, and a pharmaceutically acceptable carrier therefor.

For use in medicine, the compounds of the present invention can be administered in a standard pharmaceutical composition. The present invention therefore provides, in a further aspect, pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a Formula I compound or a pharmaceutically acceptable salt, solvate or hydrate thereof, in an amount effective to antagonize D4 receptor stimulation.

The compounds of the present invention may be administered by any convenient route, for example by oral, parenteral, buccal, sublingual, nasal, rectal or transdermal administration and the pharmaceutical compositions formulated accordingly.

Compounds of Formula I and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions, or as solid forms such as tablets, capsules and lozenges. A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable pharmaceutical liquid carrier for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier, for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.

Alternatively, the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as flurochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

Preferably, the composition is in unit dose form such as a tablet, capsule or ampoule. Suitable unit doses i.e. therapeutically effective amounts; can be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will of course vary depending on the desired clinical endpoint. It is anticipated that dosage sizes appropriate for administering the compounds of the examples will be roughly equivalent to, or slightly less than, those used currently for clozapine. Accordingly, each dosage unit for oral administration may contain from 1 to about 500 mgs, and will be administered in a frequency appropriate for initial and maintenance treatments.

For laboratory use as a ligand, the present compounds can be stored in packaged form for reconstitution and use. The present compounds can be used to distinguish dopamine receptors from other receptor types, for example glutamate and opioid receptors, within a population of receptors, and in particular to distinguish between the D4 and D2 receptors. The latter can be achieved by incubating preparations of the D4 receptor and of the D2 receptor with a D4 selective compound of the invention and then incubating the resulting preparation with a radiolabelled dopamine receptor ligand, such as ³H-spiperone. The D2 and D4 receptors are then distinguished by determining the difference in membrane-bound radioactivity, with the D4 receptor exhibiting lesser radioactivity, i.e., lesser ³H-spiperone binding.

In another aspect of the invention, a compound of Formula I is provided in labelled form, such as radiolabelled form e.g. labelled by incorporation within its structure of ³H or ¹⁴C or by conjugation to ¹²⁵I or ¹²³I. Such radiolabelled forms can be used directly to distinguish between dopamine D4 and dopamine D2 receptors. Furthermore, radiolabelled forms of the present compounds can be exploited to screen for more potent dopamine D4 ligands, by determining the ability of the test ligand to displace the radiolabelled compound of the present invention.

Example 1: (3-R,S)-4-Iodo-N-(l-benzyl-3-pyrτolidinyl)benzamide

To a solution of 4-iodobenzoyl chloride (10 g, 37 mmol) in ether (400 mL) at 0 °C was added triethylamine (4.7 g, 45 mmol). (3-R,S)-(±)-l-Benzyl-3-aminopyrrolidine (7.3 g, 41 mmol) in ether (100 mL) was then added slowly via dropping funnel at 0 °C. After the addition was complete, the mixture was further stiπed for 18 hrs. Water (200 mL) was added and the mixture extracted with methylene chloride (3x 200 mL) dried and concentrated to give the title compound (9.7 g, 64%) as beige powder. 1H NMR (CDC1₃) δ: 7.79 (m, 2H), 7.47 (m, 2H), 7.26-7.35 (m, 5H), 6.5 l(d, 1H, J = 8.8 Hz), 4.62-4.68 (m, 1H), 3.63 (s, 2H), 2.91-2.96 (m, 1H), 2.70-2.74 (m, 1H), 2.58-2.63 (m, 1H), 2.25-2.44 (m, 2H), 1.68-1.77 (m, 1H); ¹³C NMR (CDC1₃) δ: 166.0, 138.5, 137.7, 128.8, 128.6, 128.4, 127.2, 60.7, 60.0, 52.6, 49.3, 32.6.

Example 2(a): (3-R.S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(4-trifluoromethylphenyl)benzamide To a solution of (3-R,S)-4-iodo-N-(l-benzyl-3-pyπolidinyl)benzamide (Example 1, 100 mg, 0.25 mmol) in dimethoxyethane (10 mL) at room temperature was added 4- trifluoromethyl-phenylboronic acid (93 mg, 0.50 mmol), Pd (PPh₃)₄ (14 mg, 0.012 mmol), 2M Na₂CO₃ (5 mL). The mixture was stiπed and refluxed for 18 hrs. The solution was cooled to room temperature, extracted with methylene chloride (2x 100 mL), dried over MgSO and concentrated to give a white solid. Purification by silica gel chromatography (EtOAc and EtOAc: MeOH 80:20) provided a white solid (92 mg, 80%). mp 180-183 °C; HRMS (FAB): MH⁺ calculated for C₂₅H₂₃N₂OF₃: 425.18408 found: 425.18344; 1H NMR (CDC1₃) δ: 7.26- 8.09 (m, 17H), 4.73 (bs, 1H), 3.75 (s, 2H), 3.11 (m, 1H), 3.02 (m, 2H), 2.69-2.74 (m, 1H), 2.36-2.48 (m, 2H), 1.81-1.88 (m, 1H); ¹³C NMR (CDCI₃) δ: 167.0, 143.4, 142.7, 136.2, 133.7, 132.3, 131.9, 129.3, 128.7, 128.0, 127.3, 125.8, 124.1, 60.3, 59.6, 52.6, 48.8, 32.1.

In a like manner, the following additional compounds were prepared: (b) (3-R,S)-4-(3-Aminophenyl)-N-(l-benzyl-3-pyπolidinyl)benzamide: from 3- aminophenylboronc acid; 62%; yellow oil; HRMS (FAB): MH⁺ calculated for C₂ H₂₅N₃O: 372.20758 found: 372.20754; 1H NMR (CDC1₃) δ: 782 (m, 2H), 7.57 (m, 2H), 6.96-7.34 (m, 8H), 6.88 (s, 1H), 6.69 (d, 1H, J = 7.4 Hz) 4.73 (bs, 1H), 3.77 (s, 2H), 3.00 (m, 1H), 2.82 (m, 1H), 2.64-2.69 (m, 1H), 2.30-2.42 (m, 2H), 1.73-1.81 (m, 1H); ¹³C NMR (CDCI3) δ: 166.6, 146.9, 144.4, 141.2, 137.7, 133.1, 129.8, 129.1, 128.5, 127.4, 127.1, 117.6, 114.7, 113.8, 60.6, 60.0, 52.7, 48.9, 32.5.

(c) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(2-thienyl)benzamide: from 2-thiopheneboronic acid; 73%; beige powder; mp 118-120 °C; HRMS (FAB): MH⁺ calculated for C₂₂H₂₂N₂OS:

363.15311 found: 363.15309; 1H NMR (CDC1₃) δ: 7.09-7.79 (m, 12H), 6.67 (d, 1H, J = 7.4), 4.69 (m, 1H), 3.65 (s, 2H), 2.96 (m, 1H), 2.76 (m, 1H), 2.60-2.66 (m, 1H), 2.27-2.43 (m, 2H), 1.74-1.76 (m, 1H); ¹³C NMR (CDC1₃) δ: 166.2, 143.2, 138.4, 137.3, 128.9, 128.4, 128.2, 127.6, 127.2, 125.9, 125.7, 124.1, 60.8, 60.1, 52.7, 49.1, 32.7.

(d) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(2-formylphenyl)benzamide: from 2- formylphenylboronic acid; 92%; yellow oil; HRMS (FAB): MH⁺ calculated for C₂₅H₂₄N₂O₂: 385.19159 found 385.19350; 1H NMR (CDCI₃) δ: 9.93 (s, 1H), 7.26-8.04 (m, 13H), 7.06 (d, 1H, J = 7.5 Hz), 4.74 (bs, 1H), 3.68 (s, 2H), 2.99-3.06 (m, 1H), 2.83 (m, 1H), 2.67-2.72 (m, 1H), 2.33-2.44 (m, 2H), 1.76-1.85 (m, 1H); ¹³C NMR (CDCI₃) δ: 191.9, 166.2, 144.8, 140.9, 137.6, 133.7, 132.1, 132.0, 130.2, 129.1, 128.6, 128.5, 128.3, 127.9, 127.5, 127.2, 60.5, 59.9, 52.7, 49.2, 32.4.

(e) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(4-methylthiophenyl)benzamide: from 4- methylthiophenylboronic acid; 95%; white powder; mp 168-170 °C; HRMS (FAB): MH⁺ calculated for C₂₅H₂₆N₂OS: 403.18442 found 403.18410; 1H NMR (CDCI3) δ: 7.26-7.89 (m, 13H), 6.91 (d, 1H, J = 7.4 Hz), 4.75 (bs, 1H), 3.70 (s, 2H), 3.01-3.04 (m, 1H), 2.83 (m, 1H), 2.65-2.70 (m, 1H), 2.53 (s, 3H), 2.32-2.48 (m, 2H), 1.74-1.83 (m, 1H); ¹³C NMR (CDCI3) δ: 166.4, 143.49, 138.68, 136.72, 132.99, 129.05, 128.47, 127.59, 127.50, 126.82, 126.79, 60.61, 59.91, 52.63, 48.91, 32.55, 15.74. (f) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(3-thienyl)benzamide: from 3-thiopheneboronic acid; 55%; off-white powder; mp 128-130 °C; HRMS (FAB): MH⁺ calculated for C₂₂H₂₂N₂OS: 363.1511 found: 363.16928; Η NMR (CDC1₃) δ: 7.26-7.82 (m, 12H), 6.64 (d, 1H, J = 7.5 Hz), 4.70 (bs, 1H), 3.66 (s, 2H), 2.98 (m, 1H), 2.77 (d, 1H), 2.61-2.67 (m, 1H), 2.30-2.43 (m, 2H), 1.25-1.76 (m, 1H); ¹³C NMR (CDC1₃) δ: 166.0, 132.2, 132.2, 128.9, 128.6, 128.4, 127.6, 127.3, 126.6, 126.4, 126.2, 121.5, 60.8, 60.1, 52.7, 49.0, 32.7.

(g) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(4-fluorophenyl)benzamide: from 4- fluorophenylboronic acid; 60%; white powder; mp 124-128 °C; HRMS (FAB): calculated for C₂₄H₂₃FN₂O: 375.18726 found: 375.19010; 1H NMR (CDCI₃) δ: 7.07-7.91 (m, 13H), 6.99 (bs, 1H), 4.72 (bs, 1H), 3.70 (s, 2H), 3.05 (m, 1H), 2.77 (m, 1H), 2.62-2.74 (m, 1H), 2.30-2.50 (m, 2H), 1.74-1.93 (m, 1H); ¹³C NMR (CDCI₃) δ: 166.5, 163.0, 143.3, 136.3, 132.8, 132.2, 132.0, 131.3, 129.2, 128.9, 128.7, 127.7, 127.0, 115.8, 61.0, 59.0, 53.0, 48.0, 32.0.

(h) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)l-4-(3-fluorophenyl)benzamide: from 3- fluorophenylboronic acid; 91%; white powder; mp 118-122 °C; HRMS (FAB): MH⁺ calculated for C₂₄H₂₃FN₂O: 375.18726 found: 375.18901, Η NMR (CDCI₃) δ: 7.00-7.87 (m, 14H), 4.70 (bs, 1H), 3.71 (s, 2H), 3.03 (m, 1H), 2.86 (m, 1H), 2.70-2.76 (m, 1H), 2.34-2.43 (m, 1H), 1.86 (m, 1H); ¹³C NMR (CDC1₃) δ: 166.6, 163.0, 142.8, 142.2, 136.8, 133.5, 132.1, 130.4, 129.2, 128.6, 127.8, 127.1, 122.8, 114.8, 60.3, 59.7, 52.7, 48.9, 32.1.

(i) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(4-chlorophenyl)benzamide: from 4- chlorophenylboronic acid; 100%; white powder; mp 159-163 °C; HRMS (FAB): MH⁺ calculated for C₂₄H₂₃ClN₂O: 391.15771 found: 391.15616; 1H NMR (CDC1₃) δ: 7.21-7.85 (m, 14H), 4.67 (bs, 1H), 3.69 (s, 2H), 3.02 (m, 1H), 2.84 (m, 1H), 2.68 (m, 1H), 2.33-2.38 (m, 2H), 1.83 (m, 1H); ¹³C NMR (CDC1₃) δ: 166.6, 142.9, 138.4, 132.4, 129.2, 129.1, 128.7, 128.6, 128.4, 127.8, 127.7, 127.0, 60.3, 59.7, 52.6, 48.9, 32.2.

(j) (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(4-formylphenyl)benzamide: from 4- formylphenylboronic acid; 74%; white solid; mp 144-145 °C; 1H NMR (CDC1₃) δ: 10.05 (s, 1H), 7.20-7.96 (m, 13H), 4.72 (d, 1H, J = 7.5 Hz), 3.66 (s 2H), 2.94-2.99 (m, 1H), 2.80 (m, 1H), 2.65-2.70 (m, 1H), 2.30-2.45 (m, 2H), 1.77-1.85 (m, 1H), ¹³C NMR (CDCI₃) δ: 191.9, 166.2, 145.9, 142.5, 138.0, 135.6, 134.3, 132.0, 130.3, 129.0, 128.7, 128.4, 127.4, 60.7, 60.0, 52.7, 49.1, 32.5.

Example 3(a): (3S)- N-(l-Benzyl-3-pyπolidinyl)-4-phenylbenzamide

To a solution of 4-biphenyl carboxylic acid (0.50 g, 2.52 mmol) in CH₂C1₂ (10 mL) at room temperature, was added EDCI (0.53 g, 2.77 mmol), DMAP (0.03 g, 0.25 mmol) and (3S)-(+)-l-benzyl-3-aminopyπolidine. The reaction mixture was stiπed at room temperature for 18h, concentrated to dryness, and diluted with EtOAc. The organic phase was successively washed with water (3x 20 mL), saturated NaHCO₃ (25 mL) and brine (20 mL). This was followed by drying (MgS0₄) and concentrating to yield an off-white solid. The crude product was purified by silica gel chromatography (5% MeOH/CH₂Cl₂) to give an off white solid (0.7980 g, 89%). mp 161-163°C; HRMS (FAB): MlT for C₂₄H₂₄N₂O: calc'd: 357.4743, found 357.1978; 1H NMR (CDCl₃)δ: 7.84 (d, 2H, J = 9 Hz), 7.65 (d, 2H, J = 9 Hz), 7.61 (d, 2H, J = 8 Hz), 7.49-7.24 (m, 8H), 6.70 (d, IH, J = 8 Hz), 4.71 (m, IH), 3.65 (s, 2H), 2.95 (dt, IH, J = 9, 3 Hz), 2.76 (dd, IH, J = 10, 2 Hz), 2.64 (dd, IH, J = 10, 6 Hz), 2.46-2.27 (m, 2H), 1.77 (m, IH); ¹³C NMR (CDC1₃) δ: 166.4, 144.2, 140.5, 138.5, 134.0, 128.9, 128.4, 128.0, 127.5, 127.2, 60.9, 60.1, 52.7, 49.1, 32.7.

In a like manner, the following additional compounds were prepared:

(b) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-3-phenylbenzamide: from (3-R,S)-(±)-l-benzyl-3- aminopyπolidine; 34%; HRMS (FAB): MH⁺ for C₂₄H₂₄N₂O: calc'd: 357.4743, found 357.1932; 1H NMR (CDCl₃)δ: 8.04 (s, IH), 7.75 (d, IH, J = 8 Hz), 7.69 (d, IH, J = 8 Hz), 7.61 (d, 2H, J = 7 Hz), 7.48-7.23 (m, 9H), 7.08 (d, IH, J = 8 Hz), 4.71 (m, IH), 3.63 (s, 2H), 2.92 (dd, IH, J = 8, 3 Hz), 2.75 (dd, IH, J ■= 10, 3 Hz), 2.65 (dd, IH, J = 10, 7 Hz), 2.43-2.04 (m, 2H), 1.76 (m, IH); ¹³C NMR (CDC1₃) δ: 166.9, 141.6, 140.3, 138.3, 135.2, 130.0, 128.9, 128.4, 127.7, 127.3, 127.2, 125.9, 125.8, 60.7, 60.1, 52.7, 49.1, 32.5.

(c) (3R)-N-(l-Benzyl-3-pyπolidinyl)-4-phenylbenzamide: from (3R)-(-)-l-benzyl-3- aminopyπolidine; 81%; white powder; mp 161-162 °C; HRMS (FAB): MH⁺ for C₂ H₂₄N₂O: calc'd: 357.4743, found 357.1980; Η NMR (CDCl₃)δ: 7.84 (d, 2H, J = 9 Hz), 7.65 (d, 2H, J = 9 Hz), 7.61 (d, 2H, J = 8 Hz), 7.49-7.25 (m, 8H), 6.67 (d, IH, J = 8 Hz), 4.71 (m, IH), 3.65 (s, 2H), 2.95 (dt, IH, J = 9, 3 Hz), 2.76 (dd, IH, J = 10, 2 Hz), 2.65 (dd, IH, J = 10, 6 Hz), 2.46- 2.27 (m, 2H), 1.75 (m, IH); ¹³C NMR (CDC1₃) δ: 166.4, 144.2, 140.5, 138.5, 134.0, 128.9,

128.4, 128.0, 127.5, 127.2, 60.9, 60.1, 52.7, 49.1, 32.7.

(d) (4-R,S)-N-(l-Benzyl-4-piperidinyl)-4-phenylbenzamide: from (4-R,S)-(±)-l-benzyl-4- piperidine; 96%; white powder; mp 180-181 °C; LRMS (FAB): 371.8 (M+l); Η NMR (CDCl₃)δ: 7.82 (d, 2H, J = 8 Hz), 7.66-7.59 (m, 4H), 7.49-7.28 (m, 8H), 6.05 (d, IH, J = 8 Hz), 4.04 (m, IH), 3.53 (s, 2H), 2.87 (d, 2H, J = 12 Hz), 2.20 (dt, 2H), 2.05 (d, 2H, J = 12 Hz), 1.59 (dq, 2H); ¹³C NMR (CDC1₃) δ: 167.0, 145.0, 140.5, 139.0, 134.0, 129.1, 128.9, 128.3, 128.0, 127.4, 127.2, 127.1, 63.1, 52.3, 47.1, 32.3.

(e) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-phenylbenzamide: from (3-R,S)-(±)-l-benzyl-3- aminopyπolidine; 89%; white powder; mp 155-156 °C; 1H NMR (CDCl₃)δ: 7.83 (d, 2H, J = 9 Hz), 7.65 (d, 2H, J = 9 Hz), 7.61 (d, 2H, J = 8 Hz), 7.47-7.26(m, 8H), 6.63 (d, IH, J = 8 Hz), 4.70 (m, IH), 3.65 (s, 2H), 2.95 (dt, IH, J = 9, 3 Hz), 2.76 (dd, IH, J = 10, 2 Hz), 2.65 (dd, IH, J = 10, 6 Hz), 2.46-2.27 (m, 2H), 1.75 (m, IH); ¹³C NMR (CDCI₃) δ: 166.4, 144.2, 140.5,

138.5, 134.0, 128.9, 128.8, 128.4, 128.0, 127.5, 127.2, 60.9, 60.1, 52.7, 49.1, 32.7.

(f) (4-R,S)-N-(l-Benzyl-4-piperidinyl)-3-phenylbenzamide: from (4-R,S)-(±)-l-benzyl-4- piperidine; 75%; white powder; mp 167-169 °C; 1H NMR (CDCl₃)δ: 7.97 (s, IH), 7.70 (d, 2H, J = 7 Hz), 7.60 (d, 2H, J = 7 Hz), 7.51-7.23 (m, 9H), 6.14 (d, IH, J = 8 Hz), 4.04 (m, IH), 3.52 (s, 2H), 2.86 (d, 2H, J = 12 Hz), 2.18 (dt, 2H), 2.03 (d, 2H, J = 12 Hz), 1.59 (dq, 2H, J = 11, 3 Hz); ^,3C NMR (CDCI3) δ: 166.9, 141.7, 140.3, 138.4, 135.4, 130.1, 129.1, 129.0, 128.9, 128.3, 127.8, 127.2, 127.1, 125.8, 125.5, 63.1, 52.3, 47.2, 32.3.

(g) (3-R,S)-N-( 1 -Benzyl-3-pyπolidinyl)-4-(4-ethylphenyl)benzamide: from (3-R,S)-(±)- 1 - benzyl-3-aminopyπolidine and 4'-ethyl-4-biphenylcarboxylic acid.

(h) (3-R,S)-N-(l-Benzyl-3-pyπolidinyl)-4-(4-hydroxyphenyl)benzamide: from (3-R,S)- (±)-l-benzy 1-3 -aminopyπolidine and 4'-hydroxy-4-biphenylcarboxylic acid. Example 4: Receptor Binding Assay

D2 and D4 receptor-binding affinities of the compounds of examples 1 and 2 were evaluated according to their ability to reduce binding of ³H-spiperone as compared to the reference compound clozapine. The potency of the test compound to reduce H-spiperone binding directly coπelated to its binding affinity for the receptor.

D4 Receptor Preparation

HEK 298 (human embryonic kidney) cells stably transfected with human D4 receptor (D4.2 sub-type) were grown in NUNC cell factories for 5 days (75% confluency) without a media change and removed with versene (approximately 19 mg of cells per cell factory tray). The cells were then centrifuged in a Sorval centrifuge for 10 min, 5000 rpm (GS3 rotor) and the pellets quickly frozen in liquid nitrogen and stored at -80°C until used in binding assay. When used in the assay, cells were thawed on ice for 20 min and then 10 ml of incubation buffer (50 mM Tris, 1 mM EDTA, 4 mM MgCl₂, 5 mM KC1, 1.5 mM CaCl₂, 120 mM NaCl, pH 7.4) was added. The cells were then vortexed to resuspend pellet and homogenized with a Kinematica CH-6010 Kriens-LU homogenizer for 15 seconds at setting 7. Concentration of receptor protein was determined using the Pierce BCA assay.

D2 Receptor Preparation GH/_tCi (rat pituitary) cells stably transfected with the human D2 receptor (short isoform) were grown in HAM'S F10 media in NUNC cell factories for 5 days. 100 mM ZnSO₄ was added to the cells (the D2 promoter being zinc inducible). After 16 hours, fresh media was added to allow the cells to recover for 24 hours. The cells were harvested using versine and then centrifuged in a Sorval centrifuge for 10 minutes, at 5000 rpm (GS3 rotor). Pellets were quickly frozen in liquid nitrogen and stored at -80°C until used in the binding assays. When used in the assay, cells were thawed on ice for 20 minutes. Each cell factory produced approximately 72 mg of protein. 10 ml of incubation buffer was added to the pellets which were then vortexed, resuspended and homogenized with a Kinematica CH-6010 Kriens-LU homogenizer for 15 seconds at setting 7. The receptor protein concentration was determined using the Pierce BCA assay. Total Spiperone Binding Assay

The incubation was started by the addition of 100 ml (50 mg protein) membrane homogenate to a solution of 300 ml incubation buffer and 100 ml (0.25 nM final cone.) ³H- spiperone (90 Ci/mmol Amersham diluted in borosilicate glass vial) in 96-well polypropylene plates (1 mL per well). The plates were vortexed and incubated at room temperature for 90 minutes. The binding reaction was stopped by filtering using a Packard Harvester. The samples were filtered under vacuum over glass fibre filter plates (Whatman GF/B) presoaked for 2 hours in 0.3% polyethylenimine (PEI) in 50 mM Tris buffer (pH 7.4). The filters were then washed 6 times with 1 ml ice cold 50 mM Tris buffer (pH 7.4). The filter plates were dried overnight and 35 ml of Microscint-O (Packard) was added. The plates were sealed and counted in the Packard Top Count (3 minutes per well).

Non-Specific Binding Assay for D4 The incubation was started by the addition of 100 ml (50 mg protein) membrane homogenate to a solution of 200 ml incubation buffer, 100 ml ³H-spiperone (90 Ci/mmol Amersham diluted in borosilicate glass vial to 0.25 nM final cone.) and 100 ml (30 mM final cone.) of fresh dopamine (Research Biochemicals Inc., light protected and dissolved in incubation buffer) in 96-well polypropylene plates (1 mL per well). The plates were vortexed and incubated at room temperature for 90 minutes at which time the binding reaction was stopped by filtering. The filters were washed and counted using the same procedure as in the total binding assay described above to give the non-specific binding value (NSB).

Non-Specific Binding Assay for D2 This assay employed the same procedures as the non-specific binding assay for D4 with the exception that 2 mM (final cone.) of (-) sulpiride (Research Chemicals Inc.) was used in place of dopamine.

Displacement Binding Assay The incubation was started by the addition, in 96-well polypropylene plates (1 mL per well), of 100 mL (50 mg protein) membrane homogenate to a solution of 200 ml incubation buffer, 100 ml (0.25 final cone.) ³H-spiperone (90 Ci/mmol, Amersham, diluted in borosilicate glass vial) and 100 ml of test compound that was prepared from 1 mM stock dissolved in DMSO and stored at -20°C in polypropylene cryogenic storage vials until dilution in incubation buffer in 96-well polypropylene plates. The plates were vortexed and incubated at room temperature for 90 minutes at which time the binding reaction was stopped by filtering. The filters were washed and counted using the same procedure as in the total binding assay described above to give the displacement binding value (B_D).

Calculations

The test compounds were initially assayed at 1 and 0.1 mM and then at a range of concentrations chosen such that the middle dose would cause about 50% inhibition of ³H- spiperone binding. Specific binding in the absence of test compound (B₀) was the difference of total binding (B_T) minus non-specific binding (NSB) and similarly specific binding (in the presence of test compound) (B) was the difference of displacement binding (B_D) minus nonspecific binding (NSB). IC₅₀ was determined from an inhibition response curve, logit-log plot of %B B₀ VS concentration of test compound.

Ki was calculated by the Cheng and Prusoff transformation:

Ki= IC₅₀ / (l + [L]/K_D) where [L] is the concentration of ³H-spiperone used in the assay and K_D is the dissociation constant of ³H-spiperone determined independently under the same binding conditions.

Assay results are reported in the following Table, and show clearly the advantage in terms of D4 selectivity and or binding affinity of compounds of the invention over clozapine. The reference compound, (4-R,S)-N-(l-benzyl-4-piperidinyl)-2-phenylbenzamide (Desai, et al EP 416581) showed no binding to the D4 receptor at a concentration of 1000 nM.

D4 AFFINITY AND SELECTIVITY

Example 5: Functional Assay

The D4 receptor responds to dopamine and other agonists by reducing adenyl cyclase mediated production of cyclic AMP. Particular test compounds were assayed for their ability to reverse dopamine inhibition of adenyl cyclase by the following procedure. Forskolin was used to elevate the basal adenyl cyclase activity.

CHO Pro 5 cells stably expressing human D4.2 receptors were plated in 6 well plates in

DMEM (Dulbecco's Modified Eagle Medium)/F12(Nutrient Mixture F12 (Ham)) media with 10% FCS (fetal calf serum) and G418 (Geneticen Disulfate), and incubated at 37°C in a CO₂ incubator. The cells were allowed to grow to about 70% confluence before use in the assay. Antagonist Assay

The culture media of each well was removed, and the wells were washed once with serum free media (SFM) (DMEM/F12). Then 2 mL of fresh SFM+IBMX media (SFM with 0.5 mM IBMX, 3-isobutyl-l-methylxanthine, 0.1% ascorbic acid and 10 mM pargyline) was added to each well and then incubated at 37°C for 10 minutes in a CO₂ incubator. Following incubation, SFM+IBMX media was removed and fresh SFM+IBMX media was added to wells separately with one of a) forskolin (10 mM final cone); b) dopamine and forskolin (both 10 mM final cone); and c) test compound (10^"4 to 10^"6 M), and dopamine and forskolin (both 10 mM final cone). Basal adenyl cyclase activity was determined from wells with only SFM+IBMX media added.

The cells were then incubated at 37°C for 30 minutes in a CO₂ incubator. Following incubation the media was removed from each well and then washed once with 1 mL of PBS (phosphate buffered saline). Each well was then treated with 1 mL cold 95% ethanol:5 mM EDTA (2: 1) at 4°C for 1 hr. The cells from each well were then scraped and transfeπed into individual Eppendorf tubes. The tubes were centrifuged for 5 min at 4°C, and the supernatants were transfeπed to new Eppendorf tubes. The pellets were discarded and the supernatants stored at 4°C until assayed for cAMP concentration. cAMP content measured in fmoles/well for each extract was determined by EIA (enzyme-immunoassay) using Amersham Biotrak cAMP EIA kit (Amersham RPN 225).

Total inhibition (lo) of forskolin-stimulated adenyl cyclase activity by dopamine was determined as the difference in concentration of cAMP in the forskolin-treated cells (C_f) and dopamine-forskolin treated cells (C_d). Io = Cf - C_d Net inhibition (I) of forskolin-stimulated adenyl cyclase activity by dopamine in the presence of an antagonist was determined as the difference in concentration of cAMP in the forskolin-treated cells (Cf) and test compound, dopamine and forskolin treated cells (C). I = C_f - C The ability of the test compounds to reverse the dopamine inhibition (% reversal, %R) was determined by the formula: %R = (l - IZIo) X 100

The compound of Example 3e was able to reverse the dopamine inhibition of forskolin- stimulated adenyl cyclase activity with an IC₅₀ of 1.5 μM and therefore acts as an antagonist.

It is predicted based on structural and biological functional similarities that the remaining compounds of the invention would also exhibit dopamine antagonist activity.

Agonist Assay

To D4.2 stably expressing CHO cells prepared as previously described were added test compound and forskolin (10 mM final concentration). The cells were incubated, extracted and measured for cAMP concentration as above. Agonist activity of a test compound would result in a decrease in cAMP concentration compared to cells treated with forskolin (C_f) only. The compound of Example 3e produced no decrease in cAMP, therefore exhibiting no dopamine agonist activity.

Claims

WE CLAIM:

1. A compound of Formula I:

wherein

R¹ is a 5- or 6-membered aromatic or heteroaromatic ring, attached at the meta- or para- position of the benzamide phenyl group, wherein said ring is optionally substituted with 1 or 2 groups independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H₂N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl; R² is selected from an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, and an optionally substituted 6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle; n is 1 or 2; and salts, solvates, stereoisomers or hydrates thereof; with the proviso that R is not imidazolo when n is 2 and R is phenyl,.

2. A compound according to claim 1, wherein

R¹ is phenyl, substituted phenyl or a heterocycle having one S, O or N atom; wherein the substituted phenyl is substituted at one or two positions independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H₂N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl.

3. A compound according to claim 2 wherein R² is an optionally substituted phenyl.

4. A compound according to claim 1 wherein R¹ is phenyl, substituted phenyl or a heterocycle having one S, O or N atom; wherein the substituted phenyl is substituted at one or two positions independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, loweralkyl-SO-, loweralkyl-SO₂-, (loweralkyl)₂NSO₂-, H(O)C-, HO₂C-, trifluoromethyl, trifluoro methoxy, H₂N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl; and

R² is an optionally substituted phenyl.

5. A compound according to claim 1 wherein R¹ is phenyl and is substituted at the meta or para position thereof by halo, trifluoromethyl, NH₂, C(O)H, SCH₃, thienyl or phenyl.

6. A compound according to claim 1, wherein R¹ is selected from an optionally substituted 6-membered aromatic ring and an optionally substituted 6-membered heteroaromatic ring containing 1 or 2 N atoms.

7. A compound according to claim 1, wherein R¹ is selected from phenyl, 4- trifluoromethylphenyl, 3-aminophenyl, 2-formylphenyl, 4-formylphenyl, 3-thiomethylphenyl, 4-fluorophenyl, 3-fluorophenyl and 4-chlorophenyl.

8. A compound according to claim 1, wherein R¹ is selected from phenyl, 3-fluorophenyl,

4-chlorophenyl and 4-trifluoromethylphenyl.

9. A compound according toclaims 1 through 8 wherein R¹ is attached at the para position of the benzamide phenyl group.

10. A compound according claims 1 though 8 wherein n is 1.

11. A compound according to claim 1 , which is selected from: (3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-trifluoromethylphenyl)benzamide;

(3-R,S)-4-(3- Aminophenyl)-N-( 1 -benzyl-3-py╧Çolidinyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(2-thienyl)benzamide;

(3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(2-formylphenyl)benzamide;

(3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-methylthiophenyl)benzamide; (3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(3-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-fluorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)l-4-(3-fluorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-chlorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-fora╬╣ylphenyl)benzamide; (3S)- N-(l-Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-3-phenylbenzamide;

(3R)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide;

(4-R,S)-N-( 1 -Benzyl-4-piperidinyl)-4-phenylbenzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide; (4-R,S)-N-( l-Benzyl-4-piperidinyl)-3-phenylbenzamide;

(3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-ethylphenyl)benzamide; and

(3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-hydroxyphenyl)benzamide.

12. A compound according to claim 1 which is selected from:

(3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-trifluoromethylphenyl)benzamide; (3-R,S)-4-(3-Aminophenyl)-N-( 1 -benzyl-3-py╧Çolidinyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(2-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(3-thienyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)l-4-(3-fluorophenyl)benzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-chlorophenyl)benzamide; (3S)- N-(l-Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide; and

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-ethylphenyl)benzamide.

13. A compound according to claim 1 , which is selected from: (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(2-thienyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(3-thienyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)l-4-(3-fluorophenyl)benzamide; (3-R,S)-N-(l-Benzyl-3-py╧Çolidinyl)-4-(4-chlorophenyl)benzamide; (3S)- N-(l-Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide;

(3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-phenylbenzamide; and (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-ethylphenyl)benzamide.

14. A compound according to claim 1, which is selected from: (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(2-thienyl)benzamide; (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(3-thienyl)benzamide; and (3-R,S)-N-( 1 -Benzyl-3-py╧Çolidinyl)-4-(4-chlorophenyl)benzamide.

15. A compound according to claim 1, which is (3-R,S)-N-(l-benzyl-3-py╧Çolidinyl)-4-(4- chlorophenyl)benzamide.

16. A pharmaceutical composition, comprising a pharmaceutically acceptable carrier and therapeutically effective amount of a compound of Claim 1.

17. A method for treating a patient having a medical condition for which a D4 receptor antagonist is indicated, comprising the step of administering to the patient a therapeutically effective amount of a a compound of claim 1.

18. A method for treating a patient for schizophrenia comprising administering to the patient a therapeutically effective amount of a compound of claim 1.

19. A method for treating a patient for anxiety comprising administering to the patient a therapeuctially effective amount of a compound of claim 1.

20. A process for making a compound of formula I:

wherein R¹ is a 5- or 6-membered aromatic or heteroaromatic ring, attached at the meta- or para-position of the benzamide phenyl group, wherein said ring is optionally substituted with 1 or 2 groups independently selected from loweralkyl, loweralkoxy, hydroxy, halo, cyano, loweralkyl-S-, H(O)C-, HO₂C-, trifluoromethyl, trifluoromethoxy, H₂N-, loweralkyl-NH-, (loweralkyl)₂N- and loweralkylcarbonyl; R² is selected from an optionally substituted 5- or 6-membered aromatic or heteroaromatic ring, and an optionally substituted 6-membered aromatic or heteroaromatic ring fused to a 5- or 6-membered hetero- or carbocycle; n is 1 or 2; and salts, solvates, stereoisomers or hydrates thereof, with the proviso that R¹ is not imidazolo when n is 2 and R² is phenyl, comprising the step of: coupling benzoic acid of Formula A

wherein Rl is as defined above, or a reactive derivative thereof, with an amine of formula B

to produce the compound of Formula I.