WO1999002990A1 - Combinatorial process for preparing fused substituted pyrimidine libraries - Google Patents

Abstract

This invention relates to a novel diverse combinatorial library of fused substituted pyrimidine compounds and to an apparatus providing a readily accessible source of individual members of the library. The apparatus can be used in assay kits and as a replaceable element in automated assay machines.

Description

COMBINATORIAL PROCESS FOR PREPARING FUSED SUBSTITUTED PYRIMIDINE LIBRARIES

This Application claims the benefit of U.S. Provisional Application No. 60/052,251, filed July 11, 1997.

The present invention relates to diverse libraries of fused pyrimidine compounds, methods of making such libraries, and an apparatus for storing and providing a readily accessible source of diverse fused pyrimidine compounds. The apparatus harboring the present combinatorial libraries is a useful component of assay systems for identifying compounds for drug development.

Research and development expenses account for a large outlay of capital in the pharmaceutical industry. Synthesis of compounds is an expensive and time consuming phase of research and development. Historically, research chemists individually synthesized and analyzed high purity compounds for biological screening to develop pharmaceutical leads. Although such methods were successful in bringing new drugs to the market, the limitations of individual synthesis and complete compound characterization considerably slowed the discovery of new pharmaceutically active compounds. The need for more rapid and less expensive drug discovery methodology is increasingly important in today's competitive pharmaceutical industry.

Recently, modern drug discovery has utilized combinatorial chemistry to generate large numbers (10² - 10⁶) of compounds generically referred to as "libraries". An important objective of combinatorial chemistry is to generate a large number of novel compounds that can be screened to generate lead compounds for pharmaceutical research.

Theoretically the total number of compounds which may be produced for a given library is limited only by the number of reagents available to form substituents on the variable positions on the library's molecular scaffold. The combinatorial process lends itself to automation, both in the generation of compounds and in their biological screening, thereby greatly enhancing the opportunity and efficiency of drug discovery. Combinatorial chemistry may be performed in a manner where libraries of compounds are generated as mixtures with complete identification of the individual compounds postponed until after positive screening results are obtained. However, a preferred form of combinatorial chemistry is "parallel array synthesis", where individual reaction products are simultaneously synthesized, but are retained in separate vessels. For example, the individual library compounds can be prepared, stored, and assayed in separate wells of a microtiter plate, each well containing one member of the parallel array. The use of standardized microtiter plates or equivalent apparatus, is advantageous because such an apparatus is readily accessed by programmed robotic machinery, both during library synthesis and during library sampling or assaying. Typically, completion of the solution phase reactions in combinatorial chemistry schemes are ensured by selecting high yielding chemical reactions and/or by using one reagent in considerable excess. When one reagent is used in excess, completion of the reaction produces a mixture of a soluble product with at least one soluble unreacted reagent. The excess soluble reagent is separated from the product by using solid phase scavengers or by classical work-up procedures dependent on the chemical characteristics of the excess reagent and the product. Combinatorial chemistry may be used at two distinct phases of drug development. In the discovery phase diverse libraries are created to find lead compounds. In a second optimization phase, strong lead compounds are more narrowly modified to find optimal molecular configurations. The condensation of amino acids with 2-ethoxycarbonyl- 3 - isothiocyanatopyridine to form the corresponding pyrido (3,2-d) pyrimidine derivatives has been described. Methylation of the resulting thioxo group with N, N- dimethylformamide dimethyl acetal was also investigated (See, Urleb, U. , Stanovnik, B., and Tisler, . , The Reaction of 2-Ethoxycarbonyl -3 -isothiocyanatopyridine with Alpha- Amino Acids: The synthesis of 3 -Substituted 2-Thiooxo-2, 3 - dihydropyri do (3, 2- d) pyrimi din- 4 (3H) -ones. J. Het. Chem. , vol. 27, pp. 413-415 (1990)).

Synthesis of certain 3 -substituted 2-thioxo- pteridinones by reaction of methyl 3-isothiocyanato-2- pyrazinecarboxylate with primary amines has also been described (See, Urleb, U. , Neidlein, R. , and Kramer, W. , A New Approach for the Synthesis of Pteridines : The Synthesis of 3 -Substituted- 2 - thioxo -1 , 2 -dihydro -4 (3H) -pteridinones . J. Het. Chem., vol. 27, pg. 433-437 (1990)). More recently Buchman, et. al . , described a solid phase synthesis of 1,3-dialkyl quinazoline-2, 4-diones from anthranilate. Alkylation of cyclized intermediates using alkyl halides is described (See, Buckman, B., and Mohan, R. , Solid-Phase Synthesis of 1 , 3 -Dialkyl Quinazoline-2, 4 -Diones . Tetrahedron Letters, vol. 37, pp. 4439-4442 (1996)).

The method of the present invention is based on a simplified solution phase synthesis scheme utilizing solid support bound scavengers to separate intermediates and/or products from unreacted starting materials to provide a novel diverse library of fused pyrimidines useful in the identification of new lead compounds. The library is created, stored, and used as an apparatus comprising of a two-dimensional array of reservoirs, each reservoir containing a predetermined library reaction product differing from those in adjacent reservoirs.

The present invention provides combinatorial libraries of structurally related compounds having fused pyrimidine structures of the general formulas

wherein X is oxygen or sulfur, ring R¹ is aryl, cycloalkenyl or heterocycle; R² is hydrogen or an organic moiety; and R³ is hydrogen or an organic moiety. The invention further provides a method for preparing fused pyrimidine libraries generally in accordance with Scheme 1 as set forth below.

Another embodiment of the present invention provides an assay kit for the identification of pharmaceutical lead fused pyrimidine compounds . The kit comprises assay materials and a well plate apparatus or equivalent apparatus providing a two-dimensional array of defined reservoirs. The well plate apparatus provides a diverse combinatorial library, wherein each well (reservoir) contains a unique reaction product of the fused pyrimidine library. The well plate apparatus is used to provide multiple reaction zones for making the library, to store the library and to provide a readily accessible source of library compounds.

Pig. 1 is a top view of a well plate in accordance with this invention. Fig. 2 is a side view of a well plate apparatus for use in the process of this invention.

The term "assay kit" as used in accordance with the present invention refers to an assemblage of two cooperative elements, namely (1) a well plate apparatus and (2) biological assay materials. "Biological assay materials" are materials necessary to conduct a biological evaluation of the efficacy of any library compound in a screen relevant to a selected disease state. A "library" is a collection of compounds created by a combinatorial chemical process, said compounds having a common scaffold with one or more variable substituents. The scaffold of the present invention is a fused pyrimidine.

A "library compound" is an individual reaction product, a single compound or a mixture of isomers, in a combinatorial library.

A "lead compound" is a library compound in a selected combinatorial library for which the assay kit has revealed significant activity relevant to a selected disease state. A "diverse library" means a library where the substituents on the combinatorial library scaffold or core structure, are highly variable in constituent atoms, molecular weight, and structure, and the library, considered in its entirety, is not a collection of closely related homologues or analogues (compare to "directed library"). A "directed library" is a collection of compounds created by a combinatorial chemical process, for the purpose of optimization of the activity of a lead compound, wherein each library compound has a common scaffold, and the library, considered in its entirety, is a collection of closely related homologues or analogues to the lead compound (compare with "diverse library").

The term "scaffold" as used in accordance with the present invention refers to the invariable region (a fused pyrimidine core in the present invention) of the compounds which are members of the combinatorial library.

"Substituents" are chemical radicals which are bonded to or incorporated onto the fused pyrimidine scaffold through the combinatorial synthesis process. The different functional groups account for the diversity of the molecules throughout the library and are selected to impart diversity of biological activity to the scaffold in the case of diverse libraries, and optimization of a particular biological activity in the case of directed libraries.

"Reagent" means any chemical compound used in the combinatorial synthesis to place substituents on the scaffold of a library.

"Solid support bound scavenger" means an insoluble composition comprising surface bound functional groups capable of reacting with and bonding to a target reagent in solution to form an insoluble reaction product and to remove said reagent from the solution.

"Parallel array synthesis" refers to the method of conducting combinatorial chemical synthesis of libraries wherein the individual combinatorial library compounds are separately prepared and stored without prior and subsequent intentional mixing.

"Simultaneous synthesis" means making of library compounds within one production cycle of a combinatorial method (not making all library compounds at the same instant in time) . The "reaction zone" refers to the individual vessel location where the combinatorial chemical library compound preparation process of the invention is carried out and where the individual library compounds are synthesized. Suitable reaction zones are the individual wells of a well plate apparatus.

"Well plate apparatus" refers to the structure capable of holding a plurality of library compounds in dimensionally fixed and defined positions.

"Non- interfering substituents" are those groups that do not significantly impede or interfere with the process of the invention and yield stable fused pyrimidine library compounds .

"Aryl" means one or more aromatic rings and includes substituted aryl having one or more non- interfering substituents. Multiple aryl rings may be fused, as in naphthyl, or unfused, as in biphenyl . "Alkyl" means straight or branched chain or cyclic hydrocarbon having 1 to 20 carbon atoms.

"Substituted alkyl" is alkyl having one or more non- interfering substituents. "Halo" means fluoro, chloro, bromo or iodo.

"Heterocycle" or "heterocyclic radical" means one or more rings of 5, 6 or 7 atoms with or without unsaturation or aromatic character, optionally substituted with one or more non-interfering substituents, and at least one ring atom which is not carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen. Suitable non-interfering substituents include, but are not limited to halo, Ci-Cio alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C₁₂ aralkyl, C7-C₁₂ alkaryl, Cχ-Cχo alkylthio, arylthio, aryloxy, arylamino, C3-C₁0 cycloalkyl, C₃-C₁₀ cycloalkenyl, di (C₁-C₁₀) -alkylamino, C2-C₁₂ alkoxyalkyl, Cχ-C₆ alkylsulfinyl, C₁-C₁₀ alkylsulfonyl , arylsulfonyl, aryl, hydroxy, hydroxy (Cχ-Cχo) alkyl, aryloxy (C1-C10) alkyl, C₁-C₁0 alkoxycarbonyl , aryloxycarbonyl, Cχ-Cχo alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (C₁-C₁₀) lkyl, C₁-C₁0 alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, carbamido, carboalkoxy, heterocyclic radical, nitroalkyl, and - (CH2)m^_Z- (Cχ-Cχo alkyl) , where m is 1 to 8 and Z is oxygen or sulfur. Multiple rings may be fused, as in quinoline or benzofuran, or unfused as in 4-phenylpyridine.

"Cycloalkenyl" means a closed ring structure containing 5, 6 or 7 carbon atoms with at least one carbon- carbon double bond. The ring is optionally substituted with one or more non-interfering substituents. Such substituents for cycloalkenyl groups include, but are not limited to halo, Ci-Cχo alkyl, C2-Cχo alkenyl, C2-Cχo alkynyl, Cχ-Cχo alkoxy, C7-CX2 aralkyl, C7-CX2 alkaryl, Cχ-Cχo alkylthio, arylthio, aryloxy, arylamino, C3-C 0 cycloalkyl, C₃-CX₀ cycloalkenyl, di (Cχ-Cχo) -alkylamino, C2-Cχ2 alkoxyalkyl, Cχ-C6 alkylsulfinyl, Cχ-Cχo alkylsulfonyl , arylsulfonyl , aryl, hydroxy, hydroxy (Cχ-Cχo) alkyl, aryloxy (Cχ-Cχo) alkyl, Cχ-Cχo alkoxycarbonyl , aryloxycarbonyl, Cχ-Cχo alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (Cχ-Cχo) alkyl, Cχ-Cχo alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, carbamido, carboalkoxy, heterocyclic radical, nitroalkyl, and - (CH2)m-Z- (Cχ-Cχo alkyl) , where m is 1 to 8 and Z is oxygen or sulfur.

"Organic moiety" means a substituent comprising a non- interfering substituent covalently bonded through at least one carbon atom. Suitable non-interfering substituents include, but are not limited to hydrogen, halo, Cχ-Cχo alkyl, C2-Cχo alkenyl, C2-Cχo alkynyl, Cχ-Cχo alkoxy, C7-CX2 aralkyl, C7-CX₂ alkaryl, Cχ-Cχo alkylthio, arylthio, aryloxy, arylamino, C3-C_X0 cycloalkyl, C₃-C_X0 cycloalkenyl, di (Cχ-Cχo) -alkylamino, C₂-Cχ₂ alkoxyalkyl, Cχ-Cβ alkylsulfinyl, Cχ-Cχo alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy (Cχ-Cχo) alkyl, aryloxy (Cχ-Cχo) alkyl, Cχ-Cχo alkoxycarbonyl , aryloxycarbonyl, Cχ-Cχo alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (Cχ-Cχo) lkyl, Cχ-Cχo alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, carbamido, carboalkoxy, heterocyclic radical, nitroalkyl, and - (CH2)m-Z- (Cχ-Cχo alkyl) , where m is 1 to 8 and Z is oxygen or sulfur.

A diverse library of fused substituted pyrimidines is provided in accordance with the present invention. The present library embodied as an apparatus of this invention serves as a readily accessible source of diverse fused pyrimidine compounds for use in identifying new biologically active fused pyrimidine compounds through pharmaceutical and agricultural candidate screening assays, for use in studies defining structure/activity relationships, and/or for use in clinical investigation.

The library provided in accordance with the present invention includes fused pyrimidine compounds of the formulas

wherein X is oxygen or sulfur, ring R¹ is aryl, cycloalkenyl or heterocycle; and R² and R³ are each independently hydrogen or an organic moiety.

In another embodiment of the present invention there is provided a library of compounds of Formulas I and II above, wherein X is oxygen or sulfur; ring R¹ is aryl, cycloalkenyl or heterocycle derived from an isocyanate or isothiocyanate of the formula

R² is hydrogen or an organic moiety derived from a primary amine of the formula R²NH2; and R³ is hydrogen or an organic moiety derived from an alkylating agent of the formula R³Y, wherein Y is a leaving group subject to nucleophilic displacement from the group R³.

In still another embodiment of the present invention there is provided a library of compounds of Formulas I and II above, wherein R² and R³ are each independently hydrogen or an organic moiety with a non- interfering substituent selected from the group consisting of halo, Cχ-Cχo alkyl, C2-Cχo alkenyl, C2-Cχo alkynyl, Cχ-Cχo alkoxy, C7-CX2 aralkyl, C7-CX2 alkaryl, Cχ-Cχo alkylthio, arylthio, aryloxy, arylamino, C3-CX0 cycloalkyl, C3-C ₀ cycloalkenyl, di (Cχ-Cχo) -alkylamino, C2-Cχ2 alkoxyalkyl, Cχ-C₆ alkylsulfinyl, Cχ-Cχo alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy (Cχ-Cχo) alkyl, aryloxy (Cχ-Cχo) alkyl, Cχ-Cχo alkoxycarbonyl , aryloxycarbonyl, Cχ-Cχo alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (Cχ-Cχo) alkyl, Cχ-Cχo alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, carbamido, carboalkoxy, heterocyclic radical, nitroalkyl, or - (CH₂)m-Z- (CχCχo alkyl) , where m is 1 to 8 and Z is oxygen or sulfur.

The present invention also provides a method for preparing the library of fused pyrimidine compounds of Formulas I and II above, using combinatorial chemistry in a parallel array synthesis technique illustrated in the following reaction scheme:

Scheme 1.

+ R²NH₂ + Ci-Cioalkanol (Excess)

Add insolu e scavenger \

Remove solids

STEPdJi) ✓ « °

Acidify /

aque OUSΗSO3

STEP e)

Optional - Remove Amine/Hydroxy Protecting Groups

STEP f) STEP f)

Evaporate Evaporate

H R³ R"

II The method comprises the steps of reacting series of isocyanates and/or isothiocyanates of the formula

wherein X is oxygen or sulfur and R¹ is aryl, cycloalkenyl or heterocycle, with series of primary amines of the formula ²NH2 wherein R² is an organic moiety, to form an intermediate compound of the formula

wherein any primary amino groups forming part of R¹ or R² are preferably in the form of amino protected derivatives . Cyclization of intermediate IV is then accomplished by addition of a base, typically an alkali metal hydroxide or alkoxide, to provide fused pyrimidine compounds of the formula

and a Cχ-Cχo alkanol by-product, wherein Formula V, M⁺ is a counter ion generally provided by the base. An insoluble solid support bound scavenger is added to the reaction solution to react with and covalently bond any unreacted primary amine from the first step of the process and the Cι-Cχo alkanol by-product. The insoluble scavenger is then separated from the reaction solution. The fused pyrimidine intermediate of Formula V is either acidified to produce library products of Formula I wherein R³ is hydrogen, or that intermediate is reacted with an alkylating agent to produce library products of Formula I and/or Formula II. When X is sulfur, the library compound is of Formula II.

When X is oxygen, the library compound is a mixture of the compounds of Formula I and Formula II. Unreacted alkylating agent is separated by washing the product with saturated aqueous bisulfite. Thereafter, any protecting groups forming a part of groups R¹, R² or R³ are removed, and the resulting library product solution is dried. Combinatorial use of this process provides a library of fused pyrimidine compounds of the Formulas I and II above, with three sites of diversity, R¹, R² and R³. Each compound is typically prepared in a separate reaction zone (i.e. parallel array synthesis), and the predetermined library compound is identified by the plate and reaction well number.

The isocyanate and isothiocyanate reagents, the primary amine reagents and the alkylating reagents used in preparation of the present library are either commercially available or prepared from commercially available starting materials. To minimize unwanted side reactions and consequent complex product mixtures, it is preferred that any primary alkylamino groups which form part of the groups R¹, R² or R³ be in the form of protected amino, utilizing any of the art-recognized amino protecting groups. Hydroxy and carboxy substituents forming part of groups R¹, R² or R³ may also be protected. However, slower reaction kinetics of reactions of hydroxy and carboxy functional groups relative to that of amino groups, renders need for protecting such groups less significant in the preparation of the present library products. When protecting groups are utilized on reactants for forming the present library products, the nature of the protecting groups are not critical. Practitioners in the art will readily appreciate what protecting groups are appropriate and the method by which they can be used and removed in the preparation of the present library compounds.

Isocyanates and isothiocyanates for use in accordance with this invention are compounds of the formula

wherein X is oxygen or sulfur and ring R¹ is aryl, cycloalkenyl or heterocycle. The isocyanates or isothiocyanates have a molecular weight of about 160 to about 800, and more typically about 180 to about 450. Illustrative of suitable isocyanates and isothiocyanates for use in preparation of the fused pyrimidine library of this invention include compounds of the following formulae:

Other suitable isocyanate and isothiocyanate reagents include those represented by the following wherein x and L2 are the points of bonding to the alkenyl bond in the partial formula

Primary amines for use in accordance with this invention are compounds of the formula R²NH2 wherein R² is hydrogen or an organic moiety. Suitable primary amines have a molecular weight of about 30 to about 600, more typically, about 50 to about 250. Illustrative of suitable primary amines for use in preparation of the fused pyrimidine library of this invention are the following:

cyclopropylamine cyclobutylamine

(-) -cis-myrtanylamine cyclopentylamine eyelohexylamine

2-methylcyclohexylamine 2 , 3 -dimethylcyclohexylamine

4-methylcyclohexylamine (aminomethyl) cyclohexane

3 -aminomethyl-3 , 5, 5-trimethylcyclohexanol 1,2,3,4-tetrahydro- 1-naphthylamine cyclooctylamine

1-tyrosine methyl ester

2- (2-aminoethyl) -1-methylpyrrolidine n- (2-aminoethyl)pyrrolidine n- (3 ' -aminopropyl) -2 -pyrrolidinone furfurylamine cyclododecylamine

1-aminoindan dl-1- (1-naphthyl) ethylamine 1-naphthalenemethylamine cycloheptylamine

(Is, 2s) - (+) -2-amino-l-phenyl-l, 3-propanediol dl-2-amino-3 -methyl- 1-butanol

1-isoleucinol 1-phenylalaninol dl-4-chlorophenylalaninol d- (- ) -leucinol

1-methioninol histamine tetrahydrofurfurylamine dl-alpha-methyltryptamine tryptamine

5-methoxytryptamine

6-methoxytryptamine piperonylamine n- (2-aminoethyl)morpholine n- (3 -aminopropyl) morpholine

2- (2-aminoethylamino) -5-nitropyridine

2- (aminomethyl)pyridine 2- (2-aminoethyl) pyridine

3- (aminomethyl) pyridine

4- (aminomethyl) pyridine ethyl 4-amino-l-piperidinecarboxylate

4-amino-1-benzylpiperidine

1- (2-aminoethyl)piperidine

1- (3 -aminopropyl) -2-pipecoline l,2-diamino-2-methylpropane benzhydrylamine d- (-) -alpha-phenylglycinol

1,2-diphenylethylamine dl-1-phenylethylamine ( - ) -norephedrine

1,2-dimethylpropylamine isopropylamine

2 -methoxyisopropylamine dl-2-amino- 1-propanol ethyl-3-aminobutyrate

1,3-dimethylbutylamine

3 -amino- 1-phenylbutane

2 -amino-5-diethylaminopentane 1,5-dimethylhexylamine sec-butylamine

(+/- ) -2 -amino- 1-butanol

3 -aminopentane

2 -aminopentane

3 -aminoheptane 2-aminoheptane

2 -aminooctane benzylamine 2-fluorobenzylamine 2 -chlorobenzylamine 2, 4-dichlorobenzylamine 2-methoxybenzylamine

2 -ethoxybenzylamine 2-methylbenzylamine

3 - fluorobenzylamine 3, 4-dichlorobenzylamine 3,4-dimethoxybenzylamine 3- (trifluoro ethyl) benzylamine 3 -methylbenzylamine 4- fluorobenzylamine 4-chlorobenzylamine 4-methoxybenzylamine 4-methylbenzylamine

2,2,2-trifluoroethylamine

2-amino-1-phenylethanol 1-amino-2 -propanol

3 - mino-1,2 -propanediol 2,2-diphenylethylamine beta-methylphenethylamine isobutylamine

2 -methylbutylamine

2 -ethylhexylamine n-decylamine n-undecy1amine dodecy1amine tridecylamine

1-tetradecylamine hexadecylamine octadecylamine ethylamme

2- (2-aminoethylamino) ethanol

2 -methoxyethylamine 2- (2-aminoethoxy) ethanol ethanolamine phenethylamine

2- (2-chlorophenyl) ethylamine

2- (2-methoxyphenyl) ethylamine 3-methoxyphenethylamine

2 - (3,4-dimethoxyphenyl) ethylamine

4-bromophenethyla ine

2 - (4- chlorophenyl) ethylamine

2 - (4-methoxyphenyl) ethylamine tyramine

2 - (4-aminophenyl) ethylamine

2- (p-tolyl) ethylamine taurine propargylamine allylamine

3 , 3 -dimethylbutylamine 3,3-diphenylpropylamine isoamylamine propylamine

3 -dimethylaminopropylamine 3 -diethylaminopropylamine 3- (di-n-butylamino) propylamine 3 - isopropoxypropylamine 3 -ethoxypropylamine

3 -amino- 1-propanol

3 -phenylpropylamine 4-amino-l-butanol

4-phenylbutylamine n-amylamine

5-amino-1-pentanol hexylamine 6-amino-l-hexanol n-heptylamine n-octylamine n-nonylamine dl-2 -amino- 1-pentanol dl-2-amino-l-hexanol

1- (3 -aminopropyl) imidazole

3, 5-bis (trifluoromethyl) benzylamine

2,4-difluorobenzylamine

2,5-difluorobenzylamine 2, 6-difluorobenzylamine

3,4-difluorobenzylamine

4- (trifluoromethyl) benzylamine

2- (trifluoromethyl) benzylamine

4- (2-aminoethyl) benzenesulfonamide n- (4-aminobutyl) -n-ethylisoluminol n-butylamine

2 - (1- cyclohexenyl) ethylamine 3 -methoxypropylamine

3,4,5-trimethoxybenzylamine

3 -butoxypropylamine aminomethylcyclopropane pentadecylamine

4- (2 , 4-di-tert-amylphenoxy) butylamine

3 -chlorobenzylamine

4-fluoro-alpha-methylbenzylamine

(r) - (+) -bornylamine n,n-di-n-butylethylenediamine

(r) - (-) -1-cyclohexylethylamine n,n, 2 , 2-tetramethyl-1,3 - ropanediamine

1-phenylalanine beta-naphthyl-amide

2- (3 -chlorophenyl) ethylamine 2-amino-l,3-propanediol

2- (2-thienyl) ethylamine

2 , 3 -dimethoxybenzylamine

3 , 5-dimethoxybenzylamine

2 , 4-dichlorophenethylamine 2,5-dimethoxyphenethylamine

3-fluoro-5- (trifluoromethyl) benzylamine

4- (trifluoromethoxy) benzylamine

1-leucinol

1-leucine-4-nitroanilide (r) - (+) -1- (1-naphthyl) ethylamine

(s) - (-) -1- (1-naphthyl) ethylamine

1-valinol d-valinol d-phenylalaninol 1- (+) -alpha-phenylglycinol d- (+) -alpha-methylbenzylamine

1 ( - ) -alpha-methylbenzylamine

(ls,2r) - (+) -phenyl-propanolamine

(s) - (+) -2 -amino- 1-propanol d-alaninol

(r) - (-) -sec-butylamine

(s) - (+) -sec-butylamine (s) - (+) -2-amino-l-butanol

(r) - (-) -2-amino-l-butanol

(r) - (- ) -1-amino-2 -propanol

(s) - (+) -1-amino-2 -propanol (s) - (-) -2-methylbutylamine

(s) - (+) -1-cyclohexylethylamine oleylamine

1-adamantanemethylamine

(ls,2r) - (+) -2-amino-l,2-diphenylethanol (lr,2s) - (-) -2-amino-l,2-diphenylethanol s-benzyl - 1 - cysteinol

2- (2- (aminomethyl) phenylthio) benzyl alcohol

3 - fluorophenethylamine

2 -aminobenzylamine 2- fluorophenethylamine

4-aminobenzylamine d-glucamine

(+/- ) -2 , 5-dihydro-2, 5-dimethoxyfurfurylamine

(s) - (+) -tetrahydrofurfurylamine 4 - fluorophenethylamine

(Is, 2s) - (+) -thiomicamine

( - ) -3,4-dihydroxynorephedrine

(r) - (+) -1- (p-tolyl) ethylamine

(s) - (-) -1- (p-tolyl) ethylamine (s) - (-) -2 -amino- 1, 1-diphenyl-l-propanol

(+/- ) -exo-2 -aminonorbornane

(s) - (+) -2- (aminomethyl) pyrrolidine

3-amino- 1-propanol vinyl ether geranylamine 4- (hexadecylamino) benzylamine

(lr,2r,3r,5s) - (-) -isopinocampheylamine

(ls,2s,3s,5r) - (+) -isopinocampheylamine nl- isopropyldiethylenetriamine

(s) -tert-leucinol (r) - (-) -tetrahydrofurfurylamine dehydroabietylamine

2 -bromo-4,5-dimethoxyphenethylamine (ls,2r) - (-) -cis-l-amino-2-indanol (lr,2s) - (+) -cis-l-amino-2-indanol.

Other suitable primary amines include compounds of the following formulae wherein L is amino:

Alkylating reagents for use in the present invention are compounds of the formula R³Y wherein R³ is an organic moiety and Y is a leaving group subject to nucleophilic displacement from R³. Typically, R³ is a primary or secondary alkyl group, optionally substituted with a non- interfering substituent. Suitable R³ groups have a molecular weight of about 15 to about 400, more typically about 40 to about 200. Preferably, R³ is an optionally substituted Cχ-C4 primary alkyl group. Illustrative of suitable alkylating reagents are compounds of the above formulas, wherein L is a leaving group Y. Leaving groups known for fascile nucleophilic displacement are halo, particularly bromo or iodo, and sulfate. Exemplary of alkylating agents for use in preparing library products in accordance with this invention are methyl iodide, dimethylsulfate, iodoacetic acid esters, iodoacetamide, 1- bromopropane, n-butylbromide, and the like. When Y is chloro or bromo, the alkylation reaction is preferably conducted in the presence of a source of I" such as sodium iodide or potassium iodide.

The preparation of the fused pyrimidine library compounds of Formulas I and II comprises a multi-step process wherein an isocyanate or isothiocyanate of Formula III is reacted with a primary amine and the intermediate product is cyclized by reaction with a base and then optionally alkylated. The progress/completion of each of the involved reactions can be assessed by any of a number of conventional techniques including thin layer chromatography (TLC) .

With reference to Scheme I above, Step a) of the process comprises the reaction of an isocyanate or isothiocyanate compound of the formula

wherein X is oxygen or sulfur and ring R¹ is aryl, cycloalkenyl or heterocycle, with a primary amine of the formula R²NH2, wherein R² is an organic moiety, to provide a compound of the formula

Primary alkylamino groups which form a part of groups R¹ or R² should be in the form of protected amino to avoid undesirable side reactions with the isocyanate or isothiocyanate group in Step a) and in the subsequent cyclization reaction in Step b) , infra. The nature of the amino protecting group is not critical provided that the protecting group (s) are removable under reaction conditions which will not cause degradation of product library compounds. Skilled practitioners in the art will readily appreciate what amino protecting groups can be employed and the methods by which they are removed.

Hydroxy, carboxy and secondary alkylamino substituent groups on R¹ or R² can also be subject to side reactions with the starting isocyantes and isothiocyanates in Step a) and as well in the removal of residual amine reagent and displaced alkanol by the insoluble scavenger material in Step c) , infra. Thus, such groups can optionally be protected by appropriate protecting groups recognized in the art, which protecting groups are later removed in the process. It is noted, however, that reactions with hydroxy, carboxy and secondary amine substituents forming part of group R² can be controlled by selection of reaction conditions which kinetically favor the desired reaction between the primary amino group and the isocyanate or isothiocyanate reagents.

The reaction conditions for Step a) are not critical. A wide variety of reaction media, temperature, time, and agitation conditions are acceptable. Most typically, the reaction Step a) is carried out in a polar, aprotic organic solvent, for example, dimethylformamide, dioxane, dimethylsulfoxide, tetrahydrofuran (THF) , methylene chloride or ethyl acetate, at ambient temperature or at slightly elevated temperatures, for example, from about 20°C to about 60°C, for about 12 to about 48 hours, with continuous or frequent mixing.

In another embodiment, Step a) is initiated by first charging the reaction zones with an insoluble acid scavenger, for example, polyvmylpyridine, in about a 1.5 to

2 fold molar excess relative to the amount of isocyanate or isothiocyanate. Solutions of the isocyanate or isothiocyanate and the primary amine, both in an organic solvent, are then added to the reaction zone to initiate the reaction.

Typically, an excess of the primary amine reagent is used relative to the amount of isocyanate or isothiocyanate.

For example, the molar ratio of primary amine to the isocyanate or isothiocyanate in the reaction mixture is preferably about 1.1 to about 2.0, more preferably about 1.2 to about 1.5.

In Step b) , a proton extracting base, typically an alkali metal hydroxide or alkoxide of the formula M⁺0H^~ or M⁺0R^" is added to the reaction zone. The added base catalyzes ring closure with the displacement of an alkanol to provide a compound of the formula

M⁺

wherein M⁺ is a counter ion corresponding to the cationic component of the base. Depending on the pH of the reaction medium, all or some portion of Intermediate VI can be in the form of Formula I wherein R³ is hydrogen. Again, the reaction conditions are not critical, but the Step b) reaction is typically carried out by adding the base to the Step a) reaction product mixture, at ambient temperature or at slightly elevated temperatures, for example from about 20°C to about 60°C, for about 12 to about 48 hours, with constant or frequent mixing. Suitable bases include aqueous alkali metal hydroxides, such as sodium or potassium hydroxide. In one embodiment, 1.3 M NaOH is added to each reaction zone in an amount corresponding to about three to five fold molar excess relative to the amount of isocyanate or isothiocyanate. The reaction carried out at ambient temperature for about 12 to 48 hours. Step c) accomplishes removal of excess unreacted reagents, by-product alkanol produced in Step b) and insoluble acid scavenger, if any, used in Step a) . An insoluble support bound scavenger for amines and alkanols is added to the reaction mixture and allowed to react. Suitable support bound scavengers are insoluble under the reaction conditions and easily separated from the reaction solution. They are selected to have functional groups that readily react with and covalently bond to primary amines and alkanols. In one embodiment of the invention, the insoluble scavenger is a polymeric resin with carbonyl chloride functionality, for example, polystyrene carbonyl chloride. The insoluble support bound scavenger is added to the reaction mixture resulting from Step b) and reacted at ambient temperature or at slightly elevated temperatures, for example from about 20°C to about 60°C, for about 12 to about 48 hours, preferably with constant or frequent mixing.

Step c) is completed by separating solids from the reaction mixture. Such can be accomplished by any of a number of separation techniques including, decantation, filtration or centrifugation to provide a reaction solution containing an intermediate compound of the formula

M⁺

wherein X is oxygen or sulfur, ring R¹ is aryl, cycloalkenyl or heterocycle; R² is hydrogen or an organic moiety; and M⁺ is a counter ion. Again, depending on the pH of the solution , all or some of Intermediate VI can be in the forms of Formula I wherein R³ is hydrogen.

In Step d) the solution of Intermediate VI is either acidified to provide compounds of Formula I, wherein R³ is hydrogen (Step d) i) ) , or Intermediate VI is reacted with an alkylating agent to prepare library compounds of Formula I or Formula II wherein R3 is other than hydrogen (Step d)ii)). Suitable acids for carrying out Step d)i) are protic acids, for example HC1, acetic acid, or sulfuric acid. In one embodiment, 1 - 2 N HC1 in THF is added to the reaction solution in an amount corresponding to a 1 - 3 molar excess relative to the amount of Intermediate VI present in the reaction solution.

In Step d)ii), a 1.5 -3 fold molar excess of an alkylating reagent of the formula R³Y, wherein Y is a leaving group subject to nucleophilic displacement from the group R³ , is added to the reaction mixture. The reaction is typically at ambient temperature or at slightly elevated temperatures, for example from about 20°C to about 60°C, for about 12 to about 48 hours, preferably with constant or frequent mixing. Suitable leaving groups, include halo and sulfate. Preferred leaving groups are bromo or iodo anions. In one embodiment, two equivalents of an alkyl iodide in THF are added to the reaction mixture containing about one equivalent of Intermediate VI, and reacted at ambient temperature for 24 hours with continuous mixing.

Alkylation in accordance with Step d)ii) provides compounds of the Formulas I and II:

wherein R³ is an organic moiety. Where X is sulfur, only compounds of Formula II are formed. Where X is oxygen, alkylation produces a mixture of compounds of Formulas I and II.

Excess alkylating reagent is removed from the library product by standard work-up techniques. In one embodiment of the invention, excess alkyl iodide is removed by extraction with saturated sodium bisulfite in 70:30, methanol:methylene chloride. The organic phase containing the library product substantially free of unreacted alkylating agent is separated and dried. Alternatively, the reaction mixture can be evaporated to dryness and the resulting product mixture can be triturated with ether and saturated sodium bisulfite to separate the library product from the unreacted alkyl iodide.

Any amino or hydroxy protecting groups on the reaction products are optionally removed in Step e) and the product solution dried in Step f) to provide the library compounds of Formulas I and II above.

Conditions used for removal of protecting groups in Step e) are dependent on the nature of the protecting groups and are well known in the art. Skilled practitioners will readily appreciate what conditions are appropriate for removal of each protecting group used in the process. The drying technique used in carrying out Step f) is not critical and may include any conditions under which the library compounds are stable. Examples of drying techniques include the use of a Speed-Vac, passive and vacuum assisted evaporation.

Samples of each library compound can be analyzed by chromatographic, or more preferably chromatographic and mass spectral techniques .

The process of the present invention utilized in preparation of a library of fused pyrimidines of Formulas I and II above may be carried out in any vessel capable of holding the liquid reaction medium. In one embodiment, the process of the invention is carried out in containers adaptable to parallel array synthesis. In particular, the present fused pyrimidine library can be formed in a 96-well plate as illustrated in Figures 1 and 2. That apparatus provides multiple reaction zones most typically in a two- dimensional array of defined reservoirs, wherein one member of the fused pyrimidine library of this invention is prepared in each reservoir. Thus the diverse fused pyrimidine library of the present invention comprises a plurality of reservoir arrays (e.g. well plates) , each reservoir or well containing a library compound of the fused pyrimidine library. Accordingly the library compounds are typically identified by reference to their well plate number and their X column and Y row well plate coordinates.

Following simultaneous preparation of the library member compounds in the reservoir array, the compounds can be transferred in whole or in part to other reservoir arrays (e.g. well plates) , to prepare multiple copies of the library apparatus or to subject the library to additional reaction conditions. Copies of the library apparatus (daughter well plates, each comprising a 2 -dimensional array of defined reservoirs with each reservoir containing a predetermined member of the library) are useful as replaceable elements in automated assay machines. The apparatus of this invention allows convenient access to a wide variety of structurally related fused pyrimidine compounds . One preferred reservoir array for use in making and using this invention is a multi-well titer plate, typically a 96-well microtiter plate. Figure 1 illustrates the top surface of a well plate apparatus of the present invention. The well plate (1) is a plastic plate with 96-wells (depressions) capable of holding liquids for parallel array synthesis. Individual reaction products are prepared in each well and are labeled by the well plate coordinates. For example, the library compound at location (2) , is identified by the alpha numeric coordinate, "A6".

Figure 2 illustrates a side view of a modified well plate apparatus for use in preparation of the library of the present invention. Well plate (3) contains wells (4) with a filter (5) , and a retaining frit (6) , and a liquid reaction medium used in carrying out the process (7) . The wells have an outlet at the bottom which is sealed by gasket (8) held in place by a top cover (9) and bottom cover (10) maintained in position by clamps (11) .

Such well plates are typically prepared using standard 96-well plates. A hole is drilled in the bottom of each well in the plates and a porous frit is placed in the bottom of each well . The plate is then placed in the clamp assembly to seal the bottom of the wells. This apparatus is commonly called a modified plate or modified titer plate.

Synthesis is initiated by adding reagents to their individual wells according to their assigned plate coordinates. The plate is then capped and tumbled to mix the reagents. Following completion of process Steps a) through c) , the reaction solution is separated from the insoluble scavengers by filtration. The filtrate containing the reaction product is then further processed as per Steps e) through g) . The residual products may then be re- dissolved in appropriate liquid solvent and analyzed, for example, by thin layer chromatography, mass spectrometry and/or nuclear magnetic resonance spectrometry. One embodiment of the present invention is an assay kit for the identification of pharmaceutical lead compounds. The assay kit comprises as essential parts, (1) a well plate apparatus (containing one member of the fused pyrimidine library in each of its individual wells) , and (2) biological assay materials . The biological assay materials are generally known to be predictive of success for an associated disease state. Illustrative of biological assay materials useful in the kit of this invention are those required to conduct the following assays:

In vitro assays:

Enzymatic inhibition Receptor-ligand binding Protein- rotein interaction Protein-DNA interaction

Cell based, functional assays: Transcriptional regulation Signal transduction/Second messenger Viral Infectivity

Add, Incubate, & Read assays:

Scintillation Proximity Assays Angiotensin II IPA receptor binding assay

Endothelin converting enzyme (¹²⁵I) SPA assay

HIV proteinase (¹²⁵I) SPA enzyme assay

Cholesteryl ester transfer (CETP) (³H) SPA assay

Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy

Calorimetric biosensors

Ca2⁺ - EGTA dyes for Cell-based assays

Receptor Gene Constructs for cell based assays Luciferase, green fluorescent protein, Beta-lactamase Electrical cell impedance sensor assays Exa ple 1. Fused pyrimidine Library Plates; General Procedure.

An unmodified 96 -well titer plate (1 ml well volume) was loaded with polyvmylpyridine (PVP) (~7 mg/well, 67 μmol/well) . Isocyanates and isothiocyanates were added across the rows (200 μl of a 0.20 M solution in THF, 40 μmol/well) then primary amines were added down the columns (200 μl of a 0.25 M solution in THF, 50 μmol/well). The plates were capped and agitated for 24 hours.

A solution of NaOH (125 μl/well of a 1.28 N aqueous solution) was added and the plates capped and agitated for an additional 24 hours.

Polystyrene carbonyl chloride resin ("13 mg/well, 33 μmol/well) was added using a solid addition plate. The plates were capped and agitated for 24-48 hours.

The contents of each well were filtered through a well of a modified titer plate (modified with a hole and frit in each well as per Figure 2) into a fresh titer plate (1 ml well volume) . The retained solids were rinsed with THF (125 μl/well) and the filtered wash was added to the initial filtrate. An aliquot of the filtrate was submitted for TLC analysis. The remainder of the filtrate was neutralized with HC1 (125 μl/well of a 1.0 N solution in THF, 125 μmol) , evaporated. Portions of the products were submitted for screening assays, and the remaining portions of the respective library products were stored in well plates for use as a source of library compounds.

Optionally, prior to acid neutralization, an alkylating agent, typically an optionally substituted alkyl iodide (125 μl/well of a 0.64 M solution in THF, 80 μmol), was added to the filtrate and the plates were capped and agitated for 24 hours. The resulting product mixture is neutralized with HC1 (125 μl/well of a 2.0 N solution in THF, 250 μmol) and evaporated. The products were purified by extracting from 70:30 methanol:methylene chloride (500-625 μl/well) and saturated sodium bisulfite (125 μl/well) . The organic layer was removed, typically by robotic manipulation, and filtered through a modified titer plate (see Figure 2) containing anhydrous MgSθ₄ (30 mg/well) , rinsed through with methylene chloride (125-250 μl/well) and analyzed by TLC. The products were dried under vacuum and a portion of each submitted for screening assays. The remaining portions of the respective library products were stored in well plates for use as a source of library compounds.

Example 2 ;

An unmodified 1 ml 96-well titer plate was loaded with polyvmylpyridine (PVP) (~7 mg/well, 67 μmol/well) . Methyl 2-carboxylate-3-isothiocyanato-4-methylthiophene (200 μl of a 0.20 M solution in THF, 40 μmol) was added to a well. Methyl amine was added (200 μl of a 0.25 M solution in THF, 50 μmol) . The well was capped and agitated for 24 hours. A solution of NaOH (125 μl of a 1.28 N aqueous solution) was added and the well capped and agitated for an additional 24 hours.

Polystyrene carbonyl chloride was added to the well ("13 mg/well, 33 μmol/well) . The well was capped and agitated for 24-48 hours.

The contents of the well were filtered through a modified titer plate (modified with a hole and frit in each well as per Figure 2) into a fresh titer plate (1 ml well volume) . The solids retained were rinsed with THF (125 μl/well) and the wash added to the initial filtrate. An aliquot of the filtrate was submitted for TLC analysis. The remainder was neutralized with HC1 (125 μl/portion of a 1.0 N solution in THF, 125 μmol) , evaporated and submitted for screening assays.

Example 3 :

An unmodified 1 ml 96-well titer plate was loaded with polyvmylpyridine (PVP) ("7 mg/well, 67 μmol/well) . Methyl 2-carboxylate-3-isothiocyanato-4-methylthiophene (200 μl of a 0.20 M solution in THF, 40 μmol) was added to a well. Methyl amine was added (200 μl of a 0.25 M solution in THF, 50 μmol) . The well was capped and agitated for 24 hours. A solution of NaOH (125 μl of a 1.28 N aqueous solution) was added and the well capped and agitated for an additional 24 hours.

Polystyrene carbonyl chloride was added to the well ("13 mg/well, 33 μmol/well) . The well was capped and agitated for 24-48 hours.

The contents of the well were filtered through a modified titer plate (modified with a hole and frit in each well as per Figure 2) into a fresh titer plate (1 ml well volume) . The solids retained were rinsed with THF (125 μl/well) and the wash was added to the initial filtrate. Methyl iodide (125 μl/well of a 0.64 M solution in THF, 80 μmol) was added to the filtrate and the well was capped and agitated for 24 hours.

The resulting product was neutralized with HC1 (125 μl of a 2.0 N solution in THF, 250 μmol) and evaporated. The product was purified by extracting with 70:30 methanol:methylene chloride (500-625 μl/well) and saturated sodium bisulfite (125 μl/well) . The organic layer was removed and filtered through a modified titer plate (modified as per Figure 2) containing anhydrous MgSθ₄ (30 mg/well) , rinsed with methylene chloride (125-250 μl/well) and an aliquot removed for analysis by TLC. The products were dried under vacuum and submitted for screening assays.

Claims

We claim:

1. A library of fused pyrimidine compounds wherein said library contains a plurality of diverse library compounds of the formula

wherein X is oxygen or sulfur; ring R¹ is aryl, cycloalkenyl or heterocycle; and R² and R³ are each independently hydrogen or an organic moiety.

2. The library of claim 1 wherein R² and R³ are each independently hydrogen or an organic moiety comprising a non- interfering substituent selected from the group consisting of hydrogen, halo, C╧ç-C╧çrj alkyl, C2-C╧ço alkenyl, C2-C╧ço alkynyl, C╧ç-C╧ço alkoxy, C7-CX2 aralkyl, C7-CX2 alkaryl, C╧ç-C╧ço alkylthio, arylthio, aryloxy, arylamino, C3- C╧ço cycloalkyl, C3-CX0 cycloalkenyl, di (C╧ç-C╧ço) -alkylamino, C2"C 2 alkoxyalkyl, C╧ç-C6 alkylsulfinyl, C╧ç-C╧ço alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydrox (C╧ç~ C╧ço) alkyl, aryloxy (C╧ç-C╧ço) alkyl , C╧ç-C╧ço alkoxycarbonyl , aryloxycarbonyl, C╧ç-C╧ço alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (C╧ç-C╧ço) alkyl, C╧ç~ C╧ço alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, carbamido, carboalkoxy, heterocyclic radical, nitroalkyl, and - (CH2)m-Z- (C╧ç-C╧ço alkyl) , where m is 1 to 8 and Z is oxygen or sulfur.

3. The library of claim 1 wherein R¹ is derived from an isocyanate or isothiocyanate of the formula

having a molecular weight of about 160 to about 800.

4. The library of claim 1 wherein R² is derived from a primary amine of the formula R²NH₂ having a molecular weight of about 30 to about 600.

5. The library of claim 1 wherein R³ is derived from an alkylating agent of the formula R³Y having a molecular weight of about 40 to about 600, wherein Y is a leaving group subject to nucleophilic displacement from the group R³.

6. A compound selected from the group consisting of the library compounds of the library of claim 1.

7. A process for preparing a combinatorial library of compounds of the formula R³

having diversity in substituent groups R¹, R² , and R³ , wherein each library compound is made in a separate reaction zone, said process comprising the steps of a) reacting an isocyanate or isothiocyanate compound of the formula

with a primary amine of the formula R²NH2 to provide an intermediate compound of the formula

provided that any primary amino group forming part of the groups R¹ and R² is in the form of protected amino; b) reacting the intermediate compound of Formula IV with a base effective to cyclize the intermediate compound to provide a solution comprising a compound of the formula

or salt thereof, a (Cχ-Cχo) alkanol and unreacted primary amine, if any from step a) above;

c) separating the compound of Formula V from the

(Cχ-Cχo) alkanol and any unreacted primary amine;

d) either i) acidifying the filtrate solution to provide a solution of library compounds of the Formula I above wherein R³ is hydrogen; or

ii) reacting the filtrate with an alkylating reagent of the Formula R³Y, wherein R³ is an organic moiety and Y is a leaving group subject to nucleophilic displacement from the group R³ , to provide a solution of a compound of Formula I or II above; e) optionally removing any amino or hydroxy protecting groups forming part of any one of the groups R¹, R² and R³; and f) evaporating the solution to provide the compound of Formula I or Formula II,

wherein in the above formulas, X is oxygen or sulfur, ring R¹ is aryl, cycloalkenyl or heterocycle; and R² and R³ are each independently hydrogen or an organic moiety.

8. The process of claim 7 wherein the primary amine in step a) is used in excess of the stoichiometric amount corresponding to the amount of the isocyanate or isothiocyanate compound of Formula III used in each reaction zone .

9. The process of claim 7 wherein the reaction of the isocyanate or isothiocyanate reagent with the primary amine reagent is carried out in the presence of an insoluble acid scavenger.

10. The process of claim 7 wherein the separation of the compound of Formula V from the (Cχ-Cχo) alkanol and any unreacted primary amine is accomplished by adding to the solution from step b) , a solid support bound scavenger having functional groups reactive with amines and alcohols, and separating the solid support bound scavenger from the solution.

11. The process of claim 10 wherein the solid support bound scavenger includes acyl halide groups.

12. The process of claim 7 wherein Step d)ii) further comprises the step of separating unreacted alkylating agent from the product by triturating or extracting in the presence of aqueous bisulfite.

13. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a well plate apparatus wherein each well in said apparatus contains a library compound of the library of claim 1.

14. The assay kit of claim 13 wherein the biological materials are selected for performing at least one assay test selected from the following group of assay tests: In vitro assays:

Enzymatic inhibition Receptor-ligand binding Protein-Protein interaction Protein-DNA interaction

Cell based, functional assays: Transcriptional regulation Signal transduction/Second messenger Viral Infectivity

Add, Incubate, & Read assays:

Scintillation Proximity Assays

Angiotensin II IPA receptor binding assay Endothelin converting enzyme (¹²⁵I) SPA assay

HIV proteinase (¹²⁵I) SPA enzyme assay

Cholesteryl ester transfer (CETP) (³H) SPA assay

Fluorescence Polarization Assays

Fluorescence Correlation Spectroscopy Calorimetric biosensors

Ca2⁺ -EGTA dyes for Cell-based assays

Receptor Gene Constructs for cell based assays Luciferase, green fluorescent protein, beta- lactamase Electrical cell impedance sensor assays.

15. An apparatus suitable as a replacement element in an automated assay machine as a source of individual members of a library of structurally related compounds, said apparatus comprising a 2-dimensional array of defined reservoirs, each reservoir containing a library compound of said library, wherein said structurally related compounds are of the formula

wherein X is oxygen or sulfur, ring R¹ is aryl, cycloalkenyl or heterocycle; and R² and R³ are each independently hydrogen or an organic moiety.

16. The apparatus of claim 15 wherein the library compound in each reservoir is prepared in accordance with the process of claim 7 and wherein each reservoir provides one reaction zone.

17. The apparatus of claim 15 wherein the 2- dimensional array of defined reservoirs is a multi-well microtiter plate.