COMBINATORIAL PROCESS FOR PREPARING FUSED PYRIMIDINE LIBRARIES

Abstract: This invention relates to a novel diverse combinatorial library of fused 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 PYRIMIDINE LIBRARIES This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/034,295, filed December 18, 1996. Field of the Invention The present invention relates to 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. Background of the Invention Research and development expenses account for the large outlay of capital in the drug 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 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 drug industry. Recently modern drug discovery has utilized combinatorial chemistry to generate large numbers (10 2 - 10°) of compounds generically referred to as "libraries". An important objective of combinatory 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. In a preferred embodiment a single reaction product is prepared in each separate vessel. 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/assaying . Combinatorial chemistry is typically conducted on a solid support, normally a polymer. The library scaffold is releasably tethered to the solid support through a chemically cleavable divalent linking group. Reactions are carried out to modify the scaffold while it remains tethered to the solid support. In a final step the product is cleaved and released from the solid support. However, combinatorial libraries can also be prepared in solution phase. To ensure the completion of the solution phase reactions one reagent is typically added in considerable excess. Thus completion of the reaction produces a soluble product with at least one soluble unreacted reactant . Accordingly, as a final step a polymer scavenger having functional groups reactive with the excess reagent is used to remove the excess reagent . 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 method of the present invention is based on the preparation of 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 a 2-dimensional array of reservoirs, each reservoir containing one predetermined compound of the library differing from those in laterally adjacent reservoirs in only one substituent group. Summary of the Invention The present invention provides combinatorial arrays of structurally related compounds having fused pyrimi ) wherein R is an organic group derived from a primary amine of the formula RNH2 , and the fusion ring ("A") is an aromatic ring or a non-aromatic ring, either of which can be heterocylic. The invention further provides a method for generating fused pyrimidine libraries generally in accordance with Scheme 1 or Scheme 2 as set forth below. One embodiment the present invention provides an assay kit for the identification of pharmaceutical lead fused pyrimidine compounds, said kit comprising 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 member of the fused pyrimidine library. The well plate apparatus is used to provide multiple reaction zones for making the library and to store and provide a readily accessible source of library member compounds . Detailed Description of the 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 "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 fused pyrimidine 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"). A "diverse library" means a library where the substituents on the fused pyrimidine combinatorial library scaffold 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"). "Lead compound" means a compound in a selected combinatorial library for which the assay kit has revealed significant activity 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 ring system. A "library compound" is an individual reaction product (usually a single compound) in a combinatorial library. "Parallel array synthesis" refers to the method of conducting combinatorial chemical synthesis of libraries wherein the individual combinatorial library reaction products are separately prepared and stored without prior and subsequent intentional mixing. 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 wellplate apparatus. 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. "Simultaneous synthesis" means making of a library of compounds within one production cycle of a combinatorial method (not making all library compounds at the same instant in time) . "Substituents" are chemical radicals which are bonded to or incorporated in the 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 biologically 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 a reactant, any chemical compound used in the combinatorial synthesis to place substituents on the scaffold of a library. " ellplate apparatus" refers to the structure capable of holding a plurality of library compounds of dimensionally fixed and defined positions. "Non-interfering substituent" are those groups that do not significantly impede the process of the invention and yield stable fused pyrimidine library compounds. Suitable non-interfering radicals 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, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, 2- 12 alkoxyalkyl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, - (CH2 )m~0- (C1-C10 alkyl), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, aryloxyalkyl, heterocyclic radical, substituted heterocyclic radical, and nitroalkyl, where m is from 1 to 8. Preferred non-interfering radicals are Cχ-Cιo alkyl, C2-C10 alkenyl, C1-C10 alkoxy, C7-C12 aralkyl, C7- C12 alkaryl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, phenyl, - (CH2 )m~0- (C1-C10 alkyl), aryl, and substituted aryl . "Aryl" means one or more aromatic rings, each of 5 or 6 carbon atoms. Multiple aryl rings may be fused, as in naphthyl or unfused, as in biphenyl. "Substituted aryl" is aryl having one or more non-interfering groups as substituents. "Alkyl" means straight or branched chain or cyclic hydrocarbon having, typically 1 to 20 carbon atoms. "Substituted alkyl" is alkyl having one or more non-interfering substituents. "Halo" means chloro, fluoro, iodo or bromo . "Heterocycle" or "heterocyclic" means one or more rings of 5, 6 or 7 atoms with or without unsaturation or aromatic character and at least one ring atom which is not carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen. Multiple rings may be fused, as in quinoline of benzofuran. "Substituted heterocycle" is heterocycle having one or more side chains formed from non-interfering substituents. A diverse library of fused pyrimidines is provided in accordance with the present invention. The fused pyrimidine library embodied as an apparatus of this invention serves as a readily accessible source of a wide variety of structurally related compounds for use in identifying new biologically active compounds through drug/agricultural candidate screening assays, for use in defining structure activity relationship studies, and/or for use in clinical investigation. Abbreviations used in the description of the process of the invention are as follows : DMF is dimethylformamide THF is tetrahydrofuran The library of fused pyrimidine compounds of Formula I or Formula II is prepared using combinatorial chemistry parallel array synthesis techniques illustrated in the following reaction schemes: Scheme 1 : General Scheme for the Preparation of the Fused Pyrimidine Library (via Formamidine) + RNH 2 ( excess ) 1) 2) polystyrene cat carboxaldehyde 1 , 2-dichloroethane/ methanol II (exclusively) or Scheme 2 : General Scheme for the Preparation of the Fused Pyrimidine Library (via Formamidate) Trimethyl orthoformate, polystyrene sulfonic acid resin 70°C, 1-3 days ent temp, 1-3 days Either structure I or structure II may be isolated The method comprises the steps of preparing aromatic or aliphatic formamidine or formamidate intermediates from the illustrated cyanoamine compounds and reacting each intermediate with a series of primary amines (RNH2) to prepare a library of fused pyrimidine compounds. While dimethylformamide and trimethylorthoformate are preferred reactants for forming the respective intermediates, other formamidine and formimidate forming reactants, e.g., di(Cι-C4 alkyl) formamide acetals and tri (C1-C4 alkyl) orthoformates, can be employed. Each formamidine/formimidate intermediate is prepared and further reacted with a primary amine in a separate reaction chamber (i.e. parallel array synthesis), and the reaction product is identified by the reaction well number. The starting cyanoa ine compounds of the formula are either commercially available or prepared from commercially available starting materials. In the above formula the fused ring A is an aromatic ring or a non- aromatic ring, either of which can be heterocyclic. Illustrative of the divalent group A forming the ring structure above and in the other formulas defining the present invention, including Formulas I and II above, include, but are not intended to be limited to, the following groups : H 3 C Li ■ L 2 N ^ UI wherein Li and 2 are the points of bonding with the rest of the molecule. The primary amines used in accordance with the process of this invention in preparation of the present fused pyrimidine libraries are compounds of the formula RNH2 wherein R is an alkyl or substituted alkyl group having substituents which do not interfere with the pyrimidine forming reaction. Preferably the primary amine reactants have a molecular weight of about 30 to about 600. Illustrative of R groups in the definition of the present invention include, but are not intended to be limited to, the following radicals: *Υ ' CH„ and wherein L is the point of bonding to the nitrogen atom to which the group is bound. Thus, exemplary of primary amines useful in the process of this invention are the compounds represented by those formulas wherein L is - NH2. Other primary amines useful in preparing the fused pyrimidine libraries of this invention include, but are not intended to be limited to: cyclopropylamine cyclobutylamine (-) -cis-myrtanylamine cyclopentylamine cyclohexylamine 2 -methylcyclohexylamine 2 , 3-dimethylcyclohexylamine 4-methylcyclohexylamine (aminomethyl ) cyclohexane 3 -aminomethyl-3 , 5, 5-trimethylcyclohexanol 1,2,3, 4-tetrahydro-l-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 ) orpholine 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-1-piperidinecarboxylate 4-amino-l-benzylpiperidine 1- ( 2 -aminoethyl ) piperidine 1- ( 3 -aminopropyl ) -2-pipecoline 1 , 2 -diamino-2 -methylpropane benzhydrylamine d- ( - ) -alpha-phenylglycinol 1, 2-diphenylethylamine dl-1-phenylethylamine ( - ) -norephedrine 1 , 2-dimethylpropylamine isopropylamine 2-methoxyisopropylamine dl-2-amino-l-propanol ethyl-3 -aminobutyrate 1 , 3 -dimethylbutylamine 3-amino-l-phenylbutane 2-amino-5-diethylaminopentane 1 , 5-dimethylhexylamine sec-butylamine (+/-) -2-amino-l-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 - ( trifluoromethy1 ) benzylamine 3 -methylbenzylamine 4-fluorobenzylamine 4-chlorobenzylamine 4-methoxybenzylamine 4-methylbenzylamine 2,2, 2-trifluoroethylamine 2-amino-l-phenylethanol l-amino-2-propanol 3-amino-l, 2-propanediol 2 , 2 -diphenylethylamine beta-methylphenethylamine isobutylamine 2-methylbutylamine 2-ethylhexylamine n-decylamine n-undecylamine dodecylamine tridecylamine 1-tetradecylamine hexadecylamine octadecylamine ethylamine 2- (2-aminoethylamino) ethanol 2-methoxyethylamine 2- (2-aminoethoxy) ethanol ethanol mine phenethylamine 2- (2-chlorophenyl) ethylamine 2 - ( 2 - ethoxyphenyl ) ethylamine 3-methoxyphenethylamine 2 - (3 , 4-dimethoxyphenyl) ethylamine 4-bromophenethylamine 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-l-pentanol hexylamine 6-amino-l-hexanol n-heptylamine n-octylamine n-nonylamine dl-2-amino-l-pentanol dl-2-amino-1-hexano1 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-l , 3-propanediamine 1-phenylalanine beta-naphthyl-amide 2- (3-chlorophenyl) ethylamine 2-amino-1 , 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 l-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 (Is, 2r) - (+) -phenyl-propanolamine (s) - (+) -2-amino-l-propanol d-alaninol (r) - ( -) -sec-butylamine (s) - (+) -sec-butylamine (s) - (+) -2-amino-l-butanol (r) - (-) -2-amino-l-butanol (r) - (-) -l-amino-2-propanol (s) - (+) -l-amino-2-propanol ( s ) - ( - ) -2-methylbutyla ' mine (s) - (+) -1-cyclohexylethylamine oleylamine 1-adamantanemethylamine (Is, 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)-(-)-l- (p-tolyl) ethylamine (s) - (-) -2-amino-l, 1-diphenyl-l-propanol (+/-) -exo-2-aminonorbornane (s) - (+) -2- (aminomethyl) pyrrolidine 3-amino-l-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 (Is, 2r) - (-) -cis-l-amino-2-indanol (lr, 2s) - (+) -cis-l-amino-2-indanol . Additional primary amines suitable for use in the process of the invention for preparing fused pyrimidine libraries are those represented by the following formulae: where CBz is benzyloxycarbonyl . The preparation of the fused pyrimidine library compounds of Formula II comprises a two step process wherein a formamidine intermediate is first formed and subsequently reacted with a primary amine to prepare a broad array of fused pyrimidine compounds in accordance with scheme 1. Alternatively those compounds can be prepared in accordance with Scheme 2 via a formimidate intermediate and thermal rearrangement of the library compounds of Formula I . In one embodiment of the process of the invention a library of fused pyrimidines of Formula I are prepared, and a portion of each of the product compounds are converted to the corresponding compounds of Formula II to produce a separate library. The completion of the reactions between the primary amine and the formamidine/formimidate intermediates and the conversion of the compounds of Formula I to those of Formula II can be determined by a number of conventional techniques including thin layer chromatography (TLC) . The process of the present invention may be carried out in any vessel capable of holding the liquid reaction medium and having an inlet and outlet means. In one embodiment the process of the invention is carried out in containers adaptable to parallel array synthesis. The process of this invention is carried out in an apparatus providing multiple reaction zones, most typically a two-dimensional array of defined reservoirs, wherein one member of the fused pyrimidine libraries 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 unique member 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 to prepare multiple copies of the library apparatus or to subject the library to additional reaction conditions. Copies of the library apparatus (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. In one embodiment of the present invention an assay kit is provided for the identification of pharmaceutical lead compounds. The assay kit comprises as essential parts, (1) a well plate apparatus (containing one of the fused pyrimidine compounds 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 assays known in the art, which include, but are not intended to be limited to: In vitro assays such as: Enzymatic inhibition, Receptor-ligand binding, Protein-Protein interaction, Protein-DNA interaction, and the like; Cell based, functional assays such as: Transcriptional regulation, Signal transduction/Second messenger, Viral Infectivity, and the like; and Add, Incubate, & Read assays such as: Scintillation Proximity Assays, Angiotensin II IPA receptor binding assay, Endothelia converting enzyme [125χ] SPA assay, HIV proteinase [ 125 I] SPA enzyme assay, Cholesteryl ester transfer (CETP) [ 3 H] SPA assay, Fluorescence Polarization Assays, Fluorescence Correlation Spectroscopy, Colorimeric biosensors, Ca^+ -EGTA for Cell-based assays, Receptor Gene Constructs for cell based assays, Cellular reporter assays utilizing, for example, reporters such as luciferase, green fluorescent protein, B-lactamase, and the like; Electrical cell impedance sensor assays, and the like. Preparation 1: Aromatic Formamidine Intermediate per (Scheme I) 5-Chloro-2- (dimethylamino) methyleneaminobenzonitrile A mixture of 2-amino-5-chlorobenzonitrile (3.82g, 25 mmol) and dimethylformamide dimethyl acetal (4.7 mL) in anhydrous tetrahydrofuran (10 mL) was refluxed for 2h, then concentrated in vacuo . The residue was taken up in a mixture of methylene chloride and hexane, and passed through a short column of silica gel (eluted with 1:1 hexane/ethyl acetate) to afford 5- chloro-2- (dimethylamino) methyleneaminobenzonitrile (5.18g, 99.8%) as a pale yellow solid; mp 75 - 77°C. This compound was made into a 1.0 N solution in methylene chloride/methanol (20 mL) for use in plate synthesis. Other formamidine intermediates were similarly prepared. Preparation 2 : 4-Amino-3-cyano-l, 2,5, 6-tetrahydropyridine A cooled (-65°C) mechanically stirred solution of 3 , 3 ' -iminodipropionitrile (41. Og, 300 mmol, weight corrected for 90% purity) under nitrogen was treated dropwise with 1.0 N lithium bis (trimethylsilyl) amide/tetrahydrofuran (330 mL, 330 mmol) at a rate to keep pot temp below -50°C (a precipitate immediately began to form) . The solution was stirred at -60°C for lh after addition was complete, then warmed to -20°C over 30 min and quenched with 5N aqueous ammonium chloride (75 mL, 375 mmol) . Water (100 mL) and 2-propanol (100 mL) were added, and the mixture was separated into two layers. The aqueous layer was extracted with 3:1 ethyl acetate/2 -propanol containing a little methanol (6X150 mL; alternatively, continuous extraction can be used) , and the combined organic extracts and initial organic layer were dried (MgS04) , filtered, and concentrated in vacuo . The residual solid was triturated from acetonitrile/2-propanol (two crops) to afford 4-amino-3-cyano-l, 2 , 5, 6-tetrahydropyridine (25.9g, 70%) as a white solid; mp 160 - 162°C. Preparation 3 : Non-aromatic Formamidine Intermediate per (Scheme I) 3-Cyano-l- (4-fluorobenzoyl) -4- (dimethylamino)methyleneamino-l, 2 , 5, 6-tetrahydropyridine An ice-cooled (5°C) suspension of 4-amino-3- cyano-1, 2, 5, 6-tetrahydropyridine (4.31g, 35 mmol) and triethylamine (7 mL, 50 mmol) in 4:1 tetrahydrofuran/DMF (50 mL) was treated dropwise with 4-fluorobenzoyl chloride (6.66g, 42 mmol) so that the pot temperature was maintained below 20°C. The suspension was stirred at room temperature for one hour, then added to ice water (250 mL) . The mixture was stirred for a few minutes to allow a precipitate to solidify, then the solid was removed by filtration, washed with water, partially air- dried, and dissolved in methanol/methylene chloride. The organic solution was dried ( gSO^) , filtered, and the filtrate concentrated in vacuo to afford 4-amino-3-cyano- 1- (4-fluorobenzoyl) -1,2,5, 6-tetrahydropyridine (8.44g, 98%) as an off-white solid. A suspension of this solid (7.36g, 30 mmol) in anhydrous tetrahydrofuran (20 mL) was treated with DMF dimethyl acetal (7.0 mL, 49 mmol), then heated to reflux for four hours (solid dissolved) . The mixture was concentrated in vacuo and recrystallized from ethyl acetate/hexane to afford 3-cyano-l- (4- fluorobenzoyl) -4- ( dimethylamino)methyleneamino-l, 2 ,5,6- tetrahydropyridine (7.g, 80%) as a tan solid; mp 148 - 150°C. Preparation 4: 5-Amino-4-cyanooxazoles The titled compounds were prepared from the appropriate acid chloride and aminomalononitrile tosylate in N-methylpyrrolidin-2-one according to the general procedure of Freeman, F.; Kim, D. S. H. L. Tetrahedron Let t . 1989, 30, 2631-2. Preparation 5: 5-Amino-4-cyanoimidazoles The titled compounds were prepared from the appropriate primary amine, aminomalononitrile tosylate, triethyl orthoformate, triethylamine, and acetonitrile according to the general procedure of Birkett, P. R. ; Chapleo, C. B.: Mackenzie, G. Synthesis 1991, 822 - 4. Preparation 6: Primary amine reactant solution. 2-Phenylethylamine in 0.2N HCl/Ethanol Concentrated hydrochloric acid (1.0 mL, 12 mmol) was diluted with reagent ethanol to a total of 60 mL. 2-Phenylethylamine (0.70g, 5.75 mmol) was dissolved in 5.0 mL of this 0.2N HC1 solution. Other primary amine solutions were prepared in similar fashion. Example 1 Fused Pyrimidine Library Plates per (Scheme I) A 96-well 1 mL deep well glass well titer plate was charged with 50 uMol of each formamidine solution in rows, with care taken that all liquid was added to the bottom of the wells, with minimum splattering. The solvents were evacuated, then acetonitrile (200 uL) was added to each well, and the plate was gently heated in a 96-well heat block (USA Scientific) with moderate shaking at 60°C with no caps for about 15 min in order to dissolve most material. The appropriate amine solutions were added (50 uL each well) in columns, followed by more acetonitrile (300 uL each well) , and the plate was topped with strip caps underneath a whole plate cap and clamped into the heat block at 70°C with moderate shaking for 6h. The caps were removed, more acetonitrile (500 uL, less if solvent remains) was added, the caps were replaced, and heating (75-80°C) with gentle shaking was continued for 42 - 90h. The reaction plates were then cooled to room temperature, uncapped, and the plate was evacuated of solvent. The well residues were taken up in dichloroethane/methanol (4:1, 500 uL each well) and heated (50°C) uncovered with shaking for 15 min. A slurry of 2.2g of polystyrenecarboxaldehyde resin (loading=0.8 mmol/g) in dichloroethane/THF (3:11, 24 mL) was added (200 uL each well) , and the plate was capped with strip caps and shaken at 55°C for 6h, then at room temperature for 18h. The caps were removed and the contents of each well transferred through a drilled filter plate (with frits placed at the well bottoms) to a square well 2 mL titer plate. Each well in the reaction plate was washed with methylene chloride/methanol (4:1, 500 uL per well) , and the washing was added to the filter plate. Finally, an additional 500 uL of 4:1 methylene chloride/methanol was added directly to the filter plate in order to elute all possible product. Silica thin layer chromatography was performed on each well of the receiving plate (eluted with 4-10% isopropanol/methylene chloride) , then the contents of each were evacuated of solvent to afford plates containing about 40 uMol of material per well. Material samples (ca. 1 mg) were extracted from four wells and submitted for low resolution mass spectral analysis. In one plate, for example, the following four products were analyzed in this manner: 4- (2-phenylaminoethylamino) quinazoline : calcd. mass 264.3; observed 265.4. 4- (2- (4-chlorophenyl) ethylamino) -lH-pyrazolo [3 , 4-d] - pyrimidine: calcd. mass 273.7; observed 274.2. 4- (2- (4-hydroxyphenyl) -ethylamino) -6, 7-dihydro-5H- cyclopentapyrimidine: calcd. mass 255.3; observed 256.0 4- (2- (2-methoxyphenyl) ethylamino) -7-methylquinzoline : calcd. mass 293.4; observed 294.4. Example 2 Fused Pyrimidine Library Plates per (Scheme 2) A 96-well deep well glass plate was charged with 0.25N solutions of eight different aminonitriles in methanol or methylene chloride/methanol, by rows (300 uL, 75 uMol per well) . The plate was evacuated of solvent, and each well was charged with a slurry (400 uL, 10 mg contained) made from taking up 1.5g of dried sulfonic acid resin (DOWEX® type, 5 mmol/g dry loading) in 9 mL of methanol and diluting the mixture to 60 mL with methylene chloride. The wells were again evacuated of solvent, then charged with trimethyl orthoformate (400 uL/well) , and the plate was capped with strip caps, clamped into a heat block, and shaken at 65 - 70°C for 40 -72 h. The plate was cooled and uncapped, and the contents were transferred by robot through a 96-well filter plate into another 96-well deep well glass plate. Methylene chloride (250 uL per well) was added to the original plate, and the contents were again transferred into the filter plate. The receiving plate was evacuated of solvent, then acetonitrile (200 uL per well) was added. A solution of the appropriate amines, (1.0N in ethanol, 100 uL per well) was added by columns, followed by another 100 uL of ethanol. The plate was capped with strip caps, clamped into a heat block, and shaken for one hour at 60°C and for 40 -72 h at room temperature, then uncapped. At this point the unrearranged fused pyrimidines could be isolated as is, or carried on to the fused aromatic pyrimidines. In the latter case, Biorad AG1-X8 hydroxide resin (1.8 mmol/g wet form, 50 mg per well) was added by solid addition to each well, and shaking was resumed at 80°C for 48-90 h. The plate was cooled and uncapped, and the contents were transferred by robot through a 96-well filter plate into another 96-well deep well glass plate. Methylene chloride (250 uL per well, DMF substituted if solubility is a problem) was added to the original plate, and the contents were again transferred into the filter plate. The receiving plate was evacuated of solvent, then each well was treated with a slurry made by suspending 3.5g of polystyrene carboxaldehyde resin (1.7 mmol/g) in 5:1 dichloroethane/methanol (70 mL) . Strip caps were added, and the plate was clamped into the heat block and shaken at 50-55°C for 4-6h, then at room temperature for 16h. The contents of each well were transferred by robot through a 96-well filter plate into a plate suitable for compound storage, then methylene chloride (250 uL per well) or DMF (if needed) was added to the original plate, and the contents were again transferred into the filter plate. TLC analysis was performed on each well, and the product plate was evacuated of solvent.

Claims: We claim: 1. A library of fused pyrimidine compounds wherein said library contains a plurality of diverse library compounds of the formula wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group derived from a primary amine of the formula RNH2. 2. The library of claim 1 wherein fused ring A is a 5 or 6-member heterocylic ring containing 1 to 3 heteroatoms selected from the group of 0, N and S. 3. The library of claim 1 wherein R is alkyl or substituted alkyl derived from a primary amine of the formula RNH2. 4. An individual fused pyrimidine library compounds of claim 1. 5. A library of fused pyrimidine compounds wherein said library contains a plurality of diverse library compounds of the formula wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group derived from a primary amine of the formula RNH2. 6. The library of claim 5 wherein fused ring A is a 5 or 6-member heterocylic ring containing 1 to 3 heteroatoms selected from the group of O, N and S. 7. The library of claim 5 wherein R is alkyl or substituted alkyl derived from a primary amine of the formula RNH2. 8. An individual fused pyrimidine library compounds of claim 5. 9. The library of claim 3 wherein the primary amine has a molecular weight of about 30 to about 600. 10. The library of claim 7 wherein the primary amine has a molecular weight of about 30 to about 600. 11. A process for preparing a combinatorial library of fused pyrimidine compounds of the formula having diversity in fused ring A and the substituent R group wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group and wherein each library compound is made in a separate reaction zone, said process comprising the steps of reacting a compound of the formula with a di(Cι-C4 alkyl) formamide acetal to form an intermediate formamidine compound of the formula N(C r C 4 alkyl) 2 in each reaction zone and reacting the intermediate in each zone with a primary amine of the formula RNH2. 12. A combinatorial process for preparing a library of fused pyrimidine compounds of the formula: having diversity in fused ring A and the substituent R group wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group and wherein each library compound is made in a separate reaction zone, said process comprising the steps of reacting a compound of the formula with a tri(Cι~C4 alkyl) orthoformate to form an intermediate formimidate compound of the formula OC r C 4 alkyl in each reaction zone and reacting the intermediate in each zone with a primary amine of the formula RNH2. 13. The process of claim 12 further comprising the step of forming a solution of the fused pyrimidine product in each reaction zone and heating each said solution to produce a library of fused pyrimidine compounds of the formula 14. The process of claim 12 wherein a portion of the product fused pyrimidine in each reaction zone is transferred to a corresponding second reaction zone and heated in solution to produce a second library of fused pyrimidine compounds of the formula: 15. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a wellplate apparatus, where each well in said apparatus contains a unique library compound of claim 1. 16. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a wellplate apparatus, where each well in said apparatus contains a unique library compound of claim 5. 17. The assay kit of claim 15 containing biological materials for performing assay tests selected from the group consisting of in vi tro assays, cell based functional assays, and add, incubate, and read assays. 18. The assay kit of claim 16 containing biological materials for performing assay tests selected from the group consisting of in vi tro assays, cell based functional assays, and add, incubate, and read assays. 19. 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 unique compound of said library, wherein said structurally related compounds are of the formula: wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group derived from a primary amine of the formula RNH2. 20. The apparatus of claim 19 wherein the library compound in each reservoir is prepared in accordance with the process of claim 12 and wherein each reservoir provides one reaction zone. 21. 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 unique compound of said library, wherein said structurally related compounds are of the formula: wherein fused ring A is an aromatic or non-aromatic ring and R is an organic group derived from a primary amine of the formula RNH2. 22. The apparatus of claim 19 wherein the library compound in each reservoir is prepared in accordance with the process of claim 11 and wherein each reservoir provides one reaction zone. 23. The apparatus of claim 19 wherein the library compound in each reservoir is prepared in accordance with the process of claim 13 and wherein each reservoir provides one reaction zone. 24. The apparatus of claim 19 wherein the library compound in each reservoir is prepared in accordance with the process of claim 14 and wherein each reservoir provides one reaction zone. 25. The apparatus of claim 19 in the form of a multi-well microtiter plate. 26. The apparatus of claim 21 in the form of a multi-well microtiter plate.

Inventors: JAGDMANN GUNNAR E JR, JAGDMANN, GUNNAR, E., JR.
Applicants: JAGDMANN GUNNAR E JR, LILLY CO ELI, ELI LILLY AND COMPANY, JAGDMANN, GUNNAR, E., JR.
Application year: 1997
Main classification: C07D403
IPC codes: C07D239, C07D471, C07D473, C07D487