This invention relates to a method for preparing combinatorial libraries of lactams having desired pharmaceutical or other biological utility. More particularly, the present invention relates to structurally diverse libraries of lactams, methods for preparing such lactams, and an apparatus for storing such libraries and for use as a component of assay systems for discovery of lead compounds for drug development.

Research and development expenses account for the largest 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 hundreds of high purity compounds for biological screening to develop potentially useful pharmaceutical drugs. Although past methods brought new drugs to market, the limitations of individual synthesis and insistence on compound characterization considerably slowed the discovery process.

The need for more rapid and less expensive drug discovery methodology is increasingly important in today's competitive drug industry.

More recently, modern drug discovery has used the methods of combinatorial chemistry to generate large numbers (i. e., about 102 to 106) of compounds generally referred to as"libraries."An important objective of

combinatorial chemistry is to generate lead compounds for pharmaceutical research. In particular, combinatorial chemistry may be used at two distinct phases of drug development. In the discovery phase, highly diverse libraries are created to find lead compounds. In a second optimization phase, strong lead compounds are much more narrowly modified to find optimal molecular configurations.

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 their biological screening.

Combinatorial chemistry may be performed in a manner where libraries of compounds are generated as mixtures with complete identification of individual compounds postponed until after the compounds are screened with positive results.

However, a preferred form of combinatorial chemistry is"parallel array synthesis"where individual reaction products (most often individual compounds) are synthesized together, but are retained in separate vessels.

For example, the library compounds are held in the individual wells of 96 well microtiter plates. Use of standardized microtiter plates or equivalent apparatus is advantageous because such an apparatus is readily manipulated by programmed robotic machinery.

Conventionally, combinatorial chemistry is conducted on a solid phase support, normally a polymer. The library scaffold reactant is cleavably tethered to the solid support by a chemical linker. Reactions are carried out

to modify the scaffold reactant while tethered to the solid support. In a final step, the product is cleaved and released from the solid support. A general procedure for conventional solid phase synthesis is illustrated by the following scheme where the shaded circle symbol is a solid support, for example, a high molecular weight polymer, and X, A and B are reactants or reagents: Solid-Phase Synthesis: excess excess X A ÇX-A Variations in reagents (e. g.,"A","B", in the general scheme, supra.) produce the desired structural diversity. Separation of solid phase product and unreacted soluble reactant is accomplished by simple separation techniques such as filtration, decantation, or washing. These separation solid phase synthesis techniques have general applicability to a wide variety of combinatorial reactants and lend themselves to large scale simultaneous/automated library creation.

The rate determining step in small molecule synthesis is typically not actual construction of the desired new chemical entities. Rather, the difficulty of synthesis is frequently caused by the task of isolating reaction product from unreacted starting materials, by-products or other impurities.

Unfortunately, it is not always practicable to tether a desired combinatorial scaffold to a solid support. A significant number of combinatorial reaction schemes are desirably performed in solution phase.

Moreover, not all desired solution phase reactions are driven to completion

using near stoichiometric ratios of reactants. Frequently, one reagent is added in considerable excess to drive a solution phase reaction to completion, which results in a reaction medium with soluble product and soluble unreacted co-reactant. Consequently, traditional organic synthetic methods often require complex purification strategies which limit their use, particularly in combinatorial syntheses.

Traditional organic synthetic methods often follow complex pathways designed for making single compounds. In contrast, combinatorial synthesis requires adaptation of new synthetic pathways to enable the simultaneous substitution of a variety of different moieties under nearly identical reaction conditions to create a library of diverse compounds. This may demand using a new synthetic pathway to construct the molecules in the reverse order from traditional methods, or using creative means to avoid interfering reactions from excess reactants during later steps.

To date, one group of therapeutical useful compounds, the lactams, has evaded optimal adaptation to combinatorial techniques. Lactams are therapeutically useful as angiotension converting enzyme inhibitors. For example, U. S. Patent No. 4,666,901, describes solution phase synthesis of monocyclic lactams having this utility. The available methods for synthesizing these compounds can be laborious. Therefore, it is desirable to develop solution phase combinatorial processes for making libraries of lactams.

The present invention is directed to a solution phase process for making diverse combinatorial libraries, where excess reagent (s) are used to promote the formation of library compounds. The present invention includes

the use of a solid supported reducing agent in a combinatorial library forming process to make the diverse library compounds.

Further, a preferred embodiment of the present invention is directed to the use of a solid supported scavenger in a combinatorial library forming process to remove an excess of soluble reagent (s).

Furthermore, the present invention is directed to an improved combinatorial process for making a library of lactam compounds. The method of the present invention can be utilized for making both diverse libraries of lactam compounds useful for finding new lead compounds, and directed libraries of lactams useful for optimizing a particular desired biological activity.

Moreover, the present invention is directed to a novel wellplate apparatus containing the novel lactam library compounds of the present invention.

This invention is also directed to an assay kit for identification of pharmaceutical lead lactam compounds, said kit comprising (i) wellplate apparatus, and (ii) biological assay reagents and, said wellplate apparatus having a combinatorial library compound in each well, wherein the improvement comprises using as a wellplate a combinatorial lactam wellplate apparatus where each well contains a lactam compound prepared by the process of the invention.

FIG. 1 is a top view of a wellplate apparatus.

FIG. 2 is a side view of a wellplate apparatus.

The present invention is directed to a solution phase process for making diverse combinatorial libraries, where excess reagent (s) are used to promote the formation of library compounds. The present invention includes the use of a solid supported reducing agent in a combinatorial library forming process to make the diverse library compounds.

I. Definitions: The following terms have the meaning defined below when used in the specification of the present invention: "Directed Library"means a collection of compounds created by a combinatorial chemistry 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 to"Diverse library").

"Diverse Library"means a library where the substituents on the 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 an Assay Kit has revealed significant activity relevant to a selected disease state.

"Library"means a collection of compounds created by a combinatorial chemical process, wherein the compounds have a common scaffold with one or more variable substituents.

"Library compound"means an individual reaction product (usually a single compound) in a library produced by the method of the present invention.

"Parallel array synthesis"means a method of conducting combinatorial chemical synthesis of libraries wherein the individual combinatorial library reaction products are separately prepared and stored without prior or subsequent intentional mixing.

"Reaction zone"means the individual vessel location where the combinatorial chemical library compound preparation process of the present invention is carried out and individual library compounds are synthesized.

Suitable reaction zones may be the individual wells of a wellplate apparatus, capped glass vials, and the like, in an ordered array.

"Scaffold"means the invariant region (i. e., core) of the compounds which are members of a library.

"Simultaneous synthesis"means making a library of compounds within one production cycle of a combinatorial method (i. e., not making all library compounds at the same instant in time).

"Solid support"means a reaction medium insoluble solid substrate capable of containing chemical functionality, and is represented by the symbol :

"Solid supported reducing agent"means a reaction medium insoluble solid substance containing chemical functionality reactive with a reactant or intermediate.

"Solid supported scavenger"means a reaction medium insoluble solid substance containing chemical functionality reactive with the soluble impurity (e. g., excess reactant) desired to be removed from the reaction medium in the presence of soluble product.

"Substituents"are chemical radicals which are bonded to the scaffold through the combinatorial synthesis process. The different functional groups account for the diversity of 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. Substituents may be further substituted with additional substituents.

"R"means a reactant which can be any chemical compound used in the combinatorial synthesis to place substituents on the scaffold of a library.

"Wellplate apparatus"means a structure capable of holding a plurality of library compounds in dimensionally fixed and defined positions.

"Non-interfering substituent"refers to an organic (non-hydrogen) radical suitable for substitution as, for example, Ri, R2, R3, R4 or R5 in the reactants used in the process of making a combinatorial lactam library, and which do not significantly impede the solution phase processes of the invention or interfere with the use of a solid phase scavenger in said processes. Suitable non-interfering radicals include, but are not limited to,

C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C12 aralkyl, C7- C12 alkaryl, Cs-Cio cycloalkyl, C3-C10 cycloalkenyl, phenyl, substituted phenyl, toluyl,xylenyl, biphenyl, C2-C, 2 alkoxyalkyl, C,-C6 alkylsulfinyl, C,-C10 alkylsulfonyl,-(CH2) m-0-(C1-C10 alkyl), thiol, aryl, substituted aryl, substituted alkoxy, amidino, fluoroalkyl, aryloxyalkyl, fluoroalkyl, aryloxalkyl, heterocyclic radical, substituted heterocyclic radical, and nitroalkyl ; where m is from 1 to 8.

Preferred non-interfering radicals are C1-C10 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.

"Cx-Cy alkyl"means a straight or branched chain hydrocarbon of between x and y carbon atoms.

"Cx-Cy cycloalkyl"means a ring of between x and y carbon atoms having at least one fully saturated bond.

"Alkyl"as used herein is a lower alkyl group which can be a branched, cyclic or straight chain alkyl group containing 1-6 carbon atoms. Suitable alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso- butyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, hexyl, cyclohexyl, etc.

"Amino acid"means a compound that contains an amino group and a carboxylic acid group. Typically, the phrase refers to oc-amino acids, although other amino acids (such as 0-and 9-amino acids) can be used.

Natural amino acids include those which are find in nature. Unnatural amino acids include synthetic amino acid residues. Synthetic amino acids can include oc-amino acids containing aryl or hetaryl groups attached as side chains. Synthetic amino acids also include oc. oc-disubstituted amino acids

such aminoisobutyric acid (AIB) and those described by Scott et al, Tetrahedron Lett. 1997,38 (21): 3695 ; incorporated herein by reference.

"Amino acid side chain"means the group attached to the oc-carbon of an oc-amino acid. Suitable amino acid side chains include the oc-side chain of the naturally encoded amino acids such as alanine (Ala, A), arginine (Arg, R), asparagine (Asn. N), aspartic acid (Asp, D), cysteine (Cys, C), glutamin (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), ornithine (Orn, O), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Alternatively, any side chain of a synthetic amino acid can be used, including lower alkyl, alkyloxy, alkylamino, alkylthiol, etc.

"Aryl"means any monovalent aromatic carbocyclic group of 5-10 carbon atoms in a single ring (i. e. phenyl) or multiple condensed rings (i. e. naphthyl), which can be unsubstituted or substituted with 1 or more, preferably 1-5, substituents including alkyl, alkylamino, alkylcarbonyl, alkoxy, amino, dialkylamino, halo, hydroxyalkyl, hydroxyl, merapto, thiol, etc.

"Electron donating groups"are those groups that can stabilize an adjacent electron deficiency. Suitable electron donating substituents include hydroxyl, alkoxy, amine, thiol, alkylamine, dialkylamine, amido, alkyl.

"Halogen"or"Halo"means fluorine, chlorine, iodine or bromine. The preferred halogen is fluorine or chlorine.

"Heterocycle"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 or benzofuran.

"Substituted heterocycle"means heterocycle with one or more side chains formed from non-interfering substituents.

"Hetaryl"means any monovalent unsaturated aromatic carbocylic group having 4 to 10 carbon atoms in a single ring (i. e. pyridyl or furyl) or multiple fused rings (i. e. quinolinyl or indolzinyl) and having at least one heteroatom, such as N, O or S, within the ring. The group can be unsubstituted or substituted with alkyl, alkylamino, alkylcarbonyl, alkoxy, amino, dialkylamino, halo, hydroxyalkyl, hydroxyl, mercapto, thiol, etc.

Preferably, the group is a five-or six-membered ring.

"Linkers"means any molecule that provides spatial distance between the support and the peptide to be synthesized. Linkers can be covalently attached on the solid phase support prior to coupling with a Noe-Boc or Nove- Fmoc or otherwise appropriately protected amino acids. Examples of linkers include aminobutyric acid, aminocaproic acid, 7-aminoheptanoic acid, and 8- aminocaprylic acid. In a further embodiment, linkers can additionally comprise p-alanine or polymeric 0-alanine.

"Monomer"means a molecule which is not substantially comprised of repeating molecular subunits.

"Polymer"means a molecule which is composed of monomer units. As used herein, polymer refers to the polymer resin to which the compounds in the combinatorial library are attached. In an alternate embodiment, a linker is first attached to the polymer resin such that the compound of the

combinatorial library is linked to the polymer resin via the linker. Suitable polymer resins for use in the present invention must be inert to the reaction conditions for peptide synthesis and include Merrifield type resins, Wang type resins and polyamide type resins. Examples of these include polystyrene (e. g., PAM-resin obtained from Bachem Inc.), polyamide resin, POLYHIPETM resin (obtained from Aminotech, Canada), p-alkoxybenzyl alcohol resin, polystyrene resin grafted with polyethylene glycol or polydimethylacrylamide resin. Preferably, polystyrene resin is used. These and other resins are discussed in"Solid Phase Peptide Synthesis,"J. M. Sheppard & J. D. Young, 2nd ed., 1984, Pierce Chemical Co., Illinois.

"Protecting group"means a material which is chemically bound to a monomer unit and which may be removed upon selective exposure to an activator such as a selected chemical activator such as an acidic or basic environment, or to another selected activator such as electromagnetic radiation, including light. Protecting groups are used to prevent the reactive functional groups (hydroxy, amino, thiol, carboxy, guanidino, etc.) in amino acid monomers from undergoing side reactions during the coupling reactions.

N-protecting groups are groups which are designed to protect amino groups.

Examples of N-protecting groups include carbobenzoyl (Cbz), t- butoxycarbonyl (Boc), fluorenyimethyloxycarbonyl (FMOC), nitropiperonyl, pyrenylethoxycarbonyl, nitroveratryl (NV), nitrobenzyl, etc. These and other protecting groups are discussed in"Solid Phase Peptide Synthesis,"J. M.

Sheppard & J. D. Young, 2nd ed., 1984, Pierce Chemical Co., Illinois.

Solvents useful in the present invention include both aqueous and organic solvents and mixtures thereof. Suitable organic solvents include halogenated hydrocarbons (chloroform, methylene chloride, carbon

tetrachloride), acetonitrile, ether, toluene, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, xylene and benzene.

Substituents may include lower alkyl, alkylamino, alkylcarbonyl, alkyloxy, amino, aralkyl, aryl, dialkylamino, halogen, hetaryl, hydroxy, hydroxylalkyl, mercapto, thiol groups and the like, and as defined otherwise herein.

II. General Description of the Lactam Library The lactam library of the present invention comprises a combinatorial library formed from (i) aldehyde scaffold reactants and (ii) primary amine reactants. Individual lactam library compounds according to the present invention are represented by the following formula: where m is 1,2,3 or4; R'is hydrogen or C, 10 alkyl ; R2 is (i) selected from the group consisting of hydrogen, C1 10 alkyl, C3 12 cycloalkyl, carbonyl, C5 12 aryl, and 5-10 membered heterocyclic group containing at least one N, S or O ; (ii) an amino acid residue; or (iii) an N- protected peptide with 2-8 amino acid residues; R3 is (i) selected from the group consisting of hydrogen, C1 10 alkyl, C2 10 alkenyl, C2-10 alkynyl, C3-12 cycloalkyl, C5-12 aryl, and 5-10 membered heterocyclic group containing at least one N, S or 0 ; or (ii) together with the N which it substitutes may comprise an amino acid residue or C-protected peptide containing 2 to 8 amino acid residues;

R4 is hydrogen or C1 10 alkyl ; R5 and R6 are each independently hydrogen, Cl-lu alkyl, Calo cycloalkyl, halogen, hydroxy, oxo, thiol, sulfinyl, sulfonyl, amino, thiol, carbonyl, C5-12 aryl, 5-10 membered heterocyclic group containing at least one N, S or O ; and when m is >1, each R5 may be different and each R6 may be different; and R7 is hydrogen, C1-10 alkyl, C3-10 cycloalkyl, halogen, C5-12 aryl, or a 5-10 membered heterocyclic group containing at least one N, S or O ; wherein each of R2, R3, R4, R5, R6 and R7 may be substituted one to three times with a substituent selected from the group consisting of C110 alkyl, C3-10 cycloalkyl, halogen, hydroxy, oxo, thiol, sulfinyl, sulfonyl, C5-12 aryl, 5-10 membered heterocyclic group containing at least one N, S or O, amino, nitro, cyano, amidino, carbonyl, and wherein said substituents may themselves be further substituted with one to three further substituents.

By providing for futher substitutions on the substitutents, a diverse range of lactam library compounds may be synthesized. For example, R2 may be a substitutent such as p-methoxy-phenyl-carbonyl. According to the above definition of R2, this substituent comprises a carbonyl that is substituted with phenyl (a C5-12aryl) that is further substituted at the para position with an oxo that is further substituted with methyl (a C1 10alkyl).

Preferably, m is 1 or 2.

Preferably, R3 is selected from C1 C10 alkyl, C2-C, 0 alkenyl, C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10 cycloaikyl, C3-C10 cycloalkenyl, phenyl,-(CH2) m-O-(C1-C10 alkyl), aryl, and substituted aryl.

The final form of the library compounds may be as a product solute dissolved in a solvent (e. g., the reaction medium) or the solvent may be removed and the final product retained as a powder, paste or oil.

III. General Process For Making The Lactam Library The reaction zone for forming each library compound of the lactam libraries by the method of the invention contains a reaction medium solvent.

The reaction medium solvent is typically a solvent for the library compound desired, as well as for impurities such as, for example, (i) unreacted reagent, and/or (ii) by-product (s). The impurities in the reaction mixture are generally soluble in the solution phase since they frequently have molecular weights lower than or equal to the product and share broad solubility characteristics of the product.

One method of driving a multiplicity of reactions with diverse reactants to completion is to use a stoichiometric excess of one reagent, preferably a large stoichiometric excess. For such methods the efficient removal of excess reagent may be accomplished with a solid supported scavenger as taught herein. Thus, for the lactam libraries, removal of excess amine reagent is typically the principal concern.

Combinatorial techniques are preferably robust for highly diverse groups of reactants. The solid supported scavengers employed by the processes of this invention remove excess unreacted reactant in a manner readily adapted to the diversity of reagents employed in combinatorial chemistry, particularly parallel array synthesis.

A general scheme for the use of solid supported scavengers is as follows (wherein represents the solid support): Solid Supported Scavengers: excels C filtrer A---o A-B + B v A-B + v filter, A-B

The solid supported scavenger is filtered from the liquid reaction media and the purified product A-B retained.

The solid supported reducing reagent is employed in a similar manner, wherein it also may be removed from the reaction medium by filtering and the like, so that substantially purified product is retained.

One use for the method of the invention and the lactam libraries created thereby is for developing new drugs. Pharmaceutical drug discovery relies heavily on studies of structure-activity relationships wherein the structures of discovered"lead compounds"are the basis for new drug development. The method of the invention systematically and simultaneously generates either a diverse library of lactam molecules useful as a source of lead compounds, or a directed library of lactam molecules useful for the optimization of lead compounds. The combinatorial lactam libraries of the invention may be screened for pharmacologically active compounds using conventional screen protocols known in the art for any targeted disease state.

The process for making the library requires the preparation of the scaffold reactant and the amine reactant. The novel scaffold reactant may be custom synthesized to build a unique framework for targeted or diverse library formation.

A. Aldehyde Scaffold Reactant: According to one embodiment, the aldehyde scaffold reactant is an amino acid aldehydic derivative represented by the general formula : Preferably, the scaffold reactant is represented by the formula : where R2 and R4 are as defined above, and Y is as defined above.

Preferably, Y is methyl or ethyl. Preferably, R2 is a substituted carbonyl group.

Examples of Preparation of Aldehyde Scaffold Reactant The aldehyde scaffold reactant may be prepared according to the procedures used in the following examples where R4 is CH2Phe (i. e. starting compound (1) is phenyl alanine) and R is selected from the group consisting of alkyl, cycloalkyl, aralkylalkyl, aryl, substituted aryl, amino, alkylamino, dialkylamino, an amino acid residue and an N-protected peptide with 2-8 amino acid residues; or R2 is as defined;

Preparation of 2.

To commercially available L-phenyl alanine (199 g, 1.2 mole) in a 3 liter three-necked flask, was added methanol (2 liters) with mechanical stirring. HCI gas was bubbled into the reaction mixture until all the solid dissolved. The reaction was heated to reflux for 5 hours then let stand at room temperature overnight. Reaction was 90% complete so additional HCI gas was bubbled in and reaction was refluxed for 3 hours. After standing at room temperature for 2 days the methanol was evaporated to a white solid which was triturated with diethyl ether. The solid was collected by filtration and dried under vacuum to afford 257 grams (99% yield) of (2), a white solid with a melting point of 155.5°C to 158°C. Structure was confirmed by NMR (CDC13). Elemental Analysis-Theoretical: C 55.69,11 6.54, N 6. 49. Found: C 55.44, H 6.57, N 6.47. FD+ mass spec 179.

Preparation of 3.

To a 3 liter three-necked flask equipped with a mechanical stirrer was added (2) (257 g, 1.19 mole) followed by a solution of freshly distilled benzaldehyde (126 g, 1.19 mole) in 600 mi CH2CI2. To the stirring suspension was added a solution of triethyl amine (115 g, 1.19 mole) in 200 ml CH2CI2. An additional 500 ml CH2CI2 was added to the reaction to

facilitate stirring. After stirring overnight at room temperature the solvents were removed by evaporation and the solid residue triturated with 2 liters of diethyl ether stirring with the mechanical stirrer to create a homogenous suspension. The solid (Et3N-HCI) was removed by filtration and the filtrate evaporated to afford 282 grams (89% yield) of (3), a white solid with a melting point of 43°C to 47°C. Structure was confirmed by NMR (CDC13). Elemental analysis-Theoretical: C 76.38, H 6.41, N 5.24, O 11.97. Found: C 76.10, H 6.66, N 5.15, O 12.21. FD+ mass spec 267.

Preparation of 4.

To a 1 liter three-necked flask equipped with a mechanical stirrer and nitrogen inlet was added sodium hydride (3.2 grams, 0.08 mole) as a 60% dispersion in mineral oil. The sodium hydride was slurried with hexane (3 x 50 ml) to remove the mineral oil using a pipet to remove the hexane between slurries. A solution of (3) (20 grams, 0.075 mole) in tetrahydrofuran (60 ml) was added dropwise over an hour. Stirring was continued for 30 minutes after addition was complete. A solution of allyl bromide (9.1 grams, 0.075 mole) in tetrahydrofuran (20 ml) was added dropwise-a precipitate formed.

After 45 minutes of stirring the reaction was let stand overnight at room temperature. To the reaction was added water (200 ml) and the reaction extracted with diethyl ether (3 x 100 ml). After drying the combined extracts over sodium sulfate, filtering and evaporating the filtrate, 15.5 grams (67% yield) of (4) was obtained. Structure was confirmed by NMR (CDC13). FD+ mass spec 307.

Preparation of 5.

A solution of (4) (40.4 grams, 0.13 mole) in diethyl ether (200 ml) in a 1 liter flask was stirred vigorously with 2N HCI (200 ml) at room temperature for 3 hours. The layers were separated and the aqueous extracted with diethyl

ether (200 ml). The ether layers were discarded and sodium chloride (approximately 90 grams) was added to the saturate the aqueous solution which was then basified to pH 9.5 with sodium hydroxide (50% w/w) and then extracted with dichloromethane (3 x 200 ml). The dichloromethane layers were combined, dried (Na2SO4), filtered, and evaporated to yield 20.9 grams (73% yield) of the free base of (5) as a colorless oil. NMR (CDC13) was good for product. To a solution of this material in diethyl ether (250 ml) was bubbled HCI gas. The solution was evaporated and ethyl acetate added.

The crystals which formed were collected by filtration 17.9 grams (74% yield) of (5) as white crystals.

Preparation of 6.

(6a): R2 = CH2CH2Phe (6b): R2 = CH2 OPhe (6c): R = CH2Phe (6d): R = o-methoxy Phe (6e): R = p-methoxy Phe (6f): R = cyclohexane (6g): R = CH2C (CH3) 3 (6h): R2 = (CH2) 3 (CH3) Eight reactions were run simultaneously. To a solution of (5) (1.7g, 6.7 mmole) in tetrahydrofuran (50 ml) in each of eight 250 ml Erlenmeyer flasks was added triethylamine (1.9 g, 13.4 mmole) at which time a precipitate formed. The acid chlorides were added (7.4 mmole, (6a): 1.25 g hydrocinnamoyl chloride ; (6b): 1.26 g phenoxyacetyl chloride ; (6c): 1.14 g phenyl acetyl chloride ; (6d): 1.26 g o-anisoyl chloride ; (6e): 1.26 g panisoyl chloride ; (6f): 1.08 g cyclohexane carbonyl chloride ; (6g): 1.0 g t-butyl acetyl

chloride ; (6h): 0.89 g valeryl chloride). Reactions were capped and shaken at room temperature overnight. To each reaction was added ethyl acetate (50 ml). Each was extracted with 1 N HCI (2 x 50 ml), saturated NaHCO3 (50 ml), and saturated NaCI (50 ml). Ethyl acetate layers were dried over Na2SO4, filtered, and evaporated to yield (6a) (2.31 g, 98%), (6b) (2.05 g, 87%), (6c) (2.15 g, 95%), (6d) (2.19 g, 92%), (6e) (1.98 g, 84%), (6f) (2.07 g, 94%), (6g) (1.94 g, 91%), (6h) (1.86 g, 92%). Products were each one spot by TLC, had good NMRs (CDC13), and the correct molecular ion by FD+ mass spec.

Preparation of 7.

(7a): R2 = CH2CH2Phe (7b): R2 = CH2OPhe (7c): R2 = CH2Phe (7d): R 2= o-methoxy Phe (7e): R2 = p-methoxy Phe (7f): R = cyclohexane (7g): R2 = CH2C (CH3) 3 (7h): R2 = (CH2) 3 (CH3) Products (6a) through (6h) (5.6 mmole to 6.9 mmole) above were dissolved in 20 ml CH2CI2 in 100 ml round bottomed flasks. They were sequentially cooled to-78°C under a stream of nitrogen and treated with a stream of ozone until the reaction solution turned blue. Methyl sulfide (5 ml, 4.23 g, 68 mmole) was added to each reaction after which the reactions were allowed to stand at room temperature overnight. The reactions were each extracted with saturated NaHC03 (2 x 25 ml), and saturated NaCI (25 ml), then dried over Na2SO4, filtered and evaporated to yield (7a) (2.27 g, 97%), (7b) (1.76 g, 85%), (7c) (1.98 g, 91%), (7d) (1.93 g, 87%), (7e) (1.79 g, 90%),

(7f) (2.08 g, 99%), (7g) (1.81 g, 93%), (7h) (1.67 g, 90%) as oils. Products are good by NMR (CDC13) and FD+ mass spec.

Preparation of intermediates with R2 groups that are not stable to ozonolysis To a solution of (7i) (3.5g, 0.014 mole) in CH2CI2 was bubbled in HCI gas for 5 minutes with stirring. The solution was evaporated to give (11) as a foam with quantitative mass recovery.

To a solution of 2-quinoxaloyl chloride (0.93 g, 0.0048 mole) in THF (30 ml) in a round bottomed flask was added (11) (0.8 g., 0.0044 mole) with stirring. Triethylamine (1.22 ml, 0.0088 mole) was added dropwise to the mixture at which time (11) dissolved and a new solid formed. After stirring for one hour the reaction was extracted with CH2CI2 (2 x 30 ml). The extracts were washed with saturated NaHCO3 (2 x 30 ml), dried over Na2SO4, filtered

and evaporated to yield, after silica gel flash chromatography (EtOAc), (7k) in 55% yield. Product was good by NMR (CDC13) and ion spray mass spec.

B. Primary Amine Reactant: Each amine reactant used in the process of making a lactam combinatorial library is a primary amine. Suitable primary amines are of the formula: H2N-R3 where R3 is (i) selected from the group consisting of hydrogen, C110 alkyl, C2 10 alkenyl, C210 alkynyl, C312 cycloalkyl, C512 aryl, and 5-10 membered heterocyclic group containing at least one N, S or O ; or (ii) together with the N which it substitutes may comprise an amino acid residue or C-protected peptide containing 2 to 8 amino acid residues; and wherein R3 may be substituted one to three times with a substituent selected from the group consisting of C110 alkyl, C310 cycloalkyl, halogen, hydroxy, oxo, thiol, sulfinyl, sulfonyl, C5 12 aryl, 5-10 membered heterocyclic group containing at least one N, S or O, amino, nitro, cyano, amidino, carbonyl, and wherein said substituents may themselves be further substituted with one to three further substituents.

Preferably the amine reactant is selected from the group consisting of primary aliphatic, aromatic, and heterocyclic amines having a molecular weight of from 50 to 600.

Examples of amine reactants suitable for use in the process of the invention include, but are not intended to be limited to: cyclopropylamine cyclobutylamine (-)-cis-myrtanylamine cyclopentylamine cyclohexylamine 2,3-dimethylcyclohexylamine (aminomethyl) cyclohexane 1,2,3,4-tetrahydro-1-naphthylamine 1-tyrosine methyl ester <BR> <BR> N- (2-aminoethyl) pyrrolidine<BR> furfurylamine 1-aminoindan 1-naphthalenemethylamine (1s, 2s)- (+)-2-amino-1-phenyl-1, 3- propanediol t-isoteucino ! d, 1-4-chlorophenylalaninol I-methioninol tetrahydrofurfurylamine tryptamine 6-methoxytryptamine N- (2-aminoethyl) morpholine <BR> <BR> 2- (2-aminoethylamino)-5-nitropyridine 2- (2-aminoethyl) pyridine 4- (aminomethyl) pyridine 4-amino-1-benzylpiperidine <BR> <BR> 1- (3-aminopropyl)-2-pipecoline<BR> benzhydrylamine 1, 2-diphenylethylamine (-)-norephedrine isopropylamine d, 1-2-amino-1-propanol 1,3-dimethylbutylamine 2-methylcyclohexylamine 4-methylcyclohexylamine 3-aminomethyl-3, 5,5- trimethylcyclohexanol cyclooctylamine<BR> 2-(2-aminoethyl)-1-methylpyrrolidine<BR> N- (3x-aminopropyl)-2-pyrrolidinone cyclododecylamine d, 1-1-(1-naphthyl) ethylamine cycloheptylamine d, 1-2-amino-3-methyl-1-butanol I-phenylalaninol d- (-)-leucinol histamine d, I-alpha-methyltryptamine 5-methoxytryptamine piperonylamine<BR> N- (3-aminopropyl) morpholine 2- (aminomethyl) pyridine 3-(aminomethyl) pyridine ethyl 4-amino-1-piperidinecarboxylate 1- (2-aminoethyl) piperidine 1,2-diamino-2-methylpropane d- (-)-alpha-phenyglycinol d, 1-1-phenylethylamine 1,2-dimethylpropylamine 2-methoxyisopropylamine ethyl-3-aminobutyrate 3-amino-1-phenylbutane 2-amino-5-diethylaminopentane sec-butylamine 3-aminopentane 3-aminoheptane 2-aminooctane aniline 2-chlorobenzylamine 2-methoxybenzylamine 2-methylbenzylamine 3,4-dichlorobenzylamine 3- (trifluoromethyl) benzylamine 4-fluorobenzylamine 4-methoxybenzylamine 2,2,2-trifluoroethylamine 1-amino-2-propanol 2,2-diphenylethylamine <BR> <BR> isobutylamine<BR> 2-ethylhexylamine n-undecylamine tridecylamine hexadecylamine ethylamin 2-methoxyethylamine ethanolamine <BR> <BR> 2- (2-chlorophenyl) ethylamine 3-methoxyphenethylamine <BR> <BR> 4-bromophenethylamine<BR> 2- (4-methoxyphenyl) ethylamine<BR> 2- (4-aminophenyl) ethylamine 1,5-dimethylhexylamine (+/-)-2-amino-1-butanol 2-aminopentane 2-aminoheptane benzylamine 2-fluorobenzylamine 2,4-dichlorobenzylamine <BR> <BR> 2-ethoxybenzylamine<BR> 3-fluorobenzylamine 3,4-dimethoxybenzylamine 3-methylbenzylamine 4-chlorobenzylamine <BR> <BR> 4-methylbenzylamine<BR> 2-amino-1-phenylethanol 3-amino-1,2-propanediol <BR> <BR> beta-methylphenethylamine<BR> 2-methylbutylamine<BR> n-decylamine<BR> dodecylamine 1-tetradecylamine <BR> <BR> octadecylamine<BR> 2- (2-aminoethylamino) ethanol 2- (2-aminoethoxy) ethanol phenethylamine 2- (2-methoxyphenyl) ethylamine <BR> <BR> 2- (3, 4-dimethoxyphenyl) ethylamine<BR> 2- (4-chlorophenyl) ethylamine tyramine 2- (p-tolyl) ethylamine taurine allylamine 3,3-diphenylpropylamine propylamine 3-diethylaminopropylamine 3-isopropoxypropylamine 3-amino-1-propanol 4-amino-1-butanol n-amylamine hexylamin n-heptylamine n-nonylamine d, 1-2-amino-1-hexanol 3,5-bis (trifluoromethyl) benzylamine 2,5-difluorobenzylamine 3,4-difluorobenzylamine 2- (trifluoromethyl) benzylamine <BR> <BR> n- (4-aminobutyl)-n-ethylisoluminol<BR> 2- (1-cyclohexenyl) ethylamine 3,4,5-trimethoxybenzylamine aminomethylcyclopropane 4- (2, 4-di-tert- amylphenoxy) butylamine 4-fluoro-alpha-methylbenzylamine N, N-di-n-butylethylenediamine N, N, 2,2-tetramethyl-1,3- propanediamine <BR> <BR> 2- (3-chlorophenyl) ethylamine<BR> 2- (2-thienyl) ethylamine 3,5-dimethoxybenzylamine propargylamine 3,3-dimethylbutylamine isoamylamine 3-dimethylaminopropylamine 3- (di-n-butylamino) propylamine 3-ethoxypropylamine 3-phenylpropylamine<BR> 4-phenylbutylamine 5-amino-l-pentanol 6-amino-1-hexanol n-octylamine d, 1-2-amino-1-pentanol 1- (3-aminopropyl) imidazole 2,4-difluorobenzylamine 2,6-difluorobenzylamine 4-(trifluoromethyl) benzylamine 4- (2-aminoethyl) benzenesulfonamide n-butylamine 3-methoxypropylamine 3-butoxypropylamine<BR> pentadecylamine 3-chlorobenzylamine (r)- (+)-bornylamine<BR> (r)-(-)-1-cyclohexylethylamine 1-phenylalanine beta-naphthyl-amide 2-amino-1,3-propanediol 2, 3-dimethoxybenzylamine 2,4-dichlorophenethylamine 2,5-dimethoxyphenethylamine 4- (trifluoromethoxy) benzylamine 1-leucine-4-nitroanilide <BR> <BR> (s)-(-)-1-(1-naphthyl) ethylamine<BR> d-valinol<BR> 1- (+)-alpha-phenylglycinol 1 (-)-alpha-methylbenzylamine (s)- (+)-2-amino-1-propanol<BR> (r)- (-)-sec-butylamine<BR> (s)- (+)-2-amino-1-butanol<BR> (r)-(-)-1-amino-2-propanol<BR> (s)- (-)-2-methylbutylamine<BR> oleylamine 1-adamantanemethylamine (1 r, 2s)- (-)-2-amino-1, 2- diphenylethanol <BR> <BR> 2- (2- (aminomethyl) phenylthio) benzyl alcohol 2-aminobenzylamine 4-aminobenzylamine (+/-)-2, 5-dihydro-2,5- dimethoxyfurfurylamine 4-fluorophenethylamine (-)-3,4-dihydroxynorephedrine (s)-(-)-1-(p-tolyl) ethylamine (+/-)-exo-2-aminonorbornane 3-amino-1-propanol vinyl ether 4- (hexadecyfamino) benzylamine 3-fluoro-5- (trifluoromethyl) benzylamine 1-leucinol <BR> <BR> (r)-(+)-1-(1-naphthyl) ethylamine 1-valinol d-phenylalaninol d- (+)-alpha-methylbenzylamine<BR> (1 s, 2r)- (+)-phenyl-propanolamine d-alaninol (s)- (+)-sec-butylamine (r)-(-)-2-amino-1-butanol<BR> (s)- (+)-1-amino-2-propanol<BR> (s)- (+)-1-cyclohexylethylamine<BR> trimethoxysilylpropyl<BR> diethylenetriamine (1 s, 2r)- (+)-2-amino-1, 2- <BR> <BR> diphenylethanol<BR> s-benzyl-1-cysteinol 3-fluorophenethylamine 2-fluorophenethylamine d-glucamine<BR> (s)- (+)-tetrahydrofurfurylamine (1 s, 2s)- (+)-thiomicamine (r)- (+)-1- (p-tolyl) ethylamine<BR> (s)- (-)-2-amino-1, 1-diphenyl-1- propanol (s)- (+)-2- (aminomethyl) pyrrolidine geranylamine (1 r, 2r, 3r, 5s)- (-)-

(1 s, 2s, 3s, 5r)- (+)- isopinocampheylamine <BR> <BR> (s)-tert-leucinol<BR> dehydroabietylamine (1 s, 2r)- (-)-cis-1-amino-2-indanol isopinocampheylamine<BR> nl-isopropyidiethylenetriamine<BR> (r)- (-)-tetrahydrofurfurylamine 2-bromo-4,5- dimethoxyphenethylamine (1 r, 2s)- (+)-cis-1-amino-2-indanol and the like.

Additional primary amines suitable for the process of the invention include, but are not intended to be limited to, those represented by the following formulae :

, and the like.

Typically, from about 8 to about 800 diverse amine reactants are employed to synthesize the lactam library of the invention.

C. Solid Supported Reducing Agent: The solid supported reducing agent used in the process according to the preferred embodiment of the present invention is represented by the formula : where X can be borohydride, cyanoborohydride, triacetoxyborohydride, or the like, preferably borohydride, and where the solid support is insoluble in the liquid reaction medium used in the solution phase lactam library making process.

The preferred reducing agent is a resin-bound borohydride. The resin may be any non-interfering anion exchange resin, such as Amberlyst A-26 or Amberlite IRA-400. Preferably, Amberlite IRA-400 is used. The resin-bound borohydride may be prepared according to published procedures (see, Sande, A. R., Jagadale, M. H., Mane, R. B. and Salunkhe, M. M., Tetrahedron Letters, 25 (32): 3501-04 (1984), which is incorporated herein by reference).

D. Solid Supported Scavenger: The solid supported scavenger is used in the lactam library forming process of the invention. The solid supported scavenger is represented by the formula : where the solid support is insoluble in the liquid reaction medium used in the solution phase lactam library making process. Examples of organic solid supports include, but are not intended to be limited to, polymers such as polystyrene/divinylbenzene copolymer, polyacrylamide, cellulose and chloromethylated 2% crosslinked polystyrene. Examples of inorganic solid supports include, but are not intended to be limited to, silica gel, alumina, and controlled pore glass. The aldehyde substituent may be any lower molecular weight aldehyde, such as benzaldehyde, and the like.

The aldehyde substituent on the solid supported scavenger readily reacts with excess amine reagent in the lactam library forming process of the invention and binds the excess reagents to the solid support to thereby permit their removal as a solid phase by methods known in the art, such as filtering, centrifugation, decantation, and the like. The effective available aldehyde content of the solid supported scavenger may be readily determined by conventional chemical analysis techniques.

Alternatives to solid supported scavengers include the use of cation exchange resins. Strong cation exchange resins are commercially available.

One resin sold under the trade name SCX resin by Varian Sample Preparation Products, Inc., located in Harbor City, California, USA, may be used. Preferably, this SCX resin would be packed in a chromatography column. The reaction medium containing the excess amine reactant would be run through the column to absorb any unreacted amines.

Ideally, an array of columns could be arranged to match up with a 96 well microtiter plate so that the reaction media in one wellplate could simultaneously be run through 96 columns into another wellplate positioned below the columns.

IV. Process For Preparing Lactam Library: The solution phase combinatorial process of this invention provides lactam library compounds represented by the formula : where m is 1,2,3 or 4; R'is hydrogen or C, 10alkyl ; R2 is (i) selected from the group consisting of hydrogen, Cl-lu alkyl, C3- 12 cycloalkyl, carbonyl, C5 12 aryl, and 5-10 membered heterocyclic group containing at least one N, S or O ; (ii) an amino acid residue; or (iii) an N- protected peptide with 2-8 amino acid residues;

R3 is (i) selected from the group consisting of hydrogen, C110 alkyl, C2 10 alkenyl, C210 alkynyl, C312 cycloalkyl, C512 aryl, and 5-10 membered heterocyclic group containing at least one N, S or O ; or (ii) together with the N which it substitutes may comprise an amino acid residue or C-protected peptide containing 2 to 8 amino acid residues; R4 is hydrogen or Cl-lu alkyl ; R5 and R6 are each independently hydrogen, C110 alkyl, C310 cycloalkyl, halogen, hydroxy, oxo, thiol, sulfinyl, sulfonyl, amino, thiol, carbonyl, C512 aryl, 5-10 membered heterocyclic group containing at least one N, S or O ; and when m is >1, each R5 may be different and each R6 may be different; and R 7is hydrogen, C110 alkyl, C310 cycloalkyl, halogen, C512 aryl, or a 5-10 membered heterocyclic group containing at least one N, S or O ; wherein each of R2, R3, R4, R5, R6 and R7 may be substituted one to three times with a substituent selected from the group consisting of C110 alkyl, C310 cycloalkyl, halogen, hydroxy, oxo, thiol, sulfinyl, sulfonyl, C512 aryl, 5-10 membered heterocyclic group containing at least one N, S or O, amino, nitro, cyano, amidino, carbonyl, and wherein said substituents may themselves be further substituted with one to three further substituents.

The process, according to a preferred embodiment, comprises the following steps: a) adding to each reaction zone containing a liquid reaction medium (n) equivalents of a scaffold reactant represented by the formula :

where R1 through R7 and m are as defined above; and Y is a leaving group such as lower alkyl or selected from other non-interfering substituents. b) adding to each reaction zone of step (a) at least about 1.1 (n) equivalents of a solvent soluble amine reactant represented by the formula, H2N-R3 where R3 is selected from a non-interfering substituent as defined above, and maintaining the reaction zone at a temperature and for a time sufficient to permit reaction of said amine and scaffold reactants; c) adding to the reaction zone of step (b) a solid supported reducing reagent represented by the formula : where X can be borohydride, cyanoborohydride, triacetoxyborohydride, or the like, preferably borohydride; and wherein said reducing reagent is added in an amount at least equal to four equivalents of the scaffold reactant; and maintaining the reaction zone at a temperature and for a time sufficient to permit reaction of intermediates to form lactam products; d) removing the unreacted excess amine added in step (b), wherein the amine is preferably removed by adding to the reaction zone of step (b) a solid supported aldehyde functional scavenger represented by the formula:

wherein said scavenger is added in an amount at least equal to about the excess equivalents of unreacted amine reactant used in step (b), and maintaining the reaction zone at a temperature and for a time sufficient to permit reaction of said excess amine reactant and said scavenger, or wherein the amine is removed by applying the reaction solution to an ion exchange chromatography to remove the excess amines; e) separating the solid supported reducing reagent and scavenger, if used, from the reaction solution of step (d); f) heating the solution to effect cyclization to form a lactam ; and g) recovering in solution each substantially purified lactam library compound.

Alternatively the excess amine may be removed by applying the solution of product obtained in f) to an ion exchange chromatography column, provided the lactam product is not cationic at acidic pH.

It is understood that the phrase"adding to each reaction zone"in steps (a) and (b) means that different aldehyde scaffold reactants and different amines may be optionally added to each reaction zone in the library forming process. In one embodiment of the process of the invention, each combination of scaffold and amine reactants added to each library reaction zone (e. g., wells of a wellplate, vial, or other solid substrate) is different.

Thus, the same aldehyde may be added to each row of a wellplate apparatus (as shown in Fig. 1) and the same amine may be added to the same column of a wellplate apparatus to give a different combination of reactants in each well (i. e., reaction zone) that will expectantly yield a different library compound. Alternatively, where it is desirable to have replicate samples, the

same combination of aldehyde and amine reactants may be added to different reaction zones.

A. Details of the Lactam Library Process-Step (a): Reaction Medium The reaction medium may be any liquid which has the following characteristics: (1) The amine and scaffold reactants are capable of forming a reaction product which is substantially soluble in the reaction medium, and (2) The solid supported reducing reagent and scavenger, both in unreacted and reacted form, are substantially insoluble in the reaction medium.

Typical reaction media useful in the processes of the invention include, but are not intended to be limited to, methanol, toluene, chloroform, methylene chloride, dichloroethane, tetrahydrofuran, and the like.

The Reaction Zone The process of the invention may be carried out in any vessel capable of holding the liquid reaction medium. Preferably the process of the invention is carried out in containers adaptable to parallel array syntheses, such as 96 well microtiter plates, and the like. Most preferably, the lactam library is formed in an array of glass vials with teflon lined caps. Each vial may be filled by multiple delivery apparatus, automated or robotic apparatus, any of which may be either manually or computer controlled.

The diverse lactam library of this invention may take the form of a plurality of arrays, each array having glass vials containing a separate reaction product (library compound). In such cases, the library compounds are conveniently identified by their array number and"x"column and"y"row coordinates.

Selection of scaffold reactants A preferred technique for practicing the process of the invention is parallel array synthesis. According to parallel array synthesis individual reaction products are prepared in each of multiple reaction zones. The amount of scaffold reactant introduced into each reaction zone will depend on the desired amount of each library compound that is needed for conducting biological assays, archival storage and other related needs. Typically, the desired amount of individual reaction product is from 1 microgram to 50 milligrams.

The amount of scaffold reactant in each reaction zone is represented by the symbol" (n)", where (n) represents the equivalents of scaffold reactant.

B. Details of the Lactam Library Process-Step (b): In the process for making a diverse lactam library, as described herein, the amine reactant is the reactant used in excess. The method of the present invention contemplates solution phase reactions where a stoichiometric excess of amine reactant is used. The amount of amine reactant used to ensure an excess is defined as at least about 1.1 (n) and preferably a larger excess in the range of from about 1.25 (n) to about 5 (n), where the variable (n) is as previously defined. The 1.1 multiplier is used to ensure at least about a 10% stoichiometric excess of the amine reactant to drive the reaction

to completion, thereby reacting substantially all of the aldehyde scaffold reactant from each reaction zone used to create the lactam library. Thus, for example, if 1.25 (n)-a 25% excess-of the amine reactant is desired, then 28 mg of aldehyde scaffold reactant (7a) would be used in step (a) and 10.7 mg. of benzylamine would be used in step (b) of the process.

The reaction zone is maintained at a temperature and for a time sufficient to permit reaction of the scaffold and amine reactants, that is, to complete consumption of the scaffold reactant and form an amount of lactam library compound necessary to conduct biological assays to determine the efficacy of the prepared library compounds.

The time, temperature, and pressure of the combinatorial reaction zones used for the creation of library compounds are not critical aspects of the invention. Reaction times for a single step of the reaction are generally from about 0.1 seconds to about 24 hours, with times from between about three hours and about twelve hours being preferred. The temperature of the reaction may be any temperature between the freezing point and the boiling point of the liquid reaction medium, but is generally from about-10°C to about +60°C, with from about 10°C to about 60°C being preferred and ambient temperatures (about 20°C-30°C) being most preferred.

Endpoint determination The completion of the reaction between the scaffold and amine reactant may be determined by a number of conventional techniques. For example, one method is to use thin layer chromatography to determine if the scaffold reactant is substantially removed from the reaction zones.

Sequence of Operation The addition of the scaffold and amine reactants to the reaction zone may take place in any order. For example, the amine reactant may be initially added to the reaction zone followed by addition of the scaffold reactant, or vice versa. Alternatively, the amine and scaffold reactants may be simultaneously added to each reaction zone.

When the amine reactant is an HCI salt, it is preferred to add a solid supported base, such as, for example, polyvinyl pyridine, or piperidine polymer bound through its amine.

C. Details of the Lactam Library Process-Step (c): The solid supported reducing reagent addition step requires adding to the reaction mixture of step (b) a solid supported hydride functional scavenger represented by the formula : where X can be borohydride, cyanoborohydride, triacetoxyborohydride, or the like, preferably borohydride; wherein the reducing reagent is added in an amount at least equal to about two equivalents of the scaffold reactant. The amount of reducing reagent added should preferably be from about four to about 16 equivalents.

The reaction zone is maintained at a temperature for a time sufficient to permit reduction to form. Although the reaction is allowed to proceed overnight, it typically may require less time. The selection of reaction conditions that may be used is the same as set out in the preceding section.

D. Details of the Lactam Library Process-Step (d):

The solid supported scavenger addition step may involve, but does not require, adding to the reaction mixture of step (c) a solid supported aldehyde functional scavenger represented by the formula : where the amount of scavenger added to the reaction product of step (c) is based on the scavenger's available aldehyde functionality. The scavenger may also be added at the same time as the addition of the reducing agent in step (c), provided that they are added after the scaffold and amine reactants have had sufficient time to complete the reaction. The scavenger is added in at least about an amount equal to the theoretical excess equivalents of unreacted amine reactant (i. e., at least 0.1 equivalents used in step (b)). Preferably the solid supported scavenger is used in an amount that is from about 1.25 to about 5 times the theoretical excess equivalents of unreacted amine reactant.

Alternatively, other methods for removing the excess amine may be used. For example, ion exchange chromatography may be used as described above.

The reaction zone is maintained at a temperature for a time sufficient to permit reaction of said excess amine reactant and said scavenger.

Although the reaction is allowed to proceed overnight, it typically may require less time. The selection of reaction conditions that may be used is the same as set out in the preceding section (see, Details of the Lactam Library Process-Step (b)).

Alternatively, other methods for removing the excess amine may be used. For example, ion exchange chromatography may be used as described above.

E. Details of the Lactam Library Process-Steps (e) and (F): Purification of the library compounds is accomplished by separating the resin bound reducing reagent and the reacted and unreacted solid supported scavenger, if used, from the reaction medium of step (c) and then heating the filtrate to produce a solution of each substantially purified lactam library compound.

The separation of the solid supported reducing reagent and scavenger from the library compound dissolved in the solvent phase of the reaction may be done by any conventional chemical or physical method. Such physical methods, which are applicable to all members of a diverse library, include, but are not limited to: (i) filtration, (ii) centrifugation, (iii) decantation, (iv) washing and the like. Filtration is a particularly preferred form of purification.

It is practiced by transporting each solution of library compound through a filter medium which retains the scavenger and transfers the solution phase into a separate vessel.

The heating is performed in glass vials with teflon lined caps, typically for a period of about 12 hours at about 60°C. The heat helps promote the cyclization of the reaction product into a lactam compound.

The purification step of the process may optionally be supplemented by a solvent removal step in which the solute library compound is removed from its solvent by conventional processes known in the art, such as solvent evaporation, distillation, salting out, solvent extraction, and etc.

V. Alternate Process for Making Lactam Library:

The lactam library and/or wellplate apparatus containing the lactam library of the present invention may be prepared according to an alternative parallel synthesis process. In contrast to the process of the present invention, the alternate process performs the synthesis of the lactam library with the scaffold reactant bound to a solid-substrate and the borohydride reducing agent unbound. The details of this alternate process is described and claimed in the co-pending application titled"Peptidomimetic Template- Based Combinatorial Libraries"by Scott and Cwi filed on the same date as this present application. This co-pending application is incorporated herein by reference.

In applying the alternate process to prepare the lactam library of the present invention, the aldehyde scaffold reactant described at III. A., supra, is bound to a resin or polymer which takes the place of the leaving group"Y" shown in the general formula.

The alternate process may be carried out in any vessel which can hold the resin bound scaffold reactant and which allows for introduction of unbound primary amine reactant in solution and removal of the lactam product in solution.

The library can be assembled by running each reaction sequentially, but is preferably run in parallel. For small scale synthesis of multiple lactams, the process of the invention is preferably carried out in containers adaptable to parallel array syntheses. With parallel array synthesis, individual reaction products are prepared in physically separated multiple reaction zones. The reaction zone contains at least one type of resin bound scaffold reactant and at least one type of unbound primary amine reactant. Suitable reaction zones include solid supports such as wellplates, silicone, or agar. Resin bound

scaffold reactant is either directly attached, absorbed or incorporated into the surface of the solid support.

To illustrate this alternative process, a resin bound scaffold reactant has the structure: 0 o O-polymer 0 O This scaffold reactant can be synthesized in a variety of ways, depending on the nature and reactivity of R4.

When R4 is a side chain of a natural amino acid, the scaffold reactant can be synthesized as follows. Polymer bound amino acids are commercially available or can be attached to a solid support using conventional methods.

The polymer bound amino acid is N-protected, for example, as an imine with a benzaldehyde derivative. This intermediate is then reacted with a haloallyl, such as bromoallyl. Thereafter, the activating group is removed, in the case of an imine with acid. The N-terminus is then substituted with an appropriate R3 group. Finally, this resin bound allylic peptide derivative can be directly ozonolyzed to the scaffold reactant, for example with Os in dimethyl sulfoxide.

When R4 is not a side chain of a naturally occurring amino acid, the scaffold reactant can be synthesized as follows. Briefly, the methodology involves activation, deprotonation/alkylation, deprotonation/alkylation and hydrolysis. A resin bound glycine is activated as a benzophenone imine ester. Thereafter, the oc-carbon of the glycine is deprotonated with base, such as the organic-soluble, non-ionic iminophosphorane base, 2- [ (1, 1-

dimethylethyl) imino]-N, N-diethyl-2, 2,3,4,5,6-hexahydro-1, 3-dimethyl-1,3,2- diazaphosphorin-2 (1 H)-amine (BEMP) and alkylated with an allyl halide. This reaction is preferably conducted in an organic solvent such as 1-methyl-2- pyrrolidinone.

The second substitutent can then be added in either of two ways.

First, the second substituent can be added directly by deprotonating the oc- carbon with a base, such as potassium hexamethyldisilazide (KHMDS) (see Griffith, Tetrahedron Letters, 38 (51): 8821 (1997)). Alternatively, the activating group can be exchanged by replacing the imine activating group with an aromatic aldehyde derivative, such as 4,5-dichlorobenzaldehyde.

Thereafter, a base such as BEMP can be used to deprotonate the oc-carbon, which can then be reacted with an alkyl halide.

Alternatively, a scaffold reactant can be formed from a unbound amino acid derivative containing an allyl group by binding the amino acid to a solid support and ozonolzying, as shown below. I 1 0 zum m R4 (R. m H RR2N RR QPdY'' aPdYrra RlR'2N O O 2 One of the key steps in the above described syntheses is the ozonolysis to form the resin bound scaffold reactant.

Lactam library compounds can be synthesized from these resin bound scaffold reactants by reacting and coupling with unbound primary amine reactants. The aldehydic group of the resin bound scaffold reactant is

coupled to the nitrogen in the amine reactant. An excess amount of resin bound scaffold reactant is used to ensure complete reaction of the primary amine reactant and avoid the need of further purification. This intermediate is reduced, for example, by using unbound sodium borohydride, which then undergoes intramolecular cyclization to a lactam structure with concomitant cleavage from the polymer resin. The excess resin bound scaffold reactant and cleaved resin are removed by conventional means, such as filtering and the like.

VI. The Wellplate Apparatus of the invention : A wellplate apparatus inoculated with the novel diverse lactam compounds of the invention is itself a new construct or apparatus which has particular utility in an assay kit used to discover lead compounds. The wellplate apparatus having multiple zones where each zone independently contains a lactam library compound prepared according to the process of the present invention may be suitable as a replacement element in an automated assay machine.

A suitable system of operation and related apparatus are made as follows : 1. Reaction zones are made by drilling 96 holes in the bottom of 96 deepwell titer plates and putting a porous frit in the bottom of each well.

2. The plate is put in a clamp assembly to seal the bottom of the wells.

3. Synthesis is begun by adding reagents to their assigned plate coordinates (reaction zone).

4. The plate is capped then tumbled to mix the reagents.

5. Solid supported scavenger and solid supported borohydride is added to each reaction zone after completion of the reaction is shown by thin layer chromatography.

6. After sufficient reaction time the plate is removed from the clamp and the resin washed.

7. The solution containing product is filtered and the solution collected by transfer into another 96 wellplate.

8. The reaction products (library compounds) are analyzed by thin layer chromatography.

Referring now to the figures, FIG. 1 illustrates the top surface of a wellplate apparatus of the invention. The weliplate (3) is a plastic plate with 96 wells (depressions) capable of holding liquids. When used in the parallel array synthesis individual reaction products are prepared in each well and are labeled by the wellplate coordinates. The shaded circles in the Figure represent wells filled with urea or thiourea library compounds prepared by the solution phase combinational process of the invention. The compound at location (1), for example, is identified by the alphanumeric coordinate,"A6." FIG. 2 illustrates a side view of a wellplate apparatus used in the Assay Kit of the invention. The wellplate (5) contains wells (7) with a filter (9)

and liquid reaction medium containing scavenger (11). The wells have an outlet at bottom which is sealed by gasket (13) held in place by top cover (15) and bottom cover (17) maintained in position by clamp (19).

VII. The Assay Kit of the Invention : This invention is an assay kit for identification of pharmaceutical lead compounds, comprising biological assay materials and wellplatte apparatus.

The assay kit comprises as essential part, (i) wellplate apparatus (containing in its wells the novel lactam library compounds of the invention), and (ii) biological assay materials.

The wellplate apparatus in the kit may comprise a set of wellplate apparatus such as illustrated in Fig. 1. The lactam library compounds contained in each wellplate are prepared by the processes taught herein.

Preferably the wellplate apparatus has the form of a standard 96 well microtiter plate.

Typically, the wellplate apparatus is in the form of a rigid or semi-rigid plate, said plate having a common surface containing openings of a plurality of vessels arranged in rows and columns. A standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions on its surface. A wellplate apparatus may optionally have other elements of structure such as a top or cover (e. g., plastic or foil), a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds.

The assay kit also contains biological assay materials. These biological assay materials are generally in vitro tests 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 ; Add, Incubate, & Read assays such as: Scintillation Proximity Assays (SPA), Fluorescence Polarization Assays, Fluorescence Correlation Spectroscopy, Colorimetric Biosensors, Ca2+-EGTA Dyes for Cell-based assays, Reporter Gene Constructs for cell-based assays Cellular reporter assays utilizing, for example, reporters such as luciferase, green fluorescent protein, ß-lactamase, and the like ; Electrical cell impedance sensor assays, and the like.

EXAMPLE The following example depicts a preferred method of making selected lactam library compounds according to the process of the present invention.

The scheme shown below depicts the preferred method of making lactam libraries of the invention using a solid supported scavenger derived by DMSO/NaHCO3 oxidation of chloromethylated 2% crosslinked polystyrene as described in published procedures (see, Frechet, J. M. and Schuerch, C., Journal of the American Chemical Society, 93: 2, p. 492-496,1971, incorporated herein by reference).

Reactions were generally run 96 at a time in 4 mi screw cap vials.

Reactions were arranged in an 8 x 12 grid with the rows labeled A through H and the columns numbered 1 through 12. In these examples one aldehyde scaffold reactant was placed in each of the 12 vials in a row. One amine reactant was placed in each of the 8 vials in a column. Thus each of 8 aldehyde scaffold reactants was reacted with each of 12 amines to give 96 unique products.

Preparation of 10 Aldehyde (7a): R2 he CH2CH2Phe (7b): R2 = CH2OPhe (7c): R2 = CH2Phe (7d): R2 = o-methoxy Phe (7e): R = p-methoxy Phe (7f): R = cyclohexane (7g) : R2 = CH2C (CH3) (7h): Ruz = (CH2) 3 (CH3) Amines 1) tryptamine 2) 4-aminobenzyl piperidine 3) veratrylamine 4) isoamyl amine 5) octylamin 6) benzylamine 7) tolylethylamine 8) aminoindan 9) 3-amino-N-ethyl piperidine 10) 5-amino-1-pentanol 11) 2- (2-aminoethyl) pyridine 12) 2,2 diphenylethyl amine To aldehydes (7a) through (7h) (0.55 mmole) each in glass vials was added methanol (2.5 ml). The solutions were divided into 12 reaction vials (190 EH each), additional methanol (1.2 ml) added to the stock solution and this resulting solution divided into the 12 reaction vials (90 SEl each) for 0.046 mmole reagent per reaction. Stock solutions of the amines were made in methanol (100 mg/ml) and the appropriate volume to equal 0.069 mmole was

added into each of eight reaction vials in a column. The reaction vials were sealed and the solutions were shaken at room temperature for three hours allowing intermediate (8) to form. Into each vessel was then added dichloromethane (0.3 ml) followed by Amberlite IRA 400 borohydride resin (0.09 mmole, 36 mg) and aldehyde resin (0.046 mmole, 60 mg). After sealing the vials with Teflon backed caps the mixtures were shaken at room temperature overnight then filtered into tared vials through pipets fitted with Kimwipe plugs to yield (9) in solution. The vials were capped and heated at 60°C overnight on a thermostatically controlled hot plate without agitation.

The caps were removed and the solvent allowed to evaporate to yield products (10). Ion spray mass specs obtained on 25% of the products showed product in each of those cases. In addition, five of those spectra show contamination with intermediate (9). These products, d8, h8, a9, e12, and h12 represented the bulkier more sterically crowded amines. Yields ranged from 50% to 100%. Purity was about 70%.

Having now fully described the invention, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.