WO1998008813A1 - Combinatorial process for preparing substituted pyrrolidine libraries - Google Patents

Abstract

This invention relates to a novel solid phase process for the preparation of pyrrolidine combinatorial libraries. These libraries have use for drug discovery and are used to form wellplate components of novel assay kits.

Description

COMBINATORIAL PROCESS FOR PREPARING SUBSTITUTED PYRROLIDINE

LIBRARIES

This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/024,559, filed August 26, 1996.

Field of the Invention This invention relates to the preparation of libraries of substituted pyrrolidines by combinatorial processes.

These libraries are useful for discovery of lead compounds for drug development and improved assay kits.

Background of the Invention Traditional chemical synthesis for drug discovery is done by individually creating, isolating, and identifying candidate compounds. Companies have long relied on their historical collections of compounds and compound collections from exchange agreements as sources of diverse structures for generating lead pharmaceutical compounds.

All of these historical approaches have drawbacks. Corporate collections of compounds may have a certain bias and medicinal chemists using traditional synthetic techniques cannot synthesize hundreds or thousands of diverse compounds to find promising leads.

Combinatorial chemistry is a relatively new technique for chemical synthesis. It fills the longfelt need for a method to quickly generate highly diverse non-peptide compound libraries. Generally, diverse libraries contain compounds with a common core or scaffold which are substituted with a great variety of substituents. More recently, modern drug discovery has used the methods of combinatorial chemistry to generate large numbers (viz., about 10^ to 10°) of compounds generically referred to as "libraries . "

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 positive screening results are obtained. 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 apparatus is readily manipulated by programmed robotic machinery.

Generally, combinatorial chemistry is conducted on a solid phase support, normally a polymer. A selected scaffold is cleavably tethered to the solid support by a chemical linker. Reactions are carried out to modify the scaffold while tethered to the solid support. In a final step, the product is cleaved and released from the solid support .

Combinatorial chemistry evidences its utility by commercial success. Millions of dollars have been spent for recent purchases or cooperative associations of major pharmaceutical companies with small companies specializing in combinatorial chemistry (e.g., Glaxo' s acquisition of Affymax, Marion Merrell Dow's purchase of Selectide, Proctor & Gamble with Houghten, Astra with Alanex, Pfizer with Oxford Asymmetry, Sandoz with Pharmacopeia, Solvay with Arqule, CIBA with Chiron, and Eli Lilly with Sphinx Pharmaceutical) .

Certain chemical reactions of pyrrolidines are known. Various aspects of pyrrolidine chemistry are known in the prior art as set out below: A) The article, "Synthesis of Enantiomerically Pure Pyrrolidines by Stereospecific Cycloaddition of Azomethine Ylides with Enones , " by Patzel, M. et al . , Tetrahedron Letters., Vol. 34, No. 36, pp. 5707-5710, 1993 describes the synthesis of pyrrolidines via cycloaddition onto enones.

B) The article, "Improved Synthesis of 4-Alkoxybenzyl Alcohol Resin" by Gui-shen Lu, et . al . , J. Org. Chem., 1981, vol. 46, pp. 3433-3436, describes the preparation of a Wang resin for solid-phase peptide synthesis. C) The article, "Combinatorial Organic Synthesis of Highly Functionalized Pyrrolidines: Identification of a Patent Angiotensin Converting Enzyme Inhibitor from a Mercaptoacyl Proline Library", by Martin M. Murphy, e . al., J. Am. Chem. Soc. 1995, Vol 117, pp. 7029-7030 describes the preparation of selected functionalized pyrrolidines using 1,3-dipolar cycloaddition reactions.

D) The article, "Solid-Phase Synthesis of Proline Analogs via a Three Component 1,3-dipolar cycloaddition" by Bruce C. Hamper, et . al . Tetrahedron Letters, Vol. 37, No. 21, pages 3671-3674, 1996, describes the preparation of selected highly substituted pyrrolidines by solid phase synthesis using 1,3-dipolar cycloaddition of a resin bound azomethine ylide.

To continue exploration of new libraries for pharmaceutical and agricultural lead compouncTs it is necessary to develop new chemistries which permit chemical novel scaffolds to be functionalized with highly diverse groups . Summary of the Invention

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. The method of this invention has applicability for making both diverse libraries of pyrrolidine compounds useful for finding new lead compounds and directed libraries of pyrrolidine compounds useful for optimizing a particular desired biological activity.

This invention is an improved combinatorial process for making a library of pyrrolidine compounds. This invention is also the combinatorial library of pyrrolidine compounds.

This invention is also a library of intermediate substituted solid supported pyrrolidine library compounds. This invention is also the individual pyrrolidine compounds in the pyrrolidine combinatorial library of the invention.

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

FIG. 1 is a top view of a wellplate apparatus. FIG. 2 is a side view of a wellplate apparatus

Detailed Description of the Invention

I. Definitions:

The following terms have the meaning defined below when used in this specification of the invention:

"Acidic group" means a proton donor substituent typified by -CO2H, -SO3H, and -P(0)(OH)2-

"Aromatic group" means a substituted or unsubstituted heterocyclic group derived from pyrrolyl , furanyl , thiophenyl, pyrazolyl, imidazolyl, phenylimidazolyl, triazolyl, isoxazolyl, oxazolyl, thiazolyl, thiadiazolyl, indolyl, carbazolyl, norharmanyl, azaindolyl, benzofuranyl, dibenzofuranyl, dibenzothiophenyl, indazolyl, imidazod .2- pyridinyl, benzotriazolyl, anthranilyl, 1, 2-benzisoxazolyl, benzoxazolyl, benzothiazolyl, purinyl, pryidinyl, dipyridylyl. phenylpyridinyl , benzylpyridinyl, pyrimidinyl, phenylpyrimidinyl, pyrazinyl, 1, 3 , 5-triazinyl , quinolinyl, phthalazinyl, quinazolinyl, and quinoxalinyl ; or a carbocyclic group derived from phenyl, naphthyl, tolulyl, xylenyl, indenyl, stilbenyl, terphenylyl, diphenylethylenyl, phenyl-cyclohexenyl, acenaphthylenyl, and anthracenyl, biphenyl , bibenzylyl and related bibenzylyl homologues represented by the formula (bb) ,

where n is a number from 1 to "Assay kit" means an assemblage of two cooperative elements, namely, (i) a wellplate apparatus, and (ii) 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.

"Directed Library" is 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").

"Electrophile" means an electron seeking reagent.

"Enone" means an α, β-unsaturated ketone.

"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. "Leaving group" means a group capable of substitution by a nucleophile.

"Library" is a collection of compounds created by a combinatorial chemical process, said compounds having a common pyrrolidine 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 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 invention is carried out and individual library compounds synthesized. Suitable reaction zones are the individual wells of a wellplate apparatus.

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

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

"Solid support" means a Wang resin in its hydroxyl or halogenated form. Wang resins are represented by the symbols, ^ and ^ , and are prepared as described in the article by Gui-shen Lu, referenced in the "Background of the Invention" section, supra.

"Substituents" are chemical radicals (excluding hydrogen) 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.

"Reagent" means a reactant, 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.

"Ylide" means a species which in its ground state has charges of opposite sign on adjacent atoms.

"Non-interfering substituent" means those groups, other than hydrogen, that do not significantly impede the solid phase process of the invention and yield stable pyrrolidine library compounds. Suitable non-interfering radicals include, but are not limited to, 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, toluyl, xylenyl, biphenyl, C2-C12 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 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.

"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" having one or more non-interfering groups as substituents.

"Halo" means chloro, fluoro, iodo or bromo. "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. Selected Abbreviations used in this specification: "DBU" - diazobicycloundecane "TFA" - trifluoroacetic acid "DMAP" - dimethyl amino pyridine

II. General description of the pyrrolidine combinatorial library:

The pyrrolidine library of the invention is a diverse combinatorial library comprising individual substituted pyrrolidine library compounds represented by the general formula (I) :

wherein; the internal numbers in the pyrrolidine ring are used to denote substituent positions,

Rl is an electrophilic group;

R2 is a group represented by the formula:

(^L2) ^R6 where divalent linking group -(L2)- is selected from the group consisting of,

where "L" is the point of attachment of the pyrrolidine ring, and where Rβ is a non-interfering substituent, and Ri and R2 may join together to form a hydantoin ring on the pyrrolidone nucleus as represented by formula (la) ,

wherein R7 is a non-interfering substituent; R3 is an aromatic group; R4 is a group of the general formula,

where -(L4)- is a divalent linking group, R8 is hydrogen or a non-interfering substituent; and R5 is an aromatic group.

The pyrrolidine library compounds of this invention are non-peptide, substantially non-naturally occurring molecules having a molecular weight range of from about 100 to about 700.

Preferred libraries contain pyrrolidine library compounds wherein;

Rl is an electrophilic group derived from an electrophilic reagent having a molecular weight of from about 30 to about 600 selected from the group consisting of; organic halides, acyl halides, sulfonic acid esters, organohaloformates, organosulfonyl halides, organic isocyanates, and organic isothiocyanates . Particularly useful electrophilic groups are those listed in Section III, Step D, infra., of this specification. Other electrophilic groups for Ri include, but are not limited to C1-C10 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, toluyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl , C1-C10 alkylsulfonyl, - (CH2 )m~0- (C1-C10 alkyl), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, aryloxyalkyl, carbocyclic radical, substituted carbocyclic radical, heterocyclic radical, substituted heterocyclic radical, and nitroalkyl; where m is from 1 to 8.

R2 is a group wherein -(L2)- is,

and R is Cl to Cio alkyl; or

Rl and R2 may join together to form a hydantoin wherein R7 is Cl to Cio alkyl or an aromatic group;

R4 is preferably a group derived from the cleavage of the library compound from a Wang Resin, for example,

where R9 is a non-interfering group and m is an integer from 0 to 3.

R3 and R5 are independently carbocyclic substituted or unsubstituted aromatic groups. Preferred groups for R3 , and R5 are selected from the following:

where "L" is the point of attachment of the above electrophilic groups.

Preferred compounds of the invention are represented by Formula (la) below:

where Ri , R3 , and R5 are as defined above

III. Solid Support bound Pyrrolidine Library Compounds as Intermediates:

Products of this invention include libraries of intermediates, wherein said intermediates are the solid supported form of the substituted pyrrolidine compounds of the invention. The intermediate library contains a plurality of diverse compounds, wherein each intermediate has the formula (X) :

wherein;

Rl R2 , R3 and R5 and -(L4)- as previously defined and as

is a solid support.

IV. The Process for Making the Pyrrolidine Combinatorial Library of the Invention: Outline of Process Steps: 'Preparation of Starting Materials

•Step A - Methyl ketone functionalizing of the Wang resin solid support •Step B - Aromatic enone formation •Step C - 1,3-dipolar cycloaddition reaction with azomethine ylide

•Step D - Electrophilic substitution of pyrrolidine nitrogen •Step E - Library compound cleavage from solid support

PROCESS STEP DETAILS

of Starting Materials :

Starting Materials:

a) Preparation of the Solid Support --

The diverse highly functionalized pyrrolidine combinatorial libraries of this invention are prepared by solid phase reactions. A preferred solid support precursor is a "Wang resin." The detailed preparation of a suitable Wang resin for conducting the process in this invention is set out in the "EXAMPLES" section, of this specification infra., the disclosure of which is incorporated herein by reference. Preparation of a Wang resin is illustrated in the following scheme:

2-2.5mmol/g 2. Ph ₃PCI ₂ , CH ₂CI ₂

Mcrrifield resin 1 % cross-linked

Wang resins permit acid catalyzed cleavage in the final step of the process.

b) Preparation of the Azomethine Ylid Reagent

Azomethine ylids are prepared from aryl amino acid imines (See, Patzel, M. reference cited in the "Background of the Invention" section of this Disclosure) . The aryl imines, may in term, be prepared from a condensation reaction of aryl aldehydes and amino acid esters or amides. Aryl imines prepared glycine are preferred in the practice of this invention.

The azomethine ylid reactant is itself prepared by condensation from aryl aldehydes and amino acid esters or amides .

General Pyrrolidine Library Process Making Details:

Reaction Medium - The reaction medium may be any liquid which is non-reactive with the reactants used in the library synthesis and is a non-solvent for the solid support . It is generally advantageous to have the nucleophilic reagent and electrophilic reagent soluble in the reaction medium.

Typical reaction media useful in the processes of the invention are methanol, chloroform, dimethylacetamide, tetrahydrofuran, dimethylformamide, methylene chloride, and acetonitrile.

The Reaction Zone - the process of the invention may be carried out in any vessel capable of holding the liquid reaction medium and having inlet and outlet means.

Preferably the process of the invention is carried out in containers adaptable to parallel array syntheses. Most preferably, the pyrrolidine library is formed in standard wellplates, such as the 96 well wellplate illustrated in Fig. 1 and/or the wellplate apparatus illustrated in Fig. 2. Each well may be filled by multiple delivery apparatus, automated or robotic apparatus, any of which may be either manually or computer controlled.

The diverse pyrrolidine library of this invention may take the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound) . In such cases, the library compounds are conveniently identified by their wellplate number and "x" column and "y" wellplate row coordinates. A preferred technique for practicing the process of the invention is parallel array synthesis. With parallel array synthesis individual reaction products are prepared in each of multiple reaction zones. The amount of nucleophilic and electrophilic reagents reactants 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 reaction zone is maintained at a temperature and for a time sufficient to permit substantial reaction of the solid phase pyrrolidine compound and the nucleophilic and electrophilic reagents.

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 0.1 seconds to 72 hours, with times of 1 hour to 24 hours being most often used. 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 between -10°C and +60°C, with 10°C to 40°C being preferred and ambient temperatures (about 20°C-30°C) being most preferred. The reactions may be conducted at subatmospheric pressure or superatmospheric pressure (viz., 60Kg./πv2 - 21000 Kg./m^ absolute), but ambient atmospheric pressure (about 10330 Kg./m², absolute) is most often used.

Endpoint determination - The completion of the reaction may be determined by a number of conventional techniques. One method is to use thin layer chro atography . Sequence of Operation - Within each process step the addition of the reactants to the reaction zone may take place in any order. For example, the solid supported reaction product may be initially added to the reaction zone followed by addition of the electrophilic or nucleophilic reagent, or vice versa.

The principle sources for diversity in the library compounds of the invention are the groups Ri, R2 , R3 and R5.

The groups R2 and R5 are provided in Step C of the process, the group Rl is provided in Step D and the group R3 is provided in Step B of the process. Step A.- Methyl ketone functionalizing of the Wang resin solid support

The solid support (viz., Wang resin) must first be functionalized with methyl ketone groups to permit enone formation later in the process of the invention. This is generally accomplished by reacting the solid support with a methyl ketone bearing compound. The solid support and the methyl ketone bearing compound must each have functionalities which permit reaction. For example, acetophenone may be reacted with a halogenated Wang resin as depicted by the following scheme:

Step B. - Aromatic Enone Formation:

The solid support reaction product of step A is reacted with an aromatic aldehyde as illustrated by the following scheme:

ArCHO

0.5M NaOMe in MeOH

THF (1:1 v/v) The aryl aldehyde is the source of molecular diversity for substituent R3 on the library compounds of the invention. The aromatic aldehyde may be selected from carbocyclic and heterocyclic aromatic nuclei having reactive aldehyde functionality. Suitable aldehydes are;

2-fluorenecarboxaldehyde n-methylpyrrole-2-carboxaldehyde furfural 5-nitro-2-furaldehyde 5-methylfurfural 5-acetoxymethyl-2-furaldehyde 5-hydroxymethyl-2 -furaldehyde benzaldehyde 2-bromobenzaldehyde 2-fluorobenzaldehyde pentafluorobenzaldehyde 2-chlorobenzaldehyde 2 , 4-dichlorobenzaldehyde 2 -chloro-6-fluorobenzaldehyde

2 , 6-dichlorobenzaldehyde o-anisaldehyde 2 , 3-dimethoxybenzaldehyde 2,3, 4-trimethoxybenzaldehyde 2, -dimethoxybenzaldehyde

2,4, 5-trimethoxybenzaldehyde 2,4, 6-trimethoxybenzaldehyde 2 , 5-dimethoxybenzaldehyde 2 -ethoxybenzaldehyde salicylaldehyde

3 , 5-dibromosalicylaldehyde 3-fluorosalicylaldehyde 3 , 5-dichlorosalicylaldehyde 3 , 5-diiodosalicylaldehyde 3-ethoxysalicylaldehyde 2 , 3 -dihydroxybenzaldehyde

2,3, -trihydroxybenzaldehyde

4- (diethylamino) salicylaldehyde

2-hydroxy-4-methoxybenzaldehyde 4, 6-dimethoxy-2-hydroxybenzaldehyde

2,4, 6-trihydroxybenzaldehyde

5-bromosalicylaldehyde

5-chlorosalicylaldehyde

2-hydroxy-5-methoxybenzaldehyde 2 , 5 -dihydroxybenzaldehyde

2 -carboxybenzaldehyde

2- (trifluoromethyl) benzaldehyde o-tolualdehyde

2, 3 -dimethyl-p-anisaldehyde 2 , 4-dimethylbenzaldehyde mesita1dehyde

2 , 5-dimethylbenzaldehyde

2, 5 -dimethyl-p-anisaldehyde

3 -cyanobenzaldehyde 3-bromobenzaldehyde

3-bromo-4, 5-dimethoxybenzaldehyde

5-bromo-2-methoxybenzaldehyde

3 -fluorobenzaldehyde

3-fluoro-p-anisaldehyde 3 -chlorobenzaldehyde

3 , 4-dichlorobenzaldehyde

3 , 5-dichlorobenzaldehyde

3 -phenoxybenzaldehyde

3- (3, 4-diehlorophenoxy) benzaldehyde 3- (3 , 5-dichlorophenoxy) benzaldehyde

3- (3- (trifluoromethyDphenoxy) benzaldehyde

3- ( 4-chlorophenoxy) benzaldehyde

3 - ( 4-methoxyphenoxy ) benzaldehyde

3 - ( 4-1ert-butylphenoxy) benzaldehyde 3 - ( 4 - ethylphenoxy) benzaldehyde m-anisaldehyde

4-acetoxy-3-methoxybenzaldehyde

3 , 4-dimethoxybenzaldehyde

3,4, 5-trimethoxybenzaldehyde 4-benzyloxy-3 -methoxybenzaldehyde

3 , 5-dimethoxybenzaldehyde

3 -benzyloxybenzaldehyde

3-hydroxybenzaldehyde

3-hydroxy-4-methoxybenzaldehyde 3,4-dihydroxybenzaldehyde

3 , 4, 5-trihydroxy benzaldehyde

3- (trifluoromethyl) benzaldehyde m-tolualdehyde

3 -methyl-p-anisaldehyde 4-cyanobenzaldehyde

4-bromobenzaldehyde

4-fluorobenzaldehyde

4-chlorobenzaldehyde

4-acetamidobenzaldehyde 4-dimethylaminobenzaldehyde

4-diethylaminobenzaldehyde

4-phenoxybenzaldehyde

4-acetoxybenzaldehyde p-anisaldehyde 3 -benzyloxy-4-methoxybenzaldehyde

4-benzyloxybenzaldehyde

4-ethoxybenzaldehyde

4-n-butoxybenzaldehyde

1-naphthaldehyde 2 -methoxy-1-naphthaldehyde

2-hydroxy-1-naphthaldehyde

4-methoxy-1-naphthaldehyde

2-naphthaldehyde

1-pyrenecarboxaldehyde 3 , 4-dibenzyloxybenzaldehyde n-ethyl-3-carbazolecarboxaldehyde 2-methyl-9-acridinecarboxaldehyde pyrrole-2-carboxaldehyde 2-thiophenecarboxaldehyde 3-methylthiophene-2-carboxaldehyde 4-bromothiophene-2-aldehyde 5-bromo-2-thiophenecarboxaldehyde 5-nitrothiophene-2-carboxaldehyde 5-methyl-2-thiophenecarboxaldehyde 3-thiophenecarboxaldehyde indole-3-carboxaldehyde 5-methoxyindole-3-carboxaldehyde piperonal 6-nitropiperonal 2-pyridinecarboxaldehyde

6-methyl-2-pyridinecarboxaldehyde 3 -pyridinecarboxaldehyde 4-pyridinecarboxaldehyde 3 -quinolinecarboxaldehyde 4-quinolinecarboxaldehyde 4-hydroxybenzaldehyde 3 -ethoxy-4-hydroxybenzaldehyde 3 , 5-dimethyl-4-hydroxybenzaldehyde

^' 4-bιphenylcarboxaldehyde 4- (methylthio) benzaldehyde methyl 4-formylbenzoate 4-carboxybenzaldehyde 4-trifluoromethylbenzaldehyde 4-isopropylbenzaldehyde p-tolualdehyde

4-ethylbenzaldehyde 4-chloro-3-nitrobenzaldehyde 3 , 5-dinitro-2 -hydroxybenzaldehyde 3 -hydroxy-4-nitrobenzaldehyde 4-hydroxy-3-nitrobenzaldehyde 5-nitrovanillin

2 -nitrobenzaldehyde

2 , 6-dinitrobenzaldehyde

6-nitroveratraldehyde 3 -methoxy-2-nitrobenzaldehyde

2-chloro-6-nitrobenzaldehyde

3 -nitrobenzaldehyde

5-chloro-2 -nitrobenzaldehyde

2-chloro-5-nitrobenzaldehyde 5-hydroxy-2-nitrobenzaldehyde

5-nitrosalicylaldehyde

4-nitrobenzaldehyde

1 , 4-benzodioxan-6-carboxaldehyde

2 , 3 -dichlorobenzaldehyde -ethoxy-4-methoxybenzaldehyde

3 , 5-bis (trifluoromethyl) benzaldehyde

2,3, 6-trichlorobenzaldehyde terephthalaldehyde monodiethylacetal

2, 3 -difluorobenzaldehyde 2, 6-difluorobenzaldehyde

2 , 4-difluorobenzaldehyde

2 , 5-difluorobenzaldehyde

3 , 4-difluorobenzaldehyde

3 , 5-difluorobenzaldehyde 4-dimethylamino-1-naphthaldehyde

3-furaldehyde

3, 4-dimethoxy-5-hydroxybenzaldehyde

2,3, 5-trichlorobenzaldehyde

2 , 6-dimethoxybenzaldehyde 5-bromo-2 , 4-dimethoxybenzaldehyde

2, 4-dimethoxy-3-methylbenzaldehyde

4-stilbenecarboxaldehyde

4- (3-dimethylaminopropoxy) benzaldehyde

2, 4-dihydroxybenzaldehyde 3 -chloro-4-fluorobenzaldehyde 2-methylindole-3-carboxaldehyde 4-hydroxy-3-methylbenzaldehyde 2- (diphenylphosphino) benzaldehyde 2 , 4-dinitrobenzaldehyde 4-n-propoxybenzaldehyde l-methylindole-3-carboxaldehyde

5-bromo-2-hydroxy-3 -methoxybenzaldehyde

3 -bromo-4-methoxybenzaldehyde

4-acetoxy-3, 5-dimethoxybenzaldehyde 3, 5-dihydroxybenzaldehyde

3-methoxy-4- (4-nitrobenzyloxy) benzaldehyde 2,3- (methylenedioxy) benzaldehyde 2 -hydroxy-3 -methoxy-5-nitrobenzaldehyde 2-cyanobenzaldehyde 5-ethyl-2-furaldehyde

4-tert-butylbenzaldehyde 3-tetrafluoroethoxybenzaldehyde 3 -carboxybenzaldehyde l-acetyl-3-indolecarboxaldehyde 4- (trifluoromethoxy) benzaldehyde 3 -bromo-4-fluorobenzaldehyde 3- (trifluoromethoxy) benzaldehyde 2-chloro-4-fluorobenzaldehyde 5- (3-nitrophenyl) furfural 2-chloro-4-hydroxybenzaldehyde 2,3, 4-trifluorobenzaldehyde 2-fluoro-3 - { trifluoromethyl ) benzaldehyde 2-fluoro-6- (trifluoromethyl ) benzaldehyde 4-fluoro-2- (trifluoromethyl) benzaldehyde 4- (dibutylamino) benzaldehyde

5- (trifluoromethoxy) salicylaldehyde 3-fluoro-2-methylbenzaldehyde 3, 5-dibenzyloxybenzaldehyde 5- (4-nitrophenyl) furfural 2 -chloro-3 -quinolinecarboxaldehyde 2-chloro-5- (trifluoromethyl) benzaldehyde 5-bromo-2-furaldehyde 2,3,5, 6-tetrafluorobenzaldehyde 4-methyl-5-imidazolecarboxaldehyde 2-benzyloxy-4, 5-dimethoxybenzaldehyde

3 , 5-di-tert-butyl-2-hydroxybenzaldehyde 2 , 4-diethoxy-m-tolualdehyde 4-tert-pentylbenzaldehyde

Alternatively, aldehyde derivative of the radicals depicted in the preceding section II, definition of R3 and R5 may be used as the aromatic aldehyde reactant.

Step C. - 1,3-dipolar cycloaddition reaction with azomethine ylide

The azomethine ylid reactant has the following formula;

where R5 and R2 are as defined above.

R5 is an aromatic group and R2 is an amino acid ester or amide .

The azomethine ylid reactant is the source for diversity in the R2 and R3 substituents of pyrrolidines represented by Formula I, supra.

The solid supported reaction product of step B is reacted with an aryl imine of an amino acid ester or an amide analog thereof. The aryl imine reactant is itself prepared by condensation of aryl aldehydes and amino acid esters or amides.

This reaction is further illustrated by the reaction scheme set out below:

Illustration of the use of alternative imines is shown in the following scheme:

Step D - Electrophilic substitution of pyrrolidine nitrogen The product of Step C is reacted with an electrophile. The electrophile reacts with the nitrogen atom on the pyrrolidine nitrogen ring. Alkylation and acylation reactions are suitable, for example, as show the following scheme :

H R

Electrophilic reactants suitable for use in this step have a molecular weight of from abut 15 to 600 and are selected from organic halides, acyl halides, sulfonic acid esters, organohaloformates, organosulfonylhalides, organic isocyanates, and organic isothiocyanates . Suitable electrophilic reagents for practice of this process step of the invention are set out below: Acyl Halides --

3, 5-bis (trifluoromethyl) benzoyl chloride benzoyl chloride

2-bromobenzoyl chloride

2-fluorobenzoyl chloride pentafluorobenzoyl chloride

2 , 4-difluorobenzoyl chloride 2, 6-difluorobenzoyl chloride

2-chlorobenzoyl chloride

2 , 4-dichlorobenzoyl chloride

2 , 6-dichlorobenzoyl chloride o-acetylsalicyloyl chloride 2-methoxybenzoyl chloride

2 , 6-dimethoxybenzoyl chloride

2- (trifluoromethyl) benzoyl chloride o-toluoyl chloride

3-bromobenzoyl chloride 3-fluorobenzoyl chloride

3-chlorobenzoyl chloride

3 , 4-dichlorobenzoyl chloride m-anisoyl chloride

3 , 4-dimethoxybenzoyl chloride 3 , 4, 5-trimethoxybenzoyl chloride

3 , 5-dimethoxybenzoyl chloride

3-ethoxybenzoyl chloride isophthaloyl chloride trimesoyl chloride 3- (trifluoromethyl) benzoyl chloride m-toluoyl chloride

3- (chloromethyl) benzoyl chloride

4-bromobenzoyl chloride

4-fluorobenzoyl chloride 4-chlorobenzoyl chloride p-anisoyl chloride 4-ethoxybenzoyl chloride 4-n-butoxybenzoyl chloride 4-n-hexyloxybenzoyl chloride 4-heptyloxybenzoyl chloride 4-biphenylcarbonyl chloride terephthaloyl chloride 4- (trifluoromethyl ) benzoyl chloride 4-tert-butylbenzoyl chloride p-toluoyl chloride

4-ethylbenzoyl chloride 4-n-propylbenzoyl chloride 4-butylbenzoyl chloride 4-pentylbenzoyl chloride 4-hexylbenzoyl chloride

4-n-heptylbenzoyl chloride methyl oxalyl chloride ethyl oxalyl chloride heptafluorobutyryl chloride 2-acetoxyisobutyryl chloride pivaloyl chloride 3-chloropivaloyl chloride 2-bromopropionyl chloride 2 , 3 -dibro opropionyl chloride 2, 3-dichloropropionyl chloride o-acetylmandelic acid chloride itaconyl chloride methacryloyl chloride isobutyryl chloride 2-ethylhexanoyl chloride acetyl chloride bro oacetyl chloride chloroacetyl chloride phenoxyacetyl chloride 4-chlorophenoxyacetyl chloride methoxyacetyl chloride phenylacetyl chloride

3 , 3-dimethylacryloyl chloride cinnamoyl chloride fumaryl chloride ethyl malonyl chloride tert-butylacetyl chloride isovaleryl chloride undecanoyl chloride lauroyl chloride myristoyl chloride palmitoyl chloride heptadecanoyl chloride stearoyl chloride propionyl chloride

3-bromopropionyl chloride

3-chloropropionyl chloride hydrocinnamoyl chloride succinyl chloride 3-carbomethoxypropionyl chloride ethyl succinyl chloride butyryl chloride

4-bromobutyryl chloride

4-chlorobutyryl chloride valeryl chloride

5-chlorovaleryl chloride adipoyl chloride hexanoyl chloride

6-bromohexanoyl chloride pimeloyl chloride heptanoyl chloride suberoyl chloride octanoyl chloride

10-undecenoyl chloride 2 -chloro-2 , 2 -diphenylacetyl chloride dichloroacetyl chloride alpha-chlorophenylacetyl chloride

2-chloropropionyl chloride

2-iodobenzoyl chloride 4-iodobenzoyl chloride cyclopropanecarbonyl chloride trans-2-phenyl-1-cyclopropanecarbonyl chloride cyclobutanecarbonyl chloride cyclopentanecarbonyl chloride 3-cyclopentylpropionyl chloride cyclohexanecarbonyl chloride

4-cyanobenzoyl chloride

2-furoyl chloride

1-naphthoyl chloride 2-naphthoyl chloride pyrrolidine-2-carbonyl chloride

2-thiopheneacetyl chloride trimellitic anhydride chloride

2 , 6-pyridinedicarboxylic acid chloride 2-quinoxaloyl chloride

2-nitrobenzoyl chloride

3-nitrobenzoyl chloride

3 , 5-dinitrobenzoyl chloride

4-ni robenzoyl chloride 3, 4-dimethoxyphenylacetyl chloride

3-methyladipoyl chloride

3 , 5-dichlorobenzoyl chloride

2, 5-difluorobenzoyl chloride

3 , 4-difluorobenzoyl chloride 9-fluorenone-4-carbonyl chloride

3, 5-difluorobenzoyl chloride

(s) - (-) -n- (trifluoroacetyl)prolyl chloride benzyloxyacetyl chloride acetoxy acetyl chloride 3-cyanobenzoyl chloride 2, 5-dimethoxyphenylacetyl chloride

3-methoxyphenylacetyl chloride iminodibenzyl-5-carbonyl chloride

2, 4, 6-trimethylbenzoyl chloride tetrafluorosuccinyl chloride perfluorooctanoyl chloride diphenylacetyl chloride alpha-methyl valeroyl chloride methyl malonyl chloride ethyl glutaryl chloride

5-bromovaleryl chloride methyl adipyl chloride

3-cyclohexenecarbonyl chloride

3-isocyanato benzoyl chloride 2 , 4, 6-triisopropylbenzoyl chloride fluoroacetyl chloride

2-ethoxybenzoyl chloride piperonyloyl chloride

2 , 4-dimethoxybenzoyl chloride 2 , 3 , 5, 6-tetrachloroterephthaloyl chloride

5- (dimethylsulfamoyl) -2-methoxybenzoyl chloride

2 - (4-chlorobenzoyl) benzoyl chloride

2 , 2-bis ( chloromethyl ) ropionyl chloride cinnamylidenemalonyl chloride 2-phenoxypropionyl chloride

2 -phenylbutyryl chloride 2-ethylbutyryl chloride p-tolylacetyl chloride gamma-methylvaleroyl chloride 3 , 3-dichloropivaloyl chloride

1-methyl-l-cyclohexanecarboxylic acid chloride 2- (2, 4, 5-trichlorophenoxy) acetyl chloride 4-chloro-3-nitrobenzoyl chloride 4-methyl-3-nitrobenzoyl chloride 2 , 3-dichlorobenzoyl chloride morpholine-4-carbonyl chloride p-chlorophenylacetyl chloride bicyclo [2.2.1] heptane-2-carbonyl chloride d ( - ) -alpha-formyloxy-alpha-phenylacetyl chloride d(-) -alpha-phenylglycine chloride hydrochloride trifluoroacetyl chloride pentafluoropropionyl chloride hexafluoroglutaryl chloride

2-chlorocinnamoyl chloride o-methoxycinnamyl chloride

5-nitro-2-furoyl chloride

2-chlorobutyryl chloride

4-phenylazobenzoyl chloride

4-n-amyloxybenzoyl chloride 4-decylbenzoyl chloride

4-octylbenzoyl chloride dl-2-methylbutyryl chloride linolenoyl chloride linolelaidoyl chloride llh-eicosafluoroundecanoyl chloride

9h-hexadecafluorononanoyl chloride

2, 3 -difluorobenzoyl chloride

2 - (benzoyloxymethy1 ) benzoyl chloride

2, 2-dimethylvaleroyl chloride 3 , 5, 5-trimethylhexanoyl chloride phenothiazine-10-carbonyl chloride

3, 4-dimethyl benzoyl chloride

( + ) -p- ( 2-methylbutyl ) benzoyl chloride

2 , 4-dichlorophenoxyacetic chloride pentadecanoyl chloride nonadecanoyl chloride neoheptanoyl chloride

9-anthracenecarbonyl chloride

2 -ethoxy-1-naphthoyl chloride pyrrolidine carbonyl chloride m- (chlorosulfonyl) benzoyl chloride

2-n-propyl-n-valeroyl chloride

2-chloro-4-nitrobenzoyl chloride

2-phenoxybutyryl chloride 2-chloronicotinyl chloride

6-chloronicotinyl chloride

4- (trifluoromethoxy) benzoyl chloride

2- (trifluoromethoxy) benzoyl chloride

2, 6-dichloropyridine-4-carbonyl chloride 3-chlorobenzo [b]pyrrolidine-2-carbonyl chloride

4-chloromethylbenzoyl chloride neodecanoyl chloride

(phenylthio) acetyl chloride -carbethoxyhexafluorobutyryl chloride octafluoroadipoyl chloride

2-diazo-3 , 3 , 3-trifluoropropionylchloride

2-bromobutyryl chloride arachidoyl chloride cis-vaccenoyl chloride 11-eicosenoyl chloride behenoyl chloride petroselinoyl chloride palmitoleoyl chloride tridecanoyl chloride 2-chloro-5-nitrobenzoyl chloride

3 -methylthiopropionyl chloride methyl 4-chlorocarbonylbenzoate anthraquinone-2-carbonyl chloride carbazole-n-carbonyl chloride 2-nitrophenoxyacetyl chloride

2-bromo-2-methylpropionyl chloride

2-fluoro-3- (trifluoromethyl) benzoyl chloride

2-fluoro-4- (trifluoromethyl) benzoyl chloride

2-fluoro-5- (trifluoromethyl) benzoyl chloride 3-fluoro-5- (trifluoromethyl) benzoyl chloride 4-fluoro-2- (trifluoromethyl) benzoyl chloride 4-fluoro-3- (trifluoromethyl) benzoyl chloride 2-fluoro-6- (trifluoromethyl) benzoyl chloride 2, 3 , 6-trifluorobenzoyl chloride 2 , 4, 5-trifluorobenzoyl chloride

2 , 4-di (trifluoromethyl) benzoyl chloride 2, 6-di (trifluoromethyl) benzoyl chloride 3- (trifluoromethoxy) benzoyl chloride m- ( fluorosulfonyl ) benzoyl chloride trans-1, 2-cyclobutanedicarboxylic acid chloride 3-cyclohexylpropionyl chloride

4-ethyl-2 , 3-dioxo-l-piperazinecarbonylchloride isoxazole-5-carbonyl chloride bromodifluoroacetyl chloride erucoyl chloride

2, 4, 6-trifluorobenzoyl chloride dichlorochrysanthemic acid chloride isononanoyl chloride 1-adamantanecarbonyl chloride 2, 5-bis (trifluoromethyl) benzoyl chloride 2 , 3 , 4-trifluorobenzoyl chloride 2,3,4, 5-tetrafluorobenzoyl chloride

2 , 4 , 6-trichlorobenzoyl chloride

2 , 4-dichloro-5-fluorobenzoyl chloride 4-methoxyphenylacetyl chloride trans-3- (trifluoromethyl) cinnamoyl chloride

3- (dichloromethyl) benzoyl chloride

4-isocyanato benzoyl chloride heneicosanoyl chloride 2-chloroisobutyryl chloride trans-4-nitrocinnamoyl chloride

3 , 4, 5-trifluorobenzoyl chloride

5-fluoro-2- (trifluoromethyl) benzoyl chloride 2 , 3 , 5-trifluorobenzoyl chloride 2-chloro-4-fluorobenzoyl chloride {-) -alpha-chlorophenylacetyl chloride

2- (para-tolylsulfonyl) acetyl chloride

4-methyl-4-nitrohexanoyl chloride l-chloro-4-fluorosulfonyl-2-naphthoyl chloride 2 , 3-dibromo-3-phenylpropionyl chloride

2 -menthoxyacetyl chloride

2-phenyl-2- (phenylsulfonyl) acetyl chloride

4, 4, 4-trifluorocrotonyl chloride

4, 4, 4-trifluorobutyryl chloride 3, 4-dichloro-2, 5-thiophenedicarbonyl chloride pentachlorobenzoyl chloride

4,4,7, 7-tetranitrosebacoyl chloride alpha, alpha ' -dimethylsuccinyl chloride alpha-bromoisovaleryl chloride benzoyl chloride oleoyl chloride methyl suberyl chloride gamma-linolenoyl chloride

( - ) -camphanic acid chloride 4, 4 ' -stilbenedicarbonyl chloride chlorinated benzoyl chloride

(lr) -(+) -camphanic chloride

2- (4-nitrophenoxy) tetradecanoyl chloride

7- [ (chlorocarbonyl) methoxy] -4-methylcoumarin n,n-bis (2-chloroethyl) carbamoyl chloride

(s) - (-) -2-acetoxypropionyl chloride linoleoyl chloride

3 -chlorotetrafluoropropionyl chloride

3 , 4-dichloropentafluorobutyryl chloride 7h-dodecafluoroheptanoyl chloride

5h-octafluoropentanoyl chloride perfluorononanoyl chloride

3h-tetrafluoropropionyl chloride

2-bromo-2, 3,3, 3 -tetrafluoropropanoyl chloride arachidonoyl chloride pentachloropropionyl chloride

4-decenoyl chloride tridecafluoroheptanoyl chloride undecafluorocyclohexanecarbonyl chloride 4-n-nonylbenzoyl chloride

3- (trichlorogermyl ) propionylchloride

3, , 5-triiodobenzoyl chloride

2- (phenylthio) propionyl chloride

2 , 2 , 2-triphenylacetyl chloride d(-) -alpha-azido-phenyl acetyl chloride

4-azido-benzoyl chloride difluoroacetyl chloride

5-chloropyrazine-2 -carbonyl chloride n- (1-naphthalenesulfonyl) -1-phenylalanyl chloride n- (4-nitrophenylsulfonyl) -1-phenylalanyl chloride n- (p-toluenesulfonyl) -1-phenylalanyl chloride dimethylmalonyl chloride methyl sebacoyl chloride

2, 5-dichloropyridine-3-carbonyl chloride 3- (2, 5 xylyloxy) propionyl chloride.

Additionally, acyl chorides suitable for use in the process of the invention are represented by the following formulae:

Organic Halides -- benzyl bromide alpha-bromo-o-xylene alpha-bromo-m-xylene 4- (tert-butyl) benzyl bromide alpha-bromo-p-xylene tert-butyl bromoacetate methyl bromoacetate benzyl bromoacetate ethyl bromoacetate 2-bromoacetophenone

2-bromo-2 ' -methoxyacetophenone

2-bromo-2 ' , 4 ' -dimethoxyacetophenone

2-bromo-2 ' , 5 ' -dimethoxyacetophenone 3-methoxyphenacyl bromide

2-bromo-4 ' -methoxyacetophenone

2-bromo-4 ' -phenylacetophenone

2-bromo-4 ' -methylacetophenone ethyl bromopyruva e 1-bromopinacolone

1-bromo-2-butanone

1-bromo-2, 2-dimethoxypropane l-bromo-2 , 2-dimethylpropane bromoacetaldehyde dimethyl acetal bromoacetaldehyde diethyl acetal

1-bromo-2- ethylpropane l-bromo-2 -ethylbutane

2-ethylhexyl bromide

1-bromodecane 1-bromoundecane

2-bromoacetamide iodoacetamide

4- (bromomethyl)phenylacetic acid phenacyl ester isopropyl bromoacetate 5-bromo-2-methyl-2-pentene

3 , 4-difluorobenzyl bromide

2 , 5-difluorobenzyl bromide

3, 5-bis (trifluoromethyl) benzyl bromide

2-bromo-2 ' -nitroacetophenone 3 , 5-difluorobenzyl bromide

2, 4-bis (trifluoromethyl) benzyl bromide

8-bromo-l-octanol

4- (bromomethyl)phenylacetic acid methyl (r) - (+) -3-bromo-2 -methylpropionate 4-iodobutyl acetate 7-acetoxy-4-bromomethylcoumarin 4-bromomethy1-6,7-dime hoxycoumarin 2 , 4-difluorobenzyl bromide methyl 2 - (bromomethyl ) acrylate 3-bromopropionaldehyde dimethyl acetal (r) - ( -) -3 -bromo-2 -methyl-1-propanol

Sulfonic Acid Esters -- ethyl trifluoromethanesulfonate 2 , 2 , 2-trifluoroethyl p-toluenesulfonate

2 -chloroethyl-p-toluenesul onate

1,3 -propane sultone

5 ' -tosyladenosine

1,4-butane sultone cyanomethyl benzenesulfonate hexadecyl ethanesulfonate ethyl methanesulfonate

2-chloroethyl methanesulfonate ethyl p-toluenesulfonate trans-2-hydroxycyclohexyl p-toluenesulfonate (2r) - (-) -glycidyl tosylate

(s) - (+) -2-methylbutyl methanesulfonate

(s ) - (+) -2-methylbutyl p-toluenesulfonate

(s) - (+) -1-phenyl-l, 2-ethanediol 2-tosylate (2r) -(-) -glycidyl 3-nitrobenzenesulfonate propargyl benzenesulfonate

2 , 2-dimethyl-l, 3-dioxolan-4-ylmethyl p-toluenesulfonate (r) - (-) -2,2-dimethyl-l, 3-dioxolan-4-ylmethyl p- toluenesulfonate (s) -(+) -2, 2-dimethyl-l, 3-dioxolan-4-ylmethyl p- toluenesulfonate

1,2:5, 6-di-o-isopropylidene-3 -o- (methylsulfonyl ) -alpha- d-glucofuranose ethyl 1-2- ( (methylsulfonyl ) oxy) propionate (2s) -(+) -glycidyl tosylate (2s) - (+) -glycidyl 3-nitrobenzenesulfonate 3-o-acetyl-6-o-benzoyl-5-o- (methylsulfonyl) -1, 2-o- isopropylidene-alpha-d-glucofu

(r) - (-) -l-benzyloxy-3- (p-tosyloxy) -2-propanol (s) - (+) -l-benzyloxy-3- (p-tosyloxy) -2-propanol ethyl 1-2- ( (trifluoromethylsulfonyl) oxy)propionate 2- (2-chloroethoxy)ethyl methanesulfonate 1-cyanoethyl p-toluenesulfonate

Organohaloformates

9-fluorenylmethyl chloroformate phenyl chloroformate

4-chlorophenyl chloroformate methyl chloroformate benzyl chloroformate vinyl chloroformate isobutyl chloroformate

2-ethylhexyl chloroformate ethyl chloroformate 2-bromoethyl chloroformate

2-chloroethyl chloroformate

1-chloroethyl chloroformate allyl chloroformate n-propyl chloroformate butyl chloroformate n-hexyl chloroformate octyl chloroformate

2,2, 2-trichloro-l, 1-dimethylethyl chloroformate

2,2, 2-trichloroethyl chloroformate cholesteryl chloroformate

4-nitrophenyl chloroformate

4-nitrobenzyl chloroformate

(-)-menthyl chloroformate

4-t-butylcyclohexyl chloroformate cetyl chloroformate (+) -1- (9-fluorenyl) ethyl chloroformate isopropyl chloroformate

3-chlorocyclohexyl chloroformate decyl chloroformate oleyl chloroformate octadecyl chloroformate butenediol bischloroformate

2-chlorobenzyl chloroformate

4-chlorobutyl chloroformate (+)-menthyl chloroformate

4, 5-dimethoxy-2-nitrobenzyl chloroformate cyclopentyl chloroformate t-butylcyclohexyl chloroformate menthylchloroformate p-tolyl chloroformate

4-bromophenyl chloroformate

4-fluorophenyl chloroformate

4-methoxyphenyl chloroformate

2-nitrophenyl chloroformate 4-methoxycarbonylphenyl chloroformate

1-chloro-2-methylpropyl chloroformate

(+/-) -1,2,2, 2-tetrachloroethyl chloroformate

2 , 2-dichloroethyl chloroformate myristyl chloroformate cyclohexyl chloroformate chloromethyl chloroformate.

Organosulfonylhalides --

1-naphthalenesulfonyl chloride dansyl chloride

2-naphthalenesulfonyl chloride

2-acetamido-4-methyl-5-thiazolesulfonyl chloride

2-thiophenesulfonyl chloride

8-quinolinesulfonyl chloride benzenesulfonyl chloride pentafluorobenzenesulfonyl chloride

2, 5-dichlorobenzenesulfonyl chloride

2-nitrobenzenesulfonyl chloride

2 , 4-dinitrobenzenesulfonyl chloride 3 , 5-dichloro-2-hydroxybenzenesulfonyl chloride

2,4, 6-triisopropylbenzenesulfonyl chloride

2-mesitylenesulfonyl chloride

3-nitrobenzenesulfonyl chloride p-bromobenzenesulfonyl chloride 4-fluorobenzenesulfonyl chloride

4-chlorobenzenesulfonyl chloride

4-chloro-3-nitrobenzenesulfonyl chloride pipsyl chloride

4-nitrobenzenesulfonyl chloride 4-methoxybenzenesulfonyl chloride

4-tert-butylbenzenesulfonyl chloride p-toluenesulfonyl chloride trifluoromethanesulfonyl chloride trichloromethanesulfonyl chloride isopropylsulfonyl chloride methanesulfonyl chloride alpha-toluenesulfonyl chloride trans-beta-styrenesulfonyl chloride

2,2, 2-trifluoroethanesulfonyl chloride 1-hexadecanesulfonyl chloride ethanesulfonyl chloride

2-chloroethanesulfonyl chloride

1-propanesulfonyl chloride

3-chloropropanesulfonyl chloride 1-butanesulfonyl chloride methyl 2- (chlorosulfonyl ) benzoate

2-nitro-4- (trifluoromethyl) benzenesulfonyl chloride

3- (trifluoromethyl) benzenesulfonyl chloride

1-octanesulfonyl chloride 4- (trifluoromethoxy) benzenesulphonyl chloride (lr) - (-) -10-camphorsulfonyl chloride d- (+) -10-camphorsulfonyl chloride (+/-) -10-camphorsulfonyl chloride 2-nitro-alpha-toluenesulfonyl chloride.

Isocyanate Reagents -- trans-2-phenylcyclopropyl isocyanate phenyl isocyanate

2 -bromophenyl isocyanate 2-fluorophenyl isocyanate

2 , 4-difluorophenyl isocyanate 2 , 6-difluorophenyl isocyanate 2-chlorophenyl isocyanate 2, 3-dichlorophenyl isocyanate 2, 4-dichlorophenyl isocyanate 2, 5-dichlorophenyl isocyanate 2 , 6-dichlorophenyl isocyanate 2 -methoxyphenyl isocyanate 2 , 4-dimethoxyphenyl isocyanate 2, 5-dimethoxyphenyl isocyanate 2-ethoxyphenyl isocyanate 2- (trifluoromethyl) phenyl isocyanate o-tolyl isocyanate 2 , 6-dimethylphenyl isocyanate 2-ethylphenyl isocyanate 3 -bromophenyl isocyanate 3 -fluorophenyl isocyanate 3-chlorophenyl isocyanate 3 , 4-dichlorophenyl isocyanate 3-methoxyphenyl isocyanate

3- (trifluoromethyl) phenyl isocyanate m-tolyl isocyanate

4-bromophenyl isocyanate

4-fluorophenyl isocyanate 4-chlorophenyl isocyanate 4-methoxyphenyl isocyanate ethyl 4-isocyanatobenzoate

4- (trifluoromethyl) phenyl isocyanate p-tolyl isocyanate n- (chlorocarbonyl) isocyanate benzoyl isocyanate tert-butyl isocyanate

(s) - (-) -alpha-methylbenzyl isocyanate isopropyl isocyanate methyl isocyanate ethyl isocyanatoacetate octadecyl isocyanate ethyl isocyanate

2-chloroethyl isocyanate allyl isocyanate n-propyl isocyanate butyl isocyanate cyclohexyl isocyanate

1-naphthyl isocyanate (r) -(-) -1- (1-naphthyl) ethyl isocyanate

4-fluoro-3-nitrophenyl isocyanate

2-nitrophenyl isocyanate

3-nitrophenyl isocyanate

4-nitrophenyl isocyanate 2 , 6-diisopropylphenyl isocyanate benzyl isocyanate

3-chloropropyl isocyanate ethoxycarbonyl isocyanate

3, 5-bis (trifluoromethyl) phenyl isocyanate 2, 4, 6-tribromophenyl isocyanate

2 , 5-difluorophenyl isocyanate

2, 4, 5-trichlorophenyl isocyanate

2, 4, 6-trichlorophenyl isocyanate

2-methoxycarbonylphenyl isocyanate 2-ethoxycarbonylphenyl isocyanate 2-isopropylphenyl isocyanate

2 , 3 -dimethylphenyl isocyanate

4-methoxy-2-methylphenyl isocyanate

2 , 4-dimethylphenyl isocyanate 2 , 5-dimethylphenyl isocyanate

2-ethyl-6-methylphenyl isocyanate

3-cyanophenyl isocyanate

5-chloro-2 , 4-dimethoxyphenyl isocyanate

3-chloro-4-methylphenyl isocyanate 3 , 5-dichlorophenyl isocyanate

5-chloro-2 -methoxyphenyl isocyanate

3 , 4, 5-trimethoxyphenyl isocyanate

3 , 5-dimethoxyphenyl isocyanate

3- (methylthio) henyl isocyanate 3-ethoxycarbonylphenyl isocyanate

3-acetylphenyl isocyanate

3 , 4-dimethylphenyl isocyanate

3 , 5-dimethylphenyl isocyanate

2-methoxy-5-methylphenyl isocyanate 3-ethylphenyl isocyanate

4-chloro-2-methoxyphenyl isocyanate

4-chloro-2-trifluoromethylphenyl isocyanate

4-chloro-3-trifluoromethylphenyl isocyanate

4-iodophenyl isocyanate 4-phenoxypheny1 isocyanate

4-ethoxyphenyl isocyanate

4- (methylthio) phenyl isocyanate

4-acetylphenyl isocyanate

4-isopropylphenyl isocyanate 4-ethylphenyl isocyanate

4-n-butylphenyl isocyanate

3- (dichloromethylsilyl)propyl isocyanate octyl isocyanate

4-methyl-3-nitrophenyl isocyanate 4-chloro-2 -nitrophenyl isocyanate 2 -methyl-4-nitrophenyl isocyanate 4-methyl-2-nitrophenyl isocyanate 2-fluoro-5-nitrophenyl isocyanate 2 -methyl-5-nitrophenyl isocyanate 3 -bromopropyl isocyanate

2, 4, 6-trimethylphenyl isocyanate 2-isopropyl-6-methylphenyl isocyanate 2, 6-diethylphenyl isocyanate 5-chloro-2 -methylphenyl isocyanate 4-chloro-2-methylphenyl isocyanate

4- (trifluoromethoxy) phenyl isocyanate 4-trifluoromethylthiophenylisocyanate 2 , 4-dibromophenyl isocyanate 2, 6-dibromo-4-ethylphenyl isocyanate 2, 3, 4, 5-tetrachlorophenyl isocyanate

2-chloro-5-trifluoromethylphenyl isocyanate 2 -chloro-6-methylphenyl isocyanate 2-n-carbobutoxyphenyl isocyanate 2, 4, 5-trimethylphenyl isocyanate 2 -methyl-6- (t-butyl) phenyl isocyanate 2-ethyl-6-isopropylphenyl isocyanate 3 -chloro-2 -methoxyphenyl isocyanate 3 -chloro-2 -methylphenyl isocyanate 3-chloro-4-fluorophenyl isocyanate 4-cyanophenyl isocyanate

4-bromo-2-methylphenyl isocyanate 4-bromo-2, 6-dimethylphenyl isocyanate 2, 6-dibromo-4-fluorophenyl isocyanate 4-n-butoxyphenyl isocyanate 4-butoxycarbonylphenyl isocyanate phenethyl isocyanate 2-methyl-3 -nitrophenyl isocyanate hexyl isocyanate hexadecyl isocyanate methylene bis (o-chlorophenyl isocyanate) 4-chloro-3 -nitrophenyl isocyanate 2 -chloro-4-nitrophenyl isocyanate 4, 5-dimethyl-2 -nitrophenyl isocyanate 2-chloro-5-nitrophenyl isocyanate 2-methoxy-4-nitrophenyl isocyanate 3-fluoro-4-methylphenyl isocyanate 5-fluoro-2-methylphenyl isocyanate 3, 5-dicarbomethoxyphenyl isocyanate 2, 4-dichlorobenzyl isocyanate 2- (methylthio) phenyl isocyanate n- (me hoxycarbonyl ) isocyanate n- (phenoxycarbonyl) isocyanate 2-biphenylyl isocyanate 3-iodophenyl isocyanate 4-phenylphenyl isocyanate tetrahydro-2-pyranyl isocyanate 4- ( tert-butyl )phenylisocyanate 1- (4-bromophenyl) ethyl isocyanate isocyanatoacetic acid n-butyl ester dodecyl isocyanate

6, 7-methylenedioxy-4-isocyanate-methylcoumarin (r) - (+) -alpha-methylbenzyl isocyanate (+/-) -1- (1-naphthyl) ethyl isocyanate (s) - (+) -1- (1-naphthyl) ethyl isocyanate 3, 4-difluorophenyl isocyanate

2 -methoxy-5-nitrophenyl isocyanate undecyl isocyanate ethyl 2-isocyanato-4-methyl valerate ethyl 6-isocyanatohexanoate ethyl 2-isocyanato-4-methylthiobutyrate ethyl 2-isocyanatopropionate ethyl 3-isocyanatopropionate ethyl 2-isocyanato-3-methylbutyrate tert-butyl 3-isothiocyanatopropionate ethyl 2-isocyanato-3-phenylpropionate 1, 3 -bis (isocyanatomethyl) cyclohexane 2- (trifluoromethoxy) henyl isocyanate 4- (chloromethyl) phenyl isocyanate 1-adamantyl isocyanate 1, 3 -bis ( 2 -isocyanato-2-propyl) benzene n-amyl isocyanate n-heptyl isocyanate

2-chloroethyl isocyanate, [ethyl-1, 2-14c] 1,1,3, 3-tetramethylbutyl isocyanate 3 , 5-dinitrophenyl isocyanate

Organic Isothiocyanates -- cyclohexyl isothiocyanate 1-naphthyl isothiocyanate trimethylsilyl isothiocyanate phenyl isothiocyanate 2 -bromophenyl isothiocyanate 2-fluorophenyl isothiocyanate 2-chlorophenyl isothiocyanate o-tolyl isothiocyanate

3 -bromophenyl isothiocyanate 3-fluorophenyl isothiocyanate 3-chlorophenyl isothiocyanate m-tolyl isothiocyanate 4-bromophenyl isothiocyanate 4-fluorophenyl isothiocyanate 4-chlorophenyl isothiocyanate p-tolyl isothiocyanate ethoxycarbonyl isothiocyanate benzoyl isothiocyanate tert-butyl isothiocyanate tert-octyl isothiocyanate methyl isothiocyanate benyl isothiocyanate ethyl isothiocyanate phenethyl isothiocyanate allyl isothiocyanate

Preferred groups for acylation of the pyrrolidine nitrogen are as follows:

Part b - Formation of hydantoins

The groups Ri and R2 may form a hydantoin ring. When hydantoin structures are desired the alkylating/acylating agent is an isocyanate or isothiocyanate. A hydantoin forming reaction is illustrated by the following scheme:

Suitable isocyanate reactants for hydantoin formation were described in the preceding listings the disclosure of which is incorporated herein by reference. The solid support-pyrrolidine compounds produced at this step in the process of the invention are themselves valuable stable, and storable intermediates which may used when needed as sources of individual library compounds. Individual library compounds are made from these intermediates by cleavage as described in the following process Step (E) .

Step E. - Library compound cleavage from Solid Support.

The final step of the process for preparing combinatorial pyrrolidine libraries is separation of the library compounds from its solid support. For polymeric solid supports of the Wang Resin type the decoupling is conventionally done with strong acids. For Example, the following reaction employing TFA with a Wang resin supported pyrrolidine may be used.

The final step in the pyrrolidine library forming process of the invention may be supplemented by purification techniques such as chromatography, crystallization, distillation, solvent extraction, or combinations of such techniques.

REACTION SCHEME 1 An illustrative reaction scheme illustrating all steps of the pyrrolidine combinatorial library process in combination is shown below:

resin, 0.93mmol/g ex Advanced Chenttech

Cs₂Cθ₃, DMF

Ar = aryl

acylating agents py, DMAP, CH ₂CI₂

X . CO, SO ₂

R « alkyl, aryl

EXAMPLE The following example illustrates the preparation of a pyrrolidine combinatorial library with reference to Scheme 1 , supra .

Chlorination of Wang Resin 1

Wang resin 1 (5g, 100-200 mesh, 0.93mequiv/g, ex Advanced Chemtech) was suspended in anhydrous DMF (60ml) . To this was added triphenyl phosphine (4.88g, 18.6mmol) and then carbon tetrachloride (1.80ml, 18.6mmol). The reaction vessel was capped and placed on an orbital shaker for 2 days. At this time the reaction mixture was filtered and washed with the following: THF (200ml), THF-H0 (1:1, 200ml), THF (200ml) and finally MeOH (200ml) . The resulting white resin was dried in vacuo to provide 5.04g of chlorinated Wang resin 2. Anal, found: C, 87.46, H, 7.45, Cl, 2.83.

Coupling of 3-Hvdroxyacetophenone 3 to Chlorinated Wang Resin 2

Chlorinated Wang resin 2 (3.82g) , 3-hydroxyacetophenone (ex Aldrich) 3 (1.56g, 11.46mmol), cesium carbonate (3.73g, 11.46mmol) and sodium iodide (0.69g, 4.58mmol) were combined together in anhydrous DMF (50ml), capped and placed on an orbital shaker for 3 day. The reaction mixture was filtered and washed successively with the following solvents (50ml each) : DMF, MeOH, H2O, DMF-MeOH, DMF, CH2CI2, MeOH. Following the final wash, the resin was dried overnight in vacuo to provide an off-white resin 4 (4.20g): IR(KBr) 1675cm-¹. Anal, found C, 88,34. H, 7.32.

The loading of this resin was determined by cleavage of a known amount and HPLC analysis. Thus the resin (51.2mg) was suspended in TFA (1ml) and stirred for 20hr. HPLC analysis indicated 3.42 mg of 3-hydroxyacetophenone 3 had been cleaved. This corresponds to a loading of 0.49mmol/g.

Preparation of Enone 5 (Ar=p-CHj.OMe) bv Condensation Reaction of 3-Hvdroxvacetophenone Resin 4

A solution of NaOMe in MeOH (31.3ml of a 0.5M solution in MeOH, 15.6mmol) was added to a mixture of acetophenone resin 4 (2.66g) and p-anisaldehyde (2.36g, 15.6mmol) in anhydrous THF (30ml) . The flask was capped and placed on an orbital shaker for 4 day. The reaction mixture was filtered and washed successively with the following solvents (50ml of each) : THF, MeOH, THF, MeOH and finally THF. The resin was dried with air pulling through the Buchner funnel to give 3.0g of a light yellow resin 5 (Ar=p-C₆H θMe) .

A small sample was suspended in TFA and stirred for 20 hr. The supernatant liquid was decanted and evaporated. The resulting oil was re-evaporated from methylene chloride several times to give an off white solid. ¹H NMR (CDCI₃) of this material was identical to a sample of enone prepared independently by standard solution synthesis.

Preparation of Pyrrolidine 7 (Ar=p-C H θMe) via 1.3-Dipolar Cycloaddition of Imine 6 (Ar'=Ph)

Resin 5 (Ar=OMe) (lg) was suspended in anhydrous THF. To this was added sequentially imine 6 (Ar'=Ph) (434mg as a solution in dry THF), LiBr (255mg, 2.94mmol) and DBU (372mg, 2.45mmol). The reaction mixture was slowly stirred for 3 day at which time the resin was filtered and washed successively with MeOH, THF, MeOH, THF, MeOH, THF, CH2CI2 and air dried to give resin 7. Acetylation of Pyrrolidine Resin 7 (Ar=p-C£ 0Me, Ar'=Ph)

Resin 7 (Ar=pC₆H₄θMe, Ar'=Ph) (300mg) was suspended in anhydrous methylene chloride. To this was added DMAP (3mg) , pyridine (190μl, 2.35mmol) and acetyl chloride (1.18ml of a 1M solution in methylene chloride, 1.175mmol). The mixture was stirred at ambient temperature for 20hr, at which time the resin was filtered and washed sequentially with the following solvents (10ml each) : CH₂CI₂, DMF, MeOH, DMF, MeOH, DMF, CH2CI2, and further CH₂CI₂. The resin was air dried and suspended in a 1:1 (v/v) mixture of CH₂Cl2:TFA and stirred at ambient temperature for 20hr. The supernatant liquid was removed by filtration and the resin washed several times with methylene chloride. The filtrates were evaporated in vacuo to yield a crude brown foam (90mg) . Purified by chromatography (Siθ2, 3:2 EtOAc-hexanes) to afford pyrrolidine 9 (31mg) (Ar=pC6H4θMe, Ar'=Ph, X=CO, R=Me) . Crystallised from methanol. Anal, calcd. for C, .H, N, . Found C, .H, .N, .

Synthesis of Bicvclic Hydantoin 8 (Ar=p-C H^OMe. Ar'=Ph. R=n-Bu)

Pyrrolidine resin 7 (Ar=p-C_δH₄θMe, Ar'=Ph) was suspended in methylene chloride containing DMAP (l g) and pyridine

(103μl, 1.28mmol). Butyl isocyanate (0.40ml of a 1M solution in methylene chloride, 0.40mmol) was added and the mixture stirred at ambient temperature for 20hr. The resin was filtered and washed sequentially with the following solvents (15ml each of): CH₂CI2, DMF, MeOH, DMF, MeOH, DMF, CH₂C1₂.

The resin was air dried and suspended in a 1:1 (v/v) mixture of CH2Cl₂: FA and stirred at ambient temperature for 20hr. The supernatant liquid was removed by filtration and the resin washed several times with methylene chloride. The filtrates were evaporated in vacuo to yield a crude brown foam (19mg) . Purified by preparative TLC (Siθ₂, 3% MeOH- CH₂CI₂) to afford bicyclic hydantoin 8 (Ar=p-C6H₄θ e, Ar'=Ph, R=Bu) as a white solid. Crystallised from MeOH. FAB MS 499 (M+l) . Anal, calcd. for C30H30N2O5 C, 72.27. H, 6.06. N, 5.61. Found: C, 72.38. H, 6.12. N, 5.65.

Experimental Conditions for Combinatorial Plate Synthesis

Resin Preparation

Sodium methoxide (29.13g, 0.539mol) was added to a stirred mixture of Merrifield resin 1 (80g of 2-2.5mmol/g, ex Acros) and 4-hydroxybenzylalcohol (66.95g, 0.539mol, ex Aldrich) in DMA. The reaction mixture was heated to 55°C for 8hr and allowed to cool. Filtered and washed (400ml, 2 times) successively with dioxane, DI water, dioxane, dioxane-DI water (1:1 v/v), dioxane, MeOH and dried to give an off- white resin (81g) .

This resulting resin (81g) and dichlorotriphenylphosphorane (235g, 0.728mol) were combined in dry methylene chloride (1 litre) and stirred for 2 days, at which time it was filtered and washed successively (500ml of each) with methylene chloride, methanol, methlene chloride, methanol, methylene chloride. The resin was dried in vacuo (35°C) to afford a white resin 2 (80. Ig).

Chlorinated Wang resin 2 (70. Og), 3-hydroxyacetophenone 3 (64.2g, 0.472mol, ex Aldrich), cesium carbonate (102.5g, 0.315mol, ex Fluka) and sodium iodide (23.6g, 0.157mol, ex Fluka) were combined in dry DMF (800ml) and stirred at ambient temperature for 3 days. The mixture was filtered and washed successively with DMF, MeOH, DI water, THF, DI water, THF, MeOH, and dried in vacuo overnight to afford a light brown resin 4 (81.2g). Plate Synthesis

3 -Hydroxy acetophenone resin 4 (35.3g) was suspended in a ca . 1:1 (v/v) mixture of DMF:CH2Cl2 (650ml) to obtain an isopicnic slurry. This was distributed to 13 x 96-well plates (0.50ml to each well, corresponds to ca . 27mg/well [ ca . 29umol/well] ) . The wells were allowed to drain and were washed with THF via an 8-way manifold several times, drained and pulled dry over a vacuum plenum.

1) Condensation Reaction: To each row in a 96-well plate was added a unique aryl aldehyde (400μl of a 1M solution in THF, 14 equiv) and was followed by addition (to every well) a solution of sodium methoxide (500μl of a 0.5M solution in methanol, 8.6 equiv., ex Aldrich). The wells were capped and tumbled for 3-4 day.

The wells were uncapped, filtered and washed successively with the following solvents (500μl of each) : THF, MeOH, THF, MeOH, THF, MeOH, THF, and pulled dry under a vacuum plenum. 2) 1,3 -Dipolar Cycloaddition Reaction: To each well was added in sequence the following reagents via 8-way manifold: a) benzaldehyde imine of glycine (188μl of a 1M solution in THF, 6.5equiv.), b) LiBr (500μl of a 0.5M solution in THF, 8.6equiv.) and c) DBU (188μl of a 1M solution in THF, 6.5equiv.) . The wells were capped and tumbled for 3-4 days, at which time the resin was washed successively with the following solvents (500μl of each) : THF, MeOH, THF, MeOH, THF, MeOH, THF, CH2CI₂ and pulled dry under a vacuum plenum. 3) Acylation Reaction: To each well was added via 8-way manifold a solution of pyridine and DMAP in CH2CI₂ (35.1μl pyridine and 0.53mg DMAP in 400μl CH₂CI₂ total volume) and this was followed by a solution of unique acylating agent to each row (1-8) (400μl of 1M solution in CH₂CI₂) . The plates were capped and tumbled for 20hr, filtered and washed successively with the following solvents (500μl of each) : CH₂CI₂, DMF, MeOH, DMF, MeOH, and CH2CI2. The resin was dried under a vacuum plenum.

4) Cleavage from the Resin: To each well was added via 8-way manifold a solution of TFA in CH₂CI₂ (750μl of a 10% solution). The plates were capped and tumbled for 20hr.

The wells were then uncapped and allowed to gravity filter to a 1ml 96 well plate. The resin was washed with 125μl CH₂CI₂ (each well) and the solvents evaporated in a speed- vac. TLC ' s were obtained after re-dissolving in 10%MeOH- CH₂C1₂.

Experimental for Plate Synthesis

1.4-hydroxybenzzyyl' ^ ^ cr^α

Merrifϊled resin,

2-2.5mmol/g ex Acres

1

(distribute to plates) Ar = aryl

Ar' = aryl

R is alkyl and/or aryl X = O or S This invention is particularly well suited as a general method for preparing a structurally diverse pyrrolidine library. The final form of the library compounds in the pyrrolidine library may be as a solute dissolved in a solvent (viz., the reaction medium) or the solvent may be removed and the final product retained as a powder, paste or oil.

The reaction zone for forming each pyrrolidine library compound of this invention contains a solvent. The solvent reaction medium is typically a solvent for the reactants used.

The utility of the method of the invention and the pyrrolidine library 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 large numbers of diverse pyrrolidine molecules useful as a source of lead compounds. The combinatorial pyrrolidine libraries of the invention may be screened for pharmacologically active compounds using conventional screen protocols known in the art for any targeted disease state. Certain library compounds prepared by the process of the invention. The successful practice of combinatorial chemistry is best done by confining reactants, products, and assay materials in specially defined arrays, adaptable to automated methods. Automated methods, optionally, software driven and computer assisted, permits full exploitation of combinatorial chemistry for diverse library preparation. For example, pipetting, diluting, dispensing, data collection, storage, plate heating/cooling, plate washing, measurements ( fluorescent/radiographic/colorimetric ) , data collection and high-capacity operation are all adaptable to automation.

Wellplate Apparatus containing library compounds prepared bv the process of the invention:

The processes of making the pyrrolidine library of the invention may be conveniently carried out in a wellplate apparatus such as illustrated in Fig. 1 and Fig. 2, hereinafter described. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 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 sequence of operations to be used for library- generation with the wellplate is as follows:

The wellplate apparatus of the invention:

A wellplate inoculated with the novel pyrrolidine library compounds of the invention is itself a new construct or apparatus which has particular utility in an assay kit used to discover lead compounds.

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 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 well plate.

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

FIG. 1 illustrates the top surface of a wellplate apparatus of the invention. The wellplate (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 pyrrolidine library compounds prepared by the solution phase combinatorial processes of the invention. The library 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) .

Assay Kits using wellplates with the library compounds of the invention:

This invention includes an assay kit for identification of pharmaceutical lead compounds . The assay kit comprises as essential parts, (i) wellplate apparatus (containing in its wells the pyrrolidine 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 library compounds contained in each wellplate may be prepared by either the pyrrolidine combinatorial library forming process taught herein. Preferably the wellplate apparatus has the form of a standard 96 well microtiter plate.

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 the following assays: In vitro assays: Enzymatic Inhibition

Receptor-ligand binding Protein-protein Interaction Protein-DNA Interaction Cell-based, Functional assays: Transcriptional Regulation

Signal Transduction/ Second Messenger Viral Infectivity Add, Incubate, & Read assays:

Scintillation Proximity Assays Angiotensin II SPA receptor binding assay Endothelin converting enzyme [¹²^I] SPA assay

HIV proteinase [125j] g ^ enzyme assay Cholesteryl ester transfer protein (CETP) [³H] SPA assay

Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Colorimetric Biosensors Ca²⁺-EGTA Dyes for Cell-based assays Reporter Gene Constructs for cell based assays

Luciferase, green fluorescent protein, b-lactamase Electrical cell impedance sensor assays Strep Potentiator Assay The Strep Potentiator Assay is for antibiotic therapeutic indication.

The assay has a two plate format : Into plate 1 compounds to be tested are added with medium, methicillin, and a methicillin resistant Staphylococcus aureus. After an overnight incubation, the plates are read on a plate reader at 650 nm.

The utility of the pyrrolidine library compounds of this invention is illustrated by their expected positive impact in at least one of the assays cited above.

While the present invention has been illustrated above by certain specific embodiments, it is not intended that these specific examples should limit the scope of the invention as described in the appended claims.

Claims

What is claimed is:

1. A library of substituted pyrrolidine compounds wherein said library contains a plurality of diverse library compounds, wherein each library compound has the formula (I) :

wherein; Rl is an electrophilic group;

R2 is a group represented by the formula:

(La) R₆ where divalent linking group -(L2)- is selected from the group consisting of,

and

L is the point of attachment of the divalent group to the pyrrolidine ring, R6 is a non-interfering substituent, and Rl and R2 may join together to form a hydantoin ring; R3 is an aromatic group; R4 is a group of the general formula,

where -(L4)- is a divalent linking group, R8 is hydrogen or a non-interfering substituent; and R5 is an aromatic group.

2. The library of claim 1 represented by the formula (la) ,

wherein R7 is a non-interfering substituent.

3. The pyrrolidine library of claim 1 wherein; Rl is an electrophilic group derived from an electrophilic reagent having a molecular weight of from about 30 to about 600 selected from the group consisting of; organic halides, acyl halides, sulfonic acid esters, organohaloformates, organosulfonyl halides, organic isocyanates, and organic isothiocyanates; R2 is -Cθ2(Cι-Ciθ alkyl);

R3 and R5 are independently aromatic groups selected from the group consisting of substituted or unsubstituted heterocyclic groups derived from pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, phenylimidazolyl, triazolyl, isoxazolyl, oxazolyl, thiazolyl, thiadiazolyl, indolyl, ca-rbazolyl, norharmanyl, azaindolyl, benzofuranyl, dibenzofuranyl, dibenzothiophenyl, indazolyl, imidazo(1.2- pyridinyl, benzotriazolyl, anthranilyl, 1, 2-benzisoxazolyl , benzoxazolyl , benzothiazolyl, purinyl, pryidinyl, dipyridylyl. phenylpyridinyl , benzylpyridinyl , pyrimidinyl, phenylpyrimidinyl, pyrazinyl, 1, 3 , 5-triazinyl, quinolinyl, phthalazinyl, quinazolinyl, and quinoxalinyl and carbocyclic groups derived from phenyl, naphthyl, tolulyl, xylenyl, indenyl, stilbenyl, terphenylyl, diphenylethylenyl, phenyl- cyclohexenyl, acenaphthylenyl, and anthracenyl, biphenyl, bibenzylyl and related bibenzylyl homologues represented by the formula (bb) ,

where n is a number from 1 to 8; and R4 is

where R9 is a non-interfering group and m is an integer from

0 to 3.

4. The individual substituted pyrrolidine library compounds of the library of claim 1.

5. A library of intermediate substituted pyrrolidine compounds comprising a plurality of diverse compounds, wherein each intermediate has the formula (X) :

wherein;

Rl is an electrophilic group;

R2 is a group represented by the formula

^■(L -₂2)/- R^π,6 where divalent linking group -(L2)- is selected from the group consisting of,

L is the point of attachment of the divalent group to the pyrrolidine ring, R6 is a non-interfering substituent, and Rl and R2 may join together to form a hydantoin ring;

R3 is an aromatic group;

-(L4)- is a divalent li

is a solid support; and ønking group,

R5 is an aromatic group.

6. The intermediate substituted pyrrolidine compounds of claim 5.

7. The library of Claim 1 comprising a plurality of diverse library compounds, wherein each library compound is represented by Formula (la) :

(alkyO≥C

8. A combinatorial process for preparing a library of substituted pyrrolidine compounds, said library comprising a plurality of diverse library compounds, wherein each library compound is made in a separate reaction zone and is represented by the formula (I) :

wherein;

Rl is an electrophilic group;

R2 is a group represented by the formula:

(L₂) R₆ where divalent linking group -(L2)- is selected from the group consisting of,

L is the point of attachment of the divalent group to the pyrrolidine ring, Rβ is a non-interfering substituent, and Rl and R2 may join together to form a hydantoin ring;

R3 is an aromatic group;

R4 is a group of the general formula,

where -(L4)- is a divalent linking group, R8 is hydrogen or a non-interfering substituent; and R5 is an aromatic group; wherein said process comprises the steps of;

A) Methyl ketone functionalizing a Wang resin solid support;

B) forming an aromatic enone on the Wang resin reaction product of Step (A) ;

C) reacting an azomethine ylide with the reaction product of Step (B) to effect 1,3-dipolar cycloaddition; D) reacting an electrophile with the reaction product of Step (C) to effect electrophilic substitution on the pyrrolidine nitrogen; and E) cleaving the substituted-diamino pyrrolidine reaction product of Step (D) from the solid support with strong acid, then recovering each pyrrolidine library compound.

9. The process of step 8 wherein; in step (A) methyl phenyl ketone is used to functionalize the Wang resin; in step (B) the aromatic aldehyde used to form the enone is a phenyl or substituted phenyl aldehyde; in step (C) the azomethine ylide is a Ci-Cio alkyl ester of glycine; and in step (D) the electrophilic agent has a molecular weight of from about 15 to about 600 and is selected from the group consisting of; organic halides, acyl halides, sulfonic acid esters, organohaloformates, organosulfonyl halides, organic isocyanates, and organic isothiocyanates .

10. An assay kit for identification of pharmaceutical lead compounds, comprising biological assay materials and wellplate apparatus; wherein the improvement comprises using as wellplate apparatus a wellplate containing in each well the individual library compounds of a diverse combinatorial pyrrolidine library prepared by the process of claim 8.

11. The assay kit of claim 10 containing biological assay materials selected from the group of assay tests;

In vitro assays: Enzymatic Inhibition

Receptor-ligand binding Protein-protein Interaction Protein-DNA Interaction Cell-based, Functional assays: Transcriptional Regulation Signal Transduction/ Second Messenger Viral Infectivity Add, Incubate, & Read assays:

Scintillation Proximity Assays Angiotensin II SPA receptor binding assay

Endothelin converting enzyme [125j gp^ assay

HIV proteinase [125j] gp^ enzyme assay Cholesteryl ester transfer protein (CETP) [³H] SPA assay

Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Colorimetric Biosensors Ca²⁺-EGTA Dyes for Cell-based assays Strep Potentiator Assay.

Reporter Gene Constructs for cell based assays Luciferase, green fluorescent protein, b-lactamase, and Electrical cell impedance sensor assays.

12. Wellplate apparatus suitable as a replaceable element in an automated assay machine wherein the improvement comprises; using as the wellplate apparatus a diverse pyrrolidine combinatorial wellplate, wherein each well contains a pyrrolidine library compound prepared by the method of claim 8.

13. The apparatus of claim 12 comprising a 96 well microtiter plate.