WO2000038685A1

WO2000038685A1 - Solution phase synthesis of compounds including amine alcohol functionality

Info

Publication number: WO2000038685A1
Application number: PCT/US1999/031263
Authority: WO
Inventors: Bruce A. Dressman; Alexander G. Godfrey
Original assignee: Eli Lilly And Company
Priority date: 1998-12-30
Filing date: 1999-12-30
Publication date: 2000-07-06
Also published as: AU2400300A

Abstract

This invention relates to a process for producing a plurality of compounds of formula (I): The invention is also directed to a reaction vessel on which a plurality of compounds of the same formula are physically separated from each other. The invention is also directed to a method of optimizing the biological activity of a compound of the same formula. The process for producing the compounds is ideal for generating libraries of compounds for high throughput screening. The invention also provides a library of above compounds physically separated from one another in a reaction vessel. This library is useful for optimizing the activity of therapeutically useful compounds.

Description

SOLUTION PHASE SYNTHESIS OF COMPOUNDS INCLUDING AMLNE ALCOHOL FUNCTIONALITY

BACKGROUND OF THE INVENTION

The present invention relates to the generation of a plurality of compounds with amine alcohol functionality.

Many compounds with amine alcohol functionality have known pharmaceutical activity and have the backbone structure:

For example, Berthold et al, DE 2925448 (1980) teach compounds including an amine alcohol functionality as antiarrhythmtic agents, antihypertensive agents and cc- and β- adrenergic blocking agents. Their preferred compound is shown below.

In the 1960s, Gaertner (Tetrahedron Letters, 343 (1967); J. Org. Chem 33, 523 (1968)) described the ring-opening alkylation of l,l-dialkyl-3-substituted azetidinium cations with a variety of nucleophile, including amines, alkoxides, mercaptides and halides. A limited number of compounds were formed. Further, as none of the azetidinium cations were chiral, no chiral products were obtained.

The possibility for using this class of compounds as therapeutic drugs demands an economical preparation which can be carried out on an industrial scale. Combinatorial chemistry allows researchers to make collections, or libraries, of screenable compounds by parallel synthesis of large numbers of derivatives of therapeutically important classes of bioavailable organic compounds. By screening these compounds against key receptors or enzymes, useful structure-function data can be obtained, speeding the search for new therapeutic agents. A combinatorial approach to the synthesis of compounds with amine alcohol functionality is desirable.

SUMMARY OF THE INVENTION The present invention relates to a process for producing a plurality of compounds having the formula (I):

wherein R' and R² are each, independently, an unsubstituted or substituted alkyl, cycloalkyl, aryl or heterocyclic ring; or

R¹ and R² together with the N atom which they substitute and, if appropriate. 1 -3 further heteroatoms selected from NR^R³, O and S, form a 3 to 10 membered mono-, bi- or tricyclic ring which optionally can be substituted with 1-3 substituents; and Nu is a negative ion or a molecule that has an unshared pair of electrons.

In accordance with one aspect, the process comprises the steps of: (a) providing a quaternary azetidinium salt (QAS) of the formula (II):

wherein R¹ and R² are defined above and Y is a counterfoil, in solution; (b) providing a series of nucleophiles in solution; and

(c) sequentially mixing said QAS with said series of nucleophiles in solution for a time sufficient to produce a plurality of products of the formula (I).

In accordance with another aspect, the invention is directed to a reaction vessel on which a plurality of compounds of the formula (I) as defined above are physically separated from each other.

In accordance with yet another aspect, the invention is directed to a method of optimizing the biological activity of a compound by contacting a solid support containing a plurality of compounds of formula (I) as defined above, which compounds are physically separated from each other with an assay kit to determine if any of the compounds has biological activity.

In accordance with still yet another aspect, of the invention is directed to an assay kit for the identification of compounds having biological or other activity, this kit comprising assay materials and well plate apparatus where each well in this apparatus contains a compound of the library described above.

The process preferably produces these compounds in a parallel fashion, i.e. simultaneously, with the compounds comprising a diverse chemical library. All of the compounds in such a library have a common backbone -NCH₂CH(OH)CH,X-. Diversity is introduced via the R¹, R² and Nu groups. These side groups are selected to allow the creation of a chemically diverse library which maximizes the exploration of molecular spatial properties. Such maximization increases the likelihood of creating compounds which will be biologically active against selected targets.

As used herein and in the appended claims a "library" means a large number of chemical derivatives generally useful in screening for biological activity or other activity. In general a library will have greater than 20 members, preferably the library will have at least 50 members, more preferably the library will have at least 96 members and most preferably the library will have at least 1000 members.

These and other objects, advantages, and features of the present invention will be better understood upon review of the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows examples of quaternary azetidinium salts useful in the process of the present invention. DET AILED DESCRIPTION OF THE INVENTION The compounds of the present invention are produced by reacting a quaternary azetidinium salt (QAS) of the formula (II):

wherein Y is a counterion (such as a halide) with a nucleophile in solution for a time sufficient to produce a product of the formula (I):

where R¹ and R² are each, independently, an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; or

R¹ and R² together with the N atom which they substitute form a substituted or unsubstituted 3 to 10 membered mono- or bi-cyclic ring or together with the N atom which substitute and 1 to 4 further heteroatoms selected from NR R⁵, O and S form a substituted or unsubstituted 3 to 10 membered mono- or bi-cyclic ring; R⁴ and R⁵ are each, independently, hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; and

Nu is a negative ion or a molecule that has an unshared pair of electrons. Suitable substituents include hydroxyl, amino, oxy, carbonyl, thiol, alkyl, alkenyl, alkynyl, alkoxy, halo, nitrile, nitro, aryl and heterocyclic ring. "Substituted" means that the moiety contains at least one, preferably 1-3 substituent(s). These substituents can optionally be further substituted with 1-3 substituents. For example, examples of substituted substituents include carboxamide, alkylmercapto, alkylsulphonyl, alkylamino, dialkylamino, carboxylate, alkoxycarbonyl, alkylaryl, aralkyl, alkylheterocyclic ring, etc. When R¹ and R² , or R³ and R⁶, together with the N atom which they substitute form a substituted or unsubstituted 3 to 10 membered mono- or bi-cyclic ring or together with the N atom which substitute and 1 to 4 further heteroatoms selected from NR R⁵, O and S form a substituted or unsubstituted 3 to 10 membered mono- or bi-cyclic ring. The preferred groups include azetidine, aziridine, imidazole, indole, isothizaole, isoxazole, morphline, oxazole, piperidine, piperazine, purine, pyrazole, pyrrole, pyrrolidine, thiazole, and thiomorpholine.

"Alkyl" (or alkyl- or alk-) means a straight or branched chain hydrocarbon containing 1 to 20, preferably 1 to 6, carbon atoms. "Alkenyl" means a straight or branched chain hydrocarbon containing at least one olefin and 2 to 20, preferably 2 to 6, carbon atoms.

"Alkynyl" means a straight or branched chain hydrocarbon containing at least one triple bond and 2 to 20, preferably 2 to 6. carbon atoms.

"Cycloalkyl" means a cyclic hydrocarbon containing 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

"Aryl" means a mono- or bicyclic carbocyclic aromatic ring containing 6 to 10 carbon atoms, such as phenyl (Ph) or naphthyl.

"Heterocyclic ring" is a mono-, bi- or tricyclic ring system containing one or more N, O or S atoms. The system can contain one or more double bonds and can be aromatic. Suitable examples include benzofuran, benzothiophene, furan, imidazole, indole, isothiazole, oxazole, piperazine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, quinoline, thiazole, thiophene, and the like.

"Halo" means a halogen atom, such as chlorine, florine, iodine or bromine. "Nucleophile" or "Nu" is a negative ion or a molecule that has an unshared pair of electrons. Suitable nucleophiles include alkyl and aromatic thiols; alkyl and aromatic alcohols; aromatic heterocyclic compounds that contain a nucleophilic nitrogen (example- the nitrogen atom of an indole, pyrrole, oxazole, thiazole, imidazole, pyrazole, isothiazole, 1,2,3-oxadiazole, 1,2,3-triazole, 1, 3, 4-thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazines, isoindole, lH-indazole, benzimidazole, purine, benzthiazole, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, carbazole, acridine, phenazine, phenothiazine, phenoxazine ring system); primary, secondary and tertiary amines (both cyclic and non cyclic); anilines; anionic carbon atoms (example-butyl lithium); salts of halides; alkyl and aromatic phosphines; primary and secondary alkyl or aromatic hydrazines; alkyl and aromatic hydroxylamines; salts of cyanide anion; ammonia and salts of ammonia; salts of hydroxide anion; and cyclic hydrazines. Quaternary Azetidinium Salt (QAS)

Compounds of the formula (2) can be produced as described (see, for example, Gaerthner, Tetrahedron Letters, 343 (1967); Gaerthner, J. Org. Chem. 33, 523 (1968)). Briefly, an epichlorohydrin and a secondary amine are heated at reflux overnight in methanol as follows.

The solvent is removed and the resulting residue titrated with an organic solvent such as methylene chloride (CH₂C1₂), acetonitrile, acetone, hexane or ether. Some QAS produced using this procedure are shown in Figure 1. That list is exemplary only and is not intended to limit the nature of the invention.

Nucleophes (Nu)

Suitable nucleophiles are defined above. Preferred nucleophiles include aliphatic alcohols, aromatic alcohols, aliphatic thiols (also alkyl thiols or alkyl sulfides), aromatic thiols, and primary and secondary amines. Many of these compounds are commercially available. Syntheses of these classes of compounds are well described in the literature.

General Process In general the QAS is reacted with the nucleophile Nu in solvent for a time and at a temperature sufficient to result in product.

Suitable solvents are aprotic solvents, including, but are not limited to, tetrahydrofuran (THF), dimethyl formamide (DMF), dimethylsulfoxide (DMSO), dioxane, CH₂C1₂, toluene or acetone, or mixtures thereof. In some instances, the reaction can be conducted in mixed solvent systems which contain alcohol.

The reaction is conducted for at least about 1 hour, preferably overnight.

The reaction can be conducted at room temperature (ca. 25 °C) to the reflux point of the solvent used. Product can be separated from starting materials using any conventional method. Preferably, an ion exchange resin is used. Suitable ion exchange resins include Dowex resins, available from BioRad, and SCX resin, available from Varian Sample Preparation, CA.

If a chiral starting QAS is used, then a chiral product is formed. This aspect of the reaction can thus be used to produce enantiomerically pure or enantiomerically enriched products. This aspect of the reaction is described in a concurrently filed U.S. Application

No. , (attorney docket 7295/34), entitled "Novel Spiro-azetidinium Reagents," inventors, Alexander Godfrey, Steve Pedersen and Bruce Dressman.

Specific Processes Alkyl thiol

WhenNu is an alkyl thiol (R-CH₂-SH), the reaction of the present invention can be represented as follows:

Examples of suitable alkyl thiols are shown below. This list is intended to demonstrate the diversity of reagents which can be used and not intended to limit the nature of the reagent in any way.

WhenNu is an alkyl thiol (or mercaptan), the reaction is conducted in the presence of a base, preferably a resin bound base such as tetramethylammonium hydroxide (available in resin bound form from Biorad) or piperidine (which can be attached to Merrifield resin). The reaction of a QAS and an aliphatic thiol is preferably conducted in THF, dioxane or toluene. An excess of nucleophile is used; wherein the molar ratio of Nu:QAS is about one or greater or preferably is about two or greater. The reaction mixture is preferably refluxed overnight. The product can be purified by passing the reaction mixture through a SCX column (a cation exchange resin available from Varian Sample Preparation, CA). The product adheres to the column and can be removed with either acid or base. The preferred method for removing the product is to rinse with 20% NH₄ ^" / MeOH in methylene chloride. Aromatic thiols

WhenNu is an aromatic thiol (R-SH), the reaction of the present invention can be represented as follows:

Examples of suitable aromatic thiols are shown below. This list is intended to demonstrate the diversity of reagents which can be used and not intended to limit the nature of the reagent in any way.

WhenNu is an aromatic thiol, the reaction is conducted in the presence of a base, preferably a resin bound base such as resin bound piperidine. The reaction of a QAS and an aromatic thiol is preferably conducted in 1 :1 methanol : CH₂C1₂. An excess of nucleophiles is used; wherein the molar ratio of Nu:QAS is about one or greater and preferably is about five or greater. The reaction mixture is preferably heated to about 65°C overnight. The product can be purified by passing the reaction mixture through a SCX column and washing the product from the column with 20% NH₄ ⁺ / MeOH in methyl en e chloride. Thereafter, if the aromatic thiol contains a basic substituent, the product can be further purified by passing the material obtained off the SCX column through AG 2X hydroxide resin (an anionic resin exchange resin available from Biorad). Aromatic alcohol

When Nu is an aromatic alcohol (R-OH), the reaction of the present invention can be represented as follows:

?²

Examples of suitable aromatic alcohols are shown below. This list is intended to demonstrate the diversity of reagents which can be used and not intended to limit the nature of the reagent in any way.

WhenNu is an aromatic alcohol, the reaction is conducted in the presence of a base, preferably resin bound tetramethylammonium hydroxide. The reaction of a QAS and an aromatic alcohol is preferably conducted in toluene, THF or dioxane. An excess of nucleophile is used; wherein the molar ratio of Nu:QAS is about one or greater and preferably is about ten or greater. The reaction mixture is preferably heated to about 65°C; and generally the reaction is allowed to proceed longer than when other nucleophiles are used, preferably for at least about 48 hours. The product can be purified by passing the reaction mixture through a SCX column and washing the product from the column with 20% NH₄ ⁺ / MeOH in methylene chloride. Thereafter, the product can be concentrated and further purified, redissolved and passed tlirough a silica plug, preferably using a mixed solvent such as 15% MeOH: CH₂C1₂.

Process for preparing compounds (I)

The process of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means. For small scale synthesis of multiple products, 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 each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel. Examples of solid supports include wellplates, silicone chips, or agar. Compounds can be added to the surface of the solid support by multiple delivery apparatus, automated or robotic apparatus, any of which may be either manually or computer controlled.

A preferred embodiment of the present invention is a diverse amine alcohol compound library in 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.

The process of making the library of amine alcohol compounds may be conveniently carried out in a conventional wellplate apparatus. 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 reservoirs 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, or reservoirs, 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.

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

Typically, from about 8 to about 800 diverse QASs are employed to synthesize a library of compounds using the method of the invention. Combinatorial techniques are preferably very robust to work well for highly diverse groups of reactants. In the diverse amine alcohol compound library making process described herein the reactant is used in excess. The method of the invention contemplates solution phase reactions where a stoichiometric excess of the nucleophile is used. The amount of Nu used to ensure an excess is defined as at least 1.1 (n) and preferably a larger excess in the range of from 1.25(n) to 5(n), where the variable (n) is as previously defined. The 1.1 multiplier is used to ensure at least a 10% stoichiometric excess of Nu to drive the reaction to completion, thereby removing the QAS from each reaction zone used to create the amine alcohol compound library. The reaction zone is maintained at a temperature and for a time sufficient to permit reaction of the first and second reagents, that is, to complete consumption of the QAS and form an amount of amine alcohol 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 reaction times of 1 second to 60 minutes 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 about -10°C and about 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., about 60 Kg./m² and about 2100 Kg./m² absolute), but ambient atmospheric pressure (about 10330 Kg./m², absolute) is most often used. Endpoint determination - The completion of the reaction between the QAS and Nu may be determined by a number of conventional techniques, including, but not limited to, chromatography. The preferred method for determining if the QAS is substantially removed from the reaction zones is thin layer chromatography.

Sequence of Operation - The addition of the first and second reagents to the first reaction zone may take place in any order. For example, Nu may be initially added to the reaction zone followed by addition of the QAS, or vice versa. Alternatively, the first and second reagents may be simultaneously charged to each reaction zone.

When necessary, a solid-supported scavenger can be added to the reaction mixture to bind the unreacted nucleophile. The amount of scavenger added to the reaction mixture is based on the scavenger's available functionality. The scavenger is added in at least an amount equal to the theoretical excess equivalents of unreacted isocyanate reactant.

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 second reagent. The reaction zone is maintained at a temperature for a time sufficient to permit reaction of the excess second reagent and the scavenger. Typically, the reaction requires only seconds but the selection of reaction conditions that may be used is the same as set out above. The separation of the solid supported scavenger from the library compound dissolved in the solvent phase of the reaction may be done by any conventional chemical or physical method. Preferred are physical methods which are applicable to all members of a diverse library. Such methods include, for example: (i) ion exchange chromatography (ii) filtration, (iii) centrifugation, (iv) decantation, and (v) washing, and combination thereof. Filtration and ion exchange chromatography are particularly preferred forms of purification.

The last 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.

The library of compounds formed using the process of the invention can be used to screen for compounds with biological activity. A myriad of biological assays are known in the art and can be used to screen the library of compounds. Illustrative assays include, but are not intended to be limited to In vitro assays such as Enzymatic Inhibition,

Receptor - ligand binding, Protein - protein Interaction, and Protein - DNA Interaction;

Cell based, Functional Assays such as Transcriptional Regulation, Signal Transduction /

Second Messenger, and Viral Infectivity; Add, Incubate & Read Assays such as Scintillation Proximity Assays (SPA), Fluorescence Polarization Assay, Fluorescence

Conelation Spectroscopy, Colorimetric Biosensors, Cellular Reporter Assays using reporter genes such as luciferase, green fluorescent protein, β-lactamase, and the like; and

Electrical cell impedance sensor assays.

All of the above assays are known to be predictive of success for an associated disease state.

EXAMPLES

The following example are provided to demonstrate the underlying chemistry and the processes of the present invention. These examples are not intended to limit the scope of the invention. Preparation of starting materials

Procedures for the Synthesis of Polymer Bound Reagents

Polymer Bound Isocvanate:

To a stirring solution of 50 g (61 mmol, 1 eq) of aminomethyl polystyrene (100-

200 mesh, 1% DVB, 1.16 mmol/g) (available from Advanced Chemtech, Louisville, KY) in 800 mL of anhydrous toluene was added in one portion 193 mL (366 mmol, 6.0 eq) of a 1.9 M solution of phosgene in toluene (available from Fluka Chemie AG, Switzerland).

The reaction was stirred 10 min. and then 67 mL (482 mmol, 7.9 eq) o triethylamine was added via syringe which immediately resulted in the formation of a white precipitate. The reaction was stirred overnight, filtered under a stream of nitrogen and washed with CH,C1₂ (1.0 L). The crude resin was added to 500 mL of CH₂C1₂, stirred 15 min. and filtered under a stream of nitrogen. This step was then repeated once more. The resin was then washed with ether (500 mL) and dried overnight in a vacuum oven with light heating (40-50°C) to give 49 g of a light yellow resin.

Polymer Bound Piperidine: A solution of 50 g (215 mmol, 1 eq) of chloromethyl polystyrene (Fluka Chemie

AG, 200-400 mesh, 2% DVB, 4.1 mmol/g), 6.25 g (45 mmol, 0.2 eq) of K₂C0₃ and 65 mL (888 mmol, 4.1 eq) of piperidine in 500 mL of anhydrous DMF was heated overnight at 90°C. The resin was filtered, washed with DMF (500 mL) and water (500 mL) and then stirred for 20 min. in 400 mL of water. After filtration the resin was washed with water and dioxane and then strrred for 20 min. in 400 mL of ethanol. The resin was filtered again and then sequentially washed with ethanol, THF and ether. After overnight drying at 55°C in vacuum oven a light yellow resin was obtained.

Polymer Bound Quaternary Ammonium-Hydroxide Form (AG 2X hydroxide form):

The hydroxide counter ion form of AG 1-X2 anion exchange resin (available from BioRad, Hercules CA) was prepared by slowly washing (10-20 mL/min) the commercially available chloride form with a large excess of IN sodium hydroxide (this operation was performed on a large sintered glass filter). The hydroxide form resin was then washed with water to remove any unbound sodium hydroxide.

QAS reagents can be prepared in the following manner: A solution of secondary amine (1 eq) and epichlorohydrin (1 eq) were heated at reflux overnight in MeOH. The reaction solvent was removed under vacuum and the residue was slurried in CH_^Cl, (or acetone) to give an off white precipitate which was collected by filtration and washed with CH₂C1₂. This material typically needs no further purification and is suitable for further synthesis.

Specific examples

A B QAS 3

Compound A (described in U.S. Pat. Nos. 5,627,196 and 5,576,321) was dissolved in MeOH in a 250 mL round-bottom flask equipped with a magnetic stirrer, nitrogen bubbler, heat mantle, thermometer/thermowatch, and condenser. NaOH was added, a slurry formed and was stirred for 10 minutes, after which the epichlorohydrin B was added. The slurry was heated to 50°C and stirred at this temperature for 16 hours. The heat was removed, the reaction mixture cooled, dried with Na^SO,,, filtered and then concentrated. CH₂C1₂ was added to the concentrate and the mixture was stirred for 2 hours at room temperature. The resulting crystalline solid was filtered, rinsed and dried at 45°C overnight (18 g, 90% yield). OAS 1

QAS 2

QAS 4

QAS 1, 2, and 4 were synthesized using analogous procedures starting from the appropriate secondary amines.

EXAMPLE 1 - Solution Phase Opening of QAS with Aromatic Thiol General procedure

A solution of quaternary azetidinium salt 1 (0.05 mmol, 1 eq), aromatic thiol (0.2 mmol, 4 eq) and polymer bound piperidine (0.3 mmol, 6 eq, 75 mg) in 2 mL of 50%_> MeOH / CH2CI2 were shaken overnight at ambient temperature in a 4 mL sealed vial. The reaction mixture was then heated at 65°C overnight, cooled and filtered through a pipette stuffed with cotton. This solution was placed directly onto an SCX ion exchange column and purified as previously stated. Average product yields and HPLC purities were typically on the order of >90%. This method was used to generate over 270 compounds by systematic substitution of the substituents or the reagents. Specific example

To a solution of thiophenol (41 mg, 0.375 mmol, 5.0 eq) and polymer bound piperidine (190 mg, 0.75 mmol, 10 eq) in CH₂C1₂ (2 mL) was added intermediate 1 (20 mg, 0.075, 1.0 eq). The reaction mixture was heated overnight at 65°C, filtered, loaded onto a SCX column (500 mg) and washed with methanol (3 x 3 mL). The product was eluted from the SCX column using three 2 mL portions of 20% 2 M NH₃ MeOH in CH₂C1₂. After concentration, 17.6 mg (69%) of the desired product was obtained in >99% purity by HPLC using UV detection (220 nm).

EXAMPLE 2 -- Solution Phase Opening of QAS with an Aromatic alcohol

General procedure

A solution of QAS (0.05 mmol, 1 eq), aromatic alcohol (0.25-0.35 mmol, 5-7 eq) and AG 2X hydroxide form resin (0.5-0.9 mmol, 10-20 eq, 150-250 mg) in 2 mL of toluene were heated for 48 h at 65°C in a 4 mL sealed vial. (While other solvents such as

THF and dioxane have been successfully used, toluene appears to be the best at preventing the fonnation of a diol side product.)

The reaction mixtures were cooled, filtered through a pipette containing cotton and then added directly onto an SCX ion exchange column. The purification on ion exchange was carried out as previously stated. The eluted samples were concentrated in a speed vac, redissolved in 1 mL of 15% MeOH in CH₂C1₂ and filtered through a silica gel column (500 mg, Varian, 3 mL column size) using 6-8 mL of the same solvent. Concentration in a speed vacuum gave average product yields of >80% with average purities >90%. In cases were the aromatic alcohol contains a basic group, causing it to stick to the SCX column as well as the product, a second column containing AG 2X hydroxide form resin was used. After the concentration of the eluted samples from the SCX columns, the samples were redissolved in 1-2 mL of MeOH and slowly run, via gravity, through a 1 cm x 4 cm column of AG 2X hydroxide form resin created by filling the narrow end of a BioRad wide mouth polypropylene column (cat. # 731-1550). The column was washed with 3 x 2 mL portions of MeOH (6 mL total) and the collected eluent was concentrated in a speed vacuum. These samples were then filtered through a silica gel column as previously described. Specific examples

To a solution of phenol (66 mg, 0.7 mmol, 10.0 eq) and AG 2X hydroxide form (240 mg, 0.7 mmol, 10 eq) in toluene (2 mL was added intermediate 2 (20 mg, 0.07 mmol, 1.0 eq). The reaction mixture was heated 48 h at 65°C and then loaded onto a SCX column (500 mg) and washed with methanol (3 x 3 mL). The product was eluted form the SCX column using 3 x 2 mL portions of 20% 2 M NH₃ MeOH in CH₂C1₂. After concentration, the resulting residue was redissolved in 15% MeOH/ CH₂C1₂ and passed through a silica gel plug (500 mg) using more of the same solvent (total 10 mL). 21.0 mg (94%) of the desired product was obtained in >99% purity by HPLC (15.8 min) using UV detection (220 nm).

EXAMPLE 3 - Solution Phase Opening of QAS with Aliphatic Thiols General procedure A mixture of a QAS (0.05 mmol, 1 eq), alkyl thiol (0.5 mmol, 10 eq) and AG 2X hydroxide form resin (0.50-1.0 mmol, 10-20 eq) in 2 mL of THF were heated at 65°C for 48 h in a 4 mL sealed vial and then filtered through a pipette containing cotton onto a SCX column (500 mg, LRC 10 L column, Varian). The column was washed with 4 x 2 mL of MeOH and the product eluted from the column using a 20% solution of 2 M NH₃- MeOH in CH2CI2. This method was used to generate over 200 compounds by systematic substitution of the substituents on the QAS reagents. The average yields were >95%, with average HPLC purities of >90%. Specific example

To a solution of p-chlorobenzyl mercaptan (60 mg, 0.375 mmol, 5.0 eq) and AG 2X hydroxide form ion exchange resin (214 mg, 0.75 mmol, 10 eq) in THF (2 mL) was added intermediate 1 (20 mg, 0.075 mmol, 1.0 eq). The reaction mixture was heated overnight at 65°C, filtered and loaded onto a SCX column (500 mg) and washed with MeOH (3 x 3 L). The product was eluted from the SCX column using 3 x 2 mL portions of 20% 2 M NH₃-MeOH in CH₂C1₂. After concentration, 29.6 mg (96%) of the desired product was obtained in >99% purity by HPLC (20.6 min) using UV detection (220 nm).

The compounds shown below can be synthesized using the process of the present invention. This list is intended to demonstrate the diversity of compounds which can be synthesized and is not intended to limit the nature of the process in any way.

It should be understood that a wide range of changes and modifications can be made to the embodiments described above. It is therefore intended that the foregoing description illustrates rather than limits this invention, and that it is the appended claims, including all equivalents, which define this invention.

Claims

CLAIMS:

1. A process for preparing a plurality of compounds of the formula (I):

R¹ OH

I N -

R2

where R' and R² are each, independently, an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; or

R' and R² together with the N atom which they substitute and, if appropriate, 1 to 4 further heteroatoms selected from NR R⁵, O and S, form a substituted or unsubstituted 3 to 10 membered mono-, bi- or tricyclic ring;

R⁴ and R⁵ are each, independently, hydrogen or an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; Nu is a negative ion or a molecule that has an unshared pair of electrons; wherein said process comprises the steps of: a) providing a quaternary azetidinium salt (QAS) of the fonrmla (2):

wherein R' and R² are defined above and Y is a counterion, in solution; b) providing a series of nucleophiles in solution; c) sequentially mixing said QAS with said series of nucleophiles in solution for a time sufficient to produce a plurality of products of the formula (I).

2. The process of claim 1, which further comprises the step (d) adding a resin bound scavenger or an ion exchange resin to the solution, wherein any excess unreacted nucleophile binds thereto.

3. The process of claim 2, wherein said scavenger is an isocyanate or aldehyde.

4. The process of claim 1, wherein step (c) is conducted in a solid phase array, such that each nucleophile is mixed with the QAS in a physically separated area.

5. The process of claim 4, wherein the solid phase array is a wellplate apparatus.

6. The process of claim 1, wherein the substituted or unsubstituted 3 to 10 membered mono-, bi- or tricyclic rings are selected from the group consisting of azetidine, aziridine, imidazole, indole, isothizaole, isoxazole, morphline, oxazole, piperidine, piperazine, purine, pyrazole, pyrrole, pyrrolidine, thiazole, and thiomorpholine.

7. A reaction vessel on which a plurality of compounds are physically separated from each other, wherein the compounds are of the formula (I):

where R' and R² are each, independently, an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; or R¹ and R² together with the N atom which they substitute and, if appropriate, 1 to 4 further heteroatoms selected from NR⁴R\ O and S, form a substituted or unsubstituted 3 to 10 membered mono-, bi- or tricyclic ring;

R⁴ and R⁵ are each, independently, hydrogen or an unsubstituted or substituted alkyl, alkenyl, cycloalkyl, aryl or heterocyclic ring; and

Nu is a negative ion or a molecule that has an unshared pair of electrons.

8. The reaction vessel of claim 7, which is a wellplate apparatus.

9. The reaction vessel of claim 8, which is a 96 well microtiter plate.

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

R¹ and R² together with the N atom which they substitute and, if appropriate, 1 to 4 further heteroatoms selected from NR⁴R⁵, O and S, form a substituted or unsubstituted 3 to 10 membered mono-, bi- or tricyclic ring; R⁴ and R⁵ are each, independently, hydrogen or an unsubstituted or substituted alkyl, alkenyl, cycloalkyl, aryl or heterocyclic ring; and Nu is a negative ion or a molecule that has an unshared pair of electrons.

11. A method of optimizing the biological activity of a compound which comprises the steps of contacting a solid support containing a plurality of compounds physically separated from each other with an assay kit, and determining if any of the plurality of compounds has biological activity, wherein the plurality of compounds are of the formula (I):

where R' and R² are each, independently, an unsubstituted or substituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl or heterocyclic ring; or R' and R² together with the N atom which they substitute and, if appropriate, 1 to 4 further heteroatoms selected from NR⁴R⁵, O and S, form a substituted or unsubstituted 3 to 10 membered mono-, bi- or tricyclic ring;

R⁴ and R⁵ are each, independently, hydrogen or an unsubstituted or substituted alkyl, alkenyl, cycloalkyl, aryl or heterocyclic ring; and Nu is a negative ion or a molecule that has an unshared pair of electrons.