WO1996030393A1

WO1996030393A1 - A method for the synthesis of mixtures of compounds

Info

Publication number: WO1996030393A1
Application number: PCT/US1995/016332
Authority: WO
Inventors: Katherine Becker; Sheila Hobbs Dewitt
Original assignee: Warner-Lambert Company
Priority date: 1995-03-27
Filing date: 1995-12-08
Publication date: 1996-10-03
Also published as: AU4424496A

Abstract

Described is a method of synthesizing a plurality of compounds in a plurality of wells comprising the steps of: (a) providing a plurality of test wells in a plurality of arrays of the wells; (b) reacting in at least a one step reaction a first reagent with a plurality of reagents called building blocks in the test well to obtain a unique product designed to be the same in each array; and (c) continuing to react reagents such that there are multiple reagents resulting in mixtures of multiple different products in each well.

Description

A METHOD FOR THE SYNTHESIS

OF MIXTURES OF COMPOUNDS

Technical Field

The invention relates to a method for the synthesis of mixtures of compounds and, in particular, the multiple simultaneous synthesis of compounds.

Background Art

It is well known in the art that peptides or oligonucleotides may be multiply and simultaneously synthesized. In a basic, single synthesis of a peptide, amino acids are sequentially coupled in solution or on a functionalized solid support. Several methods have been developed by which peptides or oligonucleotides may be multiply synthesized. One such methodology for peptide synthesis was disclosed in Geysen et al., International Publication No.g WO 90/09395. Geysen's method involves functionalizing the termini of polymeric rods and sequentially immersing the termini in solutions of individual amino acids. A second method of peptide or oligonucleotide synthesis was developed by Affymax Technologies N.V. and disclosed in U.S. Patent No. 5,143,854. The Affymax method involves sequentially using light for illuminating a plurality of polymer sequences on a substrate and delivering reaction fluids to said substrate.

A further method and device for producing peptides or oligonucleotides is disclosed in Houghton, European Patent No. 196174. Houghton' s apparatus is a polypropylene mesh container, similar to a tea-bag, which encloses reactive particles.

Combinatorial strategies for the synthesis of oligonucleotide mixtures are described in the paper by David J. Ecker, Timothy A. Vickers, Ronnie Hanecak, Vickie Driver and Kevin Anderson, entitled "Rational Screening of Oligonucleotide Combinatorial Libraries for Drug Discovery", NUCLEIC ACIDS RESEARCH, 1993, Vol. 21, No. 8, 1853-1856. The reference describes a technique known as synthetic unrandomization of randomized fragments which is based on repetitive synthesis and screening of increasingly simplified sets of oligonucleotide analogue pools. The technique is limited to oligonucleotides.

Another similarly limited approach is de- scribed in a paper by Colette T. Dooley and Richard A. Houghten, entitled "The Use of Positional Scanning Synthetic Peptide Combinatorial Libraries for the Rapid Determination of Opioid Receptor Ligands", LIFE SCIENCES, Vol. 52, pp. 1509-1517. This technique requires the resynthesis of potentially active individual components of the mixtures. Furthermore, this technique is limited to the sequentially constructed compounds such as peptides.

U.S. Patent No. 5,281,540 teaches a test array for performing assays. A semi-automated biological sample analyzer is described for simultaneously performing a plurality of enzyme immunoassays for human IgE class antibodies specific to a panel of preselected allergens in each of a plurality of biological samples. The technique, while having multiple biological samples in a well, uses a coating of an elongated cellulosic body such as a strip of paper which will contact the multiple samples to ascertain which antibodies are specific for the coated allergens and which will then, in turn, bind to the appropriate bands or islands. The bands or islands are then analyzed for the presence of labeled antibodies. The technique describes testing done in a seriatim basis, namely, a number of samples, one after the other, even though multiple samples are present in a reaction vessel. The use of antibodies which bind to a specific sample is required for the system to be effective. The samples may be detected by use of optical reading capabilities.

Other patents that test multiple compounds in a seriatim fashion utilizing automated equipment are described in U.S. Patent Nos. 4,039,286 and 4,166,095. In the journal, INTERNATIONAL JOURNAL OF PEPTIDE

PROTEIN RESEARCH, 37, 1991, 487-493 entitled "General Method for Rapid Synthesis of Multicomponent Peptide Mixtures", A. Furka et al., a method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle, the resin is divided into equal parts and each portion is coupled with a different amino acid. Then, the portions are mixed and before the next coupling cycle, the resin is again distributed into equal portions.

Another method is described in the journal,

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, U.S.A., Vol .12 , pp. 5131-5135, August 1985, Immunology. The paper by

R.A. Houghton is entitled "General Method for the Rapid

Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino Acids". The paper indicates that 248 different 1,3-residue peptides representing single amino acid variance of a segment of the hemoglobulin hemaglutinin (HAI) protein were prepared and characterized in approximately 4 weeks. Through examination of the binding of these analogs to monoclonal antibodies raised against residues of HA1, it was found that a single amino acid, aspartic acid at position 101, is of unique importance to the interaction.

Publication No. WO91/17823, published November 28, 1991, describes a method and apparatus for synthesizing biopolymers such as polypeptides and polynucleotides. The apparatus includes plural reaction vessels in which subunit coupling to biopolymers in a particle suspension is carried out. The vessels are connected to common valving structure for use in mixing the suspension and removing suspension liquid.

"Encoded combinatorial chemistry" by S. Brenner et al., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, U.S.A., Vol. 89, pp. 5381-5383, June 1992, describes a method for drug screening. A process of alternating parallel combinatorial syntheses was used to encode individual members of a large library of chemicals with unique nucleotide sequences. After the chemical entity is bound to a target, the genetic tag can be amplified by replication and utilized for enrichment of the bound molecules by serial hybridization to a subset of the library. The nature of the chemical structure bound to the receptor is decoded by sequencing the nucleotide tag. International Published Patent Application No. WO 9408711 A 1 (Warner Lambert Company) discloses an apparatus and method for multiple simultaneous synthesis. The patent indicates at column 14 that the final product solutions are combined, taking care to avoid cross-mixing. The individual solutions/extracts are then manipulated as needed to isolate the final compounds which compounds are individually tested for biologic activity. The problem that has arisen in the previous prior art techniques generally requires that the materials be physically separated or resynthesized for subsequent testing. Frequently, to ascertain the identification of active ingredients of testing requires a resynthesis of the original mixture.

It is an object of the present invention to accommodate multiple simultaneous synthesis of general organic compounds including, but not limited to, non-peptide or non-nucleotide compounds. It is another object of the present invention to synthesize mixtures of compounds, especially organic compounds. These mixtures may then be tested for activity, preferably biological activity without physical separation or resynthesis of unique products. It is also an object of the present invention after synthesis of unique compounds to be able to identify the precise unique compound that is prepared from the numerous compounds that are simultaneously prepared. It is a further object of the present invention to describe a method for synthesizing a plurality of compounds utilizing a plurality of arrays of reaction wells or test zones and to be able to ascertain the unique products produced in each test zone without separating the mixture.

Summary Of The Invention

Described is a method of synthesizing and analyzing a plurality of compounds in a plurality of wells comprising the steps of: (a) providing a plurality of test wells in a plurality of arrays of the wells, (b) reacting in at least a one step reaction a first reagent with a plurality of reagents called building blocks in the test well to obtain a unique product designed to uniquely occur in each array, (c) continuing to react the same or different reagents such that there are multiple reagents resulting in multiple different products in each well. Preferably, the synthesis is by solid phase or traditional solution phase methods. Brief Description Of The Drawings

FIGURE 1 shows the combinatorial statistics for 4, 5 or 6 building blocks (reagents) and the number of compounds that may be synthesized per well. Description OfPreferred Embodiments

The present invention is directed to a method which will provide a means to synthesize, analyze, and ascertain the individual components of the mixtures of organic compounds as they are synthesized. Additionally, the synthesis of inorganic compounds, organo metallic compounds, as well as other compounds, are within the scope of this invention.

Central to the demonstration of this technique is the need to devise a "general" method for synthesis, analysis, identification, and quantitiation of individual components of compound mixtures. The compounds may be synthesized simultaneously or sequentially and compatible with standard techniques of organic synthesis. Traditional solution phase or solid-phase techniques (commonly used in peptide or oligonucleotide synthesis) enable achievement of this criteria.

Typical solid supports (resins) include cross-linked divinylbenzene-styrene (polystyrene), controlled pore glass (CPG), polyacrylamides, poly (ethylene glycol) monomethyl ether and poly (ethylene glycol) (PEG), silica gel, cellulose, acrylic acid grafted polypropylene, and the like. Additionally, the solid support contains a reactive moiety. Thus, a functionalized solid support is an insoluble material containing an accessible reactive moiety such as, for example, a carboxylic acid, alcohol, amine, halomethyl, and the like which is used to covalently attach an incoming building block.

The approach described herein greatly increases the flexibility and diversity of structures that can be produced by a parallel, solid phase synthesis tech nology. Since neither the building blocks nor the methods for connecting them are in any way limited, the chemistries compatible with this method are very broad, encompassing nearly all organic reactions. In particular, the steps to synthesize permits one to construct orthogonally overlapped compound mixtures such that each unique product within a mixture of compounds is synthesized in "n" unique mixtures, where "n" represents the number of reaction steps in a synthetic group.

The steps utilized to synthesize mixtures of compounds are: 1) development of a synthetic route that will be feasible in solution or on a solid support, 2) utilize the "mixed resin" or subequimolar reagent approach to generate mixtures of products or intermediates, and 3) construct orthogonally overlapped compound mixtures such that each unique product within a mixture of compounds is synthesized in n unique mixtures, where n represents the number of reaction steps in a synthetic route. The steps possible to analyze mixtures of compounds are: 1) construction of a compound mixture which contain products each with unique molecular weights, 2) qualitative analysis of each mixture by mass spectrometry technique to confirm the presence of each unique product, 3) analysis of each mixture by 1H NMR (nuclear magnetic resonance) or other analytical methods and comparison of spectra of different mixtures of compounds (computationally by addition or subtraction of spectra) to identify and confirm the presence of each desired product, and 4) quantitate the concentration of each unique product in the compound mixture utilizing a standard in combination with the 1H NMR. The method involves the sequential coupling of building blocks to form soluble or resin-bound intermediates.

Reference is made to U.S. Patent No. 5,324,483 for the broad scheme outline for multiple simultaneous synthesis. This patent is incorporated by reference herein. The patent indicates a very detailed description of the synthesis of various materials described below. General Description Of Experimental Procedure

U.S. 5,324,483 description taught the synthesis of individual reaction products on a solid support. Extension of this method to the synthesis of mixtures would involve the simultaneous addition of multiple reagents at one or more steps during the synthesis. The reagents may be soluble or attached to a solid support.

As described in U.S. Patent No. 5,324,483, a number of reaction tubes equal to the total number of compounds to be synthesized by the array method are loaded with 1 to 1000 mg, preferably 100 mg, of the appropriate functionalized solid support, preferably 1 to 3% cross-linked polystyrene. The individual reaction tubes are inserted into a holder block. The reaction tubes, in combination with the holder block and a manifold, are inserted into the reservoir block so that each reaction tube is submerged in a volume, preferably 3 to 5 mL, of a solvent capable of swelling the polystyrene resin (such as, but not limited to, dichloromethane, chloroform, dimethylformamide (DMF), dioxane, toluene, tetrahydrofuran (THF), ethanol and the like). The polystyrene resin within the reaction tubes is preferably agitated for 15 to 30 minutes to affect swelling. Swelling times of 5 to 600 minutes, with or without agitation are also within the scope of the invention. The reaction tubes, in combination with the holder block and manifold, are removed from the reservoir block and the excess solvent within the reaction tubes is allowed to drain, preferably by gravity, although gas pressure applied to the manifold inlet (while closing the outlet) can be used to expel the solvent, if desired.

The reaction wells are emptied of solvent and the proper reactant solutions are dispensed into the new or clean reaction wells at appropriate locations in the reservoir block. If the reactant is actually one of the building blocks that is to become covalently attached to the growing compound on the solid support, the quantity of reactant is usually 1 to 100 equivalents based on the milliequivalents per gram (meq/g) loading of functionalized solid support (typically 0.1 to 1.0 meq/g for polystyrene resins) originally weighed into the reaction tube. Additional equivalents of reactants can be used if required to drive the reaction to completion in a reasonable time. The reaction tubes, in combination with the holder block and manifold, are reinserted into the reservoir block and the apparatus is fastened together. Gas flow through the manifold is initiated to provide a controlled environment, for example, nitrogen, argon, air, and the like. The gas flow may also be heated or chilled prior to flow through the manifold. Heating or cooling of the reaction wells is achieved by immersing the reservoir block in water baths, oil baths, isopropanol/dry ice baths, sand baths, and the like to perform synthetic reactions. Agitation is achieved by shaking, sonication (preferred), or magnetic stirring (within the reaction well or within the reaction tube). Reflux is achieved by circulating chilled gas through the manifold while heating the reaction wells in the reservoir block. Reactants may be injected directly into the reaction tubes through an injectable gasket on the top of the manifold. The reaction is allowed to proceed for an amount of time deemed necessary from the preliminary validation experiments or monitored by removal and quantitative analysis of filtrate aliquots from selected wells by methods such as GC/ISTD (internal standard) or HPLC/ISTD. If necessary, the complete assembly is allowed to return to ambient temperature, then the holder block and manifold, in combination with the reaction tubes, are detached from and raised above the reservoir block and the excess reagent solution is drained by gravity followed by gas pressure applied to the manifold inlet (while closing the outlet) to expel the excess reagents, solvents, and by-products. The resin-bound intermediate within each reaction tube is washed clean of excess retained reagents, solvents, and by-products by repetitive exposure to clean solvent (s) by one of two methods: 1) the reaction wells are filled with solvent (preferably 1-5 mL), the reaction tubes, in combination with the holder block and manifold, are immersed and agitated for 5 to 300 minutes, preferably 15 minutes, and drained by gravity followed by gas pressure applied through the manifold inlet (while closing the outlet) to expel the solvent; 2) the manifold is removed from the holder block, aliquots of solvent (preferably 5 mL) are dispensed through the top of the reaction tubes and drained by gravity through the filter into a receiving vessel such as a test tube or vial. Both of the above washing procedures are repeated 1 to 50 times (preferably 10 times), monitoring the efficiency of reagent, solvent, and byproduct removal by methods such as TLC, GC, or visualization of the wash filtrates.

The above described procedure of reacting the resin-bound compound with reagents within the reaction wells followed by removal of excess reagents, byproducts, and solvents is repeated with each successive transformation until the final or penultimate resin-bound compound is prepared.

Detachment of the final product from the solid support is achieved by immersion of the reaction tubes, in combination with the holder block and manifold, in reaction wells containing a solution of the cleavage reagent (preferably 3-5 mL). Gas flow, temperature control, agitation, and reaction monitoring are implemented as above and as desired to affect the detachment reaction. The reaction tubes, in combination with the holder block and manifold, are disassembled from the reservoir block and raised above the solution level but below the upper lip of the reaction wells and gas pressure is applied through the manifold inlet (while closing the outlet) to efficiently expel the final product solution into the reservoir wells. The spent resin in the reaction tubes is then washed 2 to 5 times as above with 3 to 5 mL of an appropriate solvent to extract (wash out) as much of the detached product as possible. The final product solutions are combined, taking care to avoid cross-mixing. The individual solutions/extracts are then manipulated as needed to isolate the final compounds. Typical manipulations include, but are not limited to, evaporation, concentration, liquid/liquid extraction, acidification, basification, neutralization or additional reactions in solution. The final compounds are individually tested for biological activity once they are isolated. For example, the method of Sweetnam, et al, Molecular Pharmacology 1986;29:299 employing bovine cortical membranes as a receptor source and a radiolabeled benzodiazepine, such as Flunitrazepam, to determine competitive ligand binding to the benzodiazepine receptor (central [brain] localized) and some quantitative measure of relative binding potency such as K_D or IC₅₀ values. Performing this type of assay serves two purposes: 1) to discover new compounds with biological activity in a given biological screening assay and 2) the development of a relationship between the structural variations contained within the series and biolog- ical potency. This second utility is known as development of a structure activity relationship (SAR). This type of assay can be done using the compounds isolated from the array synthesis for any receptor binding screen or assay (screening for receptor agonists or antagonists), enzyme functional assays (measuring competitive or noncompetitive inhibition of the catalyzed reaction), and the like.

This strategy may be used to screen for pharmaceutical agents, veterinary agents, agricultural agents, diagnostic reagents, and the like.

Typical compounds and pharmaceutical applications include: (l) nitrogen containing heterocyclic compounds; imidazopyridines having antiulcer or anxioly- tic activities, dihydropyridines having calcium antagonist activity, nucleoside and nucleoside analogs having antiviral activity, indazoles having 5HT₃ antagonist activity, piperidines having antidopamine, antiserotonin, antidepressant, or antihistamine activities, benzazepines having antiparkinsonism and antidopamine activities, indoles and condensed indoles with 5HT antagonist activities, quinolines and isoquinolines having anti-infective and antiulcer activities, pyrrolidines having anti-infective and antihypertensive activities, aminopyrimidines having antihypertensive activities, pyrrolizidines having antiarrhythmic activities, guanidines having anticancer activities, tetrazoles having antiallergenic; (2) oxygen containing heterocycles; benzopyrans having potassium agonist and antagonist activities, coumarins having antiplatelet aggregating and antithrombotic activities, prostaglandins and prostacyclins having antiplatelet, antiulcer, labor inducing activities, psoralens having antipsoriasis activities, tetrahydrofurans and pyrans having antidiabetic activity; (3) nitrogen and sulfur containing compounds; beta-lactams and cephalosporins having anti- infective activities; (4) carbocyclic compounds; tocopherol analogs having antipsoriasis activities, vitamin D analogs having antipsoriasis activities, steroids having anti-inflammatory, bronchiodilating, antihyperplasia and antifertility activities, naphthalenes having antifungal activities, anthracene analogs having anticancer activities; (5) alicyclic compounds; polyunsaturated alkenes having antithrombotic activities, hydroxypropanolamines having adrenergic blocking activities, benzofused bicyclic amines having analgesic activities, aryl amides having anesthetic, gastroprokinetic, antidepressant, and anti- inflammatory activities; and (6) cyclic peptides and cyclic nucleotide having anti-infective and antiautoimmune activities; and the like.

In alternative uses, the apparatus and methods may be implemented without using a solid support, for example, standard solution techniques. An additional alternative use of the apparatus is for the optimization of chemical reactions, for example, reaction yields or reaction times, on solid supports or in solution. This use is accomplished by performing the same reaction in all reaction well locations but systematically determining and varying dependent reaction variables across the array. For example, the reagent concentration and/or reagent equivalency (mole percentage) can be varied from near zero to the maximum achievable with an undiluted reagent. Typically, one varies this variable from 0.001 to 25 mol. Reaction times can be varied within the array by withdrawal of the contents of the reaction well, followed by for example workup, and/or isolation, and/or purification, and/or quantitation of the final solution. Statistical experimental design strategies or quantitative structure activity relationship (QSAR) strategies may also be implemented to select a subset of locations within the original array which will provide the necessary information for final analysis and conclusions, therefore reducing the number of reactions necessary in the final array.

Synthesis of Dipeptides In the operation of the present invention, the synthesis of dipeptides is achieved using the apparatus and applying 9-fluorenylmethyloxycarbonyl (FMOC) strategy, as taught in Meienhofer, et al, INTERNATIONAL JOURNAL PEPTIDE PROTEIN RESEARCH 1979; 13: 35 and Atherton, et al, BIOORGANIC CHEMISTRY, 1979; 8:351 on a variety of commercially available polystyrene resins (see Table 1). For peptide acids the p-benzyloxy benzyl alcohol cross-linked divinylbenzene-styrene (WANG) resin, as taught in Wang, JOURNAL AMERICAN CHEMISTRY SOCIETY 1973; 95:1328, is utilized. Eight FMOC amino acid resins including phenylalanine, glycine, alanine, isoleucine, leucine, proline, valine, and tryptophan are deprotected, reacted with either FMOC alanine or FMOC isoleucine, deprotected again, then cleaved from the resin to generate 16 discrete dipeptides. A total of 3 to 20 mg (28-85%) of each crude dipeptide is isolated as the trifluoroacetic acid (TFA) salt and analyzed by HPLC, mass spectroscopy (MS), and proton nuclear magnetic resonance spectroscopy (¹H NMR) (see Table 1).

Synthesis of Hydantoins

The synthesis of an array of 40 hydantoins, including phenytoin, is achieved using the apparatus described herein. Five samples of eight FMOC or BOC protected amino acid resins (phenylalanine, glycine, isoleucine, leucine, alanine, valine, tryptophan, and 2,2-diphenylglycine) are deprotected then separately reacted with five isocyanates (trimethylsilyl, n-butyl, allyl, 2-trifluorotolyl, or 4-methoxyphenyl isocyanate) followed by treatment with aqueous 6N hydrochloric acid (HCl) to generate 40 discrete hydantoins (see Scheme 2 and Table 2).

The synthesis of 40 hydantoins in an array illustrates a standard protocol for a typical parallel array synthesis. In this example, a robotic sample processor (Tecan^® 5032) is used for every liquid sample handling step. Some of the key features which demonstrate the strengths, flexibility and scope of the method and apparatus are highlighted below. Forty BOC or FMOC protected resin-bound amino acids are weighed into the reaction tubes, assembled in the apparatus, and simultaneously deprotected employing GC/ISTD calibration methods to determine the completion of the FMOC deprotection reactions. The ability to simultaneously perform different reactions with vastly different reagents (for example, piperidine, DMF and TFA) further demonstrates the flexibility of the apparatus. Residual solvents, reactants, or byproducts are removed by wash cycles which include submersion and sonication of the reaction tubes in a series of solvents, followed by robotic spotting of the filtrates on a TLC plate and observation of the results under ultraviolet (UV) light to insure the thoroughness of the wash cycles. Following the wash cycles, the resin-bound amines are reacted with the desired isocyanates in DMF. Again, GC/ISTD calibration methods are used to quantitatively monitor the uptake of the isocyanates. After washing, the resin-bound ureas are cyclized by heating in aqueous 6N HCl to produce the desired hydantoins. Following the HCl treatment, the spent resins in the reaction tubes are washed with methanol to completely extract the hydantoins from the resins. All filtrates are combined, concentrated on a commercial centrifugal vacuum system

(SpeedVac^®), weighed, and analyzed to yield the expected hydantoins, in all but one example (see Table 2). A total of 0 to 11.5 mg corresponding to 0 to 81% yield of each crude hydantoin is isolated and analyzed by TLC, MS, and ¹H NMR.

Synthesis of Benzodiazepines and Biological Testing

The investigation of an array synthesis of benzodiazepines as potential targets was prompted by a brief communication by Camps, et al, ANALES DE QUIMICA

1974; 70: 848 who reported a one-step synthesis of several benzodiazepines starting with a resin-bound amino acid. A two-step route is outlined in Scheme 3. The first step in the sequence is the formation of an imine between a resin-bound amino acid and a 2-amino-benzophenone. Initially, a number of condensation methods were explored, but all proved unsatisfactory. Replacement of the imine condensation with a transimination reaction, as taught by O'Donnell and Polt, JOURNAL ORGANIC CHEMISTRY 1982 ,-47:2663, to form a mixture of E and Z imine isomers proved satisfactory. The resin-bound amino acid imines are then converted to the corresponding benzodiazepines by heating in TFA.

The synthesis of 40 benzodiazepines in the array is similar to that outlined for the dipeptides and hydantoins with several modifications, which demonstrate the flexibility of both the method and the apparatus (see Scheme 3 and Table 3). Five amino acid Merrifield resins (alanine, glycine, 2-bromobenzyloxycarbonyltyrosine, tryptophan, and valine) as their TFA salts are separately reacted with eight 2-aminobenzophenone imines. The reactions are not monitored, but are reacted long enough to accommodate the slowest example (valine resin with N-isopropyl 2 -amino-4-methylbenzophenone imine) based on previous validation studies. Following wash cycles to extract the unreacted imine, the resin bound imines are heated in TFA for a time sufficient to ensure cyclization of the slowest examples (imines of N-methyl 2-amino-5-nitro-benzophenone), while minimizing decomposition of the tryptophan derived benzodiazepines. When the reaction is complete, the spent resins in the reaction tubes are washed and the combined filtrates concentrated to dryness. In this case, an aqueous bicarbonate work-up is implemented (utilizing the Tecan^® 5032 robotic sample processor) to remove residual TFA. For each reaction tube the corresponding organic extracts are combined, dried, and concentrated to yield the expected benzodiazepines in all but one example (see Table 3). The 40 (39 desired) products are characterized by TLC, ¹H-NMR, and MS. The crude yields range from 7 to >100% and the estimated purities from NMR and TLC are greater than 60% in most cases.

To verify that the compounds produced could be used directly in a biological assay, the crude benzodiazepines are tested for activity in a benzodiazepine receptor binding assay without further purification. (The assay was performed using the commercially available NovaScreen^® assay system contracted by Scios-Nova Pharmaceutical Corp., Baltimore, MD, whereby bovine cortical membrane preparations were used as the source of receptor and the radioligand employed was [³H] Flunitrazipam while the positive control was Clonazepam.) The calculated IC₅₀'s from three concentrations (average of two determinations) for the individual compounds are shown in Table 3.

The following nonlimiting examples are offered by way of illustration and are not intended to limit the invention in any manner. Example 1

Synthesis of Dipeptides

The synthesis of 16 dipeptides is summarized in Table 1. 101 to 153 mg each of eight FMOC protected amino acids (phenylalanine, glycine, alanine, isoleucine, leucine, proline, valine, and tryptophan) on a commercially available WANG resin (loading = 0.37-0.60 meq/g, 200-400 mesh) was measured into each of 16 gas dispersion tubes (reaction tubes). The reaction tubes were fitted into the holder block with two gaskets, one above and one below the holder block. The array of reaction tubes was fitted into a matching array of reaction wells in an appropriate reservoir block, in this case a test tube rack, and 3 mL of DMF was dispensed through the aperture at the top of the reaction tubes to wash any residual resin to the bottom of the glass frit. The manifold was fitted over the holder block and a nitrogen atmosphere was initiated through the ports on the manifold. The apparatus was then agitated in a sonic bath for 15 minutes to swell the resin support in preparation for the first reaction. Following swelling of the resin, the holder block and the manifold, in combination with the reaction tubes, were raised above the reservoir block and the reaction tubes were allowed to drain by gravity.

To deprotect the FMOC amino acids, the reaction tubes were submerged in reaction wells containing 2 mL of a solution of 22% piperidine in DMF

(v/v) with an internal standard (e.g., anthracene at

1.97 mg/mL). The apparatus was fastened together with clamps and agitated in a sonic bath for 2 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress was monitored by removing a sample of the filtrate (10-100 μl ) and analyzing for the FMOC-piperidine adduct and dibenzofulvene by GC/ISTD calibration methods. At the end of the reaction, the holder block and the manifold, in combination with the reaction tubes, were raised above the reservoir block and the reaction tubes were allowed to drain by gravity, then sparged with nitrogen. The reaction tubes were sparged by introducing nitrogen into one port of the manifold and closing off the second port.

The reaction tubes were subjected to a series of wash cycles to remove residual solvents, reagents, and byproducts. A standard protocol included sequentially submerging the reaction tubes in 2 to 5 mL each of 2 x DMF, 2 × methanol, 2 × water, 2 x water:dioxane

(1:1), 2 × dioxane, and 2 × DMF, followed by agitation by sonication for 10 to 15 minutes, and finally draining by gravity and nitrogen sparge. The efficiency of the wash cycles was monitored by TLC or GC/ISTD of the wash filtrates.

In order to couple the FMOC amino acids, the following DMF solutions were sequentially admixed in two 8-unit reservoir blocks, to generate the activated esters: 1.0 mL of 0.46 M FMOC alanine or FMOC isoleucine , 0 .5 mL of 1. 02 M Benzotriazol - 1 -yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 1.0 mL of 1.02 M N-Hydroxybenzotriazole (HOBT), and 0.5 mL of 1.84 M N-methylmorpholine. The apparatus was assembled as above and agitated on a rotational platform shaker for 18 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress was monitored by removing a sample of the resin following the wash cycles and colorometrically analyzing for primary amines (Bromophenol blue or Kaiser/Ninhydrin). At the end of the reaction, the holder block and the manifold, in combination with the reaction tubes, were raised above the reservoir block and the reaction tubes were allowed to drain by gravity then sparged with nitrogen.

The reaction tubes were subjected to the same series of wash cycles as previously set forth. The deprotection of the above formed FMOC protected dipeptides was repeated following the same procedure as previously set forth.

The reaction tubes were again subjected to the same series of wash cycles as previously set forth. In order to cleave the product from the solid support, the reaction tubes were submerged in reaction wells containing 3 mL of a solution of anisole:TFA

(5:95). The apparatus was sealed with clamps and agitated in a sonic bath, while maintaining a positive nitrogen flow through the manifold. After 3 hours, the holder block and the manifold, in combination with the reaction tubes, were raised above the reservoir block and the reaction tubes were allowed to drain by gravity then sparged with nitrogen. To isolate and purify the products, a holder block was fitted with disposable glass pipets which were submerged in the TFA filtrates. The filtrates were concentrated in a well-ventilated hood or glove box fitted with a scrubber by initiating and maintaining a positive, subsurface nitrogen flow through the manifold. The TFA salt was dissolved in 2 mL of water and extracted with 2 mL of ethyl acetate. The reaction reservoirs were vortexed to affect efficient extraction and the upper organic layer was withdrawn via syringe. The extraction cycle was repeated until the organic layer contained no residues as determined by TLC or GC/ISTD (3 times). The aqueous layers were concentrated on a Speed-Vac^® in tared vials. The dipeptides generated in the apparatus including, alanylphenylalanine; alanylglycine; alanylisoleucine; alanylleucine; alanylalanine; alanylproline; alanylvaline; alanyltryptophan; isoleucylphenylalanine; isoleucylglycine; isoleucylisoleucine; isoleucylleucine; isoleucylalanine; isoleucylproline; isoleucylvaline; and isoleucyltryptophan, are set forth in Table 1.

Example 2 Synthesis of Hydantoins

The synthesis of 40 hydantoins is summarized in Scheme 2 and Table 2. 95 to 105 mg each of seven FMOC protected amino acids (phenylalanine, glycine, isoleucine, leucine, alanine, valine, and tryptophan) on a commercially available WANG resin (loading = 0.37 to 0.60 meq/g, 200 to 400 mesh) and 95 to 105 mg of BOC protected diphenylglycine loaded on a commercially available cross-linked hydroxymethyl divinylbenzene-styrene resin (loading = 1.04 meq/g, 200-400 mesh) was measured into 40 reaction tubes. The apparatus was assembled, swelled with 4 mL of DMF, and drained as set forth in Example 1.

To deprotect the FMOC amino acids, the appropriate reaction tubes were submerged in reaction wells containing 3 mL of a solution of 25% piperidine in

DMF (v/v) with an internal standard (anthracene at 1.74 mg/mL). To deprotect the BOC amino acids the appropriate reaction tubes are submerged in wells containing 3 mL of 50% TFA/DMF. The apparatus was assembled as set forth in Example 1 and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold. The reaction progress was monitored by removing a sample of the filtrate (e.g. 10-100 μl) and analyzing for the FMOC-piperidine adduct and dibenzofulvene by GC/ISTD calibration methods. The reaction was complete after 6 hours in a sonic bath. At the end of the reaction, the reaction tube array was drained and sparged as set forth in Example 1.

The reaction tubes were subjected to the standard wash cycle set forth in Example 1.

To facilitate urea formation, the appropriate reaction tubes were submerged in reaction wells containing 3 mL of a solution of 0.19 to 0.23 M isocyanate (trimethylsilyl isocyanate, butyl isocyanate, allyl isocyanate, trifluoro-o-tolyl isocyanate, and 4-methoxyphenyl isocyanate) in DMF, containing an internal standard (e.g., anthracene at 1.89-2.00 mg/mL). The apparatus was sealed as set forth in Example 1 and agitated in a sonic bath for 6 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress was monitored by removing a sample of the filtrate (10-100 μl ) , derivatization with an appropriate amine or alcohol, and analysis by GC/ISTD calibration methods. At the end of the reaction, the reaction tube array was drained and sparged as set forth in Example 1. The reaction tubes were subjected to the standard wash cycle set forth in Example 1. To facilitate cleavage of the final product from the solid support, the reaction tubes, in combination with the holder block and manifold, were submerged in reaction wells containing 3 mL each of 6N HCl. The apparatus was sealed with clamps and submerged in an oil bath (at 105°C) and heated at 85 to 100°C while maintaining a positive chilled nitrogen flow through the manifold. (The chilled nitrogen flow through the manifold was affected by submersion of the nitrogen inlet tubing in an isopropanol/dry ice bath.) After 2 hours, the reaction tube array was cooled, drained and sparged as set forth in Example 1.

To isolate and purify the products, the reaction tubes, in combination with the holder block and manifold, were submerged in reaction wells containing 3 mL each of methanol . The apparatus was agitated in a sonic bath for 10 to 15 minutes to extract the hydantoins from the resins then drained by gravity and nitrogen sparge. The methanol extraction protocol was repeated until the filtrates were free of any organic components as determined by TLC (4 times). The HCl and methanol filtrates were concentrated on a Speed-Vac^® in tared vials to afford 39 of the 40 desired hydantoins. The hydantoins generated in the apparatus, including: 5-methyl-2,4-imidazolidinedione; 5-(phenylmethyl)-2,4-imidazolidinedione; 2,4-imidazolidinedione; 5- (1-methylpropyl)-2,4-imidazolidinedione; 5- (2-methylpropyl) -2,4-imidazolidinedione; 5-(1-methylethyl)-2,4-imidazolidinedione; 5-(1H-indol-2-ylmethyl)-2,4-imidazolidinedione; 5,5-diphenyl-2,4-imidazolidinedione; 3-butyl-5-methyl-2,4-imidazolidinedione; 3-butyl-5-(phenylmethyl)-2,4-imidazolidinedione; 3-butyl-2,4-imidazolidinedione, 3-butyl-5-(1-methylpropyl)-2,4-imidazolidinedione; 3-butyl-5-(2-methylpropyl)-2,4-imidazolidinedione; 3-butyl-5-(1-methylethyl)-2,4-imidazolidinedione; 3-butyl-5-(1H-indol-2-ylmethyl)-2,4-imidazolidinedione; 3-butyl-5,5-diphenyl-2,4-imidazolidinedione; 5-methyl-3-(2-propenyl)-2,4-imidazolidinedione; 5-(phenylmethyl)- 3-(2-propenyl)-2,4-imidazolidinedione; 3-(2-propenyl)-2,4-imidazolidinedione; 5-(1-methylpropyl)-3-(2-propenyl)-2,4-imidazolidinedione; 5-(2-methyl-propyl)-3-(2-propenyl)-2,4-imidazolidinedione; 5-(1-methylethyl)-3-(2-propenyl)-2,4-imidazolidinedione;

5- (1H-indol-2-ylmethyl) -3- (2-propenyl) -2,4-imidazolidinedione; 5,5-diphenyl-3-(2-propenyl)- 2,4-imidazolidinedione; 5-methyl-3-[2-(trifluoromethyl)-phenyl]-2,4-imidazolidinedione; 5-(phenylmethyl)-3-[2-(trifluoromethyl)phenyl]-2,4-imidazolidinedione;

3-[2-(trifluoromethyl)phenyl]-2,4-imidazolidinedione; 5-(1-methylpropyl)-3-[2-(trifluoromethyl)phenyl]-2,4-imidazolidinedione; 5-(2-methylpropyl)-3-[2-(trifluoromethyl)phenyl]-2,4-imidazolidinedione; 5-(1-methylethyl)-3-[2-(trifluoromethyl)phenyl]- 2,4-imidazolidinedione; 5-(1H-indol-2-ylmethyl)- 3-[2-(trifluoromethyl)phenyl]-2,4-imidazolidinedione; 5,5-diphenyl-3- [2-(trifluoromethyl)phenyl] -2,4-imidazolidinedione; 3-(4-methoxyphenyl)-5-methyl-2,4-imidazolidinedione; 3-(4-methoxyphenyl)- 5- (phenylmethyl) -2,4-imidazolidinedione; 3- (4-methoxyphenyl)-2,4-imidazolidinedione; 3-(4-methoxyphenyl)-5-(1-methylpropyl)-2,4-imidazolidinedione; 3-(4-methoxyphenyl)-5-(2-methylpropyl)-2,4-imidazolidinedione; 3-(4-methoxyphenyl)-5-(1-methyl¬ethyl)-2,4-imidazolidinedione; 5-(1H-indol-2-ylmethyl)-3- (4-methoxyphenyl)-2,4-imidazolidinedione; 3-(4-methoxyphenyl)-5,5-diphenyl-2,4-imidazolidinedione; are set forth in Table 2. Example 3

Synthesis of Benzodiazepines

The synthesis of 40 benzodiazepines is summarized in Scheme 3 and Table 3. Five commercially available BOC amino acid

Merrifield resins (alanine, glycine, 2-bromobenzyloxy carbonyl- tyrosine, tryptophan, and valine; 0.57-0.89 meq/g, 200-400 mesh) were deprotected in bulk (1-5 g) instead of within the apparatus, using TFA:CH₂Cl₂ (1:1) at room temperature overnight. After washing with dioxane and CH₂Cl₂, the resins were dried under vacuum and used directly.

99 to 107 mg of each amino acid resin prepared above, as its TFA salt, was loaded into 40 reaction tubes. The apparatus was assembled, swelled with 3 mL of CH₂Cl₂, and drained as set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of the appropriate 2-aminobenzophenoneimines (3-6 eq) in 3 mL of dichloroethane (see Scheme 2). The apparatus was sealed as set forth in Example 1 and heated at 60°C (oil bath temp.), while maintaining a positive chilled nitrogen flow through the manifold. In this case the reactions were not monitored, but were reacted for sufficient time (24 hours) to drive the slowest reaction (valine resin with N-isopropyl 2-amino-4-methyl benzophenone imine) to completion based on earlier validation studies. The reaction tube array was allowed to cool and then drained and sparged as set forth in Example 1. In this example, the reaction tubes were washed by adding 4 mL portions of CH₂Cl₂ through the top of each reaction tube repeatedly (12 times) until the washes were no longer colored (indicating the absence of 2 -amino benzophenone imine or its corresponding ketone from hydrolysis).

The reaction tube array was then sparged as set forth in Example 1 and the reaction tubes are submerged in reaction wells containing 3 mL of 100% TFA. The apparatus is sealed as set forth in Example 1 and heated at 60°C (oil bath temp.) for 20 hours, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. Again, the reactions were not monitored, but were allowed to react for a time sufficient to ensure conversion of the slowest reactions (N-methyl2-amino-5-nitro benzophenone imine), while minimizing decomposition of the tryptophan derived benzodiazepines. The reaction tube array was allowed to cool and then drained as set forth in Example 1.

The reaction tubes are then washed as above with 3 × 2 mL portions of CH₂Cl_2.

The appropriate washes are then combined and evaporated under a stream of nitrogen as set forth in Example 1, except in this case the pipet tips were maintained above the surface of the liquid. A simple extraction procedure was implemented using the Tecan^® robot. The residues from evaporation were dissolved in 3 mL CH₂Cl₂ and mixed with 3 mL saturated sodium bicarbonate. The organic phase was withdrawn and the aqueous layer was extracted twice more with 1.5 mL of CH₂Cl₂. The combined organic extracts were dried with MgSO₄, filtered, and concentrated as before to yield the expected benzodiazepines in all but one example (see Table 3). The 40 (39 desired) products were characterized by TLC, ¹H-NMR, and MS. The crude yields range from 4 to >100% and the estimated purities from

NMR and TLC are greater than 60% in most cases. The desired products including: 1, 3-dihydro-3-methyl- 5-phenyl-2H-1,4-benzodiazepin-2-one; 7-chloro- 1,3-dihydro-3-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-5-(4-methoxyphenyl)-3-methyl-2H- 1,4-benzodiazepin-2-one; 1,3-dihydro-3-methyl-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro- 1-isopropyl-3,6-dimethyl-2H-1,4-benzodiazepin-2-one; 7-nitro-1,3-dihydro-1,3-dimethyl-5-phenyl-2H-1,4-benzodiazepin-2-one; 5-cyclohexyl-1,3-dihydro- 3-methyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro- 3-methyl-5-(2-thienyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one; 7-chloro-1,3-dihydro-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-5-(4-methoxyphenyl)-2H- 1,4-benzodiazepin-2-one; 1,3-dihydro-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-1-isopropyl-6-methyl-2H-1,4-benzodiazepin-2-one; 7-nitro- 1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one; 5-cyclohexyl-1,3-dihydro-2H-1,4-benzodiazepin- 2-one; 1,3-dihydro-5-(2-thienyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(4-hydroxyphenyl)-5-phenyl-2H- 1,4-benzodiazepin-2-one; 7-chloro-1,3-dihydro- 3-(4-hydroxyphenyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-5-(4-methoxyphenyl)-3-(4-hydroxyphenyl)-2H- 1,4-benzodiazepin-2-one; 1,3-dihydro-7-nitro- 3-(4-hydroxyphenyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-1-isopropyl-6-methyl-3-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one; 7-nitro-1,3-dihydro-1-methyl-3-(4-hydroxyphenyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 5-cyclohexyl-1,3-dihydro-3-(4-hydroxyphenyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro- 3-(4-hydroxyphenyl)-5-(2-thienyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1H-indol-2-ylmethyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 7-chloro-1,3-dihydro-3-(1H-indol-2-ylmethyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1H-indol-2-ylmethyl)-5-(4-methoxyphenyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1H-indol- 2-ylmethyl)-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1H-indol-2-ylmethyl)-1-isopropyl- 6-methyl-2H-1,4-benzodiazepin-2-one; 7-nitro- 1,3-dihydro-3-(1H-indol-2-ylmethyl)-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one; 5-cyclohexyl-1,3-dihydro-3-(1H-indol-2-ylmethyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1H-indol-2-ylmethyl)-5-(2-thienyl)-2H- 1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1-methylethyl)-5-phenyl-2H-1,4-benzodiazepin-2-one; 7-chloro- 1,3-dihydro-3- (1-methylethyl) -5-phenyl-2H-1,4-benzodiazepin-2-one; 1, 3-dihydro- 5- (4-methoxyphenyl) -3- (1-methylethyl) -2H- 1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1-methylethyl)-7-nitro-5-phenyl-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-1-isopropyl-6-methyl-3-(1-methylethyl)-2H-1,4-benzodiazepin-2-one; 7-nitro-1,3-dihydro-1-methyl-3-(1-methylethyl)-5-phenyl-2H-1,4-benzodiazepin-2-one;

5-cyclohexyl-1,3-dihydro-3-(1-methylethyl)-2H-1,4-benzodiazepin-2-one; 1,3-dihydro-3-(1-methylethyl)-5-(2-thienyl)-2H-1,4-benzodiazepin-2-one; are set forth in Table 3. Example 4

Synthesis of Quinolones

The synthesis of 36 quinolones is summarized in Scheme 4 and Table 4. Thirty-six gas dispersion tubes are loaded each with 100 ± 5 mg of hydroxymethyl polystyrene resin (1% cross-linked, 1.0 meq/g, loading) and placed in the holder block. The apparatus is assembled, swelled with 3 mL of CH₂Cl₂, and drained as set forth in Example 1.

To three sets of 12 reaction wells within the array is added 2 mL of a 0.2 M CH₂Cl₂ solution of either 3-(2,4,5-trifluorophenyl)-3-oxo-1-propanoic acid, 3-(2,3,4,5-tetrafluorophenyl)-3-oxo-1-propanoic acid, or 3-(2,4-dichloro-5-fluoropyridyl)-3-oxo-3-propanoic acid. An additional 1 mL of a CH₂Cl₂ stock solution containing 0.4 M dicyclohexylcarbodiimide and 0.4 M 1-hydroxybenzotriazole is added to all 36 reaction wells of the array and the appropriate reaction tubes are submerged in the corresponding reaction wells. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 12 hours, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is then washed using CH₂Cl₂ (10 times) as set forth in Example 3.

Into every well of the array is added 3 mL of a solution of acetic anhydride/triethyl orthoformate (1:1 v/v) and the apparatus is sealed as set forth in Example 1. The apparatus is then heated at 150°C (oil bath temp.) for 3 hours, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using CH₂Cl₂ (10 times, 3 mL.)

To three sets of 12 reaction wells within the array is added 3 mL of a t-butanol solution 0.13 M in both ethyl amine and t-butoxide, or cyclopropyl amine and t-butoxide, or 2,4-difluoroaniline and t-butoxide. The apparatus is reassembled as set forth in Example 1, immersed in an oil bath (60°C) and heated for 12 hours as set forth in Example 3. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1.

The reaction tubes are then washed as set forth in Example 3, using CH₂Cl₂ (12 times, 3 mL.) To four sets of nine reaction wells within the array is added 3 mL of a 0.26 M acetonitrile solution of either piperazine, piperidine, N-methyl piperazine, or 3- (ethylaminomethyl) pyrrolidine. The apparatus is assembled as set forth in Example 1, and heated in an oil bath (80°C) for 4 hours as set forth in Example 3. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1.

The reaction tubes are then washed as set forth in Example 3, using CH₂Cl₂ (12 times, 3 mL.) The reaction tubes are submerged in reaction wells containing 3 mL of 2N NaOH in 1:1 dioxane :water. The apparatus is assembled as set forth in Example 1, and allowed to stand at room temperature for 6 hours to affect ester hydrolysis. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are then washed as set forth in Example 3, using 3 x 3 mL of 0.1N NaOH in dioxane:water (1:1). The filtrates are then combined and the dioxane is removed by evaporation as set forth in Example 3. The resulting aqueous solutions are acidified with 5 N HCl solution and evaporated to give the 36 individual expected crude quinolones. Example 5

Synthesis of Keto-ureas

The synthesis of eight keto-ureas is summarized in Scheme 5 and Table 5.

100 g of Merrifield' s chloromethyl polystyrene resin (1-3% cross-linked, loading = 0.78 meq/g, 200-400 mesh) is heated for 20 hours at 150-155°C in 500 mL of DMSO with 5 eq of NaHCO₃ to form the resin-bound benzaldehyde. The resin is filtered and washed sequentially with excess DMSO, hot water, dioxane:water (2:1), water, dioxane, acetone, ethanol, CH₂Cl₂, and benzene. The dried aldehyde resin is then reacted with O-methylhydroxylamine hydrochloride (7.5 eq) and pyridine (7.8 eq) in refluxing ethanol to form the resin-bound oxime, which is washed with excess MeOH and CH₂Cl₂, and then directly reduced with sodium cyanoborohydride (10 eq) in EtOH saturated with HCl at room temperature for 3 hours using methyl orange as an indicator and adjusting the pH to 3.1 with additional EtOH/HCl. The resin is washed sequentially with excess MeOH, hot water, dioxane:water (1:1), dioxane, and CH₂Cl₂ and used directly below.

100 mg of O-Methyl hydroxylamine resin from above is measured into each of the eight gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of CH₂Cl₂, and drained as set forth in Example 1.

To facilitate coupling of the Boc-amino benzoic acids, the appropriate reaction tubes are submerged in reaction wells containing 3 mL of a solution of 0.08 M diisopropylethylamine, 0.16 M BOP

(Benzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), and 0.08 M Boc-amino benzoic acid

(either 3-amino-4-chlorobenzoic acid or 4-amino-

3-hydroxybenzoic acid) in CH₂Cl_2. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 8 hours while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 4 mL of sat.

HCl-Ether:dioxane (2:1). The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for

2 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 3 mL of 0.16 M phenyl isocyanate or ethyl isocyanate in dioxane with an internal standard (e.g., anthracene at 2-4 mg/mL). The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 6 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress is monitored by GC/ISTD as set forth in Example 1. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

In order to cleave the product from the solid support, the reaction tubes are submerged in reaction wells containing a solution of 3 mL of dry CH₂Cl₂ at

-78°C. The apparatus is sealed as set forth in

Example 1 and placed in an isopropanol/dry ice bath while maintaining a positive chilled nitrogen flow through the manifold. A solution of either methylmagnesium bromide 1.4 M in toluene/tetrahydrofuran or phenylmagnesium bromide 1.0 M in tetrahydrofuran

(0.2 mL) is added to the top of the appropriate reaction tubes through the gasket at the top of the manifold and the apparatus is sonicated at -78°C (maintained at -10°C by circulating chilled fluid through the sonic bath). After 2 hours, 10% HCl (0.5 mL) is added to each reaction tube through the gasket at the top of the manifold and the apparatus is allowed to warm to room temperature. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

To isolate and purify the products, the reaction tubes are washed as set forth in Example 1, using 3 mL portions of methanol. The methanol extraction protocol is repeated until the filtrates are free of any organic components as determined by TLC (1-2 times). The appropriate HCl and methanol filtrates are then combined and concentrated on a Speed-Vac^® in tared vials to afford eight discrete keto-ureas. Example 6

Synthesis of N²-Substituted Hydantoins

The synthesis of eight N²-substituted hydantoins is summarized in Scheme 6 and Table 6.

100 mg each of the commercially available FMOC- protected amino acids on WANG resins (FMOC-Alanine and FMOC-Phenylalanine, loading = 0.37-0.60 meq/g, 200-400 mesh) are measured into two sets of four gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of DMF, and drained as set forth in Example 1.

To deprotect the FMOC amino acids, the appropriate reaction tubes are submerged in reaction wells containing a solution of 4 mL of 25% piperidine in DMF (v/v) containing an internal standard (anthracene at 2-4 mg/mL). The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold. The reaction progress is monitored by removing a sample of the filtrate (e.g., 10-100 μL) and analyzing for the FMOC-piperidine adduct and dibenzofulvene by GC/ISTD calibration methods. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 4 mL of 0.10 to 0.30 M acetaldehyde or benzaldehyde in dioxane containing an internal standard (e.g., anthracene at 2-4 mg/mL). The apparatus is sealed and agitated in a sonic bath for 12 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress is monitored by GC/ISTD calibration methods as set forth in Example 1. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of 4 mL of 0.10-0.30 M sodium cyanoborohydride in methanol with an indicator (e.g., methyl orange at 2-4 mg/mL). The apparatus is sealed as set forth in Example l and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold. The reaction progress is monitored by visually observing the color of the filtrates. When neutral or basic conditions are observed, the pH is adjusted by injection of dilute aqueous HCl into the appropriate reaction tubes through the gasket at the top of the manifold. Stabilization of pH changes indicates completion of the reduction reaction. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 4 mL of 0.10-0.30 M trimethylsilyl isocyanate or allyl isocyanate in DMF containing an internal standard (e.g., anthracene at 2-4 mg/mL). The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 6 hours, while maintaining a positive nitrogen flow through the manifold. The reaction progress is monitored by removing a sample of the filtrate (10-100 μL), derivatization with an appropriate amine or alcohol, and analysis by GC/ISTD calibration methods as set forth in Example 1. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

In order to cleave the product from the solid support, the reaction tubes are submerged in reaction wells containing a solution of 4 mL of aqueous 6N HCl. The apparatus is sealed as set forth in Example 1, placed in an oil bath and heated at 95 to 100°C for 2 hours while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. To isolate and purify the products, the reaction tubes are washed as set forth in Example 1 using 4 mL portions of methanol. The methanol extraction protocol is repeated until the filtrates are free of any organic components as determined by TLC (1-2 times). The appropriate HCl and methanol filtrates are then combined and concentrated on a Speed-Vac^® in tared vials to afford eight discrete hydantoins.

Example 7

Synthesis of (R)-4-Benzamido-5-oxopentanoic Acids The synthesis of 12 oxopentanoic acids is summarized in Scheme 7 and Table 7.

5 g of α-2-Trimethysilylethyl-N-BOC-glutamic acid is linked through the γ-carboxyl group to commercially available p-benzyloxybenzyl alcohol resin (loading = 1.2 meq/g, 200-400 mesh) using

N,N'-diisopropylcarbodiimide (4 eq) and HOBT (2 eq) in

3 mL of DMF for 24 hours. The resulting resin is then washed with excess MeOH and CH₂Cl₂ and used directly below. 95 to 105 mg of the above formed resin is measured into each of 12 gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of dioxane, and drained as set forth in Example 1. The reaction tubes are submerged in reaction wells containing a solution of tetrabutylammoniun fluoride (5 eq) in dioxane (3 mL) at room temperature. The apparatus is sealed as set forth in Example l and agitated at room temperature for 8 hours in a sonic bath as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 2 × 3 mL each of the following solutions, dioxane:H₂O (1:1), dioxane and CH₂Cl₂.

The reaction tubes are submerged in reaction wells containing a solution of ethylchloroformate (3 eq) and triethylamine (3 eq) in dioxane (3 mL) at -10°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at room temperature for 4 hours as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is allowed to warm to room temperature and then drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in

Example 3, using 4 × 2 mL of anhydrous DMF. Because of the labile nature of the above formed acyl-carbonates, the reaction tubes are rapidly washed in order to minimize unwanted hydrolysis.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 4 eq of either n-butylamine, cycloheptylamine,

8-azaspiro[4.5]decane, or dipentylamine in dioxane (4 mL) at -10°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at -10°C as set forth in Example 5 for 1 hour and then 3 hours at room temperature, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in

Example 3, using 2 × 3 mL each of the following solutions, dioxane:H₂O (1:1), dioxane:1N HCl (1:1), dioxane:H₂O (1:1), dioxane:0.1N NaOH (1:1), dioxane:H₂O

(1:1), and dioxane.

The reaction tubes are submerged in reaction wells containing a solution of 20% piperidine in DMF at room temperature. The apparatus is sealed as set forth in Example 1 and agitated at room temperature in a sonic bath for 6 hours as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 2 × 3 mL each of the following solutions, DMF, dioxane:H₂O (1:1), dioxane and CH₂Cl₂.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 2 eq of either benzoyl chloride, 3-methoxybenzoyl chloride, or 3-nitrobenzoyl chloride in pyridine (3 mL) at 0°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at 0°C for 3 hours as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the apparatus is allowed to warm to room temperature, and the reaction tubes are drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 x 2 mL each of the following solutions, dioxane, dioxane:H₂O (1:1), and dioxane.

In order to cleave the product from the solid support, the reaction tubes are submerged in reaction wells containing 3 mL each of trifluoroacetic acid. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at 0°C for 2 hours and then at room temperature for 4 hours as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold. After 6 hours in a sonic bath, the apparatus is allowed to warm to room temperature, and the reaction tubes are drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 2 × 3 mL of methylene chloride. The filtrates are then combined and concentrated as set forth in Example 3. The crude products are then redissolved, transferred to tared vials, and reconcentrated to yield the final crude products.

Example 8

Synthesis of Diketopiperazines The synthesis of 40 diketopiperazines is summarized in Scheme 8 and Table 8.

95 to 105 mg of Merrifield' s resin (loading = 0.66 meq/g, 200-400 mesh) is measured into each of 40 gas dispersion tubes (reaction tubes) containing a magnetic stirring bar. The apparatus is assembled, swelled with 3 mL of DMF, and drained as set forth in Example 1. The reaction tubes are submerged into wells containing a solution of 75.0 mg BOC 4-hydroxyproline cesium salt in 5 mL of DMF. The apparatus is assembled and sealed with clamps as set forth in Example 1, and placed into an oil bath over a magnetic stirring plate while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2 and reacted at 50 ºC. After 24 hours, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1, ending however in CH₂Cl₂, to remove residual reagents and byproducts.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of 0.07 M acyl halide (either, benzoyl bromide, acetyl chloride, 4-biphenylcarbonyl chloride, p-anisoyl chloride, 4-chlorobenzoyl chloride, 4-nitrobenzoyl chloride, pivaloyl chloride, or trifluoroacetyl chloride) and 0.07 M triethylamine in CH₂Cl₂. The apparatus is sealed as set forth in Example 1 and agitated as above while maintaining a positive nitrogen flow through the manifold. After 72 hours, the reaction tube array is drained and sparged as set forth in Example 1, using DMF, methanol, and CH₂Cl₂. The reaction tubes are submerged in 5 mL of

0.08 M TFA in CH₂Cl₂ to remove the BOC protecting group from N₁. The apparatus is assembled and the reactions stirred as above. After 72 hours, the reaction tubes are drained and washed with DMF, methanol, 1.0 M TFA in CH₂Cl₂, and CH₂Cl₂ as set forth in Example 1. A solution (5 mL) of 0.07 M BOC amino acid,

0.07 M diisopropylcarbodiimide and 0.07 M TFA in CH₂Cl₂ are stirred for 3 hours at room temperature. The appropriate reaction tubes are then submerged in the above reaction wells, and the apparatus is assembled as set forth in Example 1. Five BOC amino acids are employed, BOC-glycine, BOC-alanine, BOC-valine, BOC-phenylalanine, and BOC-diphenylglycine. After 72 hours, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1, ending however in CH₂Cl₂.

Cyclization to the final product and thus cleavage from the resin is accomplished by removal of the BOC (t-butyloxycarbony) group of the amino acid. The reaction tubes are submerged in reaction wells containing a solution of 5 mL of saturated HCl in CH₂Cl₂. The apparatus is assembled as set forth in Example 1 and the reactions stirred as above at room temperature. After 24 hours, the reaction tube array is drained and sparged as set forth in Example 1. The contents in the reaction wells are concentrated to dryness using a stream of nitrogen as set forth in Example 3.

A solution of 5 mL of 1.0 M triethylamine in CH₂Cl₂ is added to each reaction well and the reaction tubes are then submerged in these wells. The apparatus is assembled as set forth in Example 1, and the reactions stirred as above. After 24 hours, the reaction tube array is drained and sparged as set forth in Example 1. The contents in the reaction wells are concentrated to dryness using a stream of nitrogen as set forth in Example 3. These crude products are partitioned between CH₂Cl₂ and a saturated solution of NaHCO₃ using a Tecan^® robot to deliver the necessary liquids and to remove the aqueous layer. The basic aqueous wash is repeated and the residual CH₂Cl₂ layers are dried by passing the samples through a bed of MgSO₄ contained in standard SPE cartridges employing the vacuum SPE apparatus described above, giving the crude diketopiperazines.

Example 9

Synthesis of Tetrahydro-4-hydroxy-6-[2-(1H-pyrrol-1yI)

ethyl]-2H-pyran-2-ones

The synthesis of three 2H-pyranones is summarized in Scheme 9 and Table 9.

Three dispersion tubes are loaded each with 100 ± 5 mg of hydroxymethyl polystyrene resin (1% cross-linked, 1.0 meq/g, loading) and placed in the holder block. The apparatus is assembled, swelled with 3 mL of acetonitrile, and drained as set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing 2 mL of a solution of either 0.2 M acetic anhydride, propanoic anhydride, or 2-phenylacetic anhydride in acetonitrile, and 1.0 mL of a 0.4 M solution of triethylamine in CH₂Cl₂. The apparatus is sealed as set forth in Example 1, and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold for 12 hours. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 10 × 4 mL of CH₂Cl₂.

The reaction tubes are submerged in reaction wells containing 3 mL of a solution of 0.2 M lithium diisopropylamide in THF and the apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at room temperature as set forth in Example 5, while maintaining a positive nitrogen flow through the manifold for 4 hours.

1 mm of a solution (in THF) of either 1.0 M 5-[2-(4-fluorophenyl)-3-phenyl-4-carboxamidophenyl-5-isopropyl-1-pyrrololyl]-3-oxo-1-pentanoic acid methyl ester (to wells #1 and #2), or 1.0 M 5- [2,3,4-triphenyl-5-methyl-1-pyrroloyl]-3-oxo-1-pentanoic acid methyl ester (to well #3) is added to the top of the appropriate reaction tubes through the gasket at the top of the manifold and sonicated at room temperature for 12 hours as set forth in Example 5. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are submerged in reaction wells containing 2.0 mL of a 0.26 M THF solution of diethyl methoxy borane and the apparatus is sealed as set forth in Example 1. 1.0 mL of a 1.0 M THF solution of sodium borohydride is added to the top of the appropriate reaction tubes through the gasket at the top of the manifold and the apparatus is sonicated at room temperature for 4 hours as set forth in Example 5. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 10 × 4 mL of CH₂Cl₂. The reaction tubes are submerged in reaction wells containing 3.0 mL of 3% hydrogen peroxide in 1:1 dioxane:water and the apparatus is assembled as set forth in Example 1, and sonicated at room temperature for 6 hours as set forth in Example 5 to affect boron oxidation. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 10 × 3 mL of dioxane:water (1:1). The reaction tubes are submerged in reaction wells containing a solution of 0.1 M HCl in 1:1 dioxane:water (3 mL). The apparatus is assembled as set forth in Example 1, and sonicated at room temperature for 6 hours as set forth in Example 5 to affect hydrolysis/ring closure. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in

Example 3, using 3 × 3 mL of dioxane:water (1:1). The appropriate filtrates are then combined and concentrated as set forth in Example 3 to give the three individual expected lactone products.

Example 10

Synthesis of N-Arylpiperazines The synthesis of 12 N-arylpiperazines is summarized in Scheme 10 and Table 10.

100 mg of hydroxymethyl polystyrene (2% cross- linked, 1.2 meq/g, loading) resin is measured into each of 12 gas dispersion tubes (reaction tubes) . The apparatus is assembled, swelled with 3 mL of DMF, and drained as set forth in Example 1.

To facilitate coupling of the acid, the appropriate reaction tubes are submerged in reaction wells containing a solution of 0.09 M 4-chlorobutyric acid or 3-chloropropionic acid; and 0.09 M N,N'-diisopropylcarbodiimide and 0.09 M 4,4-dimethylaminopyridine in DMF (3 mL) at room temperature. The apparatus is sealed as set forth in Example l and agitated in a sonic bath for 2 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of 3 mL of either 0.09 M piperazine, phenylpiperazine, or 1-(2-pyridyl)piperazine; 0.18 M potassium carbonate; and 0.18 M potassium iodide in DMF. The apparatus is sealed as set forth in Example 1, placed in an oil bath, and heated at 95 to 100°C while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. After 6 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of 3 mL of toluene-20% 1,2-dimethoxyethane at 0°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at 0'C as set forth in Example 5, while maintaining a positive argon flow.

To form the enolate, 0.5 mL of a solution (in

THF) of 0.15 M trityllithium is added to the top of the appropriate reaction tubes through the gasket at the top of the manifold and sonicated at 0°C for 15 minutes as set forth in Example 5.

After 15 minutes, 0.5 mL of 0.28 M acid chloride (either nicotinoyl chloride or naphthoyl chloride) in THF is added to the top of the appropriate reaction tubes through the gasket at the top of the manifold and sonicated at room temperature for 1 hour as set forth in Example 5. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1. In order to cleave the product from the solid support, the reaction tubes are submerged in reaction wells containing a solution of 3 mL of dry HBr in TFA. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold. After 1 hour, the reaction is stopped, and the reaction tube array is drained and sparged as set forth in Example 1.

To isolate and purify the products, the reaction tubes are washed as set forth in Example 1, using 3 mL portions of methanol. The methanol extraction protocol is repeated until the filtrates are free of any organic components as determined by TLC

(1-2 times) . The appropriate HCl and methanol filtrates are then combined and concentrated on a Speed-Vac^® in tared vials to afford 12 discrete products.

In an example of a post-cleavage reaction, a modified Wolff-Kishner reduction is done on half of the amount of each of the isolated compounds. The compounds are placed in the appropriate reaction wells containing

3 mL of 0.09 M potassium hydroxide, and 0.09 M hydrazine hydrate in diethylene glycol. The reaction tubes are filled with 0.5 g sodium sulfate (to absorb water) and submerged in the reaction wells . The apparatus is sealed as set forth in Example 1, placed in an oil bath, and heated at 80°C while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. After 12 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1. Each product is isolated by adding

4 mL of a solution of 1:1 water:CH₂Cl₂. Each organic layer is separated, dried, and concentrated as set forth in Example 8 to yield another 12 desired products. Example 11

Synthesis of Benzisothiazolones

The synthesis of nine benzisothiazolones is summarized in Scheme 11 and Table 11. 95 to 105 mg of Merrifield's resin (loading =

0.66 meq/g, 200-400 mesh) is measured into each of nine gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of dioxane and drained as set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of either 0.2 M thiosalicylic acid, 5-chloro-thiosalicylic acid, or 2-mercaptonicotinic acid; and 0.4 M triethylamine in dioxane and magnetic stirring bars. The apparatus is sealed with clamps as set forth in Example 1 and placed over a magnetic stirring plate, while maintaining a positive nitrogen flow through the manifold. After 96 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of water, 1N HCl, 1N HCL:dioxane (1:1), and finally dioxane to remove residual reagents and byproducts. The reaction tubes are submerged in reaction wells containing a solution of 5 mL of 0.20 M carbonyldiimidazole in dioxane. The apparatus is assembled and agitated as above. After 5 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 4 × 3 mL each of DMF then dioxane. The appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of either 0.20 M 4-methoxyaniline, cyclohexylamine, or benzylamine in dioxane. The apparatus is assembled as set forth in Example 1, while maintaining a flow of cold nitrogen. The apparatus is placed in an oil bath over a magnetic stirring plate and the reactions are warmed to reflux and stirred as above. After 24 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 4 × 3 mL each of DMF, methanol, CH₂Cl₂, and then dioxane.

The reaction tubes are submerged in reaction wells containing a solution of 5 mL of 0.13 M NaBrO₂ in dioxane/water (8:1). The apparatus is assembled as set forth in Example 1, while maintaining a flow of nitrogen. The apparatus is placed over a magnetic stirring plate and stirred at room temperature as above. After 1 hhur, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 4 × 3 mL each of dioxane, water, dioxane/water (1:1), methanol, and then CH₂Cl₂. Cyclization to the final product and thus cleavage from the resin is accomplished by addition of trichloroacetic anhydride to the sulfoxide and rearrangement as taught by Wright, et al, Tetrahedron Letters 1991; 33:153. Thus, reaction tubes are submerged in reaction wells containing a solution of 5 mL of CH₂Cl₂. The apparatus is assembled as set forth in Example 1, while maintaining a flow of nitrogen. The apparatus is then placed in a cold bath at 0°C and the reactions stirred as above. Trichloroacetic anhydride (0.18 mL) is then added to the top of each reaction tube through the gasket at the top of the manifold and the temperature of the reaction is allowed to warm to 25°C over 7 hours. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 2 × 3 mL each of CH₂Cl₂, dioxane, MeOH, and CH₂Cl₂. The appropriate filtrates are then combined and concentrated as set forth in Example 3. These crude products are partitioned between CH₂Cl₂ and 2N NaOH using a Tecan^® robot to deliver the necessary liquids and to remove the aqueous layer. The basic aqueous wash is repeated and the residual CH₂Cl₂ layers are dried by passing the samples through a bed of MgSO₄ contained in standard SPE cartridges employing the vacuum SPE apparatus described above to yield the crude benzisothiazolones. Exmpte 12

Synthesis of Isoindolone-Based Spirosuccinimdes

The synthesis of nine spirosuccinimides is summarized in Scheme 12 and Table 12. A solution of 0.10 mol cyanoacetic acid and

0.10 mol carbonyldiimidazole are combined in 500 mL of dioxane. After 5 hours, 10 g of benzylhydroxy polystyrene resin (loading = 1.08 meq/g, 200 400 mesh) is added. The reaction is refluxed for 8 hours, cooled to room temperature, filtered, and washed with DMF, methanol, dioxane, and finally CH₂Cl₂. The product resin, benzyl cyanoacetate polystyrene resin, is used directly in the next step.

95 of 105 mg of the resin prepared above (loading = 1.08 meq/g, 200-400 mesh) is measured into each of nine gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of DMF and drained as set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of either 0.20 M 3-imino-1-oxoisoindoline, 6-chloro-3-imino-1-oxoisoindoline, or 6-phenyl-1-oxoisoindoline in diglyme with stirring magnetic bars. The 3-imino-1-oxoisoindoline employed are prepared by the method of Wrobel, et al, JOURNAL OF MEDICINAL CHEMISTRY 1992; 35:4613. The apparatus is sealed with clamps as set forth in Example 1, and placed in an oil bath over a magnetic stirring plate while maintaining a positive flow of cold nitrogen as set forth in Example 2. The reaction vessels are warmed to reflux, and after 2 hours the reaction is stopped, and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of dioxane, CH₂Cl₂, and DMF.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 0.20 M alkyl halide (either methyl iodide, benzyl bromide and 3-bromobenzyl bromide) and 1 mmol K₂CO₃ in 5 mL of DMF. The apparatus is assembled as set forth in Example 1, while maintaining a positive flow of cold nitrogen as set forth in Example 2. The oil bath is then warmed to 100°C and agitated as above. After 24 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of DMF, water, MeOH, dioxane, CH₂Cl₂, and DMSO.

The reaction tubes are submerged in reaction wells containing a solution of 5 mL of 0.20 M potassium cyanide in DMSO. The apparatus is assembled as set forth in Example 1 and agitated as above. After 24 hours at 25°C, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of DMF, water, MeOH, water, and 10% aqueous HCl. The reaction tubes are submerged in reaction wells containing a solution of saturated dry HCl in MeOH. The apparatus is assembled as set forth in Example 1 and agitated as above. After 3 days at 25°C, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of MeOH, dioxane, and CH₂Cl₂. The appropriate filtrates are then combined and concentrated as set forth in Example 3.

The crude products are dissolved in glacial HOAc and a holder block with nine clean empty reactions tubes is attached. The apparatus is assembled as set forth in Example 1 and agitated as above. After 24 hours at 25°C, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL of MeOH.

The appropriate filtrates are then combined and concentrated as set forth in Example 3 to yield the crude spiro[1H-isoindole-1,3'-pyrolidine]-2',3,5'(2H))triones. Example 13

Synthesis of Pilocarpine Analogs

The synthesis of four pilocarpine analogs is summarized in Scheme 13 and Table 13. 95 to 105 mg of 2-Chloro-triphenylmethyl resin as taught by Barlos, et al, TETRAHEDRON LETTERS 1989 ,-30:3947 is measured into each of four gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of DMF, and drained as set forth in Example 1.

FMOC-histidine methyl ester is coupled to the resin. The reaction tubes are submerged in reaction wells containing a solution of 0.2 M FMOC-histidine methyl ester and 0.4 M pyridine in 3 mL dry THF. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 6 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

To deprotect the FMOC amino acids, reaction tubes are submerged in reaction wells containing 3 mL of a solution of 25% piperidine in DMF. The apparatus is sealed as set forth in Example 1 and placed in a sonic bath, while maintaining a positive nitrogen flow through the manifold. The reaction is stopped after 4 hours. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tube array is subjected to the standard wash cycle set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 0.2 M anhydride (either acetic anhydride or isobutyric anhydride) and 0.4 M triethylamine in 3 mL of dry THF. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 12 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The apparatus is sealed as set forth in Example 1 while maintaining a positive nitrogen flow through the manifold. A solution of lithium aluminum hydride (LAH) in dry THF (0.1 M, 3 mL) is then added to the top of each reaction tube through the gasket at the top of the manifold. The apparatus is placed in an oil bath heated at 67°C for 8 hours, while maintaining a positive chilled nitrogen flow through the manifold (as set forth in Example 2). The apparatus is cooled to room temperature and 1 mL of water is added to the top of each reaction tube through the gasket at the top of the manifold, and the apparatus sonicated at room temperature for 30 minutes to neutralize any unreacted

LAH. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1, except that a base wash cycle is added to remove the neutralized LAH salts.

The cyclic carbamate is formed according to the method of Gonzalez, et al, TETRAHEDRON LETTERS 1989; 30: 2145. The reaction tubes are submerged in reaction wells containing a solution of 0.2 M diethyl carbonate and 0.4 M NaOMe in MeOH. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath for 12 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tube array is subjected to the standard wash cycle set forth in Example 1. The appropriate reaction tubes are submerged in reaction wells containing a solution of 0.2 M iodoalkane (either 1-iodohexane, or iodomethane) in 3 mL of CH₂Cl₂. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold for 1 hour. The apparatus then placed in an oil bath heated at 37°C, while maintaining a positive chilled nitrogen flow through the manifold (as set forth in Example 2) for 6 hours. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

To isolate and purify the products, the reaction tubes are submerged in reaction wells containing a solution 3 mL each of MeOH. The apparatus is sealed as set forth in Example 1, placed in an oil bath and heated at 64°C while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 1, using 3 mL portions of methanol. The methanol extraction protocol is repeated until the filtrates are free of any organic components as determined by TLC (2 times). The appropriate methanol filtrates are then combined and concentrated on a Speed-Vac^® in tared vials to afford four discrete products. Example 14

3-Substituted-(Aminomethyl)-3.4-dihydro-5.6-dihydroxy-1H-2-benzopyrans

The synthesis of six benzopyrans is summarized in Scheme 14 and Table 14.

10 g of Merrifield' s chloromethyl polystyrene resin (2% cross-linked, loading = 1.2 meq/g,

200-400 mesh) is reacted with excess sodium ethyl acetoacetate (10 eq) in DMF at 80°C for 16 hours. After washing with DMF and dioxane the crude beta-keto ester is hydrolyzed and decarboxylated 4N HCl: dioxane (1:1) at 60°C to give the phenethyl ketone resin, after washing with dioxane. This is further reacted with excess catechol and cat. p-TsOH in toluene at 90°C for

30 hours. The resin is thoroughly washed with toluene, dioxane, and CH₂Cl₂ to remove all catechol. The crude resin is then used directly below. 95 to 105 mg of the catechol ketal resin prepared above is measured into each of six gas dispersion tubes (reaction tubes). The reaction tubes are fitted into the appropriate holder block and swelled and drained as set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of nBuLi (1 eq) in THF at 0°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath (maintained at 0°C as set forth in Example 5), while maintaining a positive nitrogen flow through the manifold. After 1 hour, the bath is warmed to room temperature and sonicated for 3 hours more. The bath is then recooled to 0°C and a solution of either cyclohexyl- or phenyl- ethyleneoxide (2 eq) in THF (0.5 mL) is then added to the top of the appropriate reaction tube through the gasket at the top of the manifold. The bath is again warmed to room temperature and sonicated for 2 hours. The apparatus is then removed from the sonic bath, and 0.5 mL of THF:H₂O (1:1) are added to the top of each reaction tube through the gasket at the top of the manifold as above, to quench any excess n-BuLi. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 3 × 3 mL each of dioxane: sat NH₄Cl (1:1), dioxane:H₂O (1:1), and dioxane.

The appropriate reaction tubes are submerged in reaction wells containing a solution of (2 eq) of either bromoacetaldehyde dimethyl acetal (4 reaction tubes) or [(formylamino)methyl]acetaldehyde dimethyl acetal (2 reaction tubes); and BF₃OEt₂ (3 eq) in Et₂O (3 mL) at 0°C. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at room temperature for 24 hours, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL each of dioxane:H₂O (1:1), dioxane:H₂O 0.1N NaOH (1:1), dioxane:H₂O (1:1), dioxane. The appropriate four reaction tubes are submerged in reaction wells containing a solution of either allylamine or benzylamine (2 eq) in dioxane (4 mL) at room temperature. The other two reaction tubes (those which are reacted with [(formylamino)methyl]-acetaldehyde dimethyl acetal above) are submerged in wells containing a solution of dioxane:MeOH: 15% NaOH (2:2:1) 5 mL at room temperature. The apparatus is sealed as set forth in Example l and agitated in a sonic bath at 60°C as set forth in Example 5, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. After 8 hours the reaction is stopped, the reaction tube array is allowed to cool, and then drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 3 × 2 mL each of dioxane, dioxane:H₂O (1:1), dioxane.

In order to cleave the products from the solid support, the reaction tubes are submerged in reaction wells containing a solution of 3 mL of 5 M HCl in dioxane: ethanol (1:1). The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at 80°C for 4 hours as set forth in Example 5, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. After 4 hours the reaction is stopped, the reaction tube array is allowed to cool, and then drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 3 mL of dioxane:ethanol (1:1). The filtrates are then combined and concentrated as set forth in Example 3. The crude products are then redissolved, transferred to tared vials, and reconcentrated. The final crude products including, cis-1-(aminomethyl)-3-cyclohexyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride; cis- 1 -(aminomethyl)-3-phenyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride; cis-1-[(allylamino)methyl]-3-cyclohexyl-3,4-dihydro-5,6-dihydroxy-1H-2 -benzopyranhydrochloride; cis-1-[(allylamino)methyl]-3-phenyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyranhydrochloride; cis -1-[(benzylamino)methyl]-3-cyclohexyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride; cis-1-[(benzylamino)methyl]-3-phenyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyran hydrochloride; are set forth in Table 14. Example 15

6,7-Dihydro-4H-pyrazolo-1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-diones

The synthesis of eight pyrimidinediones is summarized in Scheme 15 and Table 15.

15 g of Merrifield' s chloromethyl polystyrene resin (2% cross-linked, loading = 0.7 meq/g, 200-400 mesh) is reacted with excess cesium acrylate (10 eq) and Nal (0.1 eq) in DMF at 80°C for 14 hours to yield the desired ester. The resin is thoroughly washed with DMF, dioxane:H₂O (1:1), dioxane, and CH₂Cl₂ to remove excess reagents. The crude resin is then used directly below.

95 to 105 mg of the acrylate ester resin prepared above is measured into each of eight gas dispersion tubes (reaction tubes). The reaction tubes are fitted into the holder block and swelled and drained as set forth in Example 1.

The appropriate reaction tubes are submerged in reaction wells containing a solution of either cyclohexylamine or n-hexylamine (1 eq) in 3 mL of DMF at room temperature. The apparatus is sealed as set forth in Example l and agitated in a sonic bath (maintained at room temperature as set forth in Example 5), while maintaining a positive nitrogen flow through the manifold. After 24 hours, a solution of diethyl oxalate (1 eq) and Na₂CO₃ (1 eq) in 1 mL of DMF is added to the top of the each reaction tube through the gasket at the top of the manifold. The bath is warmed to 60°C and sonicated for 2 hours. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 2 mL each of dioxane: sat. NH₄Cl (1:1), dioxane:1N HCl (1:1), dioxane:H₂O (1:1), and dioxane. In order to cleave the products from the solid support, the appropriate reaction tubes are submerged in reaction wells containing a solution of (1 eq) of either 5-phenylpyrazol-3-amine or 5-(4-chlorophenyl)pyrazol-3-amine in HOAc (4 mL) at room temperature. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath at 100°C for 2 hours as set forth in Example 5, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 × 2 mL of dioxane:ethanol. The filtrates are then combined and concentrated as set forth in Example 3. The crude products are then redissolved, transferred to tared vials, and reconcentrated.

In an example of a post-cleavage reaction, a solution of either 2-chloroacetophenone (2 eq) and K₂CO₃ (1.1 eq) in DMF (3 mL) or 1-bromo-5-hexene (2 eq) and K₂CO₃ (1.1 eq) in DMSO (3 mL) is added to the appropriate reaction wells. A holder block with clean empty reaction tubes is added and the apparatus is sealed and agitated as set forth in Example 1 in a sonic bath at 40°C for 22 hours as set forth in Example 5, while maintaining a positive chilled nitrogen flow through the manifold as set forth in Example 2. At the end of the reaction, the reaction tube array is allowed to cool and then drained and sparged as set forth in Example 1

The crude reaction mixtures are transferred to the corresponding tubes in the SPE apparatus equipped with 20 mL columns and 5 μm. filters. Each reaction mixture is diluted and mixed with 15 mL of H₂O to precipitate the desired products. The suspensions are then filtered and washed with H₂O and Et₂O (3 × 2 mL, each). The resulting solids are then dissolved with CH₂Cl₂ (3 × 2 mL washes) run through the filter and collected in clean tubes. These solutions are then concentrated as set forth in Example 3. The crude products are then redissolved, transferred to tared vials, and reconcentrated. The final crude products including, 6-Cyclohexyl-6,7-dihydro-4-(phenylmethyl)-2-phenyl-4H-pyrazolo[1,5a]pyrrolo[3,4-d]-pyrimidine- 5,8-dione; 6-Cyclohexyl-6,7-dihydro-4-(2-oxo- 2-phenylethyl)-2-phenyl-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione; 6-Cyclohexyl-6,7-dihydro- 4-(phenylmethyl)-2-(4-chlorophenyl)-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione; 6-Cyclohexyl- 6,7-dihydro-4-(2-oxo-2-phenylethyl)-2-(4-chlorophenyl)-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione;6-Hexyl-6,7-dihydro-4-(phenylmethyl)-2-phenyl-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione; 6-Hexyl-6,7-dihydro-4-(2-oxo-2-phenylethyl)-2-phenyl-4H-pyrazolo[1,5-a]-pyrrolo[3,4-d]-pyrimidine-5,8-dione; 6-Hexyl-6,7-dihydro-4-(phenylmethyl)-2-(4-chlorophenyl)-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione; 6-Hexyl-6,7-dihydro-4-(2-oxo-2-phenylethyl)-2- (4-chlorophenyl)-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]-pyrimidine-5,8-dione are set forth in Table 15.

Example 16

Synthesis of Tenoxalin Derivatives

The synthesis of eight tepoxalin derivatives is summarized in Scheme 16 and Table 16. 95 to 105 mg of benzylhydroxy polystyrene resin (loading = 1.08 meq/g, 200-400 mesh) is measured into each of eight gas dispersion tubes (reaction tubes). The apparatus is assembled, swelled with 3 mL of DMF, and drained as set forth in Example 1.

A solution (5 mL) of either 0.20 M 6-phenyl-4,6-dioxohexanoic acid, or 6-(4-chlorophenyl)-4,6-dioxohexanoic acid and 0.20 M carbonyldiimdazole in CH₂Cl₂ are stirred for 6 hours at room temperature. The two6-aryl-4,6-dioxohexanoic acids employed are prepared by the method of Murray, et al, JOURNAL OF ORGANIC CHEMISTRY1990;55:3424.

The appropriate reaction tubes are submerged in the reaction wells above. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath while maintaining a positive nitrogen flow through the manifold. After 12 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 3 × 3 mm each of DMF, MeOH, dioxane, CH₂Cl₂, and MeOH.

The appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of either 0.2 M 4-methoxyphenylhydrazine hydrochloride or 4-methylphenylhydrazine hydrochloride and 0.2 M triethylamine in MeOH. The apparatus is sealed as set forth in Example 1 and agitated in a sonic bath, while maintaining a positive nitrogen flow through the manifold. After 12 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1.

The reaction tubes are washed as set forth in Example 3, using 3 x 3 mL each of DMF, MeOH, dioxane, and CH₂Cl₂.

Cleavage is accomplished by aminolysis of the resin ester linkage. Thus, the appropriate reaction tubes are submerged in reaction wells containing a solution of 5 mL of either 0.20 M methylhydroxylamine hydrochloride or benzylhydroxylamine hydrochloride and 0.2 M triethylamine in CH₂Cl₂ along with stirring bars. The apparatus is sealed with clamps as set forth in Example 1 and placed in an oil bath over a magnetic stirring plate while maintaining a positive flow of cold nitrogen as set forth in Example 2. The reaction vessels are warmed to reflux and stirred as above. After 24 hours, the reaction is stopped and the reaction tube array is drained and sparged as set forth in Example 1. The reaction tubes are washed as set forth in

Example 3, using 3 x 3 mL each of MeOH, dioxane, and CH₂Cl₂.

The appropriate filtrates are then combined and concentrated as set forth in Example 3. These crude products are partitioned between CH₂Cl₂ and 1 M HCl using a Tecan^® robot to deliver the necessary liquids and to remove the aqueous layer. The acidic aqueous wash is repeated and the residual CH₂Cl₂ layers are dried by passing the samples through a bed of MgSO₄ contained in standard SPE cartridges employing the vacuum SPE apparatus described above to yield tepoxalin and the expected tepoxalin derivatives.

BB_N represents the building blocks which are bifunctional to allow sequential attachment. The curved structure represents the solid support where the asterick is the functionality capable of covalently attaching the growing molecule to the solid support.

The three possible cleavage modes are illustrated. Cleavage 1 represents the third building block attacking the solid support linkage to cleave the final molecule. This provides structural vaiation at the former site of attachment to the support. Cleavage 2 represents a distal functionality attacking the solid support linkage to cleave the final molecule as a cyclized product. Cleavage 3 represents a cleavage by an invariant agent. This provides a constant functional group at the former site of attachment to the support. I

1

The invention of the '483 patent describes the use of apparatus which facilitates the simultaneous introduction of multiple reagents to reaction or test wells. The invention described herein is applicable to a technique which permits the multistep, multireagents to obtain mixtures of unique reaction products.

Combinatorial Mixtures

For The Synthesis Of Amides

A 2-step reaction utilizing four building blocks for the manufacture of amides can be described below (Schemes 17 and 18 and Tables 17 and 18). P indicates polymeric support and the letters A, B, C, or D indicate reactants and the numbers 1, 2, 3, or 4 likewise indicate reactants. In the X and Y-plates, there are four wells in each. The materials are prepared as follows with the reagents being placed in each of the wells as described. The individual materials made in each of the wells follows what is called a deconvolution technique. Convolute means to be coiled or wound together. Deconvolute therefore means the uncoiling or the unwinding of the mixture. In the present case, deconvolution means the ascertaining of the unique reaction product in the reaction or test wells containing the mixture of the products. Listed below (Scheme 19 and Table 17) is a proposed synthesis route and technique to deconvolute the products.

3-Step Reaction Utilizing Three Building Blocks

Following a similar technique as outlined above for the 2-step reaction with four building blocks, listed below in Schemes 20 and 21 and Table 19 is a 3-step reaction using three building blocks. The letters A, B and C, X, Y and Z are reactants and the numbers 1, 2 and 3 are likewise reactants. The reaction procedure can be outlined below:

3-Step Reaction Utilizing Four Building Blocks

Following the technique outlined above, listed below in Schemes 22 and 23 and Table 20 is a technique for a 3-step reaction utilizing four building blocks where the letters A, B, C, and D, W, X, Y and Z are reactants and the numbers 1, 2, 3, and 4 further designate reactants.

General Description of Experimental Procedure Utilizing A Solid Support

A number of reaction vessels equal to the total number of wells to be synthesized by the array method are loaded with 1-1000 mg of the appropriate functionalized solid support, preferably 1-3% cross-linked polystyrene. The individual reaction vessels are charged with a volume, preferably 3-5 mL, of a solvent capable of swelling the polystyrene resin (such as, but not limited to, dichloromethane, chloroform, dimethylformamide (DMF), dioxane, toluene, tetrahydrofuran (THF)).

The reaction vessels are emptied of solvent and the proper reactant solutions are dispensed into the reaction vessels at appropriate locations in the array. If the reactant is actually one of the building blocks that is to become covalently attached to the growing compound on the solid support, the quantity of reactant is usually 0.1-100 equivalents based on the meq/g loading of functionalized solid support (typically 0.1 - 1.0 meq/g for polystyrene resins) originally weighed into the reaction tube. If a mixture of reactants are charged to single resin-bound starting material or intermediate to generate a mixture of resin-bound or cleaved products, equimolar amounts of each reagent are preferable. For example, if four reactants are charged to a single resin-bound intermediate, 0.25 equivalents of each reactant should provide some quantity of each target compound in the final mixture , allowing for differing reaction kinetics of individual reactants. Additional equivalents of single or multiple reactants can be used if required to drive the reaction to completion in a reasonable time. Agitation is achieved by shaking, sonication, or magnetic stirring. The reaction is allowed to proceed for an amount of time deemed necessary from the preliminary validation experiments or monitored by removal and quantitative analysis of filtrate aliquots from selected wells by methods such as GC/ISTD (internal standard) or HPLC/ISTD. The resin-bound intermediates within each reaction vessel are washed clean of excess retained reagents, solvents, and by-products by repetitive exposure to clean solvent (s). The washing procedure is repeated 1-50 times, monitoring the efficiency of reagent, solvent, and byproduct removal by methods such as TLC, GC, or visualization of the wash filtrates.

The above described procedure of reacting the resin-bound compounds with reagents within the reaction vessels followed by removal of excess reagents, byproducts, and solvents is repeated with each successive transformation until the final or penultimate resin-bound compounds are prepared.

Detachment of the final products from the solid support is achieved with a solution of a cleavage reagent (preferably 3-5 mL). Gas flow, temperature control, agitation, and reaction monitoring are implemented as above and as desired to affect the detachment reaction. The spent resin in the reaction vessels is then washed 2-5 times as above with 3-5 mL of an appropriate solvent to extract (wash out) as much of the detached products as possible. The final product solutions are combined, taking care to avoid cross-mixing. The individual solutions/extracts are then manipulated as needed to isolate the final compounds.

Typical manipulations include, but are not limited to, evaporation, concentration, liquid/liquid extraction, acidification, basification, neutralization or additional reactions in solution.

Example 17 Synthesis of Substituted Amides Approximately 800 mg of commercially available cross-linked hydroxymethyl divinylbenzene-styrene resin

(loading = 0.65 meq/g, 200-400 mesh, 1% Divinylbenzene

Crosslinked) was weighed into each of 8 reaction tubes.

The apparatus was assembled, nitrogen atmosphere was initiated, and 10 mL of DMF was injected into each reaction tube. A 1.0 mL solution of diisopropylcarbodiimide (DIC, 263 mg, 2.08 mmol, 4 eq) and N,N-dimethylaminopyridine (DMAP, 6.25 mg, 0.053 mmol, 0.1 eq) in DMF was delivered to each reaction tube. The appropriate carboxylic acids were injected as solutions into each reaction tube. 1.0 mL each of 0.52M 2-ethyl butyric acid in DMF (1 eq), 0.52M 2-propyl pentanoic acid in DMF (1 eq), 0.52M diphenylacetic acid in DMF (1 eq), and 0.52M bis-(p-chlorophenyl)-acetic acid in DMF 1 eq) were delivered to reaction tubes 1, 2, 3, and 4 of plate X respectively (X1-X4). A solution of the mixture of the carboxylic acids was made by combining 1.0 mL each of 0.52M 2-ethyl butyric acid in DMF, 0.52M 2-propyl pentanoic acid in DMF, 0.52M diphenylacetic acid in DMF, and 0.52M bis-(p-chlorophenyl)-acetic acid in DMF. The resultant mixture contained an equimolar concentration of each carboxylic acid. 1.0 mL of the dissolved acid mixture was dispensed into each reaction tube in plate Y (Y1-Y4) to provide 0.25 equivalents each of 2-ethyl butyric acid, 2-propyl pentanoic acid, diphenylacetic acid, and bis-(p-chlorophenyl)-acetic acid in each reaction tube, based upon indicated loading of commer cial resin. The apparatus was agitated at 20-25 ºC in a sonic bath for 48 h, while maintaining a positive nitrogen flow through the manifold. At the end of the reaction, the reaction tube array was drained and sparged with nitrogen.

The reaction tubes were subjected to a series of wash cycles to remove residual solvents, reagents, and byproducts. A standard protocol included sequentially submerging the reaction tubes in 10-15 mL each of 2 × DMF, 2 × methanol, 2 × water, 2 × tetrahydrofuran (THF), and 2 × dichloromethane (DCM) followed by agitation by ultrasound for 10-15 minutes, and finally draining by gravity and nitrogen sparge.

To facilitate cleavage and amide formation, the apparatus was assembled, argon atmosphere was initiated, and 10 mL of anhydrous toluene was delivered into each reaction tube. A 1.0 mL solution of boron tribromide solution in DCM (1.0 m) was injected into each reaction tube. The apparatus was agitated in a sonic bath for 1.5 h, while maintaining a positive argon flow through the manifold. Following activation of the resin-bound ester with boron tribromide, the appropriate amines were injected as solutions in toluene into each reaction tube. A solution mixture of the amines was made by combining 1.0 mL each of 0.52M benzylamine in toluene, 0.52M diethylamine in toluene, 0.52M aniline in toluene, and 0.52M cyclohexylamine in toluene. The resultant mixture contained an equimolar concentration of each amine. 1.0 mL of the dissolved amine mixture was dispensed into each reaction tube in plate Y (Y1-Y4) to provide 0.25 equivalents each of benzylamine, diethylamine, aniline, and cyclohexylamine in each reaction tube, based upon indicated loading of commercial resin. The apparatus was agitated at 20-25 ºC in a sonic bath for 48 h, while maintaining a positive argon flow through the manifold. At the end of the reaction, the reaction tube array was drained and sparged with nitro- gen.

To isolate and purify the products, the reaction tubes, were sequentially submerged in reaction wells containing 10 mL each of toluene and DCM. The apparatus was agitated in a sonic bath for 10-15 minutes to extract the amides from the resins then drained by gravity and nitrogen sparge. A second extraction protocol with 5 mL each of 1M HCl was executed to thoroughly remove ureacted amines.

The filtrates were combined and the upper organic layer was separated. The combined organic extracts were washed with 1M HCl (2 × 10 mL), 1M NaOH (1 × 10 mL), and saturated NaCl (1 × 10 mL), dried with MgSO₄, filtered, and concentrated to yield the expected mixtures of substituted amides. The 8 unique mixtures were characterized by ¹H-NMR, and MS. The crude yields range from 6 to 23%. See Table 21.

Shown in Figure 1 are proposed combination statistics demonstrating the number of building blocks per step, namely, 4, 5 or 6 building blocks. Further indicated are the total number of reaction wells that may be utilized as well as the total number of compounds that may be produced. The small black box indicates the number of compounds per well. Table 18 shows the combinatorial statistics that may be obtained utilizing the techniques of the present invention, namely that combinations of mixtures of materials are synthesized and a rapid technique can be obtained for testing high numbers of different materials for biological or other activity.

In copending application Serial No.

(Case # PD 5137-01) filed concurrently herewith, entitled "Method and Apparatus for Simultaneously Testing a Plurality of Compounds to Detect Their Activity", commonly assigned to the assignee of the present invention, there is a discussion as to a large number of activities or biological assays that compounds can be tested for. Because the technique as described herein for the synthesis of mixtures of compounds are likewise useful for a rapid screening technique, the present invention is equally applicable toward the screens or activities as described therein. In other words, through the synthesis of mixture of compounds described herein, the mixtures may be tested without separating the materials. The screens can be used for determining a variety of biological activities as well as other utilities such as rust remover activity, ability to die textile materials, ability to clean substrates, ability to decompose in the presence of soil bacteria, ability to hybridize to a library of genes, ability to detect sunscreen activity, ability to detect immune response to mammals, ability to detect the presence or absence of a gene associated with a malady, ability to detect the activity of a gene associated with a biological response in mammals, ability to detect permeability of a membrane such as a cell, ability to detect signal transduction of a cell, ability to bind to a cell, ability to bind to an enzyme, ability to bind to an antibody, and the like. For a more detailed listing of screens or assays, see the aforementioned S.N.

, filed concurrently.

The present invention is equally applicable to determining an optimized dosage or weight for active compounds. In other words, the present invention can assist in quantitatively determining active materials and their degree of activity. In this manner, identical or differential amounts of a compound(s) to be tested are placed in various unique well locations.

Further, by the present invention, one may ascertain from the mixture of different compounds with unique molecular weights in the wells which materials were synthesized and are particularly active. One could therefore confirm and select for appropriate target molecules by the present invention. For example, as part of an analysis by mass spectrometry of a mixture of products in the wells one could confirm the synthesis of individual components within a matrix. The particular desired synthetic compounds would be uniquely designed and identified.

Further, by utilizing NMR techniques one could quantitate activities for individual materials within each matrix. At the end of the synthesis and NMR analysis, one would be able to quantify the desirable materials made, the desirable target molecular weights (in combination with mass spectrometry) and the concentration of the desired products. NMR, in combination with internal or external standards, could be used to quantitate the amounts of each individual compound in a synthetic mixture of products. Following synthesis, analysis, and testing, identification of potency by quantitative deconvolution would be achieved.

A general way of reviewing the ascertainment of compounds that are biologically active after the mixture has been detected to have biologic activity is described below. This is utilization of the concept "deconvolution", namely, the ascertainment of an individual compound that is reactive in a mixture. The key concept behind the method of deconvolution is that each well corresponds to a unique building block used in forming the compounds. Suppose each compound to be analyzed is formed in N steps; in other words, each compound consists of N building blocks. Each of the building blocks can be represented by a variable B_ij, where i is an index variable which indicates the step for which the building block B_ij is employed in forming a compound. An index variable j is used to distinguish between m different building blocks which can be used for each step i.

In order to further clarify the definition of B_ij, the different building blocks used at each step are grouped as follows:

All building blocks All building blocks All building blocks

used in step 1 used in step 2 used in step N

B ₁₁, B₁₁, ..., B_1m B₂₁, B₂₂. . .. B_2m . . . B_N1, B_N2, ..., B_Nm Each compound can be represented by a string formed by concatenating N building block variables, i.e., exactly one building block from each of the above groups. For example, {B₁₁ B₂₄ B₃₃ ... B_N2} represents one of the compounds.

In the step of creating the plates, each building block B_ij is assigned to a corresponding well W_ij. More specifically, the well Wij contains all possible compounds formed using building block B_ij. This is illustrated by the following example using N=3 steps and m=2 building blocks per step.

Example (N = 3 , m = 2 )

Though not necessary, the current embodiment arranges the wells on separate plates as follows:

Hence, plate 1 corresponds to the first building block, plate 2 corresponds to the second building block, and plate N corresponds to the Nth building block.

If a "hit" (or positive cavity) occurs for well W_ij, it can be concluded that there exists an active compound having a B_ij as a building block. Hence, one active compound will result in N hits, with each plate having one hit.

The method of deconvolution can be summarized as follows: using the observed hits, form all possible combinations of these observed hits using exactly one hit per plate. The potentially active components are then represented by these combinations.

Example (N = 3, m = 2):

Suppose the hits are in wells W₁₁, W₂₁, and W₃₂. The potentially active compound is {B₁₁ B₂₁ B₃₂}.

Next, suppose the hits are in wells W_u, W_2J, W₂₂, and W₃₂. The potentially active compounds are {B₁₁ B₂₁ B₃₂} and {B₁₁ B₂₂ B₃₂}.

In general, the method of the present invention allows the testing of m^N different compounds using mN total wells. The resulting fraction of wells per compound is mN/m^N, which simplifies to N/m^N-1. The number of compounds in each well is m^N-1. Using N plates to separate the wells results in m wells per plate. The above can be applied to the testing shown in Tables 20 and 21 as follows.

Plate Creation

A. Make up N "plates", numbers P=l to N.

B. Each plate has B_max wells, numbered W=l to B_max.

C. Into well "W" of plate "P", put exactly one copy of each string (compound) being Building Block B_ij = Brø. If max (By) for each step i = B_max, there will be (B_max)^N-1 such strings. For any string of length N, with j building blocks at step i = B_ij, max (B_ij) ≡ B_max. Note: Each string (compound) will occur exactly once on each plate in this scenario.

Plate Deconvolution A. Let the property be binary (1= Hit, 0= No Hit), contain no missing data.

B. Following testing, a list of wells with hits:

C. Matrix of Hits

If exactly one string is a hit and since each string occurs exactly once on each plate, then each row of the matrix W^ will have values = 0 except for a single "1" in the well containing the active string. Similarly, each column will have only one value = 1. Therefore, the non-zero entries of W_ij matrix = W_ij ^hit. For plate 1 = row 1 = W_ij ^hit, the hit occurs in column = j corresponding to well,

, corresponding to building block

Therefore, for each row (plate) i of W_ij,

For each column (well) j of W_ij;

If W_ij =∅, ignore it

otherwise (i.e., W_ij = 1).

Note that position (row) of the hit string must be building block,

End: otherwise,

next j

next i. D. Example

Each well :

cmpds. per well (Step C)

(See Table 20)

If "231" is the hit, then:

where = plate X (1)

plate Y (2)

plate Z (3)

or analogous values, for example:

where hit = "B2X".

Application of Algorithm:

For row 1

For column 2 (hit), position 1 of string - B₁₂ For row 2

For column 3 (hit), position 2 of string = B₂₃=2

For row 3

For column 1 (hit), position 3 of string = B₃₁=X So string = B2X Multiple Hits; Quantitative Deconvolution

A. Qualitative Hits: Uncertainty of Deconvolution

If in previous example, both "B2X" and "C2Y" are active and generate hits:

The basic algorithm breaks down and any of the following could produce this result:

B2X + C2Y

B2X + C2Y + C2X

B2Y + B2X + C2X + C2Y

B2Y + C2X + C2Y

B2Y + C2X

Worst case: if A0X, B1Y, and C2Z all hits

compatible with all 27 possible strings.

B. Quantitative Hits (Based on Analytical Calibration)

Suppose B2X has hit value = 3

& C2Y has hit value = 2

& values sum linearly

Therefore, "5" is clearly a sum, since its a single value in rows that generally have 2 values. However, B2X with hit value = 2

& C2Y with hit value = 2

& C2Z with hit value = 2

Ambiguous because hits could also be:

Other alternative combinations may be possible.

While the forms of the invention herein disclosed constitute presently preferred embodiments, others may be possible. It is not intended herein to mention all of the possible equivalent forms or ramifications of the invention. It is understood that terms used herein are merely descriptive rather than limited, and that various changes may be made without departing from the spirit or scope of the invention.

Claims

What Is Claimed Is:

1. A method of synthesizing a plurality of compounds in a plurality of wells comprising the steps of:

(a) providing a plurality of test wells in a plurality of arrays of the wells;

(b) reacting in at least a one step reaction a first reagent with a plurality of reagents called building blocks in the test well to obtain a unique product designed to be the same in each array; and

(c) continuing to react the same or additional reagents such that there are multiple reagents resulting in mixtures of multiple different products in each well.

2. The method of claim 1 wherein there are at least two steps to the reaction scheme.

3. The method of claim 1 wherein there are at least three steps to the reaction scheme.

4. The method of claim 1 wherein there are at least three building blocks for each step.

5. The method of claim 1 wherein there are at least four building blocks for each step.

6. The method of claim 1 wherein there are at least two arrays of test wells.

7. The method of claim 1 wherein there are at least three arrays of test wells.

8. The method of claim 1 wherein there are at least four arrays of test wells.

9. The method of claim 1 wherein the compounds synthesized are dipeptides.

10. The method of claim 1 wherein the compounds synthesized are hydantoins.

11. The method of claim 1 wherein the compounds synthesized are benzodiazepines.

12. The method of claim 1 wherein the com- pounds synthesized are quinolones.

13. The method of claim 1 wherein the compounds synthesized are keto-ureas.

14. The method of claim 1 wherein the compounds synthesized are N²-substituted hydantoins.

15. The method of claim 1 wherein the compounds synthesized are (R)-4-benzamido-5-oxopentanoic acids.

16. The method of claim 1 wherein the compounds synthesized are diketopiperazines.

17. The method of claim 1 wherein the compounds synthesized are tetrahydro-4-hydroxy-6-[2-(1H-pyrrol-lyl)ethyl]-2H-pyran-2-ones.

18. The method of claim 1 wherein the compounds synthesized are N-arylpiperazines.

19. The method of claim 1 wherein the compounds synthesized are benzisothiazolones.

20. The method of claim 1 wherein the compounds synthesized are isoindolone-based spirosuccinimdes.

21. The method of claim 1 wherein the compounds synthesized are pilocarpine analogs.

22. The method of claim 1 wherein the compounds synthesized are 3substituted 1-(aminomethyl)-3,4-dihydro-5,6-dihydroxy-1H-2-benzopyrans.

23. The method of claim l wherein the compounds synthesized are 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-diones.

24. The method of claim 1 wherein the compounds synthesized are tepoxalin derivatives.

25. The method of claim 1 further comprising analyzing the mixtures of reaction products to identify the unique products produced.

26. The method of claim 25 wherein the analysis is performed by nuclear magnetic resonance.

27. The method of claim 25 wherein the analysis is performed by mass spectroscopy.

28. The method of claim 25 wherein the analysis of the mixtures is performed by comparing the spectra of different mixtures of known compounds to confirm the presence or absence of desired products.

29. A method of simultaneously testing a plurality of compounds for activity comprising the steps of:

(a) providing a plurality of test wells in a plurality of arrays of the wells;

(b) reacting in at least a one step reaction a first reagent with a plurality of reagents called building blocks in the test well to obtain a unique product designed to be the same in each array;

(c) continuing to react reagents such that there are multiple reagents resulting in mixtures of multiple different products in each well;

(d) determining the location of each compound in each test well;

(e) subjecting the arrays of mixtures of products to a testing screen; and

(f) ascertaining those compounds that had a positive response to the testing screen.

30. The method of claim 29 further comprising ascertaining quantitative data from the testing screen.

31. The method of claim 29 wherein there are at least two steps to the reaction scheme.

32. The method of claim 29 wherein there are at least three steps to the reaction scheme.

33. The method of claim 29 wherein there are at least three building blocks for each step.

34. The method of claim 29 wherein there are at least four building blocks for each step.

35. The method of claim 29 wherein there are at least two arrays of test wells.

36. The method of claim 29 wherein there are at least three arrays of test wells.

37. The method of claim 29 wherein there are at least four arrays of test wells.

38. The method of claim 29 further comprising analyzing the mixtures of reaction products to identify the unique products produced.

39. The method of claim 38 wherein the analysis is performed by nuclear magnetic resonance.

40. The method of claim 38 wherein the analysis is performed by mass spectroscopy.

41. The method of claim 39 wherein the analysis of the mixtures is performed by comparing the spectra of different mixtures of known compounds to confirm the presence or absence of desired products.

42. The method of claim 40 further comprising designing molecular weights of products and analyzing the mixtures to confirm those compounds which were synthetically achieved.

43. The method of claim 41 further comprising utilizing quantitative methods in combination with NMR for the compounds synthesized and ascertaining individual product yields and concentrations.

44. The method of claim 43 wherein the product mixtures are tested to ascertain quantitative activity for individual components.