USOO5874589A United States Patent (19) 11 Patent Number: 5,874,589 Campbell et al. 45) Date of Patent: Feb. 23, 1999

54 METHODS FOR SYNTHESIZING DIVERSE El Marini et al., 1992, Synthesis pp. 1104-1108 Synthesis of COLLECTIONS OF TETRAMIC ACIDS AND enantiomerically pure B-and Y-amino acids from aspartic DERVATIVES THEREOF and glutamic acid derivatives. Evans et al., 1982, J. Amer. Chem. Soc. 104: 1737–1739 75 Inventors: David A. Campbell, San Mateo; Todd Asymmetric alkylation reactions of chiral imide enolates. A T. Romoff, San Jose, both of Calif. practical approach to the enantioselective Synthesis of C-Substituted carboxylic acid derivatives. 73 Assignee: GlaxoWellcome, Inc., Research Fontenot et al., 1991, Peptide Research, 4: 19-25A Survey Triangle Park, N.C. of potential problems and qulaity control in peptide Synthe sis by the flourenylmethocvarbonyl procedure. 21 Appl. No.: 896,799 Giesemann et al., 1982, J. Chem. Res. (S) pp. 79 Synthesis 22 Filed: Jul.18, 1997 of chiral C-isocyano esters and other base-Sensitive isocya nides with 51) Int. Cl...... C07D 211/40; CO7D 207/00 oxomethylenebis-(3H-Imidazolium)Bis(methanesulphonate), 52 U.S. Cl...... 548/540; 546/220; 548/539 a versatile dehydrating reagent. 58 Field of Search ...... 546/220; 548/539, Geysen et al., 1987, J. Immunol. Meth. 102: 259-274 548/540 Strategies for epitope analysis using peptide Synthesis. Giron-Forest et al., 1979, Analytical Profiles of Drug Sub 56) References Cited stances, 8: 47-81 Bromocriptine methaneSulphonate. U.S. PATENT DOCUMENTS Gokeletal, 1971, Isonitrile Chemistry, Ugi, I. ed., Academic 3,299.095 1/1967 Harris et al...... 548/531 Press, NY pp. 145-199 Four-component condensations and 3,361,626 1/1968 Harris et al...... 514/425 related reactions. 3,752,814 8/1973 Fluckiger et al. ... 544/346 Gordon et al., 1995, Bioorganic & Medicinal Chemistry 3,752,888 8/1973 Fluckiger et al. ... 514/250 Letters, 5: 47–50 Reductive alkylation on a solid phase: 3,923,790 12/1975 Imanaka et al...... 540/456 Synthesis of a piperazinedione combinatorial library. 3,929,790 12/1975 Leimgruber et al. . ... 544/347 3,984.559 10/1976 Weinstock ...... 514/343 Hasumi et al., 1993, J. Antibiotics 46: 1782–1787 Lateritin, 4,522,911 6/1985 Clecak et al. . ... 430/192 a new inhibitor of acyl-CoA: cholesterol acyltransferase 4,683.202 7/1987 Mullis ...... 435/91.2 produced by Gibberella. 4,694,081 9/1987 Miller et al...... 544/385 Ho et al., 1993, Peptide Research, 6: 10-12 An improved 4,965,188 10/1990 Mullis et al...... 435/6 low racemization Solid-phase method for the Synthesis of 5,147,875 9/1992 Coates et al. . ... 514/259 5,164,388 11/1992 De et al. ... 514/235.8 reduced dipeptide (YCH2NH) Bond Isosteres. 5,288,514 2/1994 Ellman ...... 435/4 Horwell et al. (1993), Bioorganic & Medicinal Chemistry 5,324.483 6/1994 Cody et al...... 422/131 Letters, 3(5):799-802 The Design of a dipeptide library for 5,475,013 12/1995 Talley et al...... 514/311 Screening at peptide receptor Sites. 5,498,732 3/1996 Loffet ...... 549/274 Hwang et al., 1983, Biochemistry 22: 4756–4763 Specific FOREIGN PATENT DOCUMENTS receptor Sites for 1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

2 266 888 11/1993 United Kingdom ...... CO7D 207/44 (platelet activating factor) on rabbit platelet and guinea pig WO 92/00091 1/1992 WIPO ...... A61K 37/02 Smooth muscle membranes. WO95/12608 5/1995 WIPO ...... CO7K 1/04 Jung and Beck-Sickinger (1992), Angewandte Chemie OTHER PUBLICATIONS 31(4):367-486 Multiple peptide synthesis methods and their applications. Armstrong et al., 1996, Acc. Chem. Res. 29: 123-131 Multiple-component condensation Stratgies for combinato Keating et al., 1996, J. Am. Chem. Soc. 118:2574–2583 rial library synthesis. Postcondensation modifications of Ugi four-condensation Bertho et al., 1991, Tetrahedron Ltrs. 32: 1303–1306 Amino products: 1-isocyanocyclohexene as a convertible isocya acid flourides: Their preparation and use in peptide Synthe nide. SS. Brenner et al. (1992), Proc. Natl. Acad. Sci. USA (List continued on next page.) 89:5381-5383. Encoded combinatorial chemistry. Primary Examiner John Kight Burger et al., 1993, J. Fluorine Chem. 65: 149-152 3,3, 3-trifluoro-2-isocyanopropionates, new versatile building ASSistant Examiner-Garth M. Dahlen blooks for the introduction of trifluoromethyl groups into Attorney, Agent, or Firm-Lauren L. Stevens organic molecules. 57 ABSTRACT Carpino et al. (1990), J. Am. Chem. Soc. 112:9651-9652 ((9-Fluorenylmethyl)oxy)carbonyl (FMOC)) amino acid Disclosed are methods for Synthesizing very large collec fluorides. tions of diverse diverse tetramic acids, tetronic acids, pen Chaturvedi et al., 1970, J. Med. Chem. 13: 177-181 Analogs tamic acids, pentonic acids, and derivatives thereof and of angiotensis II. I. Solid Phase Synthesis. Synthetic compound libraries comprising compounds pre Coy et al., 1988, Tetrahedron, 44: 835–841 Solid phase pared by Such methods. reductive alkylation techniques in analogue peptide bond and Side chain modification. 7 Claims, No Drawings 5,874,589 Page 2

OTHER PUBLICATIONS Shimazaki et al., 1987, Chem Parm. Bull., 35: 3527-3530 Kim et al. (1992), Tetrahedron Asymmetry, 3: 1421-1430 Diketopiperazine derivatives, A new Series of platelet-acti Polymer attached cyclic dipeptides as catalysts for enanti Vating factor inhibitors. oSelective cyanohydrin formation. Kucharczyk et al., 1993, J. Med. Chem., 36: 1654–1661 Shimazaki et al., 1987, J. Med. Chem. 30: 1706–1709 Tetrapeptide tachykinin antagonists: Synthesis and modula Diketopiperazines as a new class of platelet-activating fac tionof the physicochemical and pharmacological properties tor inhibitors. of a new Series of partially cyclic analogs. Leznoff (1978), Account of Chemical Research, pp. Shiosaki et al., 1990, Peptides. Chemistry Structure and 327–333. The use of insoluble polymer supports in general Biology ESCOM Science Publishers B.V., The Netherlands, organic Synthesis. pp. 978-980 Toward development of peptidomimetics: Palom et al., 1993, Tetrahedron Letters, 34: 2195-2198 An acid-labile linker for Solid-phase oligoribonucleotide Syn Diketopiperazine templates for the Trp-Met Segment of thesis using Fmoc group for 5'-hydroxyl protection. CCK-4. Rajappa et al., 1993, Advances in heterocyclic chemistry 57: 187-289 Piperazine-2,5-diones and related lactim Shiosaki et al., 1993, Bioorganic & Medicinal Chemistry ethers. Letters, 3: 855-860 Toward development of peptidomimet Sammes et al., 1975, Chem. Org. Naturst., 32: 51-118 ics: Diketopiperazine templates for the Trp-met Segment of Naturally occuring 2,5-dioxopiperazines and related com CCK-4. pounds. Scott et al., 1995, Molecular Diversity 1(2): 125-134 Solid Williams et al., 1988, Chem Rev., 88: 511–540 Bicyclomy phase organic Synthesis (SPOS):a novel route to diketopip cin: Synthetic, Mechanistic, and Biological Studies. erazines and diketomorpholines. Shen et al., Feb. 1985, Proc. Natl. Acad. Sci. USA. 82: Winitz et al., 1956, J. Amer. Chem. Soc. 78:2423–2430 672-676 Characerization of a platelet-activating factor Studies on diastereoisomeric C-amino acids and corre receptor antagonist isolated from haifenteng (piper futokad sponding O-hydroxy acids. VII. Influence of B-configura Sura): Specific inhibition of in vitro and in vivo platelet-ac tion on enzymic Susceptibility. tivating factor-induced effects. Shih et al., 1990, J. Am. Chem. Soc., 112: 9652-9654 Zuckermann et al., 1992, J. Am. Chem. Soc. 114: Biosynthesis of 3,6-Dideoxyhexoses: Strereochemical 10646-10647 Efficient method for the preparation of pep Analysis of the Deprotonation Catalyzed by the Pyriodxam toidsoligo(N-substituted glycines) by submonomer solid ine 5'-Phosphate Dependent Enzyme phase Synthesis. CDP-4-keto-6-deoxy-D-glucose-3-dehydrase Isolated from Yersinia Pseudotuberculosis. Jouin, P. et al., J. Chem. Soc. Perkin Trans. I, 1987, p. 1177. 5,874,589 1 2 METHODS FOR SYNTHESIZING DIVERSE and bacterial RNA polymerase enzymes. BU2313A and COLLECTIONS OF TETRAMIC ACIDS AND BU2313B are broad-spectrum antibiotics effective against DERVATIVES THEREOF both Gram-positive and Gram-negative anaerobic bacteria as well as Some aerobic bacteria including Streptococci. BACKGROUND OF THE INVENTION Tirandamycin and the BU2313 compounds have exhibited efficacy in mouse protection tests, and tirandamycin in a 1. Field of the Invention mouse abscess model for Bacteroides fragilis. This invention is directed to methods for Synthesizing very large collections of diverse tetramic acids (i.e., 2,4- s - O s s pyrrolidinediones) and derivatives thereof, including s tetronic acids, thiotetronic acids, pentamic acids, pentonic acids, and thiopentonic acids. This invention is further directed to methods for identifying and isolating tetramic acid compounds and derivatives thereof with useful and OH diverse activities from Such collections. 15 2. State of the Art Compounds having biological activity can be identified by Screening diverse collections of compounds (i.e., libraries Streptolydigin of compounds) produced through either molecular biologi Streptomyces lydicus cal or Synthetic chemical techniques. Such Screening meth ods include methods wherein each member of the library is tagged with a unique identifier tag to facilitate identification of compounds having biological activity or where the library comprises a plurality of compounds Synthesized at Specific locations on the Surface of a Solid Substrate wherein a 25 receptor is appropriately labeled to identify binding to the compound, e.g., fluorescent or radioactive labels. Correla Tirandamycin tion of the labeled receptor bound to the substrate with its Streptomyces tirandis location on the Substrate identifies the binding compound. Central to these methods is the Screening of a multiplicity of compounds in the library and the ability to identify the Structures of the compounds which have a requisite biologi cal activity. Preferably, in order to facilitate synthesis and identification, the compounds in the library are typically formed on Solid Supports wherein the compound is 35 covalently attached to the Support via a cleavable or non BU2313A (R = H) cleavable linking arm. In this regard, libraries of diverse BU2313B (R = CH3) compounds are prepared and then Screened to identify “lead compounds' having good binding affinity to the receptor. 40 Although the complicated polycyclic Side chains of the Pharmaceutical drug discovery relies heavily on Studies of above antibiotics may be required for inhibition of RNA Structure-activity relationships wherein the Structure of polymerase and DNA transferase, the 3-acyl-2,4- “lead compounds” is typically altered to determine the effect pyrrolidinedione unit itself offerS Significant potential as a of the alteration on activity. Alteration of the structure of the drug scaffold. With a pKa of 5, the ability to chelate metals, lead compounds permits evaluation of the effect of the 45 and the two ketone units, acyltetramic acids possess a Structural alteration on activity. Thus libraries of compounds number of potential binding modes with biological targets. derived from a lead compound can be created by including Tetramic acids and derivatives thereof are also valuable derivatives of the lead compound and repeating the Screen chiral Starting materials for the Synthesis of other natural and ing procedures. unnatural products. Ideally, the compounds are Synthesized in Situ on the Solid 50 A variety of Solution phase techniques have been devel Support So that the Support can be tagged to identify the oped to prepare tetramic acids. For example, it is known to Synthetic Steps employed and/or the derivative incorporated condense activated N-protected alpha-amino acids with onto the Support. However, relatively simple Synthetic meth Meldrum S acid to yield the corresponding ods to produce a diverse collection of Such derivatives on the 1-hydroxyalkylidene Meldrums acid. Upon heating in solu Supports are often not available. 55 tion acetone and CO2 are eliminated to form N-protected One particular class of compounds which would be useful 5-substituted tetramic acids. See Jouin et al. (1987).J. Chem. for inclusion in Screening libraries are tetramic acids com Soc. Perkin Trans. I 1177. Alternatively, alpha-amino acid pounds. 3-Acyl-2,4-pyrrolidinediones (acyltetramic acids) esters can be reacted with malonic acid ester chlorides to are a class of natural products that have attracted Significant yield the corresponding N-(alkoxycarbonylacetyl)-alpha attention over the years as a result of their biological 60 amino acid esters. The latter are cyclized to the activities and the Synthetic challenges presented by their 3-alkoxycarbonyl tetramic acids. See Mulholland et al. complex Structures. Representative examples of this struc (1972) J. Chem. Soc. Perkin Trans. I 2121. See, also, U.S. tural class include Streptolydigin, tirandamycin A, BU2313A Pat. No. 5,008,402, U.S. Pat. No. 5,534,540, U.S. Pat. No. and BU2313B which are natural products extracted from 5,468,774, U.S. Pat. No. 4,996,227, and U.S. Pat. No. fungi that exhibit potent antibacterial activities. Streptoly 65 5,498,732. digin and tirandamycin exhibit potent activity against Gram However, the incorporation of a multiplicity of tetramic positive organisms by inhibiting terminal DNA transferase acid derivatives on Solid Supports is not previously known. 5,874,589 3 4 The ability to synthesize a multiplicity of tetramic acid b) contacting the amine component with a f-keto ester derivatives on a Solid Support or on different Solid Supports equivalent to yield a B-keto amide; and would enhance the Structural variation of a library and c) treating the B-keto amide under conditions Suitable for provide important Structure-activity information. the cyclization and concomitant release of the tetramic acid SUMMARY OF THE INVENTION group from the Solid Support. This invention is directed to general Synthetic methods for In a particularly preferred embodiment, Step a) of the preparing tetramic acids and derivatives thereof, including method further comprises the Step of reductively alkylating thiotetronic acids, pentamic acids, pentonic acids, and thio the covalently bound amine component. pentonic acids, which methods can be employed in conjunc DETAILED DESCRIPTION OF THE tion with known Stochastic methods for preparing libraries INVENTION of compounds comprising one or more tetramic acid or related groups. In one embodiment, the compounds can be I. Terminology further derivatized thereby elaborating their structure. Prior to discussing this invention in further detail, the Accordingly, in one of its method aspects, this invention following terms will first be defined: is directed to a method for Synthesizing a tetramic acid or 15 “Alkoxy' refers to the group alkyl-O-. pentamic acid group or derivative thereof which method comprises: “Alkyl” refers to a cyclic, branched, or Straight chain (a) providing an amine component immobilized on a Solid chemical group containing only carbon and hydrogen, Such Support, as methyl, heptyl, -(CH2)2-, and adamantyl. Alkyl groups (b) contacting the amine component with a f-keto ester can either be unsubstituted or Substituted with one or more equivalent to yield a B-keto amide; and Substituents, e.g., halogen, alkoxy, acyloxy, amino, aryl, (c) treating the f-keto amide under conditions Suitable for hydroxyl, mercapto, carboxy, benzyloxy, phenyl, benzyl, or the cyclization and concomitant release of the compound other functionality which may be suitably blocked, if nec from the Solid Support. essary for purposes of the invention, with a protecting group. The amine component may comprise a primary amino 25 Typically, alkyl groups will comprise 1 to 12 carbon atoms, group having the formula: R-NH, wherein R is selected preferably 1 to 10, and more preferably 1 to 8 carbon atoms. from the group consisting of alkyl, amino, aryl, heteroaryl, “Amino” or “amine group” refers to the group -NR'R", and arylalkyl or Salts thereof. According to the latter where R' and R" are independently selected from the group embodiment, R can also be hydrogen. More preferably, the consisting of hydrogen, alkyl, Substituted alkyl, aryl, Sub amine component will have the formula: Stituted aryl, aryl alkyl, Substituted aryl alkyl, heteroaryl, and Substituted heteroaryl. In a primary amino group, both R" and R" are hydrogen, whereas in a Secondary amino group, either, but not both, R' or R" is hydrogen. --> NH An “C.-amino acid” consists of a carbon atom, called the 35 C-carbon, to which is bonded an amino group and a carboxyl wherein R' and R are independently selected from the group. Typically, this C-carbon atom is also bonded to a groups consisting of hydrogen, alkyl, aryl, heteroaryl, hydrogen atom and a distinctive group referred to as a "side carboxyl, carboxyalkyl, and arylalkyl. More preferably, the chain.” The hydrogen atom may also be replaced with a amine component will comprise an alpha- (i.e., to produce group Such as alkyl, Substituted alkyl, aryl, Substituted aryl, a tetramic acid) or beta-amino acid (i.e., to produce a 40 heteroaryl, and other groups. The Side chains of naturally pentamic acid). In a particularly preferred embodiment, the occurring amino acids are well known in the art and include, amine group of the amine component will be alkylated. for example, hydrogen (as in glycine), alkyl (as in alanine Preferably, the B-keto ester equivalent will comprise an (methyl), Valine (isopropyl), leucine (sec-butyl), isoleucine acyl Meldrums acid, a lower alkyl ester of a B-keto car (iso-butyl), and proline (-(CH2) )), Substituted alkyl (as boxylic acid, or an acyl chloride of a B-keto carboxylic acid. 45 in Serine (hydroxymethyl), cysteine (thiomethyl), aspartic Most preferably, the B-keto ester equivalent will comprise an acid (carboxymethyl), asparagine, arginine, glutamine, acyl Meldrums acid. glutamic acid, and lysine), aryl alkyl (as in phenylalanine, Other embodiments of the invention are drawn to the histidine, and tryptophan), Substituted aryl alkyl (as in Synthesis of analogs of tetramic acids, Such as thiotetronic tyrosine and thyroxine), and heteroaryl (as in histidine). See, acids, pentamic acids, pentonic acids, and thiopentonic acids 50 e.g., Harper et al. (1977) Review of Physiological Chemistry and derivatives thereof. According to this embodiment, a 16th Ed., Lange Medical Publications, pp. 21-24. hydroxyl or thiol component is contacted with a B-keto ester In addition to naturally occurring Side chains, the amino to yield a Second B-keto ester. AS in the Synthesis of tetramic acids used in the present invention may possess Synthetic acids, Diekmann cyclization and concomitant release from Side chains. A “synthetic Side chain' is any Side chain not the support yields the desired compound. If the hydroxyl 55 found in a naturally occurring amino acid. For example, a component comprises an O-hydroxy acid, tetronic acids are Synthetic Side chain can be an isostere of the Side chain of formed. Likewise, if B-hydroxy acids are used, pentonic a naturally occurring amino acid. Naturally occurring and acids are produced. If an O-mercapto acid is used, Synthetic Side chains may contain reactive functionalities, thiotetronic acids result. Likewise, if a B-mercapto acid is Such as hydroxyl, mercapto, and carboxy groups. One used, thiopentonic acids result. 60 skilled in the art will appreciate that these groups may have In another of its method aspects, this invention is directed to be protected to carry out the desired reaction Scheme. AS to a method for preparing a library of tetramic acids, Stated above, the hydrogen at the C-carbon can also be pentamic acids or derivatives thereof, which library is Syn replaced with other groups, those of skill in the art recognize thesized in a proceSS comprising: the medicinal importance of C.-methyl amino acids and other a) apportioning the Supports comprising a covalently 65 Cl-, C.-disubstituted amino acids. bound amine component, preferably, an alpha- or beta “Aryl' or “Ar” refers to an aromatic carbocyclic group amino acid, among a plurality of reaction vessels, having a single ring (e.g., phenyl) or multiple condensed 5,874,589 S 6 rings (e.g., naphthyl or anthryl), which can optionally be “Pentamic acid” refers to the basic ring structure: unsubstituted or substituted with amino, hydroxyl, lower alkyl, alkoxy, aryloxy, chloro, halo, mercapto, aryl, and other Substituents. Preferred aryl groups include phenyl, 1-naphthyl, 2-naphthyl, biphenyl, phenylcarboxylphenyl (i.e., derived from benzophenone), and the like. “Aryloxy” refers to the group aryl-O- or heteroaryl-O-. “Arylalkyl” refers to the groups R'-Ar and R-HetAr, where Ar is an aryl group, HetAr is a heteroaryl group, and wherein the nitrogen and carbon atoms may bear a variety of R" is Straight-chain or branched-chain aliphatic group. Substituents as described hereinbelow. Examples of arylalkyl groups include benzyl and furfuryl. “Pentonic acid” refers to the basic ring structure: “Carboxy” or “carboxyl” refers to the group-R(COOH) where R is alkyl, Substituted alkyl, aryl, Substituted aryl, aryl alkyl, Substituted aryl alkyl, heterocyclic, heteroaryl, or substituted heteroaryl. 15 “Carboxyalkyl” refers to the group -(CO)-R" where R' is alkyl or substituted alkyl. “Carboxyaryl” refers to the group -(CO)-R' where R' wherein the carbon atoms may bear a variety of Substituents is aryl, heteroaryl, or Substutited aryl or heteroaryl. as described hereinbelow. “Chemical library” or “array' is an intentionally created “Protecting group' refers to a chemical group that exhib collection of differing molecules which can be prepared its the following characteristics: (1) reacts Selectively with either Synthetically or biosynthetically and Screened for the desired functionality in good yield to give a derivative biological activity in a variety of different formats (e.g., that is Stable to the projected reactions for which protection libraries of soluble molecules; and libraries of compounds 25 is desired; 2) can be selectively removed from the deriva tethered to resin beads, Silica chips, or other Solid Supports). tized Solid Support to yield the desired functionality; and 3) The term is also intended to refer to an intentionally created is removable in good yield by reagents compatible with the collection of Stereoisomers. other functional group(S) generated in Such projected reac “Combinatorial synthesis strategy” or “combinatorial tions. Examples of protecting groups can be found in Greene chemistry” refers to an ordered Strategy for the parallel et al. (1991) Protective Groups in Organic Synthesis, 2nd Synthesis of diverse compounds by Sequential addition of Ed. (John Wiley & Sons, Inc., New York). Preferred pro reagents which leads to the generation of large chemical tecting groups include photolabile protecting groups (Such libraries. Thus, combinatorial chemistry refers to the sys as methylnitropiperonyloxycarbonyl (Menpoc), methylni tematic and repetitive, covalent connection of a Set of tropiperonyl (Menp), nitroveratryl (Nv), nitroveratryloxy 35 carbonyl (Nvoc), or nitroveratryloxymethyl ether (Nvom)); different “building blocks” of varying structures to each acid-labile protecting group (Such as Boc or DMT); base other to yield large arrays of diverse molecular entities. labile protecting groups (such as Fmoc, Fm, phosphonioet “Heteroaryl' or “HetAr” refers to a monovalent unsatur hoxycarbonyl (Peoc, see Kunz (1976) Chem. Ber ated aromatic carbocyclic group having a single ring (e.g., 109:2670); groups which may be removed under neutral pyridyl or furyl) or multiple condensed rings (e.g., indoliz 40 conditions (e.g., metal ion-assisted hydrolysis ), Such as inyl or benzothienyl) and having at least one hetero atom, DBMB (see Chattopadhyaya et al. (1979) J.C.S. Chem. Such as N, O, or S, within the ring, which can optionally be Comm. 987–990), allyl or alloc (see, e.g., Greene and Wuts, unsubstituted or Substituted with amino, hydroxyl, alkyl, “Protective Groups in Organic Synthesis”, 2nd Ed., John alkoxy, halo, mercapto, and other Substituents. Wiley & Sons, Inc., New York, N.Y. (1991), 2-haloethyl (see “Identifier tag denotes a physical attribute that provides 45 Kunz and Buchholz (1981) Angew. Chem. Int. Ed. Engl. a means whereby one can identify a chemical reaction. The 20:894), and groups which may be removed using fluoride identifier tag Serves to record a step in a Series of reactions ion, such as 2-(trimethylsilyl)ethoxymethyl (SEM), used in the Synthesis of a chemical library. The identifier tag 2- (trimethylsilyl)ethyloxycarbonyl (Te o c) or may have any recognizable feature, including for example: 2-(trimethylsilyl)ethyl (Te) (see, e.g., Lipshutz et al. (1980) a microscopically or otherwise distinguishable shape, Size, 50 Tetrahedron Lett. 21:3343-3346)); and groups which may mass, color, optical density, etc., a differential absorbance or be removed under mild reducing conditions (e.g., with emission of light, chemical reactivity; magnetic or electronic sodium borohydride or hydrazine), such as Lev. Id. at 30–31, properties, or any other distinctive mark capable of encoding 97, and 112. Particularly preferred protecting groups include the required information, and decipherable at the level of Fmoc, Fm, Menpoc, Nvoc, NV, Boc, CBZ, allyl, alloc, one (or a few) molecules. A preferred example of Such an 55 Npeoc (4-nitrophenethyloxycarbonyl) and Npeom identifier tag is an oligonucleotide, because the nucleotide (4-nitrophenethyloxy-methyloxy). Sequence of an oligonucleotide is a robust form of encoded “Solid Support' or “Support” refers to a material or group information. Identifier tags can be coupled to the Solid of materials having a rigid or Semi-rigid Surface or Surfaces. Support. Alternatively, the “identifier tag can be coupled In many embodiments, at least one Surface of the Solid directly to the compound being Synthesized, whether or not 60 Support will be Substantially flat, although in Some embodi a Solid Support is used in the Synthesis. In the latter ments it may be desirable to physically Separate Synthesis embodiment, the identifier tag can conceptually be viewed regions for different compounds with, for example, Wells, as also Serving as the “Support' for Synthesis. raised regions, pins, etched trenches, or the like. According "Linker” refers to a molecule or group of molecules to other embodiments, the solid support(s) will take the form attached to a Solid Support and Spacing a synthesized com 65 of beads, resins, gels, microSpheres, or other geometric pound from the Solid Support, Such as for exposure/binding configurations. The Solid Support is alternatively referred to to a receptor. herein as a Support. 5,874,589 7 8 "Stereoisomer' refers to a chemical compound having the BOC=t-butyloxycarbonyl Same molecular weight, chemical composition, and consti BOP=benzotriazol-1-yloxytris(dimethylamino) phospho tution as another, but with the atoms grouped differently. That is, certain identical chemical moieties are at different nium hexafluorophosphate orientations in Space and, therefore, when pure, has the DCC=dicyclohexylcarbodiimide ability to rotate the plane of polarized light. However, Some Ddz=dimethoxydimethylbenzyloxy pure Stereoisomers may have an optical rotation that is So Slight that it is undetectable with present instrumentation. DIC=diisopropylcarbodiimide The compounds of the instant invention may have one or DCM=dichloromethane more asymmetrical carbon atoms and therefore include various Stereoisomers. All Stereoisomers are included within 1O DIAD=diisopropyl azodicarboxylate the Scope of the invention and within the Scope of the term DMAP=4-dimethylaminopyridine “tetramic acid”. “Tetramic acid” refers to the basic ring structure: DMT=dimethoxytrityl Fmoc fluorenylmethyloxycarbonyl O O 15 M Fmoc-Cl=fluorenylmethyloxycarbonyl chloride R HBTU = 2 - (1 H-benzo triazol-1-yl)-1,1,3,3- N tetramethyluronium hexafluorophosphate O HOBt=1-hydroxybenzotriazole Menpoc=methylnitropiperonyloxycarbonyl wherein the nitrogen and carbon atoms may bear a variety of Menp=methylnitropiperonyl Substituents as described hereinbelow. “Tetronic acid” refers to the basic ring structure: Nv=nitroveratry1 25 Nvoc=6-nitroveratryloxycarbonyl and other photoremov O O able groups R OPfp=pentafluorophenyloxy O OSu=N-Succinimidyloxy (also known as NHS) O PG=protective group PPh=triphenyl phosphine wherein the carbon atoms may bear a variety of Substituents as described hereinbelow. TFA=trifluoroacetic acid “Thiopentonic acid” refers to the basic ring structure: 35 O O II. Overview The present invention, in one aspect, includes a highly efficient and Versatile method of Synthesizing and Screening, O 40 preferably in parallel and Simultaneous fashion, large num bers of tetramic acids or derivatives thereof. Thus, according wherein the carbon atoms may bear a variety of Substituents to one aspect, the present invention provides a Synthesis and the Sulfur may be alkylated, oxidized and the like as method for tetramic acids in which variable Substituent described hereinbelow. groups are attached to a common central Structure using the “Thiotetronic acid” refers to the basic ring structure: 45 procedure shown below:

O O O

R S 50 O wherein the carbon atoms may bear a variety of Substituents and the Sulfur may be alkylated, oxidized and the like as described hereinbelow. 55 Isolation and purification of the compounds and interme diates described herein can be effected, if desired, by any Suitable Separation or purification procedure Such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer 60 (preparative) chromatography, distillation, or a combination of these procedures. Specific illustrations of Suitable Sepa ration and isolation procedures can be had by references to the examples hereinbelow. However, other equivalent Sepa ration or isolation procedures can, of course, also be used. 65 Abbreviations: The following abbreviations are intended to have the following meanings: 5,874,589 9 10 -continued -continued OH O R1 N N-/ Rs O R1 wherein R and R are derived from the hydroxyl compo nent and R is derived from the beta-keto ester equivalent. wherein R is derived from the amine component, Rs results Likewise, if the hydroxyl component comprises an alpha from the alkylation of the amine component, and R is hydroxy acid, tetronic acids result. derived from the beta-keto ester equivalent. The use of structures of these kinds for certain multiple If the amine component comprises a beta-amino acid (or Simultaneous reactions is known in the art, and its applica alkylated beta-amino acid), pentamic acids (i.e., 3-acyl-2,4- tion to the present invention will become apparent from the piperidinediones) can be prepared: 15 description which follows.

O O III. The Components A. The Solid Support R1 1. Nature of the Support Typically, the libraries or arrays of the invention are initially composed of a collection of “solid supports”. Such Solid Supports may be of any shape, although they preferably will be roughly Spherical. The Supports need not necessarily be homogenous in size, shape or composition; although the Supports usually and preferably will be uniform. In Some embodiments, Supports that are very uniform in Size may be particularly preferred. In another embodiment, two or more distinctly different populations of Solid Supports may be used for certain purposes. “Solid Support' embraces a particle with appropriate Sites RSCH2 R2 for compound Synthesis and, in Some embodiments, tag attachment and/or synthesis. Solid Supports may consist of O O many materials, limited primarily by capacity for derivati R 35 Zation to attach any of a number of chemically reactive R3 2 groupS and compatibility with the Synthetic chemistry used to produce the array and, in Some embodiments the methods O N R1 used for tag attachment and/or Synthesis. Suitable Support RSCH2 materials typically will be the type of material commonly 40 used in peptide and polymer Synthesis and include glass, lateX, heavily croSS-linked polystyrene or Similar polymers, wherein R, R2 and Rs are derived from the amine gold or other colloidal metal particles, and other materials component, and R is derived from the beta-keto ester known to those skilled in the art. Except as otherwise noted, equivalent. the chemically reactive groups with which Such Solid Sup Alternatively, if a beta-hydroxy acid is used as the 45 ports may be derivatized are those commonly used for Solid hydroxyl component, tetronic acids can be prepared: phase synthesis of the polymer and thus will be well known to those skilled in the art, i.e., carboxyls, amines and

O O hydroxyls. To improve Washing efficiencies, one can employ nonpo 50 rous Supports or other Solid Supports less porous than typical peptide Synthesis Supports, however, for certain applications of the invention, quite porous beads, resins, or other Supports work well and are often preferable. A preferred Support is glass, as described in U.S. Pat. No. 5,143,854, Supra. 55 Another preferred Solid Support is resin, Such as the beads described in co-pending U.S. patent application Ser. No. 07/946,239, filed Sep. 16, 1992, Supra. In general, the bead Size is in the range of 1 nm to 100 um, but a more massive Solid Support of up to 1 mm in Size may Sometimes be used. 60 Particularly preferred resins include Merrifield resin, pref erably pre-loaded with an amino acid ester, hydroxymethyl polystyrene resins, ArgoGel (available from Argonaut, S. San Francisco, Calif.); Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland); and 65 TentaGel S AC, TentaGel PHB, or TentaGel S NH resin (polystyrene-polyethylene glycol copolymer resins avail able from Rappe Polymere, Tubingen, Germany). Other 5,874,589 11 12 preferred Supports are commercially available and described arylalkyl or salts thereof. More preferably, the amine com by Novabiochem, La Jolla, Calif. ponent will have the formula: Another preferred Solid Support comprises a “Soluble' polymer Support. Typically, polyethylene glycol, polyvinylalcohol, polyvinylalcohol co-polymerized with polyvinyl pyrrolidine or derivatives thereof is used as the -N NH soluble support. See Janda and Hyunsoo (1996) Methods Enzymol. 267:234-247; Gravert and Janda (1997) Chemical wherein R and R are independently selected from the Reviews 97:489–509; and Janda and Hyunsoo, PCT publi groups consisting of hydrogen, alkyl, aryl, heteroaryl, cation No. WO 96/O3418. carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl. The 2. Linkers amine component, if not commercially available, can be When bound to a solid support, the tetramic acid prepared by Standard chemical procedures. precursors, e.g., the amine component or the B-keto amide, In a preferred embodiment, the amine component will are usually attached by means of one or more molecular 15 comprise an amino acid, and more preferably, an amino acid linkers. The linker molecules preferably have lengths suffi cient to allow the compounds to which they are bound to bearing a Substituent on the alpha carbon. The amino acids interact freely with any molecules exposed to the Solid finding utility in the present invention include the twenty Support Surface, Such as Synthetic reagents or receptors naturally occurring C.-amino acids, in either their D- or which are an object of Study. The linker molecule, prior to L-enantiomeric forms. Unnatural amino acids Such as C, attachment, has an appropriate functional group at each end, C.-disubstituted amino acids, N-alkyl amino acids, lactic one group appropriate for attachment to the Support and the acid, and other unconventional amino acids are also Suitable other group appropriate for attachment to the compound components. Examples of unconventional amino acids being prepared. include, but are not limited to: 4-hydroxyproline, O-phosphoSerine, 3-methylhistidine, 5-hydroxylysine, and One can, of course, incorporate a wide variety of linkers, 25 depending upon the application and the effect desired. For other Similar amino acids. Since peptides are composed of instance, one can Select linkers that impart hydrophobicity, amino acid Subunits, one of skill in the art will appreciate hydrophilicity, or steric bulk to achieve desired effects on that peptides can also serve as amine components. properties Such as coupling or binding efficiency. In one Preferably, the amino group of the amine component is aspect of the invention, branched linkers, i.e., linkers with alkylated. bulky side chains such as the linker, Fmoc-Thr(tBu), are If the amine component possesses reactive Sites other than used to provide rigidity to or to control Spacing of the the amino group, it may be necessary to protect them during molecules on the Solid Support in a library. In Some the synthesis. Suitable protecting groups include acid-labile, embodiments, cleavable linkers will be used to facilitate an base-labile, photoremovable, or removable under neutral assay or detection Step as discussed more fully below. 35 conditions. See, e.g., Green, Protecting Groups in Organic 3. Immobilization Synthesis, Wiley 1985, pp. 218-288, which is incorporated The choice of functionality used for binding a molecule to herein by reference. The choice of a particular protecting the Solid Support will depend on the nature of the compound group will be determined generally by the conditions under to be Synthesized and the type of Solid Support. Conditions which the tetramic acid thereof is formed and by the types for coupling monomers and polymers to Solid Supports 40 of protecting groups used on the Side chains of the other through a wide variety of functional groups are known in the components to be used in Synthesis. In a most preferred art. See, e.g., U.S. Pat. No. 4,542,102; U.S. Pat. No. 4,282, embodiment, the protecting groups are photoremovable and 287; Merrifield, “Solid Phase Peptide Synthesis,” J. Am. Chem. Soc., (1963) 85:2149-2154; Geysen et al., “Strategies their removal is accomplished by exposing the Surface or for Epitope Analysis. Using Peptide Synthesis,” J. Imm. 45 Selected regions thereof to light (e.g., from a light Source Meth., (1987) 102:259-274; Pirrung et al., U.S. Pat. No. through a mask) or removable under neutral conditions. 5,143,854; and Fodor et al., “Light-Directed Spatially Such protecting groups and techniques are described in U.S. Addressable Parallel Chemical Synthesis,” Science (1991) Pat. No. 5,148,854 and co-pending U.S. patent applications 251:767-773, each of which is incorporated herein by Ser. No. 07/624,120, filed Dec. 6, 1990, and 07/971,181, reference. 50 filed Nov. 2, 1992. If the amine component comprises a beta-amino acid B. The Amine Component rather than an alpha-amino acid, a pentamic acid derivative According to the present invention, an amine component is formed upon contact of the amine component with a is coupled to a 3-keto ester equivalent to yield a B-keto B-keto ester and Subsequent cyclization and cleavage: amide which can then be cyclized with concomitant cleav 55 age of the resulting tetramic acid from the Solid Support. The amine component typically will be attached to a Solid Support and thus, should include a functionality which can covalently bind the molecule to the Solid Support (e.g., an activated carbonyl, acyl halide, ester, amide, thioester, or 60 activated hydroxyl) as well as the amino group or a protected derivative thereof. Typically the amine component will comprise a primary amine having the formula: R-NH2, wherein R is selected from the group consisting of hydrogen (i.e., an amine Salt as 65 a further Valence is necessary to attach the amine component to the Solid Support), alkyl, amino, aryl, heteroaryl, and 5,874,589 13 14 groups as discussed above. Among a wide variety of pro -continued tecting groups are materials. Such as Fmoc, BOC, t-butyl esters, t-butyl ethers, and the like. Various exemplary pro tecting groups are described in, for example, Atherton et al., 5 Solid Phase Peptide Synthesis, IRL Press (1989), incorpo rated herein by reference and U.S. Pat. No. 5,148,854. O O Each procedure for this reaction is preferably conducted R in a single reaction medium employing an inert diluent or a R3 mixture of inert diluents. The inert diluent employed in the reaction is not critical and Suitable diluents include, by way O N R1 of example only, acetonitrile, benzene, toluene, methylene chloride, chloroform, carbon tetrachloride, tetrahydrofuran R2 and the like. Typically, the reactions will be conducted at a temperature of from about 0° C. to about 100° C. More C. The B-keto ester equivalent 15 preferably, a temperature of about ambient temperature will The amine component discussed above is contacted with be used. a B-keto ester equivalent to yield a B-keto amide. Preferably, B. The Reductive Amination the 3-keto ester equivalent will comprise an acyl Meldrums According to Some embodiments, the amino group of the acid, a lower alkyl ester of a B-keto carboxylic acid, or an bound first amino acid is alkylated. To effect this acyl chloride of a f-keto carboxylic acid. Most preferably, transformation, the bound first amino acid can be treated the 3-keto ester equivalent will comprise an acyl Meldrums with conventional alkylating agents, such as RCH2X, acid. Acyl Meldrum's acids can be readily prepared through where X is bromine or iodine and where R can be hydrogen the reaction of a carboxylic acid or acyl chloride with or as discussed below; or with RCOOH to form the amide Meldrums acid. which can then be reduced, for example, with diborane to The B-keto ester equivalent can bear a wide variety of 25 yield the alkylamine. substitutents. For example, R can be selected from the groups consisting of hydrogen, alkyl, aryl, heteroaryl, In a preferred embodiment, the bound first amino acid is carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl, each of treated with an aldehyde of formula RCHO where R is which can be optionally substituted. alkyl, aryl, heteroaryl, aralkyl, or heteroaralky or a ketone of In a preferred embodiment, the amine component will formula R COR where R and R are independently comprise the limiting reagent and an excess of the B-keto Selected from the group consisting of alkyl, aryl, heteroaryl, ester equivalent will be used. Typically, the ratio of amine aralkyl, or heteroaralky. (One of skill in the art will appre component to B-keto ester equivalent will range from about ciate that any protecting groups present on the amine group 1:1.1 to about 1:100, preferably from about 1:1 to about of the bound first amino acid should be removed prior to the 35 reductive amination reaction.) Preferably, the reductive ami 1:10. nation is performed in the presence of a dehydrating reagent, IV. The Method-Tetramic Acids as Standard Solid phase reaction conditions (aldehyde, sodium cyanoborohydride, 1% acetic acid in DMF) may A. Immobilization of the Amine Component result in over alkylation of the amino acid and yielded the AS discussed above, the amine component will include a 40 corresponding N,N-dialkyl amino acid. Preferred dehydrat functionality that covalently binds the molecule to the Solid ing agents include molecular Sieves, magnesium Sulfate, Support, e.g., activated carbonyl, acyl halide, ester, amide, or Sodium Sulfate, trimethyl orthoformate, Zinc chloride, and thioester, in addition to the amino group. The choice of the like. More preferably, the dehydrating agent is in a form functionality depends on the nature of the monomer and the which can be easily washed away from the Solid Support or type of Solid Support. Conditions for coupling through a 45 is even used as the Solvent. Most preferably, the dehydrating wide variety of functional groups are known in the art. agent comprises trimethylorthoformate. Preferably, Mukaiyama reagent is utilized to effect the Typically, an excess of both the aldehyde and the reducing coupling. Other preferred reagents useful for coupling the agent is used. Preferably, at least 5 equivalents of aldehyde amine component to the solid support include DIC/DMAP and/or at least 5 equivalents of reducing agent is used. More and DIAD/PPh. Typically, between about 0.1 and about 0.5 50 preferably, the alkylation is performed with about 10 equiva equivalents of DMAP or between about 5-10 equivalents of lents or more of aldehyde and about 10 equivalents or more DIC, based on resin loading, will be used. These solid phase of reducing agent. Synthesis procedures are well known in the art and further The reaction times and procedures will vary with the described, for example, by Stewart and Young, Solid Phase reactivity of the aldehyde. For example, with a short alkyl Peptide Synthesis (2nd Ed., Pierce Chemical Company, 55 aldehyde with little Steric hindrance (e.g., propanal or 1984). isovaleraldehyde), the bound first amino acid derivative is Typically, the amine component is coupled via its carboxy contacted with the aldehyde, preferably in the presence of terminus to the Support. Preferably, a Support having the dehydrating agent. The reaction mixture is Shaken or hydroxyl groups at the surface will be utilized. Suitable solid stirred for 30-60 minutes and then the reducing agent is Supports include TentaGel, ArgoGel, Merrifield resin and 60 added, preferably with additional dehydrating agent. For hydroxy-PAM resin (available from Advanced Chemtech). alkyl aldehydes with more steric hindrance (e.g., heptanal, In a preferred embodiment, Merrifield resin, ArgoGel or a t-butylacetaldehyde, and cyclohexane carboxaldehyde), the TentaGel hydroxy resin is used. Most preferably, a resin above procedure is followed. However, shortly after the preloaded with amino acid esters is used. reducing agent is added (e.g., 10 minutes), to the reaction is The active sites of the Surface and/or the amine compo 65 added a dilute solution of a protic solvent, preferably a 5% nent are optionally protected initially by protecting groups or less solution of a lower alkyl alcohol in trimethyl which may be acid-, base-, or photoremovable protecting orthoformate, and more preferably about a 1% aqueous 5,874,589 15 16 solution of methanol in trimethyl orthoformate. For aromatic light at 470-480 nm with molar absorptivities in the range and Sterically hindered alkyl aldehydes (e.g., benzaldehyde, of 3-9x10°. Preferably, the assay involves the dissolution of pivaldehyde, and 2-ethylbutanal), the procedure for short the compounds to be tested in a known volume of Solvent. alkyl aldehydes is followed. However, a dilute solution of a An aliquot is removed and diluted to yield a Solution with weak acid, preferably a 5% or leSS Solution of acetic acid in 290 nm absorbance of 0.5-1.0 absorbance units. Beers law trimethyl orthoformate, more preferably about a 1% solution then allows for the calculation of the concentration of the of acetic acid in trimethyl orthoformate, is added immedi Subject compound. ately after the addition of the reducing agent. V. Methods-Analogs The reductive amination reaction can be followed by ninhydrin test or the Kaiser test. However, it should be noted Other embodiments of the invention are drawn to the that the preformed imines also give a red or brown color in Synthesis of analogs of tetramic acids, including thiotetronic the ninhydrin test and Some are quite Stable to hydrolysis acids, tetronic acids, pentonic acids, and thiopentonic acids therefore falsely indicating a completed reaction. Samples and derivatives thereof. This class of compounds can be for the Kaiser test should be heated at least for 15 minutes. produced by using a hydroxyl component or a thiol com 15 ponent instead of the amine component. C. Preparation of beta-keto acid equivalents More specifically, in a preferred embodiment, a hydroxyl Beta-keto acid equivalents may be obtained via the acy component is immobilized on the Solid Support. According lation of Meldrum's acid. Typically, Meldrums acid is to Some embodiments, the hydroxyl components can be treated with an acid chloride and DMAP in the presence of produced via a B-keto acid which has been immobilized, a base, Such as pyridine. Alternatively, the acylation may be preferably via its carboxy terminus. Reduction of the car carried out by the in Situ activation of a carboxylic acid with bonyl group yields the corresponding B-hydroxy acid, i.e., DCC and DMAP. the hydroxyl component. Esterification of the hydroxyl D. Treatment with the beta-keto acid equivalent component with a beto-keto ester derivative followed by The resin-bound secondary amine is treated with 5-10 Diekmann cyclization and concomitant release from the equivalents of the beta-keto acid equivalent in an apolar 25 Support yields the tetronic or pentonic acid acid. Analogs of Solvent, Such as toluene. Typically, the reaction is conducted this basic ring Structure can also be prepared using the at an elevated temperature, for example, between about 30 components and basic protocols described above. C. and 100° C. Care must be taken to ensure that cyclative Typically the hydroxyl component will comprise a Sec cleavage does not occur during this step. The reaction is ondary hydroxyl having the formula: usually complete within 4-6 hours. ExceSS reagents are removed and the resin is immediately Subjected to the cyclization and cleavage protocol described below. E. Cyclization and cleavage R2 OH The Diekmann intramolecular C-acylation (i.e., cyclization) and cleavage reactions can occur under either 35 wherein R and R are independently selected from the basic or acidic conditions. The preferred basic conditions groups consisting of hydrogen, alkyl, aryl, heteroaryl, involves treatment of the resin with one theoretical equiva carboxyl, carboxyalkyl, carboxyaryl, and arylalkyl. The lent of a dilute base in a polar, protic Solvent, Such as 0.1M hydroxyl component, if not commercially available, can be potassium hydroxide in methanol, diluted 1:1 with DCM. prepared by Standard chemical procedures. In a preferred Alternatively, a dilute aqueous base in a polar, protic Solvent, 40 embodiment, the hydroxyl component will comprise a car Such as 0.8% acqueous Sodium hydroxide in aqueous metha boxylic acid or ester having a hydroxyl group at the beta nol can be employed, followed by treatment with a H" carbon. Likewise, the thiol component will comprise the eXchange resin. Preferred acidic conditions include the use analogous Structure wherein the hydroxyl group has been of dilute aqueous acid in a polar protic Solvent, Such as 1% replaced with a thiol group. acetic acid in methanol or 1% TFA in methanol, or the use 45 A number of the hydroxy acids are commercially avail of H exchange resin. able. In addition, alpha-hydroxy acid analogs of the amino F. Results acids can be prepared from commercially available amino A number of Structurally diverse tetramic acids have been acids (or any hydroxy acid). For carboxylic acids having prepared using the described metholodolgy. AS evidenced by additional Side chain functional groups, Such as Serine, the uniformly high yields that were obtained this approach 50 threonine, and tyrosine, typically the Side chain functional is compatible with a variety of building blocks. group will be protected (with a protecting group PG) prior Significantly, it has been found that racemization was leSS to diazotization. Examples of Suitable Side chain protecting than 5% with the optical purity of the amino acids being groups include, but are not limited to, t-butyl esters (for Glu retained in the tetramic acids. and Asp); t-butyl ethers (for Ser, Thr, and Tyr); BOC (for 55 LyS); and trityl amides (for Gln and ASn). Preferred substituents at the 1, 3 and 5 positions of the This synthetic route involves (1) diazotization with tetramic acid ring which are prepared via the methods Sodium nitrite in mild acid, Such as acetic acid or citric acid; described herein from Starting materials either known per Se and (2) carboxy protection, preferably as the triisopropylsi in the art or which can be prepared by art recognized lyl group, and protection of the hydroxy group, typically methods are described above. 60 with Fmoc-Cl; and carboxy deprotection. This reaction G. ASSay Sequence is Stereospecific and results in retention of the An assay has been developed which provides an estimate chirality of the carbon bearing the amino group (and thus, of the amount of tetramic acid present after cleavage. More the carbon bearing the hydroxyl group). This latter multi Specifically, it has been determined that Several tetramic Step Sequence can be performed without intermediate prod acids demonstrate an unique UV absorption at 280–290 nm 65 uct isolation and purification. This reaction Sequence will with molar absorptivities in the range of 2–4x10". In generally produce the desired hydroxy acid in 35-70% addition, the complex of Fe" and tetramic acids absorbs isolated overall yield. 5,874,589 17 18 The hydroxyl component may also be formed via binding methods described in International Patent Application Pub a beta-keto ester equivalent to the Solid Support and reducing lication No. 93/06121 to prepare synthetic libraries of tet the ketone group via conventional means appreciated in the ramic acids. This application is incorporated herein by art. reference in its entirety. If the hydroxyl component possesses reactive sites other 5 than the amino group, it may be necessary to protect them It is understood, however, that the term “single com during the Synthesis. Suitable protecting groups include pound” as used herein includes different regio and Stereoi acid-labile, base-labile, photoremovable, or removable Somers of that compound. Also, the term “single compound” under neutral conditions. See, e.g., Green, Protecting does not mean that only one copy of that compound is Groups in Organic Synthesis, Wiley 1985, pp. 218-288, attached to each Support. Rather, multiple copies of that which is incorporated herein by reference. The choice of a compound can be included on the Support. particular protecting group will be determined generally by Preferably, the library will contain at least about 10° the conditions under which the tetramic acid thereof is compounds, more preferably from about 10° to about 10" formed and by the types of protecting groups used on the compounds and still more preferably from about 10 to Side chains of the other components to be used in Synthesis. about 10 compounds. In a preferred embodiment, the protecting groups are pho 15 In another preferred aspect of this embodiment, each Solid toremovable and their removal is accomplished by exposing Support is tagged with an identifier tag that can be easily the Surface or Selected regions thereof to light (e.g., from a decoded to report the compounds formed on the Solid light Source through a mask) or removable under neutral Support. The tag can be directly attached either to the Solid conditions. Such protecting groups and techniques are Support or the tag can be included on the compound itself. described in U.S. Pat. No. 5,148,854 and co-pending U.S. 2O In this latter embodiment, cleavage of the compound from patent application Ser. Nos. 07/624,120, filed Dec. 6, 1990, the solid support will still permit identification of the com and 07/971,181, filed Nov. 2, 1992. pound. Each of these embodiments is disclosed in Interna The immobilized B-keto acid, i.e., the precursor of the tional Patent Application Publication No. WO 93/06121. hydroxyl component can be prepared as shown below: 25 Alternatively, a portion of the same compounds attached to a single Support is cleaved and Subjected to mass >< Spectroscopy, nuclear magnetic resonance Spectroscopy and/ O O or other forms of direct Structural analysis So as to identify the compound on the Support. O 4N-s O -- 3O Still another method for incorporating a tag with the Solid Support is disclosed in U.S. patent application Ser. No. TMS-O 08/146,886 and entitled “METHOD OF SYNTHESIZING DIVERSE COLLECTIONS OF COMPOUNDS which application is incorporated herein by reference in its entirety. O l 35 Additionally, libraries of compounds attached to Solid R X Supports can be used for a variety of additional uses as Set forth in International Patent Application Publication No. WO 93/06121. D< O O O VII. Preparation of Derivatives and Analogs The ring Systems described above can be further manipu lated. For example, 3-acyl-2,4-thiophenediones and 3-acyl 2,4-furandiones can be readily prepared. In addition, cata HO R1 lytic hydrogenation can also be used to reduce one or more 45 of the carbonyl bonds. O O Alkylidene derivatives can also be produced by the reac

tion of a tetramic acid ring having no Substitution on the 5 -N- Oalkyl + HO ( position of the ring with an aldehyde. See, e.g., Harhashet al. X 50 (1973) Ind. J. Chem. 11:128–130. The carbonyl group of the ring System is also amenable to One of skill in the art will readily recognize that derivatives further derivitization. For example, the tetramic acids can be of the resulting beta keto ester can be produced by treatment reacted with hydroxyl amine to generate the corresponding of the compound with two equivalents of Strong base, 4-oximyl derivative. See, e.g., Diurnoet al. (1992) J. Med. followed by an alkyl halide. 55 Chem. 35:2910-1912. Likewise, the tetramic acid ring can More specifically, Meldrums acid can be contacted with be treated with a substituted hydazine to provide a source of a Solid Support bearing hydroxyl groups, followed by treat further diversity. ment with an acylating agent to yield the desired immobi Additionally, if further functionality is present on the lized B-keto acid. Likewise, the immobilized B-keto acid can tetramic acid ring System, this functionality can be modified. be produced through the treatment of a Solid Support bearing 60 For example, if one of the substituents is -CHCOOH, then hydroxyl groups with an acylated derivative of Meldrums this acid functionality may be further reacted with a variety acid or a lower alkyl ester of a beta-keto carboxylic acid. of amines, thiols, alcohols, and the like to produce amides, thioesters, esters, etc. Techniques for the further manipula VI. Method for Producing Large Synthetic tion of a Support-bound carboxyl group are known in the art Libraries 65 and can be found in copending U.S. application Ser. Nos. The above described synthetic methods can be incorpo 08/201,607, filed Feb. 25, 1994, and 08/179,741, filed Jan. rated into one or more reaction procedures in the Stochastic 11, 1994, each of which is incorporated herein by reference. 5,874,589 19 20 Alternatively, if one of the Substituents is an amino group, continued for an additional 60 min. The resin was washed 3x the corresponding tetramic acid could be derivatized with a with MeOH and 3x with DCM. Acylation. The resin was variety of isocyanates, carboxylic acids, amino acids, and suspended in toluene (3 mL) and the acyl Meldrum's acid (6 the like using techniques known in the art. eq.) was added. The mixture was heated at 65° C. for 6 hr. The above examples are illustrative; other The resin was washed 3x with toluene and 3x with DCM. transformations, Such as alkylations, acylations, and the like, Cyclative cleavage. The resin was Suspended in a 1:1 will be apparent to those skilled in the art. mixture of DCM/MeOH, and a solution of KOH in MeOH (0.1M, 1.0 eq.) was added. After shaking for 30 min the resin EXAMPLES was removed by filtration and washed with MeOH/DCM. The following examples are set forth to illustrate the The filtrates were combined and the solvent removed in claimed invention and are not to be construed as a limitation vacuo to afford the potassium Salt of the 3-acyltetramic acid thereof. 7. The potassium salt was converted to the free acid by Unless otherwise Stated, all temperatures are in degrees dissolving it in DCM, washing with 1M HCl, drying over Celsius. Also, in these examples, unless otherwise defined Na2SO, and removing the Solvent in vacuo. below, the abbreviations employed have their generally 15 Examples of tetramic acids: The following lists represen accepted meaning: tative examples of tetramic acids produced by the general FMOC=fluorenylmethyl oxycarbonyl procedures described above and isolated yields for these "H-nmr=proton nuclear magnetic resonance compounds. HPLC=high performance liquid chromatography mL=milliliter OH / O mmol=millimol NMP=N-methylpyrrolidone 1S N-R TFA=trifluoroacetic acid 25 THF=tetrahydrofuran O till-microliters R1 General procedures: Unless otherwise noted, materials were obtained from commercial Suppliers and used without Cpd R R Rs Isolated Yield further purification. 7a. (S)-PhCH, n-Propyl Ethy 69% General Procedure for the Preparation of Acyl-Meldrums 7b (S)-PhCH, n-Propyl Ph 92% 7c (S)-PhCH, n-Propyl PhCH, 51% Acids: 7d (S)-PhCH, PhCH, Ethy 74% Method A: Acyl Chlorides. A solution of Meldrum's acid (50 fe (S)-PhCH, PhCH, Ph 92% mmol) and DMAP (100 mmol) in DCM (150 mL) was 7 (R)-PhCH, PhCH, Ph 76% 35 7g (S)-PhCH, PhCH, PhCH, 66% cooled under argon in an ice-salt bath to -5°C. A Solution 7h (S)-PhCH, (CH)CH Ethy 73% of the acid chloride (60 mmol) in DCM (50 mL) was added 7i. (S)-PhCH, (CH)CH, Ph. 80% dropwise over 60 min, maintaining the temperature below 0 7 (S)-PhCH, (CH)CH, PhCH, 77% C. during the addition. The resulting Solution was Stirred at 7k (S)-i-Propyl n-Propyl Ethy 64% 7 (S)-i-Propyl n-Propyl Ph 64% O C. for 60 min, then at room temperature for an additional 7m (S)-i-Propyl n-Propyl PhCH, 68% 60 min. The reaction mixture was washed with 1M HCl 40 7n (S)-i-Propyl PhCH, Ethy 55% (3x35 mL) and water (50 mL), dried over MgSO, and the Fo (S)-i-Propyl PhCH, Ph 61% Solvent removed in vacuo. The residual Solid was recrystal 7 (S)-i-Propyl PhCH, PhCH, 59% 7q (S)-i-Propyl (CH)CH Ethy 43% lized from acetone to afford the product. 7r (S)-i-Propyl (CH)CH, Ph. 54% 5-Benzoyl-2,2-dimethyl-1,3-dioxane-4,6-dione. 7s (S)-i-Propyl (CH)CH, PhCH, 53% Method B: Carboxylic Acids. To a solution of the carboxylic 45 7 (S)-p-HOCHCH, PhCH, Ph 67% acid (20 mmol), Meldrum's acid (20 mmol), and DMAP (40 7 (S)-HOCH, PhCH, Ph 51% mmol) in DCM (60 mL) at room temperature was added DCC (20 mmol). After stirring for 60 min, the precipitated dicyclohexylurea was removed by filtration and the filtrate Preparation of C.-Hydroxy Acids from C.-Amino washed with an ice-cold solution of 10% HCl (75 mL). The 50 organic layer was dried over Na2SO and the Solvent Acids removed in vacuo. The residual Solid was recrystallized 5.1 Method A from ethyl acetate/petroleum ether to afford the product. To D-phenylalanine (2.00 g, 12.4 mmol) in water (20 5-(Phenylacetyl)-2,2-dimethyl-1,3-dioxane-4,6-dione. mL), was added 3M HCl (20 mL, 60 mmol) and acetic acid 5-Propionyl-2,2-dimethyl-1,3-dioxane-4,6-dione. 55 (10 mL). The solution was cooled to 0°C., and an aqueous General Procedure for the Solid Phase Synthesis of solution of sodium nitrite (3.92 g, 56.8 mmol, in 7.5 mL of 3- Acyl-2,4-pyrrollidine diones: Boc-Xaa-O-Resin water) was added dropwise over 10 minutes. The reaction (Merrifield, 250 mg, substitution 0.5-0.9 med/g) was swol was Stirred for eight hours, then more Sodium nitrite was len in DCM and deprotected by treatment with a 25% TFA added (3.92 g, 56.8 mmol, in 7.5 mL of water). After six in DCM Solution containing 1 mg/mL indole, once for 2 min 60 hours, the solution was washed with ether (3x25 mL), and and once for 30 min. After washing 5x with DCM, the resin the combined organic layers were extracted with Saturated was neutralized with a 10% TEA in DCM solution(2x) and sodium bicarbonate (3x30 mL). The bicarbonate washes washed 5x with DCM. Reductive alkylation. The resin was were combined, acidified to pH 1 with concentrated HCl, suspended in a 1:1 mixture of DCM/TMOF (3 mL), and and then extracted with ether (3x30 mL). The organic layers acetic acid (30 mL) and the aldehyde (20 eq.) were added. 65 were combined, dried (magnesium Sulfate), filtered, and After Shaking for 30 min at room temperature, Sodium then concentrated to afford 1.65 g of a white waxy solid cyanoborohydride (30 eq.) was added and shaking was (80%). 5,874,589 21 22 5.2 Preparation of (D)-O-nitroveratryloxy-methoxy-2-oxy We claim: 4- methylpentanoic Acid 1. A method of Synthesizing compounds comprising tet The hydroxy acid prepared above was esterified using ramic acids, pentamic acids, and derivatives thereof, com diazomethane, and to the resulting compound, methyl (D)- prising the Steps of 2-hydroxy-4-methyl valerate (1.0g, 6.85 mmol) in 10 mL of 5 (a) on the Surface of a Solid Support, providing an immo methylene chloride was added Nvom chloride (2.69 g, 10.5 bilized amine component; mmol) and diisopropylethyleneamine (DIEA, 1.8 mL). After (b) contacting said immobilized amine component with a 12 hours, the reaction mixture was diluted with methylene B-keto ester equivalent to yield a B-keto amide, and chloride (20 mL), washed with water (2x15 mL), and the (c) treating the B-keto amide under conditions Suitable for organic layer dried (over MgSO). The product was purified the cyclization and concomitant release of the com by Silica gel chromatography (4:1 petroleum ether:ethyl pound from the Solid Support. acetate) to afford 1.2 g of material (47%). The free acid was 2. The method of claim 1, wherein the step of providing obtained in quantitative yield by hydrolysis with lithium an immobilized amine component comprises the Step of hydroxide, as previously described. One could also prepare binding an amino acid to the Support to form an immobilized this compound, with possibly higher yield and Shorter reac 15 amino component. tion time, using sodium hydride/THF instead of DIEA/ 3. The method of claim 2, wherein the amino acid is an dichloromethane. alpha amino acid and the compound Synthesized by Said 5.3 Preparation of Side Chain Functionalized C-O-FMOC method is a tetramic acid. Hydroxy Acids 4. The method of claim 2, wherein the amino acid is a beta To a solution of the hydroxy acid prepared above (with the amino acid and the compound Synthesized by Said method is Side chain functionality protected, 1 mmol) in dry pyridine a pentamic acid. at 0° C. is added N-methylmorpholine (2.5 mmol) and 5. The method of claim 1 or 2, wherein the amine triisopropylsilyl chloride (1.1 mmol). The solution was component is alkylated. stirred for 15 minutes. To the reaction mixture is then added 6. The method of claim 1, wherein the solid support is a FMOC-Cl (1.1 mmol) and additional N-methylmorpholine. 25 polymer bead. The reaction is stirred for 4-8 hours until complete. To the 7. The method of claim 1, wherein the B-keto ester mixture is then added tetrabutylammonium fluoride (2.5 equivalent is an acyl Meldrum's acid. mmol). The reaction mixture is stirred for 15 minutes and quenched. k k k k k